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CD200, a “no danger” signal for hair follicles
Journal of Dermatological Science (2006) 41, 165—174
www.intl.elsevierhealth.com/journals/jods
REVIEW ARTICLE
CD200, a ‘‘no danger’’ signal for hair follicles Michael D. Rosenblum a, Kim B. Yancey b, Edit B. Olasz b, Robert L. Truitt a,* a
Department of Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226-4801, USA b Department of Dermatology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226-4801, USA Received 17 August 2005; received in revised form 21 October 2005; accepted 9 November 2005
KEYWORDS Hair follicles; Immunology; Autoimmunity; Inflammation
Summary The ‘‘danger model’’ of immune recognition proposes that the immune system does not differentiate between self and non-self when deciding whether to mount a response, but instead, discerns between that which is dangerous or not dangerous to the host. Danger signals incite inflammatory responses, which can lead to the induction of tissue-specific autoimmunity. Immunosuppressive molecules expressed on selected cells have the potential to regulate tissue-specific inflammation, and consequently, autoimmunity. Recent studies have revealed that CD200, a potent immunoregulatory protein, is expressed on Langerhans cells (LCs) and keratinocytes (KCs) in mouse epidermis. CD200 expression is concentrated on KCs comprising the outer root sheath (ORS) of murine hair follicles (HF). Skin deficient in CD200 is highly susceptible to HF-associated inflammation and immune-mediated alopecia. In this concept review, the results of recent studies on CD200 and its inhibitory receptor, CD200R, are summarized and integrated to yield a model whereby CD200—CD200R interaction attenuates perifollicular inflammation, prevents HF-specific autoimmunity and may protect epidermal stem cells from autoimmune destruction. Further elucidation of the CD200—CD200R signaling pathway in cutaneous tissues may advance understanding of how immune homeostasis is established and maintained in the skin. # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. CD200 and the CD200 receptor family. . . . . . . . . . . . . . . . . . . . . . 1.2. CD200 imparts an immunoregulatory signal to CD200R-expressing cells . 1.3. CD200 and CD200R expression in the skin . . . . . . . . . . . . . . . . . . . 1.4. CD200 suppresses inflammatory/autoimmune alopecia . . . . . . . . . . . 1.5. CD200 as a ‘‘no danger’’ signal for hair follicles . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +1 414 456 4163; fax: +1 414 456 6543. E-mail address: [email protected] (R.L. Truitt). 0923-1811/$30.00 # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2005.11.003
166 2. 3.
M.D. Rosenblum et al. Future directions . . Conclusion . . . . . . Acknowledgements. References . . . . . .
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1. Introduction Emerging concepts in immunology propose that robust regulatory mechanisms exist to actively suppress autoimmunity in the normal healthy individual. These mechanisms have been shown to inhibit the initiation of unwanted immune responses and to prevent vital immune responses from imparting excessive damage to healthy tissue. Accordingly, autoimmune disease is thought to result, at least in part, from inborn errors in immunoregulatory pathways [1]. Multiple regulatory cells of hematopoietic origin have been identified in various tissues. These cells have been shown to suppress both autoimmune and anti-tumor immune responses [2]. In addition, a growing number of immunosuppressive molecules have been found on tissue-resident non-hemopoie-
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171 173 173 173
tic cells in various organs [3,4]. Thus, tissues themselves have the capacity to actively regulate immune reactions. Recently, we showed that CD200, a potent immunoregulatory molecule, is expressed in murine epidermis and is predominantly associated with keratinocytes (KCs) comprising the outer root sheath (ORS) [5]. Here, we review our recent findings and integrate them with the work of others to propose a model whereby CD200 plays an integral role in regulating cutaneous inflammation and HFspecific autoimmunity.
1.1. CD200 and the CD200 receptor family CD200, formerly known as OX-2, is a cell surface transmembrane glycoprotein (Fig. 1). It is expressed in a variety of tissues on cells of both hemopoietic
Fig. 1 The murine CD200R family (shown here) consists of CD200R (R1) and three structurally related transmembrane glycoproteins, CD200RLa—c (R4, R3 and R2, respectively). CD200 specifically binds to CD200R1. Specific ligands for the CD200R-like homologues (CD200RLa—c) have not yet been identified. The human CD200R gene family (not shown) consists of CD200R and a closely related CD200R-like gene hCD200La (10), although different protein isoforms may be generated through alternative splicing of the CD200R gene (11). In both mice and humans, CD200R (R1) acts as an immune inhibitory receptor, whereas the CD200R-like homologues are thought to be immune activating receptors.
