Download Can the Hair Follicle Become a Model for Studying Selected

Perspectives
Can the Hair Follicle Become a Model for Studying
Selected Aspects of Human Ocular Immune Privilege?
Michael Kinori,1,2 Jennifer E. Kloepper,2 and Ralf Paus2,3
Immune privilege (IP) is important in maintaining ocular
health. Understanding the mechanism underlying this dynamic
state would assist in treating inflammatory eye diseases. Despite substantial progress in defining eye IP mechanisms, becaue of the scarcity of human ocular tissue for research purposes, most of what we know about ocular IP is based on
rodent models (of unclear relevance to human eye immunology) and on cultured human eye– derived cells that cannot
faithfully mirror the complex cell–tissue interactions that underlie normal human ocular IP in situ. Therefore, accessible,
instructive, and clinically relevant human in vitro models are
needed for exploring the general principles of why and how IP
collapses under clinically relevant experimental conditions and
how it can be protected or even restored therapeutically.
Among the few human IP sites, the easily accessible and abundantly available hair follicle (HF) may offer one such surrogate
model. There are excellent human HF organ culture systems
for the study of HF IP in situ that instructively complement in
vivo autoimmunity research in the human system. In this article, we delineate that the human eye and HF, despite their
obvious differences, share key molecular and cellular mechanisms for maintaining IP. We argue that, therefore, human
scalp HFs can provide an unconventional, but highly instructive, accessible, easily manipulated, and clinically relevant preclinical model for selected aspects of ocular IP. This essay is an
attempt to encourage professional eye researchers to turn their
attention, with appropriate caveats, to this candidate surrogate
model for ocular IP in the human system. (Invest Ophthalmol
Vis Sci. 2011;52:4447– 4458) DOI:10.1167/iovs.10-7154
I
t is now obvious that the eye possesses immune privilege
(IP) characteristics that are important for eye health. The
credit for coining this term belongs to Peter Medawar,1 who
showed over a half century ago that skin allografts are not
rejected by the host’s immune system when transplanted heterotopically into defined anatomic sites, such as the rabbit eye
or brain. However, even 130 years ago, Van Dooremaal,2 a
Dutch ophthalmologist, had already discovered that mouse
skin grafts show significantly prolonged survival if transplanted
into the anterior chamber (AC) of a dog’s eye. These pioneer-
From the 1Department of Ophthalmology, Chaim Sheba Medical
Center, Tel-Hashomer, Israel; the 2Department of Dermatology, University of Lübeck, Lübeck, Germany; and the 3School of Translational
Medicine, University of Manchester, Manchester, United Kingdom.
Supported in part by a “Cluster of Excellence” grant from
Deutsche Forschungsgemeinschaft (DFG) (“Inflammation at Interfaces”) and by a DFG Graduate College grant (“Autoimmunity”) (RP).
Submitted for publication December 31, 2010; revised April 4,
2011; accepted April 7, 2011.
Disclosure: M. Kinori, None; J.E. Kloepper, None; R. Paus,
None
Corresponding author: Michael Kinori, Department of Ophthalmology, Chaim Sheba Medical Center, Tel-Hashomer, 52621, Israel;
[email protected].
ing studies have opened the door for robust research in the
field of IP. Consequently, ocular IP has become a subject of
major recent interest, and its fundamental importance in inflammatory eye diseases is now widely accepted.3–15
However, as every investigative ophthalmologist painfully
experiences sooner or later, as a tissue on which to perform in
vitro research, the human eye is an exceptionally rare commodity that is very difficult to come by. In the rare cases in
which eyes are enucleated, the damage due to trauma, infection, or tumor growth raises questions as to how useful such
tissue is in helping us to understand the physiology of human
ocular IP. With very few exceptions (see below), therefore,
most currently available data and concepts on ocular IP are
based on the systematic analysis of rodent models.16 –26 Given
the very substantial immunologic differences between rodent
and human systems,27,28 it is inherently problematic to extrapolate from rodent to human eyes. Moreover, cultured human
eye– derived cells, yet another source of ocular IP research,29 –33 are also problematic, since IP is an in situ state
based on complex cell–tissue interactions and not a condition
displayed by isolated cell populations in vitro.15,34,35
Therefore, our understanding of human ocular IP remains
rather limited, and all extrapolation from rodent and cell culture work must be interpreted with caution.
Thus, good, clinically relevant human in vitro models are
urgently needed to enable the study of the general principles of
why and how IP collapses and how it can be protected or even
restored in situ. Although the search for such human surrogate
models meets with evident obstacles, since no other organ is
quite like the eye, at least some aspects of human ocular IP may
be studied in other, more accessible and more abundantly
available human tissues.
SEARCHING FOR
OF OCULAR IP
A
SURROGATE PRECLINICAL MODEL
The eye is not the only mammalian site of IP. Other sites
include parts of the testis and ovary, the adrenal cortex, parts
of the brain, the fetomaternal placental unit, the hamster cheek
pouch, and probably the proximal nail matrix.34,36 – 41 Notably,
the human hair follicle (HF) also qualifies as an IP site34 (for
debate, see below). This miniorgan is unique among all other
IP sites, in that it is massively distributed over the human body
and is highly accessible to experimental analysis and manipulation. Our integument has approximately 5 million HFs and
thus a correspondingly vast number of potential (human) IP
organs.42 Moreover, there are excellent human HF organ culture systems that can instructively complement in vivo research for the study of autoimmunity in the human system.43– 45
Since common mechanisms of IP in different organs have
been elucidated,46 – 48 an obvious question is whether one IP
site can hold lessons for other, less easily explored sites. Although the IP of human HFs has been much less well studied
than that of the eye, the relative ease with which human HFs
Investigative Ophthalmology & Visual Science, June 2011, Vol. 52, No. 7
Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
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can be obtained, microdissected, organ cultured, and immunologically manipulated in vitro (see below) invites one to exploit
human HF organ culture44 as an unusual, but highly instructive
and clinically relevant preclinical research model (e.g., for
following up in vitro studies of ocular IP in rodent models,
before entering into clinical trials on the human eye).
