Glucocorticoids: protectors of the brain during innate immune responses.

Abstract

The innate immune response is a coordinated set of reactions involving cells of myeloid lineage and a network of signaling molecules. Such a response takes place in the CNS during trauma, stroke, spinal cord injury, and neurodegenerative diseases, suggesting that macrophages/microglia are the cells that perpetuate the progressive neuronal damage. However, there is accumulating evidence that these cells and their secreted proinflammatory molecules have more beneficial effects than detrimental consequences for the neuronal elements. Indeed, a timely controlled innate immune response may limit toxicity in swiftly eliminating foreign materials and debris that are known to interfere with recovery and regeneration. Each step of the immune cascade is under the tight control of stimulatory and inhibitory signals. Glucocorticoids (GCs) act as the critical negative feedback on all myeloid cells, including those present within the brain parenchyma. Because too little is like too much, both an inappropriate feedback of GCs on microglia and high circulating GC levels in stressed individuals have been associated with deleterious consequences for the brain. In this review, the authors discuss both sides of the story with a particular emphasis on the neuro-protective role of endogenous GCs during immune challenges and the problems in determining whether GCs can be a good therapy for the treatment of neuropathological conditions.

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The Neuroscientist
DOI: 10.1177/1073858404263494
2004; 10; 538 Neuroscientist
Isaias Glezer and Serge Rivest

Glucocorticoids: Protectors of the Brain during Innate Immune Responses
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538THE NEUROSCIENTIST
Copyright © 2004 Sage Publications
ISSN 1073-8584
Brain Immunity and Glucocorticoids
More than one century ago, two separated systems by
which the host fights against infections were described.
Erlich identified the antitoxins, latter denominated anti-
bodies, which belong to a form of protection defined as
adaptive immunity. The phagocytes, described by
Metchnikoff, confers another kind of protection that is
present in almost all the animals, the innate immunity
(Beutler and Poltorak 2001). Although innate immunity
has for a long time been thought to be a nonspecific
reaction, adaptive immunity is characterized by the great
specificity for distinct molecules, specialization,
immunologic memory, and the ability to respond strong-
ly to repeated exposures to the same microbe. Although
conceptually the two responses diverge, innate and adap-
tive immunities compose an integrated system in which
many cells and molecules function in a cooperative man-
ner (Abbas and others 1997).
The innate immune system is based on receptors that
recognize certain molecular patterns found in microbes
but not in self-tissues. These patterns can be found in
lipopolysaccharide (LPS) present in the cell wall of
gram-negative bacteria and in many other structural
components of pathogens, such as gram-positive bacte-
ria, fungi, viruses, and so on. These molecules are
denominated pathogen-associated molecular patterns
(PAMPs). Their respective receptors are known as pat-
tern recognition receptors (PRRs) (Janeway 2001).
PRRs can be divided into three functional classes:
signaling receptors, endocytic receptors, and secreted
proteins (Kopp and Medzhitov 1999). The release of
inflammatory mediators by circulating monocytes or
tissue-resident macrophages in response to PAMPs
requires sequential signaling steps. The details of these
events have been described recently with the characteri-
zation of Toll-like receptors (TLRs), which are prototype
signaling PRRs.
The Toll protein was discovered as an essential mole-
cule to establish dorsoventral axis in Drosophila
embryos, and it was also found to be involved in anti-
fungal responses, probably being an ancestral immuno-
logic receptor. TLRs are mammalian homologues of this
protein expressed on the surface of a group of immune
cells known as antigen-presenting cells (APCs) (Akira
and others 2001). Eleven different TLRs have been
Glucocorticoids: Protectors of the Brain during
Innate Immune Responses
ISAIAS GLEZER and SERGE RIVEST
Laboratory of Molecular Endocrinology, CHUL Research Center
Department of Anatomy and Physiology, Laval University
The innate immune response is a coordinated set of reactions involving cells of myeloid lineage and a net-
work of signaling molecules. Such a response takes place in the CNS during trauma, stroke, spinal cord
injury, and neurodegenerative diseases, suggesting that macrophages/microglia are the cells that perpetu-
ate the progressive neuronal damage. However, there is accumulating evidence that these cells and their
secreted proinflammatory molecules have more beneficial effects than detrimental consequences for the
neuronal elements. Indeed, a timely controlled innate immune response may limit toxicity in swiftly elimi-
nating foreign materials and debris that are known to interfere with recovery and regeneration. Each step
of the immune cascade is under the tight control of stimulatory and inhibitory signals. Glucocorticoids (GCs)
act as the critical negative feedback on all myeloid cells, including those present within the brain parenchy-
ma. Because too little is like too much, both an inappropriate feedback of GCs on microglia and high cir-
culating GC levels in stressed individuals have been associated with deleterious consequences for the
brain. In this review, the authors discuss both sides of the story with a particular emphasis on the neuro-
protective role of endogenous GCs during immune challenges and the problems in determining whether
GCs can be a good therapy for the treatment of neuropathological conditions. NEUROSCIENTIST
10(6):538–552, 2004. DOI: 10.1177/1073858404263494
KEY WORDS Innate immune response, Inflammation, Glucocorticoids, Proinflammatory cytokines, Microglia, Neurodegeneration,
Neuroprotection
Our research on this subject is supported by the Canadian Institutes of
Health Research (CIHR, the former Medical Research Council of
Canada [MRCC]). S.R. is an MRCC scientist and holds a Canadian
Research Chair in Neuroimmunology. I.G. is supported by a postdoctoral
training fellowship from the Multiple Sclerosis Society of Canada.