CD200, a ‘‘no danger’’ signal for hair follicles and non-hemopoietic origin, including thymocytes, neurons of the central nervous system (CNS) and retina, cells of the glomeruli, cells of the syncytiotrophoblast, cells of degenerating ovarian follicles, vascular endothelium, some T cells, B cells and dendritic cells (DCs) in both peripheral blood and lymphoid tissue [6]. This pattern of CD200 expression is largely conserved in mice, rats and humans [7]. CD200 acts as a ligand (Fig. 1). It binds to a structurally similar cell surface receptor (CD200R) and induces intracellular signaling within receptorbearing cells [8]. In mice, three related CD200R-like (CD200RL) homologues have been found (Fig. 1), but their ligands remain unknown [9—11]. The human CD200R gene family consists of CD200R and a closely related CD200R-like gene hCD200La [10], but different protein isoforms may be generated by alternative splicing [11]. The human, rat and murine CD200 receptor families are emerging as a paired inhibitory and activating receptor system with CD200R acting as an inhibitory receptor [13]. The molecular mechanism by which CD200R modulates functional activity has been characterized in mouse mast cells. Unlike most immune inhibitor receptors, the cytoplasmic tail of CD200R lacks a classical ITIM (immunoreceptor tyrosine-based inhibitory motif) sequence, but contains a novel phosphotyrosinebinding motif NPXY [13]. Following ligation by CD200, CD200R is phosphorylated and binds SHIP via intermediate adaptor proteins. This results in the recruitment of the GTPase activator RasGAP and inhibition of the Ras/MAPK signal transduction pathways. Activation of ERK, JNK and p38 MAPK pathways all are inhibited by CD200R engagement, resulting in inhibition of degranulation and cytokine production in mast cells [13]. In contrast to CD200R, the CD200RL homologues appear to be activating receptors that transmit immunostimulatory signals
167 [14]. The molecular mechanisms for CD200RL signaling have not been fully elucidated, but CD200R3 has been linked to recruitment of the adaptor protein DAP12, which contains an immune activation or immunoreceptor tyrosine-based activation motif (ITAM) sequence [14]. Recent data suggest that CD200 specifically binds to CD200R and not to the other CD200RL proteins [12]. Thus, the only defined function of CD200 is to transmit an immunosuppressive signal by binding to CD200R (Fig. 1). In this review, CD200R refers specifically to the inhibitory CD200 receptor. In contrast to the rather broad expression pattern of CD200, CD200R expression is restricted to leukocytes (Fig. 1). It has been exclusively detected on macrophages, mast cells, neutrophils, basophils, DCs, B cells and some T cells [10]. Recent studies reveal that CD200R is expressed on both LCs and activated dendritic epidermal T cells (DETCs) in murine epidermis [15].
1.2. CD200 imparts an immunoregulatory signal to CD200R-expressing cells Multiple lines of evidence support a model whereby CD200 ligation of CD200R results in immune regulation of CD200R+ cells, with a downstream effect of attenuating both inflammation and autoimmunity (Fig. 2). Results from studies with CD200-deficient mice attest to the validity of such a model [15,16]. These mice have constitutively activated CD200Rexpressing leukocytes in the CNS, peripheral lymphoid tissues and skin. Although they do not develop overt autoimmunity de novo, CD200-deficient mice display an exaggerated inflammatory response to trauma and an increased susceptibility to induction of autoimmune disease, including experimental autoimmune encephalomyelitis and collageninduced arthritis [16]. Additional evidence in sup-
Fig. 2 CD200 imparts an immunoregulatory signal to CD200R-expressing cells. Various in vivo and in vitro studies have demonstrated that CD200—CD200R-mediated signaling results in an attenuation of inflammatory and/or autoimmune responses through multiple mechanisms. Relevant references are denoted in parentheses.
168 port of the concept that CD200 attenuates inflammatory and immune responses is provided by the recent identification of a viral homologue of CD200 that transmits an inhibitory signal to tissue macrophages and DCs, thereby reducing their ability to prime Tcells [17]. Collectively, these results suggest that CD200-mediated regulation of CD200R+ cells plays a role in attenuating innate inflammatory responses as well as increasing the threshold for the development of both adaptive immune responses and autoimmunity. Evidence for how CD200R ligation results in immune regulation has been elucidated in several in vitro assays utilizing various CD200R-expressing cell types. In a model of mast cell activation, Cherwinski et al. [18] have shown that signaling through CD200R inhibits both human and murine mast cell degranulation in response to FCeRI cross-linking. CD200R engagement also resulted in a potent suppression of TNFa and IL-13 secretion by mast cells [18]. Macrophages are targets of CD200—CD200Rmediated regulation, and Gorczynski [19] has shown that signaling through CD200R in purified splenic macrophages results in increased production of IL6, a cytokine implicated in immune regulation [20]. CD200R ligation on murine DCs results in increased expression of indoleamine-2,3-dioxygenase (IDO) [21], an enzyme that promotes immunosuppression of T cells through tryptophan catabolism [22]. We (Rosenblum, unpublished data) and others [10] have shown that a subset of CD4+ T cells express CD200R. The functional significance of CD200R on CD4+ Tcells is not yet clear. We found that co-culture of CD4+ T cells in vitro with autologous DCs from normal CD200+ mice (as compared to those from CD200deficient mice) resulted in diminished secretion of pro-inflammatory cytokines (TNFa and IFNg) by autoreactive T cells [23].