Following in the footsteps of earlier suggestions along this
vein,34,49 the current essay attempts to shed some light on
both the similarities and important differences between ocular
and HF IP and to attract eye researchers and clinical ophthalmologists to systematically employ, with appropriate caution
and circumspection, the human HF as an accessible, relatively
easily manipulated surrogate model for carefully selected aspects of ocular IP model.
IMMUNOLOGIC HOMEOSTASIS, IGNORANCE,
AND PRIVILEGE
Before critically exploring the usefulness of human HFs as a
candidate surrogate model for ocular IP, it helps to remember
that many tissues have their own mechanisms for preserving
the immunologic status quo for proper functioning (i.e., immune homeostasis).7 For example, the lung and the gut,
which are chronically exposed to foreign antigens that may
incite inflammatory responses must allow gas exchange and
food processing, respectively, without provoking undesired
levels of inflammation.7 Another important concept in this
context is immunologic ignorance, which emphasizes the key
role of anatomic barriers and peculiarities in a given anatomic
site (e.g., the absence of patent lymphatics), which prevents
the entry of immune cells resulting in graft rejection.8,50
In contrast, immune privilege classically describes tissue
sites within which foreign tissue grafts can survive for extended periods, whereas similar grafts placed in conventional
sites are acutely rejected by the host.46,50,51 Today, the term IP
is generally understood in a much broader sense and indicates
the presence of multiple active mechanisms for preventing the
induction and expression of both innate and adaptive immune
responses.7,8,11,34,36,50
Although there are phenomenologic indications that the HF
evades some and actively suppresses other potentially autoaggressive immune responses (see below), for evident methodological reasons, rejection and survival of heterologous tissue
transplants within this tiny miniorgan have not yet been studied. (In fact, due to insufficiently refined microsurgery and
microinjection techniques, previous attempts in our laboratory
to inject melanocytes from C57BL/6 mice into the vibrissae
hair bulb of white, immunocompetent Balb/c mice have failed
miserably.) Thus, one can only lament that Billingham’s landmark experiment and its visionary interpretation remain the
only currently available functional evidence that HFs can indeed shelter heterotransplants from immune rejection. He observed that, while donor melanocytes within the epidermis are
rapidly eliminated, heterologous epidermal melanocytes show
long-term survival if they manage to escape into the anagen
hair bulbs of (white) host guinea pigs, which thus began to
produce black hair shafts.34,52,53
OCULAR IMMUNE PRIVILEGE: BASIC CONCEPTS
CURRENT PERSPECTIVES
AND
IP has turned out to be a complex and dynamic tissue state and
the list of “players” involved in it is ever-growing (Table 1, Fig. 1).
One key mechanism of ocular IP, AC associated immune deviation (ACAID), was identified by Kaplan and Streilein et al.81– 84
ACAID means that injection of antigens into the AC of rodents
(and even of monkeys28) produces a stereotypic systemic immune response that is selectively deficient in antigen-specific
delayed-type hypersensitivity (DTH), whereas other conventional effector modalities of immunity (such as cytotoxic T
cells and non– complement-fixing antibody isotypes) are preserved.28,85
ACAID Has Ocular and Systemic Pathways
It is thought that antigens inoculated into the eye are processed
in a distinctive fashion by stromal antigen-presenting cells
(APCs) of the iris and ciliary body.14,86 This phenomenon
appears to be largely under the control of transforming growth
factor (TGF)-␤, a key immunosuppressive cytokine in the aqueous humor.61,62,87 In fact, in mice, a deficit of total TGF-␤2 in
aqueous humor correlates with loss of ACAID.51 These APCs
then emigrate from the eye by traversing the trabecular meshwork (TM) and directly enter the blood stream to reach the
spleen.88 Access to the blood stream (without an access to the
lymphatic system thorough the uveoscleral pathway) seems to
be crucial for the induction of ACAID. Indeed, monkeys treated
with topical prostaglandins (which redirects a substantial fraction of aqueous humor into the uveoscleral path89) failed to
induce ACAID.28
TABLE 1. Important Characteristics of Ocular and Hair Follicle Immune Privilege
Eye
Specific location of IP
Cyclic phenomena
Results of IP collapse
ACAID
Strong expression of immunoinhibitory
molecules
Anterior chamber, iris and ciliary body, subretinal
space7,22,54,55
No
Corneal transplant rejection, immune mediated
microbial keratitis and uveitis7,8,58,59
Yes
␣-MSH60; TGF␤261,62,63*; TSP121,64*; VIP65; SOM23;
MIF66; CGRP67; CRP32; PEDF19; IDO30; IL-1Ra68;
GITRL16; TRAIL25; B7–2(CD86)69; cortisol62
Downregulation of APC molecules
Low MHC class Ia, weak MHC class Ib, no MHC
class II cells24,59,73–76
Apoptosis induction of lymphocytes
FasL(CD95L)78⫹sFasL(sCD95L)79;
B7-H1(CD86)20,29; B7-H359
Hair Follicle
Hair bulb, hair bulge34,56,57
Yes
AA, lichen planopilaris, scleroderma, skin
manifestations of SLE, and GVHD34
No
␣-MSH, TGF␤1, IL-10, IGF-134,43,44; MIF,
TGF␤2, CD200, IDO57; cortisol70;
downregualtion of MICA71; TSP-1?72,
CGRP? (currently being investigated)
Low MHC class Ia, weak MHC class Ib
(HLA-E and G), no MHC class
II34,44,56,57,77
FasL?80
AA, alopecia areata; ACAID, anterior chamber-associated immune deviation; APC, antigen presenting cell; GVHD, graft versus host disease; IP,
immune privilege; SLE, systemic lupus erythematosus. The abbreviations for proteins are as described in Figure 1.