Address correspondence to: Serge Rivest, Laboratory of Molecular
Endocrinology, CHUL Research Center, Department of Anatomy and
Physiology, Laval University, 2705, boul. Laurier, Québec, Canada
G1V 4G2 (e-mail: serge.rivest@crchul.ulaval.ca).
REVIEW I
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Volume 10, Number 6, 2004THE NEUROSCIENTIST539
described, and the major ligands and respective recep-
tors could be briefly described as follows: bacterial
lipoproteins/lipopeptides (from a variety of microbial
products), peptidoglycan and lipoteichoic acid (from
gram-positive bacteria), lipoarabinomannan (from
mycobacteria), zymosan (from fungi), and atypical LPS
(from Leptospira interrogans and P . gingivalis), recog-
nized by TLR2 conjugated to TLR6 or TLR1; double-
stranded RNA (dsRNA, from viruses), recognized by
TLR3; LPS (from gram-negative bacteria), taxol (from
plants), fusion proteins, heat shock protein (HSP) 60 and
70 (from host and Chlamydia pneumoniae), and fibrino-
gen (from host), recognized by TLR4; flagellin, TLR5;
and CpG bacterial DNA, TLR9. Although an authentic
microbial molecule has yet to be identified, TLR7 and
TLR8 (in humans) seem to recognize small antiviral
molecules (Underhill and Ozinsky 2002; Beutler and
others 2003; Kopp and Medzhitov 2003).1
LPS from gram-negative bacteria is probably the most
frequently used stimulus to trigger the signaling path-
ways involved in the control of the innate immunity. Its
recognition involves the binding of LPS to membrane
CD14, which transfers LPS to TLR4 in an MD-
2–dependent manner. The binding of LPS to its cognate
receptor TLR4 leads to intracellular signaling, transloca-
tion of nuclear proteins, and transcriptional activation of
genes associated with the inflammatory processes
(Beutler 2002). The involvement of TLR4 in LPS recog-
nition was substantially accepted due to the identifica-
tion of a missense mutation in the lps locus correspon-
ding to TLR4 in the C3H/HeJ mouse strain, which is
largely refractory to LPS (Poltorak and others 1998).
Furthermore, TLR4-deficient mice are also unrespon-
sive to LPS but not to cell wall components derived from
gram-positive bacteria (Hoshino and others 1999). The
activation of TLR4 initiates sequential intracellular sig-
nal steps similar to those observed following interleukin
(IL)-1 receptor (IL-1R) activation, due to the conserved
domains Toll/IL-1R (TIR). The myeloid differentiation
factor 88 (MyD88) is immediately recruited to the TIR
domain after agonist binding to the receptor, resulting in
the activation of serine/threonine kinases from the IL-
1R–associated kinase (IRAK) family. This is followed
by the recruitment of the adaptor molecule tumor necro-
sis factor (TNF)-associated factor 6 (TRAF6) and acti-
vation of IκB kinase (IKK) complex, a critical step for
the release of nuclear factor NF-κB from its cytoplasmic
inhibitor IκB (Beutler 2000; Akira and others 2001).
IKK is composed of two catalytic subunits (IKKα and
IKKβ) and one regulatory subunit (IKKγ/NEMO).