1.3. CD200 and CD200R expression in the skin We first became interested in the biology of CD200 in the skin through in vivo studies examining UVBmediated suppression of contact hypersensitivity. It is well established that UVB can suppress contact hypersensitivity to cutaneously applied hapten [24]. This mechanism is thought to involve UVB-mediated apoptosis of LCs [25]. Earlier, we had reported that CD200 expression was increased on the surface of murine DCs as they underwent apoptosis [23]. Thus, we hypothesized that CD200 may play a role in UVmediated immune suppression in the skin. Strong inhibition of contact hypersensitivity was observed in WT mice exposed to UVB; however, UVB did not suppress contact hypersensitivity in CD200-deficient
M.D. Rosenblum et al. mice [23], suggesting that CD200 plays a role in cutaneous immune responses. To determine whether LCs expressed CD200, epidermal cell suspensions from healthy C57BL/6 mice were examined by multi-color flow cytometry. As expected, LCs constitutively expressed low levels of CD200, but we were surprised to find that a distinct subpopulation (10—20%) of KCs also expressed CD200 [5]. CD200 expression on KCs had not been previously reported. We confirmed that KCs expressed CD200 by performing RT-PCR and flow cytometry on primary human and murine epidermal cultures, as well as human and murine KC cell lines [5]. Both human and murine primary epidermal cell cultures expressed mRNA for CD200; however, human epidermal cells (in contrast to mouse epidermal cells) did not express CD200 glycoprotein on the cell surface. In addition, only one of three murine KC cell lines tested (line 308) expressed cell surface CD200 [5]. Taken together, these findings provided the first evidence that CD200 is expressed in murine epidermis and that expression is limited to LCs and a distinct subset of KCs. To characterize where CD200+ KCs resided within the epidermis, murine skin was examined by both immunohistochemistry and fluorescent scanning confocal microscopy. These studies revealed that CD200 was preferentially expressed on cytokeratin 14-expressing KCs that comprised the ORS of HFs [5]. Very little CD200 was observed on KCs in the interfollicular epidermis. Thus, the subpopulation of KCs that preferentially expressed cell surface CD200 (as detected by flow cytometry) were KCs that localize almost exclusively to HFs. It is tempting to speculate that the HF microenvironment induces cell surface CD200 expression. This could explain why most primary KCs cultured ex vivo did not express cell surface CD200 [5]. Accordingly, the CD200-expressing murine KC cell line may have been derived from CD200+ KCs of the ORS. Cherwinski et al. have characterized CD200R expression in the skin [18]. Using immunofluorescent microscopy, they detected CD200R on the majority of mast cells and on a significant percentage of macrophages, DCs and T cells in both human and murine skin. Recently, we reported that CD200R was expressed on LCs and activated DETCs in mouse epidermis [15]. We also found that LCs from CD200deficient mice were maintained in a heightened state of immune activation in comparison to those of WT mice, suggesting that CD200—CD200R signaling attenuates their basal level of activation [15]. Taken together, these findings indicate that leukocytes in the skin have the potential to be regulated by CD200—CD200R interactions.
CD200, a ‘‘no danger’’ signal for hair follicles
1.4. CD200 suppresses inflammatory/ autoimmune alopecia A functional role for cutaneous CD200 expression was first elucidated using gender-mismatched (male-onto-female) skin grafting. This is a wellcharacterized model of cutaneous inflammation and T cell-mediated adaptive immunity [26,27]. Female mice reject strain-matched (syngeneic) male skin due to minor histocompatibility (H) antigens encoded by the Y chromosome (i.e., H-Y mismatch). Experiments were designed to test whether CD200 influenced the rate of gender-mismatched skin graft rejection. Tail skin from either normal or CD200-deficient male mice was grafted onto the flanks of normal female recipients, and graft size was measured daily (Fig. 3A). Normal male skin was completely rejected at a mean of 59 days, whereas CD200-deficient male skin was completely rejected at a mean of 25 days (Fig. 3A). Thus, CD200 in the skin significantly attenuated graft rejection.
169 In addition to the interesting results observed in the gender-mismatched animals, gender-matched skin grafts (which were used as negative controls) yielded a novel finding [5]. Female tail skin from either normal or CD200-deficient mice was grafted onto normal female recipients (Fig. 3A). In this completely gender-matched, syngeneic system, the only known genetic difference between the two groups was presence of CD200 in the normal wildtype grafts and absence of CD200 in the CD200deficient grafts. Both normal and CD200-deficient grafts were accepted long-term with no differences in rejection between the two groups (Fig. 3). However, a pronounced alopecia was observed in the CD200-deficient skin grafts. Early post-grafting (10— 15 days), an exaggerated perifollicular and intrafollicular inflammatory cell infiltrate was observed in CD200-deficient grafts with heightened recruitment of both CD4 and CD8 T cells [5]. This infiltrate persisted long-term (>40 days), eventually leading to complete destruction of HFs within the graft, but
Fig. 3 CD200 inhibits skin graft rejection and HF destruction in syngeneic skin-grafting models. (A) Tail skin from normal (wildtype, WT) or CD200-deficient (CD200 / ) C57BL/6 (B6) male mice was grafted onto the trunks of normal female B6 recipients (gender-mismatched). As controls, gender-matched tail skin from either normal or CD200-deficient female B6 mice was grafted onto the trunks of normal female B6 recipients. *Denotes a significant difference from all other groups ( p < 0.01) by Wilcoxon rank sum test. (B) Representative views of gender-matched tail skin grafts at 80 days post-grafting [5]. Normal (WT) grafts display full density short, fine hairs typical for murine tail skin. CD200-deficient (CD200 / ) skin grafts display scarred, atrophic skin that was largely devoid of hair (photographs reproduced with permission from Blackwell Publishing, Oxford, UK).