* Main inductors of ACAID.
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The Hair Follicle as a Model for Ocular Immune Privilege
4449
FIGURE 1. Immune privilege of the eye and anagen VI hair follicle: key features. ␣-MSH, ␣-melanocyte
stimulating hormone; APC, antigen presenting cell; APM, arrector pili muscle; CGRP, calcitonin generelated peptide; CRP, complement regulatory proteins; DP, dermal papilla; E, epidermis; FasL, Fas ligand;
GITRL, glucocorticoid-induced TNF receptor family-related protein ligand; HLA, human leukocyte antigen;
HS, hair shaft; IDO, indolamine 2,3-dioxygenase; IGF, insulin-like growth factor; IL-10, interleukin 10;
IL-1Ra, interleukin 1 receptor antagonist; IRS, inner root sheath; MHC, major histocompatibility complex;
MICA, MHC class I chain-related A gene; MIF, macrophage migrating inhibitory factor; ORS, outer root
sheath; PEDF, pigment epithelium derived factor; RPE, retinal pigment epithelium; sFasL, soluble Fas
ligand; SG, sebaceous gland; SOM, somatostatin; TGF, transforming growth factor; TRAIL, TNF-related
apoptosis-inducing ligand; TSP-1, thrombospondin 1; and VIP, vasoactive intestinal peptide.
The eye-derived APCs reach the marginal zone in the
spleen. There, a complex dialog involving multiple cells, including B cells, CD4⫹ NKT cells, CD4⫹ T cells, ␥␦ T cells, and
CD8⫹ T cells, ensues.58,90 –92 When the latter recognize antigens presented by eye-derived APCs and/or marginal zone B
cells, they differentiate into ACAID-inducing regulatory T cells
(ACAID-Tregs).58,59 CD4⫹ ACAID-Tregs prevent the activation
and differentiation of antigen-specific Th1 effector cells,85
whereas CD8⫹ ACAID-Tregs inhibit the local function of effector T cells (Th1 and Th2).15,93,94
Two other elements, are also important in the induction of
ACAID: the thymus and the sympathetic nervous system.59
Although the thymus is an important source of the NKT cells
that are needed for the splenic phase of ACAID,95 the sympathetic nervous system is believed to have a role in the generation of the NKT-cell population that eventually enters the
spleen and participates in the induction of CD8⫹ ACAID
Tregs.59,96
Clinical Aspects of ACAID
In mouse models, ACAID has been demonstrated to be involved in various clinical scenarios, such as acceptance of
corneal transplants, autoimmune uveitis, acute retinal necrosis
(ARN) in a fellow eye that experienced herpes virus infection
in the anterior segment, and progression of intraocular malignant melanoma.39 This mechanism may be the eye’s way of
protecting its vital functions from immunopathogenic injury.97
ACAID has been widely studied in mice, rats, guinea pigs, and
rabbits, implying that it is not a phenomenon restricted to
laboratory rodents.98,99 Significant progress was made when
Eichhorn et al.28 demonstrated that ACAID can also occur in
primates. Even so, it should not be taken for granted that these
results (although more convenient than those obtained in rodents) can be extrapolated to conclusions about the human
eye. Evidence compatible with the existence of an ACAID-like
response in humans indeed has been shown in patients with
ARN.100 Nevertheless, the evidence that these ACAID mecha-
nisms also apply to the human condition remains rather circumstantial, since definitive experimental proof of the existence of ACAID in humans would require (e.g., the ethically
problematic transfer of regulatory T cells from one individual
to another).100 Thus, whether all the chief characteristics of
ACAID established in animal models also occur in humans
remains unknown.
IP and the Posterior Segment of the Eye
If one views the eye as an extension of the brain, it is not
surprising that there is also a close relationship between ocular
IP and the nervous system. Indeed, ocular IP may mainly be the
result of neuroimmune interactions.8,101 The retina itself represents a highly organized neuronal tissue that produces many
immunosuppressive molecules generated by neurons and glial
cells. As a cell layer, RPE can suppress the activation of bystander T cells via soluble inhibitory factors such as
TGF␤21,102,103 and can induce apoptosis of activated T cells.104
Moreover, in mice, the (neonatal) RPE105 and the neuronal
retina106 display inherent IP. It is now known that the vitreous
and subretinal space are also IP sites107 and that antigens
placed in these sites can even lead to the induction of an
ACAID-like response.22,54,55 In this context, it is interesting to
note that (in mice) IP disruption occurs when RPE cells are
damaged by laser burns.18 This finding has raised the troubling
question of whether laser-treated eyes are at increased risk of
ocular inflammation, up to the collapse of eye IP.