IKKβ is the target of upstream signals generated by
proinflammatory stimuli that give rise to phosphoryla-
tion of IκBα (the predominant and rapidly regulated IκB
isoform) at Ser32/36 and proteosomal degradation after
polyubiquitination. IκBα itself is an NF-κB target gene,
and its de novo synthesis is a crucial autoregulatory loop
(Ghosh and Karin 2002).
The recruitment of MyD88/IRAK/TRAF6 complex
can also activate mitogen-activated protein kinase kinas-
es (MKKs); in particular, the Jun kinase pathway that
leads to the formation of activator protein-1 (AP-1) com-
plex (Jones and others 2001). Nuclear translocation of
NF-κB and AP-1 is one of the most powerful mecha-
nisms to engage transcription of genes encoding most
innate immune proteins, namely, cytokines (IL-1, TNF,
IL-6, etc.), chemokines (MCP-1, IL-8, RANTES, etc.),
proteins of the complement system (C3, C3aR, C5aR,
factor B, etc.), enzymes (cyclooxygenase-2, nitric oxide
synthase, etc.), adhesion molecules (ICAM-1, VCAM-1,
P-selectin, and E-selectin, etc.) and immune response
receptors (CD14, TLR2, IL-6R, etc.). A timely con-
trolled production of these proteins is essential for a
proper innate immune response and elimination of
pathogens (Box 1).
The pathway described above illustrates the canonical
TLR signaling. It should be noted that important updates
have revealed an increasing complexity of these trans-
duction pathways (Kopp and Medzhitov 2003). Several
IRAK genes have been described: IRAK-1, IRAK-4
(both with kinase activity), IRAK-2, and IRAK-M.
Although IRAK-M seems to be a negative regulator of
the TLR signal transduction pathway and could be relat-
ed to the “LPS tolerance” phenomenon, IRAK-4 gene
loss-of-function mutations conferred hyporesponsive-
ness to LPS and IL-1 in a patient with recurrent bacteri-
al infections (Medvedev and others 2003). Actually,
IRAK-4 is believed to play a pivotal role in the develop-
ment of a normal inflammatory response initiated by
LPS, IL-1, and IL-18, which share common signaling
events. Another important role was described for TIR-
associated protein (TIRAP, also called Mal), which
could explain some preserved TLR4 signaling in
MyD88-deficient mice. However, the phenotype of
TIRAP-deficient mice is very similar to those deficient
in MyD88, which exhibit impaired cytokine production
but normal induction of co-stimulatory molecules and
no defect in interferon regulatory factor 3 (IRF3) activa-
tion in dendritic cells treated with LPS. This strongly
suggests that TIRAP is not involved in the MyD88-
independent pathway. A third TIR-containing adaptor
molecule, TIR-domain-containing adaptor inducing
interferon (IFN)-β (TRIF or TICAM-1), was character-
ized. When overexpressed, TRIF strongly induces the
IFN-β promoter, whereas MyD88 and TIRAP do not
(Kopp and Medzhitov 2003). Recently, a germline-
induced mutation in TRIF revealed a severely impaired
response to LPS and abolished cytokine production to
dsRNA, indicating that TLR3 and TLR4 share this spe-
cific proximal transducer. Absence of both TRIF and
MyD88 ablates all responses to LPS in a subpopulation
of macrophages, implying that two signaling pathways
emanate from TLR4 (Hoebe and others 2003).
1. A novel TLR (TLR11) has been characterized, which is activat-
ed by uropathogenic bacteria (Science. 2004 Mar 5;303[5663]:1522-
1526). Moreover, single-stranded RNAs (ssRNAs) from viruses (e.g.,
HIV-1 and influenza) are now believed to be the physiological ligands
for TLR7 and TLR8 (Science. 2004 Mar 5;303[5663]:1529-1531.
Epub 2004 Feb 19).
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540THE NEUROSCIENTISTBrain Immunity and Glucocorticoids
The cascade of signal transduction events triggered by
the binding of ligands to their cognate receptors leads
to the transcriptional activation through the action of
transcription factors. The extracellular domain of
Toll-like receptors contains multiple leucine-rich
repeats, whereas the intracellular domain is similar to
IL-1R: a conserved portion called TIR domain.