170 not rejection of the graft itself (Fig. 3B). In contrast, inflammation in grafts of normal skin completely resolved within 2 weeks post-grafting, and these grafts persisted long-term with hair (Fig. 3B). Interestingly, CD200-deficient grafts remained hairless, as they did not re-grow hair for the remaining life of the animals (>200 days post-grafting for some). In addition, hair was only lost within the CD200-deficient skin graft itself and not elsewhere on the grafted CD200+ normal host. Even when female CD200-deficient skin was grafted onto female CD200-deficient hosts, alopecia was confined to the graft itself [5]. Although alopecia was localized to grafted skin, cells capable of mediating alopecia were systemically distributed and not restricted to the skin graft. Both whole spleen cells [5] and purified T cells (Fig. 4) from ‘‘HF-sensitized’’ donors transferred alopecia to naı¨ve CD200-deficient hosts. These ‘‘HF-sensitized’’ T cells-induced extensive patchy alopecia (Fig. 4). Thus, destruction of HFs and alopecia in CD200-deficient skin grafts was, at least in part, mediated by systemically distributed autoreactive T cells.
1.5. CD200 as a ‘‘no danger’’ signal for hair follicles Mammalian HFs are evolutionarily conserved structures which play a major role in many important biological processes. Perhaps one of the most important functions of HFs is to house epidermal stem cells, which possess the capacity to regenerate not only HFs but the epidermis as well [28]. Given their vital importance, it has been proposed that HFs have evolved mechanisms to evade recognition by the immune system (termed ‘‘immune privilege’’) and thus thwart potential autoimmune responses [29]. A
M.D. Rosenblum et al. breakdown of this privilege can result in immunemediated hair loss, such as that observed in inflammatory alopecias and alopecia areata. The immune system maintains tissue integrity by providing protection from external as well as internal threats (e.g., infectious agents, cellular necrosis, cancer, autoreactivity, etc.). This has led to the concept of a ‘‘danger model’’ of immune recognition [30]. The danger model proposes that the immune system does not differentiate between self and non-self when deciding whether to mount a response, but instead, discerns between that which is dangerous or not dangerous to the host. A danger signal in this context is defined as any signal capable of inciting inflammation. Implicit to this model are the ideas that the immune system can and will respond to self-antigens if these antigens are presented in the context of danger (i.e., strong inflammatory mediators), and conversely, that inflammation must be regulated to avoid autoimmunity. Based on results from CD200-deficient skin grafting experiments and an emerging body of evidence on the immunosuppressive role of CD200—CD200R interactions, we propose a model in which HF-associated expression of CD200 imparts a ‘‘no danger’’ signal to the immune system. In essence, we postulate that CD200 signaling acts to both inhibit the initiation and attenuate the propagation of HF-associated inflammation. In doing so, CD200 may also play a role in suppressing HF-specific autoimmunity. Under quiescent or steady-state conditions, CD200 expression by KCs of the ORS imparts an immunoregulatory signal to CD200R+ leukocytes resident within the epidermis. This continuous signaling acts to maintain CD200R+ cells in a low or basal state of immune activation, thereby raising the threshold required to initiate an inflammatory response. Upon
Fig. 4 Tcells from a ‘‘HF-sensitized’’ B6 donor-induced alopecia in a syngeneic CD200-deficient host. Two tail skin grafts from female CD200-deficient B6 mice were placed onto a normal female WT B6 recipient 8 months apart. One year after the second graft, spleen and lymph nodes were collected, and Thy1+ Tcells were isolated by immunomagnetic separation. One day after lethal irradiation (10 Gy) of the female CD200-deficient B6 host, 5 106 enriched T cells (70% Thy1+; 38% CD4+; 30% CD8+; 17% B cells) were injected i.v. along with 10 106 normal bone marrow (BM) cells. Alopecia began to appear in the recipient within 2 weeks after injection. The animal is shown shortly before death on day +61 posttransplant.
CD200, a ‘‘no danger’’ signal for hair follicles initiation of inflammation, we speculate that CD200—CD200R signaling acts to attenuate inflammatory cell activity surrounding HFs, sparing them from excessive damage and subsequent autoimmune attack. In the syngeneic skin grafting model, we propose that epidermal and dermal CD200R+ leukocytes (e.g., LCs, dermal DCs and mast cells) are maintained at a heightened state of immune activation in CD200-deficient skin, skewing the balance of immune homeostasis towards ‘‘danger’’ (Fig. 5). This is consistent with the observation that CD200R+ cells, including LCs, display a more activated phenotype in various tissues of CD200-deficient mice [15,16]. Inflammation that is induced by the surgical procedure of skin grafting may lead to further dysregulation of CD200R+ cells resulting in increased production of pro-inflammatory mediators and a heightened recruitment of inflammatory cells. This is similar to the exaggerated inflammatory response observed when the CNS is traumatized in CD200-deficient mice [16]. In the absence of CD200 expression, HF-associated KCs have a diminished capacity to regulate multiple inflammatory cell types that have migrated into follicular epithelium, and thus perifollicular and intrafollicular inflammation persists. We speculate that a small number of HF-specific autoreactive T cells are among the peripheral T cells recruited to sites of inflammation, presumably as a consequence of trauma-induced chemokine release. In the absence of direct CD200—CD200R-mediated regulation and in the context of a heightened or dysregulated inflammatory milieu, presentation of HF-associated autoantigens may result in activation of these T cells. Consequently, they proliferate and attack HF epithelial cells that express the cognate antigen. Of note, healthy young CD200-deficient mice do not show any obvious abnormalities in skin, hair or hair follicles. Older mice appear to have slightly thinner hair, but hair density has not been quantified. After plucking, anagen phase hair growth appears to proceed normally in young animals, but older mice, especially males, show patches of alopecia. Transient alopecia has been seen around the bite wounds inflicted on males that were housed with aggressive non-littermates. Anecdotally, we have seen extensive dermatitis of unknown etiology in a small number of older CD200-deficient mice. To date, we have been able to reproducibly induce alopecia only by skin grafting and not by exogenous administration of inflammatory agents or cutaneous irritants. Destruction of HFs did not extend beyond the borders of the CD200-deficient skin grafts on either normal [Ref. [5] and Fig. 3B] or CD200-deficient
171 hosts (unpublished data). The fact that alopecia did not develop outside the skin graft suggests that other regulatory pathways may also contribute to controlling HF-associated autoimmunity. In our model, such regulatory mechanisms were apparently overwhelmed in the CD200-deficient host by systemic injection of relatively large numbers of lymphocytes [5] or enriched T cells (Fig. 4) containing autoreactive T cells.