Current Perspectives
The concept that “excessive“ ocular IP can facilitate life-threatening infections and the actual presence of autoimmune uveitis
and corneal allograft rejection underscores the importance of
viewing IP as a relative, rather than absolute status, which can
sometimes be bypassed.98 Indeed, it has been proposed that
the immune system can “decide” that, sometimes, preservation
of life supersedes preservation of vision (e.g., if there is a
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life-threatening ocular infection, such as trachoma, river blindness, or herpes simplex virus keratitis, resulting in ocular IP
collapse and inflammatory eye destruction7).
This example illustrates that IP mechanisms must generally
be carefully balanced, since we are continuously exposed to
potentially deleterious environmental agents, including microorganisms and antigenic insults; and yet, most humans do not
go blind. Moreover, corneal transplants are the least-rejected
among all organ transplants,39,108 despite the use of HLAunmatched transplants with minimal immunosuppression.109
This is probably due, not only to the anatomic characteristics
of the corneal tissue, but also to its low antigenicity.59,110
New frontiers in ocular IP research include the role of
ocular IP in eye tumor development and progression. Given
that IP can be acquired by tumor cells to evade immune
surveillance,111 it is interesting to note that modulating IP by
the injection of cytotoxic Fas ligand (FasL) in the vitreous
cavity can prevent neovascularization in a mouse model of
choroidal neovascularization.112
Thus, instructive experimental models in the human system
are urgently needed that allow deeper insights into IP biology
and pathology and that facilitate experimental manipulations.
This is where the HF, one of the defining features of mammalian species, enters our vision.
EVIDENCE
IN
SUPPORT
OF
HF IP
It has been four decades since Billingham52 discovered that the
hair bulb provides a special milieu that permits transplanted
allogeneic cells (namely, donor melanocytes) to escape limitation by the host immune system while others are attacked.52
Black skin epidermis transplanted onto skin beds of genetically
incompatible white guinea pigs quickly lost its pigmentation (a
sign that the foreign melanocytes had been rejected), whereas
black hair shafts soon thereafter began to pierce the (now
white) epidermis. This result indicates that at least some donor
melanocytes had survived in the host hair bulbs and had resumed their transfer of melanosomes to HF keratinocytes.34,52,53
Unfortunately, since then, no additional functional evidence
of the existence of HF IP has been published, perhaps because
available pointers that HFs are a site of IP have long been
ignored by the immunologic and transplantation research communities. Recently, however, HF IP has attracted more widespread interest,34,56,80,113–116 and there is an increasing community of HF immunology authorities that has embraced the
concept of that the HF enjoys a relative IP and that a collapse
of HF IP is of critical importance in alopecia areata (AA), one of
the most frequent human autoimmune diseases,43,80,114 –116
whereas a collapse of the putative IP of the epithelial stem cell
area of the HF (bulge)57 may be important in the pathogenesis
of cicatricial alopecia.117
Compared with the eye, our understanding of the functional state of resident immune cells within the HF epithelium
(Langerhans cells, T cells) and in the HF mesenchyme (macrophages, mast cells) is still very limited, and current HF immunology concepts are largely based on immunophenomenologic
analyses. However, a few important facts have surfaced that,
taken together, strongly support the concept that defined compartments of the HF represent sites of relative IP. Some key
arguments can be summarized as follows (for full discussion,
see Ref. 34).
MHC Class I and
␤2-Microglobulin Downregulation
A classic feature of IP sites is the downregulation of MHC class
Ia expression.34,44,116 The lower (proximal) epithelial com-
partments of the HF epithelium, the anagen hair bulb, shows a
striking downregulation of both MHC class Ia and associated
␤2-microglobulin gene and protein expression.44,57,77,118
Since MHC class Ia-stabilization by ␤2-microgolubin is critical
for the proper presentation of MHC class I– dependent antigens,27 any MHC class I molecules that may still be expressed
in the anagen hair bulb probably cannot effectively present
autoantigens. In humans and mice, the HF’s stem cell zone, the
bulge, even expresses nonclassic MHC class I molecules (MHC
class Ib molecules, such as Qa-2 and HLA-E), which inhibit, for
example, NK cell activities.57,77,119 To the best of our knowledge, no healthy mammalian tissue has been described thus far
that exhibits this phenomenon without enjoying relative IP.
Local Generation of Potent Immunosuppressants
A key feature of all recognized IP sites is that they express
potent, locally generated immunoinhibitory molecules.7,120,121
Therefore, it is important to note that the anagen hair bulb and
even the bulge prominently express potent immunosuppressants such as TGF␤, ␣-melanocyte-stimulating hormone (␣MSH), IL-10, and others.34,44,122,123
Functionally Impaired Langerhans Cells
Given the importance of APCs in ocular IP (see above), it is
intriguing to note that, in striking contrast to the APCs of the distal
HF epithelium (i.e., the upper outer root sheath), the very few
intraepithelial Langerhans cells that are detectable ultrastructurally or by CD1a immunohistochemistry in the proximal anagen
hair bulb of human scalp HFs do not express detectable MHC
class II or I molecules.57,123 Even the distal outer root sheath of
human HFs harbors immature Langerhans cell populations.124
That at least all professional APCs in the proximal human HF
epithelium lack evidence of full antigen-presentation capacity
perfectly fits the characteristic impaired antigen presentation
by APCs in IP sites.
Autoreactive CD8ⴙ T Cells Are Key Protagonists
in HF Autoimmunity
Since AA is a T-cell-mediated, organ-specific autoimmune disease,125 it offers an excellent model for probing the functional
relationship between autoreactive T cells and the putative HF IP.