Different structural microbial components with the
ability to activate immune response signalize via dis-
tinct TLRs. In general, although PAMPs derived from
gram-positive bacteria bind to TLR2 associated with
TLR1 or TLR6, a complex of TLR4, CD14, and MD2
recognizes lipopolysaccharide (LPS) derived from
gram-negative bacteria. Double-stranded RNA from
viruses is recognized by TLR3, whereas flagellin and
CpG DNA trigger signaling via TLR5 and TLR9,
respectively. TLRs, TNFR1, and IL-1R recruit adap-
tor molecules to their cytoplasmic domains, resulting
in a sequence of events leading to the activation of
proinflammatory effector molecules, such as NF-κB
(p50/p65), AP-1, and IRF-3 transcription factors and
p38 kinase. Whereas TNFR1
recruits TRADD through death-
domains (DDs) homophilic interac-
tions, IL-1R and TLRs interact with
adaptor molecules, such as MyD88,
TRIF (or TICAM-1), and TIRAP
(or Mal) through TIR domains.
MyD88 contains DDs that recruit
IRAK, which forms a complex with
TRAF6. After phosphorylation,
TRAF6 becomes ubiquitinated and
activates MAP3K enzymes. A role
for kinases from the MAP3K fami-
ly such as NIK (NF-κB–inducing
kinase) and TAK-1 (transforming
growth factor β–activated kinase 1)
has been proposed in the activation
of IKK complex (α, β, and
γ/NEMO). Phosphorylation of IkBa
by IKK leads to polyubiquitination
and proteasomal degradation of the
NF-κB cytoplasmic
MyD88/IRAK/ TRAF6 complex
can also activate MAPK pathway
through MKKs, leading to the acti-
vation of AP-1 components that
dimerize in the nucleus and activate
transcription. TRIF, which binds to
TLR3 and TLR4, activates IRF-3
through TBK1 kinase and also
seems to activate NF-κB/MAPK
pathways. The coordinated interac-
tion of transcription factors plays an
essential role in the production of
proinflammatory cytokines and a large family of mol-
ecules all together involved in the control of the innate
immunity. IRF-3 is involved in the induction of IFN-
β, and NF-κB activates expression of its own inhibito-
ry factor, IκBα. For selected references, please see
main text. AP-1 = activator protein-1; CpG DNA =
bacterial CpG containing motif DNA; IFN = interfer-
on; IκB = inhibitory factor κB; IKK = IκB kinase; IL-
1R = IL-1 receptor; IRAK = IL-1R-associated kinase;
IRF-3 = interferon regulatory factor 3; MAP3K =
mitogen-activated protein kinase (MAPK) kinase
kinase; MKK = mitogen-activated kinase kinase;
MyD88 = myeloid differentiation factor 88; NF-κB =
nuclear factor κB; PAMPs = pathogen-associated
molecular patterns; TBK1 = TRAF-associated NF-κB
activator (TANK)-binding kinase 1; TIR = Toll/IL-1R;
TIRAP = TIR-associated protein; TNFR1 = TNF
receptor 1; TRADD = TNFR1-associated protein with
death-domain; TRAF = TNFR-associated factor;
TRIF = TIR-domain-containing adapter inducing
IFN-β.
inhibitor.
Box 1: Innate Immunity: Intracellular Events Triggered by the Family of Toll-like Receptors
(TLRs) and Related Proinflammatory Signaling Pathways
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Volume 10, Number 6, 2004 THE NEUROSCIENTIST541
Surprisingly, the novel adaptor molecule identified as
TRIF-related adaptor molecule seems to be involved in
the MyD88-independent IFN-β production specifically
related to TLR4 signaling (Yamamoto and others 2003).
Single immunoglobulin IL-1R-related molecule is a
newly described negative regulator of TLR/IL-1R sig-
naling that is highly expressed in endothelial cells but
not macrophages (Wald and others 2003). The adaptor
protein Toll-interacting protein (Tollip) is another con-
stituent of the IL-1R-signaling pathway that associates
directly with TLR2 and TLR4 and plays an inhibitory
role in TLR-mediated cell activation (Zhang and Ghosh
2002). Moreover, Tollip coimmunoprecipitates with
TLR2 and TLR4, and overexpression of Tollip inhibits
NF-κB activation in response to TLR2 and TLR4 signal-
ing (Burns and others 2000; Bulut and others 2001).
Inhibition by Tollip is mediated by the ability of this pro-
tein to suppress the activity of specific members of the
IRAK family on TLR activation (Zhang and Ghosh
2002). Important advances have been made with regard
to the signal transduction machinery recruited to the TIR
domain proteins. These intracellular events involve sev-
eral different complexes of proteins. However, the exact
mechanisms by which these novel molecules regulate
the immune response during infection have yet to be
unraveled.