2. Future directions The findings and models discussed in this review raise a number of questions: How does CD200— CD200R signaling act to maintain immune homeostasis in skin? Mast cells are early sentinels of danger in the skin and serve as potent initiators of inflammation upon activation. CD200 inhibits degranulation and cytokine secretion by CD200R+ mast cells, and others have suggested that CD200— CD200R signaling may establish the activation threshold (‘‘danger level’’) for mast cells [18]. CD200-mediated immunosuppression in the skin may involve LCs and IDO production. As previously mentioned, IDO is an enzyme that regulates immune responses in various tissues [22]. Human LCs can be induced to produce IDO, and IDO secretion has been suggested as a mechanism by which LCs maintain epidermal immune homeostasis [31]. Interestingly, CD200—CD200R signaling is a potent stimulus for IDO expression in murine splenic DCs [21]. Constitutive CD200 signaling may promote IDO expression in LCs, and in doing so, promote an immunoregulatory environment within the epidermis. In addition, DETCs help to maintain skin homeostasis and are involved in wound healing [32]. We recently reported that expression of CD200R was increased on freshly isolated DETCs following activation in vitro and that CD200-binding inhibited proliferation and cytokine secretion of a CD200R+ DETC cell line (7—17) in vitro [15]. Thus, CD200—CD200R signaling may regulate immune homeostasis in murine skin, at least in part, by regulating DETC activity. What are the antigens recognized by HF-specific Tcells? This is a question under intense investigation by several groups, as these antigens have yet to be fully defined. Although HF autoantigens could be dominant self-antigens, it is more likely that they are among the poorly processed and poorly presented cryptic self-antigens that are recognized by self-reactive T cells only under inflammatory conditions [33]. Both dominant and cryptic selfantigens are recognized by autoreactive T cells that escape negative selection in the thymus. These T cells are recruited to sites of inflammation and must
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Fig. 5 Proposed model of CD200—CD200R signaling as a ‘‘no danger’’ signal for murine HFs. In the absence of inflammatory signals (i.e., steady-state), CD200+ KCs located in the ORS of HFs interact with CD200R+ leukocytes (e.g., LCs and DETCs). This acts as a constitutive inhibitory or ‘‘no danger’’ signal, maintaining these cells in a quiescent, non-activated state. CD200—CD200R signaling may also act to attenuate low-grade perifollicular inflammation, sparing the HF and associated stem cells from excessive tissue damage. In the absence of CD200 (i.e., CD200-deficient mice as indicated with a red X), CD200R+ leukocytes are in a heightened state of activation ( ), thereby lowering the threshold required to incite an inflammatory response. Trauma to the skin (e.g., transient ischemia and surgery associated with skin grafting) results in activation and/or dysregulation of CD200R+ leukocytes ( ) and chemokine-mediated recruitment of inflammatory cells ( ). Cells of the pro-inflammatory infiltrate express CD200R, and thus, are susceptible to CD200-mediated immunoregulation. In the absence of CD200, there is diminished attenuation of the heightened inflammatory response ( ), resulting in persistent perifollicular and intrafollicular inflammation. Either the heightened level of inflammation or the prolonged duration of inflammation (or both) is sufficient to break peripheral T cell tolerance leading to the recruitment and activation of autoreactive HF-specific T cells ( ). These T cells cause further destruction of HFs within the inflammatory milieu and mediate HF destruction when transferred into vulnerable CD200-deficient hosts as shown in Fig. 4.