AA also promises pointers to the functional relevance of HF IP.
T lymphocytes isolated from human scalp lesions and expanded in vitro with homogenates of HFs reproduce AA lesions
when transferred into scalp explants in SCID mice.126 That
CD8⫹ T cells, but not CD4⫹ T cells alone, can produce AA
lesions43,127 indicates that a prior collapse of HF IP must have
occurred, which exposes previously sequestered, MHC class
I–presented autoantigens to CD8⫹ T cells. The same is also
seen in the best-characterized mouse model of AA128 (see also
Ref. 116). This, in turn, suggests that the striking downregulation of MHC class Ia and ␤2-microglobulin in healthy anagen
hair bulbs is functionally important.
As in most autoimmune diseases, identification of the
epitopes that trigger the autoimmune response remains a major goal. In AA, much current interest centers on melanocyteassociated antigens: Melanocyte peptide epitopes (such as
Gp100-derived G9 –209 and G9 –280 and MART-1 (27–35))
injected into autologous lesional human scalp grafts on SCID
mice induce AA lesions.129 Moreover, skin-derived CD8⫹ T
cells obtained from AA patients co-cultured with MAGE3 show
a significant increase in intracellular interferon (IFN)-␥ expression compared with the control.130 IFN␥, in turn, is the most
potent stimulator of ectopic MHC class Ia expression identified
so far.34,44
Since melanocyte-associated, MHC class I–presented autoantigens recognized by CD8⫹ T cells27 are key immune
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The Hair Follicle as a Model for Ocular Immune Privilege
targets in vitiligo and melanoma, these findings in AA patients
and animal models provide functional evidence that the ectopic expression and presentation of MHC class I–presented
autoantigens within the HF entails the danger of autoaggressive
immune responses against the HF, if cognate, autoreactive
CD8⫹ T cells are present. This finding strongly supports the
concept that the prominent downregulation of MHC class Ia
and ␤2-microglobulin in healthy anagen HFs is critical to warding off deleterious immune attacks on the HF.34,131
NK Cell Activities Appear to Be Suppressed in
Healthy HFs
Since NK cells are primed to recognize and eliminate cells with
absent or low MHC class I expression,27,71,132–138 immunologically privileged HF compartments constitute a basic problem
in self-/non–self-discrimination and self-tolerance.139 One
would expect that MHC class I-negative or MHC class I ‘‘low”
anagen HFs are under constant attack by NK cells. However,
this is clearly not the case, since very few perifollicular NK
cells can ever be found around healthy human anagen HFs.123
In contrast, in AA, CD56⫹ NK cells prominently aggregate
around HFs and show an increased expression of NKG2D (NK
cell-activating receptor) and decreased expression of KIR-2D2/
2D3 (NK cell-inhibitory receptor).71 Moreover, macrophage
migrating inhibitory factor (MIF) may suppress NK cells in and
around healthy HFs71 (just like in the eye66). In contrast,
excessive NK cell stimulation by NKG2D-activating ligands
such as MHC class I chain–related A gene (MICA) by the HF
epithelium71 and/or other MICA-related NKG2D ligands114 in
and around AA HFs may contribute to HF IP collapse. Therefore, the failure to adequately suppress undesired NK cells
activities directed against MHC class I–negative HF cells during
anagen may be an important additional element in AA pathogenesis.56 A recent genome-wide association study114 identified several genetic susceptibility loci for AA. Among these,
significant associations include the ULBP genes, which encode
activating ligands for NKG2D. Normally, ULBP3 is not present
in HFs, but ULBP3 proteins were abundant in and around
human HFs affected by AA. NKG2D/ULBP3 engagement, and
thus inappropriate NK cell stimulation, may contribute to the
development of AA.
Taken together, this combination of immunophenomenologic in situ observations and functional data from the relevant
model disease (AA) constitutes sufficient evidence to suggest
that HFs do indeed enjoy relative IP.
A unique, key feature of IP in the anagen hair bulb is that it
is temporary: The HF epithelium rhythmically generates, maintains, and deconstructs an area of relative IP in the region of
the HF, which is present only during a defined segment of the
hair cycle—that is, the anagen phase (growth stage)— but
absent during HF regression (catagen) and the “resting” phase
(telogen).34,44,122,123 In contrast, the bulge, the HF’s seat of
epithelial and melanocyte stem cells, seems to continuously
enjoy a relative IP.57,140 In this anatomic HF landmark, MHC
class Ia, ␤2-microglobulin, and MHC class II molecules are
downregulated, whereas MHCIb (HLA-E) is upregulated. In
addition, immunoinhibitory molecules, like the “no danger”
signal CD200,117,141 ␣-MSH, MIF, and indoleamine-2,3-dioxygenase (IDO) are markedly overexpressed in the bulge region.57,142
All these are features of ocular IP as well.30,60,66,73,143
Thus, within the microcosmos of HF immunology, the relative IP of the bulge may actually be more closely related to
ocular IP than that of the anagen hair bulb. Bulge IP collapse
and the subsequent immunologically mediated destruction of
epithelial HF stem cells may play a key role in the pathogenesis
of irreversible, scarring alopecia,117 just as ocular IP collapse
can irreversibly destroy the eye (instead, bulb IP collapse in AA
typically only induces reversible HF damage34,43).
WHY DOES
THE
4451
HF NEED IMMUNE PRIVILEGE?