In sum, the binding of PAMPs to their specific TLRs
expressed on the surface of APCs triggers intracellular
signaling and transcription of genes encoding proteins
involved in the innate immune reaction. This concept
has, however, been challenged by a recent study showing
a critical role of LPS-binding protein (LBP) as a media-
tor of the immune response to gram-positive cell wall
components. LPB is an acute phase protein synthesized
mainly by the liver that binds LPS once in the circulation
and directs it to TLR4. Weber and colleagues (2003)
have detected high levels of LBP in the CSF of patients
with meningitis, and LBP-deficient mice failed to devel-
op meningeal inflammation to pneumococcal cell wall.
This study is of great interest because LBP—long
known for its specific interaction with LPS—also binds
an entirely different molecule produced by gram-positive
bacteria that signals through TLR2. Although the mech-
anisms explaining the interaction between LBP and
TLR2 remain to be characterized, these novel data sug-
gest that LBP may be a more general vehicle than origi-
nally thought and may be essential to mount an appro-
priate inflammatory response to both gram-negative and
gram-positive bacteria.
Immunity in the CNS:
The Good or the Bad Actor?
The mechanisms leading to proinflammatory signaling
in the brain are different from those of systemic organs
because of the blood-brain barrier (BBB) that restricts
the access of immune mediators and cells from the
blood. For this reason, the term immune-privileged
organ was used to describe the reduced exposition of the
brain to inflammatory events and its inability to mount
an immune response and process antigens. These phe-
nomena were also believed to be injurious for the neu-
ronal elements and could cause irreversible damage if
present within the cerebral environment (Nguyen and
others 2001; Wyss-Coray and Mucke 2002). Although
immigration and diapedesis are quite limited in the CNS
due to the BBB (Andersson and others 1992), the brain
exhibits its own innate immune response that resembles
in many aspects the classical immune system (Stern and
others 2000; Nadeau and Rivest 2002). Such a system is
activated in response to both cerebral and systemic
immune challenges.
The constitutive expression of CD14 and TLR4 in the
choroid plexus and circumventricular organs (CVOs)
enables immediate recognition of circulating LPS by
these organs that are devoid of BBB (Lacroix and others
1998; Laflamme and Rivest 2001). The endotoxemia
causes a rapid CD14 gene expression in these regions,
whereas a delayed response takes place in cells located
at the boundaries of the CVOs and in microglia across
the brain parenchyma (Lacroix and others 1998). As for
the systemic organs, the inflammatory wave in the brain
is dependent on NF-κB signal transduction pathway.
This response is also associated with a profound tran-
scriptional activation of a large family of immune genes
within parenchymal microglial cells (Laflamme and
Rivest 1999; Laflamme and others 2001). Microglia are
the primary sensors to microenvironment changes in the
brain, and they respond to pathogens like systemic
macrophages. They produce TNF-α that acts in an
autocrine and paracrine manner to activate the popula-
tion of immune cells across the brain parenchyma
(Nadeau and Rivest 2000). Microglial TLR4 is also cru-
cial in promoting proper immune reaction in response to
intraparenchymal LPS infusion (Glezer, Zekki, and oth-
ers 2003). The statement that the brain is an immune-
privileged organ is no longer exact, but the exact roles of
this newly described system still await to be elucidated.
In this regard, activation of microglial cells and proin-
flammatory signaling is believed to play a beneficial
role in host defense. On the other hand, these events are
becoming hallmarks of neurodegenerative processes,
and inhibition of different cytokines and chemokines
was found to have neuroprotective properties (Gonzales-
Scarano and Baltuch 1999; Allan and Rothwell 2001;
Nguyen and others 2002; Wyss-Coray and Mucke 2002).
Astrocytes and microglia are activated in the brain of
patients with Alzheimer’s, Parkinson’s, and Huntington’s
diseases; multiple sclerosis; and amyotrophic lateral
sclerosis. The serum and CSF of these patients show
elevated levels of molecules of the innate system, such
as IL-6, IL-1β, and TNF-α. IL-1β and TNF-α are secret-
ed by activated parenchymal microglia and can be potent
inducers of cell death in models of neurodegeneration,
which can be alleviated by anti-inflammatory drugs and
neutralizing antibodies (Pasinetti 1998; Gonzales-
Scarano and Baltuch 1999). Molecules of the innate
immune system are induced not only in the presence of
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