CD200, a ‘‘no danger’’ signal for hair follicles be suppressed to prevent autoimmunity. Our model of CD200-deficient skin grafting provides a system from which HF-specific T cells might be cloned and the antigens they recognize identified. Is CD200 expressed on human HFs? Although extensive characterization has yet to be reported, Cherwinski et al. [18] provide data to suggest that CD200 is expressed on human HF epithelium. Using immunofluorescent microscopy, they show that HFs in normal human skin express varying levels of CD200. This finding, taken together with our data, prompts examination of CD200 expression in chronic human cutaneous disease states, such as scarring and non-scarring alopecias. Do epidermal stem cells express CD200? The fact that KCs in the ORS of HFs show high levels of CD200 expression coupled with the fact that murine epidermal stem cells reside within the ORS [28], begs the question as to whether these cells express CD200. We found that HFs did not re-grow in CD200-deficient skin grafts after autoimmune destruction. This suggests that epidermal stem cells may have been destroyed (or significantly reduced) in the absence of CD200—CD200R signaling. HF stem cells may be protected by the CD200-expressing KCs that comprise the ORS. Alternatively, they may express CD200 themselves. This latter idea is supported by a preliminary report showing that human HF stem cells express high levels of cell surface CD200 (Dr. Manabu Ohyama, Dermatology Branch, National Cancer Institute, USA and Keio University School of Medicine, Tokyo, Japan).1
3. Conclusion Despite ongoing active investigation, much is still to be learned regarding the mechanisms responsible for establishing and maintaining immune homeostasis in the skin. The CD200:CD200R signaling pathway represents a novel mechanism by which cells of cutaneous tissues suppress inflammation, and thus, preserve vital structure and function. Little is known about the physiological role of the structurally related CD200R-like homologues except that their ligation by receptor-specific antibodies triggers degranulation and cytokine secretion in basophils and mast cells [14]. It is possible that the natural ligands for these receptors may be components of pathogens. We propose that the CD200receptor family is important for maintaining the balance between ‘‘danger’’ and ‘‘no danger’’ signals 1
Oral presentation and personal communication on May 6, 2005 at the 66th Annual Meeting of the Society for Investigative Dermatology, St. Louis, MO, USA.
173 in the skin. Further elucidation of the CD200:CD200R and related signaling pathways may have considerable impact on our understanding of both the pathophysiology and potential treatment of clinical dermatologic disease.
Acknowledgements This work was supported by United States Public Health Service Grant HL079792 from the National Heart, Lung and Blood Institute (Bethesda, MD, USA), the Midwest Athletes Against Childhood Cancer Fund (Milwaukee, WI, USA) and the Research Affairs Committee of the Medical College of Wisconsin (Milwaukee, WI, USA). The authors are grateful to Mr. Aaron Konkol (Milwaukee, WI) for preparation of the figures.
References [1] Prud’homme GJ. Altering immune tolerance therapeutically: the power of negative thinking. J Leukoc Biol 2004;75:586—99. [2] Jiang H, Chess L. An integrated view of suppressor T cell subsets in immunoregulation. J Clin Invest 2004;114:1198— 208. [3] Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annu Rev Immunol 2005;23:515—48. [4] Youngnak-Piboonratanakit P, Tsushima F, Otsuki N, Igarashi H, Machida U, Iwai H, et al. The expression of B7-H1 on keratinocytes in chronic inflammatory mucocutaneous disease and its regulatory role. Immunol Lett 2004;94:215—22. [5] Rosenblum MD, Olasz EB, Yancey KB, Woodliff JE, Lazarova Z, Gerber KA, et al. Expression of CD200 on epithelial cells of the murine hair follicle: a role in tissue-specific immune tolerance? J Invest Dermatol 2004;123:880—7. [6] Clark MJ, Gagnon J, Williams AF, Barclay AN. MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain. EMBO J 1985;4:113—8. [7] Wright GJ, Jones M, Puklavec MJ, Brown MH, Barclay AN. The unusual distribution of the neuronal/lymphoid cell surface CD200 (OX2) glycoprotein is conserved in humans. Immunology 2001;102:173—9. [8] Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown MH, et al. Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity 2000;13:233—42. [9] Gorczynski R, Chen Z, Kai Y, Lee L, Wong S, Marsden PA. CD200 is a ligand for all members of the CD200R family of immunoregulatory molecules. J Immunol 2004;172: 7744—9. [10] Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M, et al. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol 2003;171:3034—46. [11] Vieites JM, de la Torre R, Ortega MA, Montero T, Peco JM, Sanchez-Pozo A, et al. Characterization of human CD200 glycoprotein receptor gene located on chromosome 3q12— 13. Gene 2003;311:99—104.