As vision is one of the most important qualities and survival
requirements of most living creatures, it is easy to understand
conceptually why the eye must be protected against autoaggressive immune attacks, especially in its delicate, apparently
nonregenerating compartments (i.e., corneal endothelium and
retinal cells). But what about the HF? Which selection advantage might mammals have had during evolution for establishing
an area of IP in HF?
Although this may have been different for humanoids and
prehistoric early humans, in our current climates and cultures,
hair is clearly dispensable for human survival and propagation
of the species. However, during ⬎99% of the total duration of
mammalian evolution, an environmentally perfectly adapted
hair coat seems to have been vital for numerous of our mammalian ancestors and their reproduction (suffice it here to
envision the poor survival and reproduction chances of a
hairless polar bear, Arctic fox, or seal). Given that the HF is one
of the most frequent targets of immune-mediated tissue injury,34,144 the HF may therefore have established its IP as a
safeguarding system against immune injury of this important
miniorgan. Viewed from this angle, ocular and HF IP may both
be necessary (even though this is much less evident for the
latter than for the former).
SIMILARITIES
AND HF IP
AND
DIFFERENCES
BETWEEN
OCULAR
Thus, ocular and HF IP show some striking similarities—
namely, in the bulge region of the HF:
● Classic MHC class I and ␤2-microglobulin are downregulated, which renders cells relatively invisible to CD8⫹ cytotoxic T cells.34,44,57,66,74,145
● APCs are both sparse and functionally impaired (e.g., they
lack MHC class II expression).34,44,57,59,74
● IFN-␥ exposure causes ectopic upregulation of MHC class
Ia and II expression in cells lining the AC and in the
HF.44,59,146,147
● Nonclassic MHC class I molecules (e.g., HLA-E and -G) are
downrebulated. Together with MIF, they are known to inhibit
a potential attack of NK cells on MHC class Ia (i.e., HLA-A, -B,
and -C)–negative cells.57,73,75,119
● There is a strong expression of similar immunoinhibitory
molecules (see Table 1 and Fig. 1 for details).
In these specific areas, the human HF may well serve as an
attractive surrogate model for ocular IP (see below).
Of course, there are also many important differences between follicular and ocular IP that one needs to keep in mind
when studying the HF as a surrogate model. Mainly, an ACAIDlike response has not been demonstrated (yet) to be associated
with antigens introduced into the HF. Also, Fas–FasL interactions, which seem to be an important element of ocular IP,78
are unlikely to play a major role in HF IP.34,49,123 Interestingly,
however, FasL is indeed significantly decreased in lesional skin
of AA patients, compared with nonlesional skin.80
Other apparent dissimilarities between ocular and HF IP
may also be less pronounced than one may be inclined to
think. Since neuroimmune interactions play a key role in ocular
IP (see above), it is reasonable to ask whether these have any
role in HF IP. Although this question has so far only been
addressed very incompletely, the HF does represent a prototypic neuroectodermal–mesodermal tissue interaction system
and is one of the most densely and intricately innervated of all
peripheral tissues.148 In fact, HF biology has multiple neurobiological dimensions along what has been termed the “brain–HF
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axis” 149 (for a review, see Ref. 150). Namely, HF development,
growth and pigmentation are regulated in part by neurotrophins and neuropeptides, whereas HF-derived neurotrophins
control HF innervation. In addition, human scalp HFs even display
a fully functional, peripheral equivalent of the central hypothalamic–pituitary–adrenal (HPA) stress response axis.44,151 It would
be unreasonable to expect that this brain–HF axis plays no role in
HF IP.
Indeed, psychoemotional stress causes prominent perifollicular neurogenic inflammation and hair growth inhibition in
mice, which depends on mast cells, substance P, and nerve
growth factor (NGF) and goes along with phenomenologic
indications of HF IP collapse.152–156 Mice subjected to noise
stress show dense inflammatory cell infiltrates around their
HFs, as early as 24 hours after exposure, with an increase in the
number and activation of perifollicular mast cells as well as
MHC class II–positive inflammatory cell clusters. Noise stress
also increases the number of intradermal dendritic cells and
induces their maturation.152,155,157,158
Of special interest here is substance P, which is released in
response to stress by sensory skin nerves, plays a key role in the
cutaneous neuroimmune network and influences immune cell
functions through the neurokinin-1 receptor (NK-1R).153,159 In
murine AA, NK-1R is prominently expressed on CD8⫹ lymphocytes and macrophages that accumulate around lesional HFs.
Thus, currently available murine data suggest that substance P and
NK-1R are important elements in the pathogenesis of in autoimmune hair loss and the associated collapse of HF IP.159
Moreover, in organ-cultured human scalp HFs, substance P
directly induces HF IP collapse, as evidenced by ectopic MHC
class I and class II expression.45 Although the role of substance
P in ocular IP has not yet been systematically explored, the
immunoinhibitory neuropeptide calcitonin gene-related peptide (CGRP), which is co-expressed with substance P in sensory skin nerves, is involved in the maintenance of ocular IP.67
Preliminary evidence from our laboratory suggests that CGRP
may exert a similar protective function in the context of human HF IP (Kinori et al., manuscript in preparation). Thus, it is
quite likely that neuroimmune interactions are an important
component, not only of ocular, but also of HF IP.
HOW MAY INSIGHTS FROM HUMAN HF IP
GENERATE THERAPEUTIC BENEFITS FOR CLINICAL
OPHTHALMOLOGY?
Although it would be unreasonable to claim that most aspects
of ocular IP can be investigated in human HFs in vitro, we
propose here that selected IP-related insights from human HF
organ culture could be put to excellent clinical use in ophthalmology.
Figure 2 illustrates how relatively easy it is to microdissect
and organ culture human scalp HFs obtained, for example,
from excess scalp skin during routine face-lift surgery. In these,
IP collapse can easily be induced by the proinflammatory
cytokine IFN␥. This effect induces rapid, massive, ectopic
MHC class Ia and II expression in the epithelium of normal
anagen (stage VI) HFs147 (see Fig. 2), thus seriously endangering maintenance of the HF IP (see above). The same phenomenon can also be induced by adding substance P to the HF
culture medium,45 or in vivo by injecting IFN␥ into the back
skin of mice with HFs in the anagen stage of the hair cycle.147
Thus, human HF organ culture permits one to screen for
candidate agents that effectively downregulate IFN␥-induced
ectopic MHC class I expression in human anagen HFs. A “protection” or (perhaps more important) “restoration” assay design can be chosen: In the former, the candidate IP protectant
is added to the medium before IFN␥ is introduced, whereas
IOVS, June 2011, Vol. 52, No. 7
candidate “IP restoration” agents can be tested by adding them
after IFN␥ administration. In fact, three immunomodulators
known to be locally produced in the anagen hair bulb—␣-MSH,
TGF␤1, and insulin-like growth factor 1 (IGF-1)122,163,164—are
all capable of downregulating ectopic MHC class Ia expression,
on both the protein and the mRNA level, in the IP restoration
assay design.44 This human organ culture assay, therefore, is
well-suited as a clinically relevant preclinical screening system
to identify novel candidate IP-restoring or IP-protecting agents.
Once identified, these can then further be explored as candidate therapeutics for ocular IP protection and restoration.
WHICH SPECIFIC ASPECTS RELEVANT TO OCULAR IP
CAN BE STUDIED IN HUMAN HF ORGAN CULTURE?
The human HF is hardly suitable as a surrogate model for
studying ACAID or for evaluating the effects of test agents on
the delicate retinal neuronal tissue. However, given that the HF
may be more dispensable than any other human organ (privileged or not), it offers investigators interested in IP (in the eye
and elsewhere) an unparalleled opportunity to directly study
and manipulate a complex but easily accessible and widely
available human IP site. In this site, the following specific
questions that are directly relevant to ocular IP may be studied
in situ:
1. How is MHC class Ia, Ib, and II and ␤2-microglobulin
expression regulated in situ in a normal human neuroectodermal–mesodermal interaction unit?
2. How can (experimentally induced) ectopic upregulation
of these molecules be effectively downregulated again?
3. Vice versa, how can the local expression of IP-protective, immunoinhibitory molecules that are also of relevance in ocular IP (e.g., IDO; immunoinhibitory neuropeptides, such as ␣-MSH, vasoactive intestinal peptide
[VIP], and CGRP, and TGF␤1, IGF-1, and CD200) be
effectively upregulated in a normal human neuroectodermal–mesodermal interaction unit?
4. Psychoemotional stress may be implicated in the relapse
of anterior autoimmune uveitis.165 Shouldn’t it then be
possible to exploit human HF organ culture to further
explore in this human miniorgan the direct impact of
well-defined stress mediators (including neuropeptides
like VIP that have not yet been studied in a hair research
context, but are important in ocular IP) on key IP characteristics, such as MHC Ia/␤2-microglobulin expression
and the local generation of immunoinhibitory compounds?
5. What is the relative contribution of human NK cells,
NKT cells, intraepithelial T cells, Langerhans cells, and
mast cells to IP (e.g., via local tissue interactions with
the epithelium)? For example, a recent new concept in
murine AA pathobiology suggests that some NK cell
subpopulations, as opposed to IFN-␥-secreting CD49b⫹
T-cell subsets, may actually award relative protection
from AA development.125
6. How do drugs or operative techniques that are already
used in the management of inflammatory eye diseases
affect human HF IP? This may help to predict desired and
undesired effects on ocular IP, whose study in human
eyes would require enucleation and is thus essentially
impossible. For example, in mice, retinal laser burn abrogates IP in both the burned and nonburned eye.18
Applying laser burns to scalp HFs in vitro may indicate
whether laser treatment is likely to exert similar effects
on human IP. Also, at least some underlying mechanisms
of action could be studied in HF organ culture, but
hardly in human eyes.
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The Hair Follicle as a Model for Ocular Immune Privilege
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7. Along the same vein, can HFs from a given patient with
autoimmune ocular disease be used to predict the likely
response of that patient to the intended therapy, as a
potential means to predict whether that therapeutic intervention is likely to restore or protect ocular IP?
8. IP in the proximal HF epithelium is a cyclic phenomenon that appears to be restricted to a defined segment of
the hair cycle. Although the human eye is not thought to
undergo cyclic transformations in adult life, one wonders whether ocular IP also possesses some cyclic characteristics—for example, being maximal or minimal during certain periods on a circadian or perennial time
scale. Could it be that autoimmune uveitis preferentially
relapses during such hypothetical periods of constitutively “minimal ocular IP”?
ADULT EPITHELIAL STEM CELLS AND IMMUNE
PRIVILEGE: YET ANOTHER EYE–HAIR CONNECTION?