174 [12] Hatherley D, Cherwinski HM, Moshref M, Barclay AN. Recombinant CD200 protein does not bind activating proteins closely related to CD200 receptor. J Immunol 2005; 175:2469—74. [13] Zhang S, Cherwinski H, Sedgwick JD, Phillips JH. Molecular mechanisms of CD200 inhibition of mast cell activation. J Immunol 2004;173:6786—93. [14] Voehringer D, Rosen DB, Lanier LL, Locksley RM. CD200 receptor family members represent novel DAP12-associated activating receptors on basophils and mast cells. J Biol Chem 2004;279:54117—23. [15] Rosenblum MD, Woodliff JE, Madsen NA, McOlash LJ, Keller MR, Truitt RL. Characterization of CD200-receptor expression in the murine epidermis. J Invest Dermatol 2005; 125:1130—8. [16] Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, et al. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science 2000;290:1768—71. [17] Cameron CM, Barrett JW, Liu L, Lucas AR, McFadden G. Myxoma virus M141R expresses a viral CD200 (vOX-2) that is responsible for down-regulation of macrophages and T-cell activation in vivo. J Virol 2005;79:6052—67. [18] Cherwinski HM, Murphy CA, Joyce BL, Bigler ME, Song YS, Zurawski SM, et al. The CD200 receptor is a novel and potent regulator of murine and human mast cell function. J Immunol 2005;174:1348—56. [19] Gorczynski RM. Transplant tolerance modifying antibody to CD200 receptor, but not CD200, alters cytokine production profile from stimulated macrophages. Eur J Immunol 2001;31:2331—7. [20] Heinrich PC, Behrmann I, Haan S, Hermanns HM, MullerNewen G, Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 2003; 374:1—20. [21] Fallarino F, Asselin-Paturel C, Vacca C, Bianchi R, Gizzi S, Fioretti MC, et al. Murine plasmacytoid dendritic cells initiate the immunosuppressive pathway of tryptophan catabolism in response to CD200 receptor engagement. J Immunol 2004;173:3748—54. [22] Mellor AL, Munn DH. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol 2004;4:762—74. [23] Rosenblum MD, Olasz E, Woodliff JE, Johnson BD, Konkol MC, Gerber KA, et al. CD200 is a novel p53-target gene involved in apoptosis-associated immune tolerance. Blood 2004;103:2691—8. [24] Elmets CA, Bergstresser PR, Tigelaar RE, Wood PJ, Streilein JW. Analysis of the mechanism of unresponsiveness produced by haptens painted on skin exposed to low dose ultraviolet radiation. J Exp Med 1983;158:781—94. [25] Takashima A. UVB-dependent modulation of epidermal cytokine network: roles in UVB-induced depletion of Langerhans cells and dendritic epidermal T cells. J Dermatol 1995; 22:876—87. [26] Binet I, Wood KJ. In vivo models of inflammation: immune rejection and skin transplantation in vivo. Methods Mol Biol 2003;225:239—48. [27] Zelenika D, Adams E, Mellor A, Simpson E, Chandler P, Stockinger B, et al. Rejection of H-Y disparate skin grafts
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by monospecific CD4+ Th1 and Th2 cells: no requirement for CD8+ T cells or B cells. J Immunol 1998;161:1868—74. Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, et al. Defining the epithelial stem cell niche in skin. Science 2004;303:359—63. Paus R, Nickologg BJ, Ito T. A ‘hairy’ privilege. Trends Immunol 2005;26:32—40. Matzinger P. The danger model: a renewed sense of self. Science 2002;296:301—5. von Bubnoff D, Bausinger H, Matz H, Koch S, Hacker G, Takikawa O, et al. Human epidermal langerhans cells express the immunoregulatory enzyme indoleamine 2,3dioxygenase. J Invest Dermatol 2004;123:298—304. Sharp LL, Jameson JM, Cauvi G, Havran WL. Dendritic epidermal T cells regulate skin homeostasis through local production of insulin-like growth factor 1. Nature Immunol 2005;6:73—9. Moudgil KD, Sercarz EE. Understanding crypticity is the key to revealing the pathogenesis of autoimmunity. Trends Immunol 2005;26:355—9.
Robert L. Truitt received his BA and PhD degrees in microbiology from Southern Illinois University, Carbondale, Illinois, USA, in 1968 and 1973, respectively. A former scholar of the Leukemia Society of America, he has been at the Medical College of Wisconsin, Milwaukee, Wisconsin, USA, since 1984 and is currently a professor in the Department of Pediatrics and a member of the Children’s Research Institute. He also is associate director for basic science research in the Cancer Center of the Medical College of Wisconsin. He is a member of the Board of Directors of the American Society for Blood and Marrow Transplantation and serves as an associate editor for the Society’s journal Biology of Blood and Marrow Transplantation. His research interests include the biology of hematopoietic stem cell transplantation and adoptive immunotherapy as well as cellular and molecular pathways of immune regulation. He is internationally known for his research on allogeneic bone marrow transplantation and animal models of graftversus-host and graft-versus-leukemia reactions.
Michael D. Rosenblum received his BS degree in cell biology with honors from the University of Western Ontario, Canada, in 1997. He is a member of the Medical Scientist Training Program at the Medical College of Wisconsin (MCW), Milwaukee, Wisconsin, USA. He received a PhD degree in microbiology in 2004 and was recognized with the Outstanding Doctoral Dissertation Award. He is nearing completion of requirements for the MD degree at MCW, while working part-time as a post-doctoral fellow within the Department of Pediatrics. His research interests include mechanisms of immune regulation, immune tolerance and cutaneous immunology.