Stem cells rank among the most exciting current research
frontiers in experimental and clinical ophthalmology. Although
beyond the scope of this essay, it therefore should at least be
mentioned briefly that the study of adult human epithelial stem
cells (eSCs) in the bulge region of the HF166 –170 may also
benefit ophthalmology research. After all, at least in rodents,
HF-derived eSCs can differentiate into cells with a corneal
epithelial phenotype when given appropriate stimuli.171–173
On this background, it is interesting to note that physiological concentrations of thyroid hormones enhance expression
of CD200 on human HF eSCs.174 This important immunoinhibitory and tolerogenic surface molecule175 is a crucial element of bulge IP maintenance, since its targeted knockout in
mice causes massive inflammation and irreversible HF destruction.57,117,141
Therefore, one might learn from HF-associated eSCs how
cell-based therapies for the treatment of ocular disease could
be engineered so as to reduce the risk of immune rejection or
undesired immune deviation by progenitor cells introduced
into the human eye, for example, by promoting their expression of CD200. This could, for example, become important in
the treatment of limbal stem cell deficiency (LSCD)173 and
age-related macular degeneration (AMD),176 especially if one
FIGURE 2. Hair follicle isolation and culture. (A) Human temporal and
occipital uninflamed scalp skin was taken from donors with informed
consent during routine face-lift surgery, in compliance with the guidelines
in the Declaration of Helsinki. (B) After the skin is shaved, it is cut into thin
strips, approximately 5 ⫻ 10 mm. (C) Side view of a cut skin strip where
the vision of the hair follicle is complete and vertically orientated. (D) The
scalpel blade divides the epidermal– dermal part (above) from the subcutaneous (SC) layer of the skin (below). (E) If the cut is successful, a net of
white dermal collagen fibers appears, spread all over the SC fat tissue, as
shown here. The lower part of the hair follicle with its hair bulb including
the dermal papilla resides in the subcutis and is taken for further processing. (The bulge region and the sebaceous gland of the hair follicle remain
in the white dermal part) (F) The sides of the fat tissue are pressed
carefully with blunt forceps, to partially extrude the upper portion of
the hair follicles from the subcutis. At the same time, the tip of the
follicle is gently gripped with watchmaker’s forceps, and the hair
follicle is pulled from the hypodermal fat. (G) It is essential to isolate
intact hair follicle bulbs without any visible damage if the successful
maintenance of hair follicles is to be achieved. Hair follicles are freefloating in a 24-well multiwell plate (three follicles per well) filled
supplemented Williams E medium. (H) Hair follicles are maintained in
500 ␮L serum-free Williams E medium (Biochrom, Cambridge, UK)
supplemented with 2 mM L-glutamine (Invitrogen, Paisley, UK), 10
ng/mL hydrocortisone (Sigma-Aldrich, Taufkirchen, Germany), 10
␮g/mL insulin (Sigma-Aldrich) and 1% antibiotic/antimycotic mixture
(100⫻; Gibco, Karlsruhe Germany). Hair follicles are maintained freefloating in the wells at 37°C in an atmosphere of 5% CO2 and 95% air.
This permits detailed measurements to be made on the length of
individual hair follicles during the culturing period160 –162 (I) Immunofluorescent staining of a vehicle treated anagen VI hair follicle shows
very low or absent MHC class I immunoreactivity in the CTS and
proximal ORS. (J) Treatment with 75 IU/mL of IFN␥ induces the
ectopic MHC class I expression in the DP, the CTS and the proximal
ORS. (K) MHC class II expression in the anagen stage VI hair bulb is
very low or absent in the CTS and the proximal ORS. (L) Culturing with
75 IU/mL IFN␥ prominently induces MHC class II expression in the DP,
the CTS, and the proximal ORS keratinocytes. CTS, connective tissue
sheath; DP, dermal papilla; IFN␥, interferon ␥; MHC, major histocompatibility complex; ORS, outer root sheath.
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IOVS, June 2011, Vol. 52, No. 7
explores the use of autologous, easily accessible human HF
eSCs as a potential source for such cell therapy. Do HF-derived
human eSCs (or any other epithelial progenitor cell type exploited for cell-based regeneration strategies in experimental
ophthalmology) retain the relatively immune-privileged status
that they had enjoyed in situ,142 once they are isolated, propagated, and treated in cell culture?
CONCLUSIONS
AND
PERSPECTIVES
It is now widely accepted that understanding ocular IP will
contribute to the development of new therapeutic approaches
to tissue transplantation and autoimmune diseases, not only of
the eye, but also of other organs.4,7,12,59 The same may be
claimed for HF IP, and this in a clinically relevant and preclinically much more accessible and available model system. Despite the many evident differences between the eye and the
HF, the limited, but persuasive similarities between ocular and
follicular IP delineated above raise the possibility that their
respective responses to test agents, and the selected aspects of IP
listed above also are quite similar—probably at least on the same
level of similarity as that of rodent versus human ocular IP.
Collaboration between ocular and skin scientists in the joint
exploration of IP therefore promises to be very fruitful to both
communities. Let us remember: Van Dooremaal and
Medawar,1,2 the pioneers of IP research, made their seminal
discoveries on ocular IP by placing skin allografts into the AC,
thus paving the way for IP research that combines a cutaneous
and an ocular perspective. It is in this tradition that we advocate the study and manipulation of a cutaneous miniorgan, the
HF, as an unconventional, but highly instructive, accessible,
and clinically relevant preclinical surrogate model for defined
aspects of ocular IP. No doubt, the surrogate model we are
proposing here has major limitations and cannot fully satisfy a
devoted ocular IP researcher; but it is the best preclinical
surrogate model that we have so far (and may ever have) in the
human system.
Acknowledgments
MK thanks Joseph Moisseiev for his continued encouragement, professional advice, and support.
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