www.intl.elsevierhealth.com/journals/jods
REVIEW ARTICLE
CD200, a ‘‘no danger’’ signal for hair follicles Michael D. Rosenblum a, Kim B. Yancey b, Edit B. Olasz b, Robert L. Truitt a,* a
Department of Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226-4801, USA b Department of Dermatology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226-4801, USA Received 17 August 2005; received in revised form 21 October 2005; accepted 9 November 2005
KEYWORDS Hair follicles; Immunology; Autoimmunity; Inflammation
Summary The ‘‘danger model’’ of immune recognition proposes that the immune system does not differentiate between self and non-self when deciding whether to mount a response, but instead, discerns between that which is dangerous or not dangerous to the host. Danger signals incite inflammatory responses, which can lead to the induction of tissue-specific autoimmunity. Immunosuppressive molecules expressed on selected cells have the potential to regulate tissue-specific inflammation, and consequently, autoimmunity. Recent studies have revealed that CD200, a potent immunoregulatory protein, is expressed on Langerhans cells (LCs) and keratinocytes (KCs) in mouse epidermis. CD200 expression is concentrated on KCs comprising the outer root sheath (ORS) of murine hair follicles (HF). Skin deficient in CD200 is highly susceptible to HF-associated inflammation and immune-mediated alopecia. In this concept review, the results of recent studies on CD200 and its inhibitory receptor, CD200R, are summarized and integrated to yield a model whereby CD200—CD200R interaction attenuates perifollicular inflammation, prevents HF-specific autoimmunity and may protect epidermal stem cells from autoimmune destruction. Further elucidation of the CD200—CD200R signaling pathway in cutaneous tissues may advance understanding of how immune homeostasis is established and maintained in the skin. # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. CD200 and the CD200 receptor family. . . . . . . . . . . . . . . . . . . . . . 1.2. CD200 imparts an immunoregulatory signal to CD200R-expressing cells . 1.3. CD200 and CD200R expression in the skin . . . . . . . . . . . . . . . . . . . 1.4. CD200 suppresses inflammatory/autoimmune alopecia . . . . . . . . . . . 1.5. CD200 as a ‘‘no danger’’ signal for hair follicles . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +1 414 456 4163; fax: +1 414 456 6543. E-mail address: [email protected] (R.L. Truitt). 0923-1811/$30.00 # 2005 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2005.11.003
166 2. 3.
M.D. Rosenblum et al. Future directions . . Conclusion . . . . . . Acknowledgements. References . . . . . .
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1. Introduction Emerging concepts in immunology propose that robust regulatory mechanisms exist to actively suppress autoimmunity in the normal healthy individual. These mechanisms have been shown to inhibit the initiation of unwanted immune responses and to prevent vital immune responses from imparting excessive damage to healthy tissue. Accordingly, autoimmune disease is thought to result, at least in part, from inborn errors in immunoregulatory pathways [1]. Multiple regulatory cells of hematopoietic origin have been identified in various tissues. These cells have been shown to suppress both autoimmune and anti-tumor immune responses [2]. In addition, a growing number of immunosuppressive molecules have been found on tissue-resident non-hemopoie-
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171 173 173 173
tic cells in various organs [3,4]. Thus, tissues themselves have the capacity to actively regulate immune reactions. Recently, we showed that CD200, a potent immunoregulatory molecule, is expressed in murine epidermis and is predominantly associated with keratinocytes (KCs) comprising the outer root sheath (ORS) [5]. Here, we review our recent findings and integrate them with the work of others to propose a model whereby CD200 plays an integral role in regulating cutaneous inflammation and HFspecific autoimmunity.
1.1. CD200 and the CD200 receptor family CD200, formerly known as OX-2, is a cell surface transmembrane glycoprotein (Fig. 1). It is expressed in a variety of tissues on cells of both hemopoietic
Fig. 1 The murine CD200R family (shown here) consists of CD200R (R1) and three structurally related transmembrane glycoproteins, CD200RLa—c (R4, R3 and R2, respectively). CD200 specifically binds to CD200R1. Specific ligands for the CD200R-like homologues (CD200RLa—c) have not yet been identified. The human CD200R gene family (not shown) consists of CD200R and a closely related CD200R-like gene hCD200La (10), although different protein isoforms may be generated through alternative splicing of the CD200R gene (11). In both mice and humans, CD200R (R1) acts as an immune inhibitory receptor, whereas the CD200R-like homologues are thought to be immune activating receptors.
CD200, a ‘‘no danger’’ signal for hair follicles and non-hemopoietic origin, including thymocytes, neurons of the central nervous system (CNS) and retina, cells of the glomeruli, cells of the syncytiotrophoblast, cells of degenerating ovarian follicles, vascular endothelium, some T cells, B cells and dendritic cells (DCs) in both peripheral blood and lymphoid tissue [6]. This pattern of CD200 expression is largely conserved in mice, rats and humans [7]. CD200 acts as a ligand (Fig. 1). It binds to a structurally similar cell surface receptor (CD200R) and induces intracellular signaling within receptorbearing cells [8]. In mice, three related CD200R-like (CD200RL) homologues have been found (Fig. 1), but their ligands remain unknown [9—11]. The human CD200R gene family consists of CD200R and a closely related CD200R-like gene hCD200La [10], but different protein isoforms may be generated by alternative splicing [11]. The human, rat and murine CD200 receptor families are emerging as a paired inhibitory and activating receptor system with CD200R acting as an inhibitory receptor [13]. The molecular mechanism by which CD200R modulates functional activity has been characterized in mouse mast cells. Unlike most immune inhibitor receptors, the cytoplasmic tail of CD200R lacks a classical ITIM (immunoreceptor tyrosine-based inhibitory motif) sequence, but contains a novel phosphotyrosinebinding motif NPXY [13]. Following ligation by CD200, CD200R is phosphorylated and binds SHIP via intermediate adaptor proteins. This results in the recruitment of the GTPase activator RasGAP and inhibition of the Ras/MAPK signal transduction pathways. Activation of ERK, JNK and p38 MAPK pathways all are inhibited by CD200R engagement, resulting in inhibition of degranulation and cytokine production in mast cells [13]. In contrast to CD200R, the CD200RL homologues appear to be activating receptors that transmit immunostimulatory signals
167 [14]. The molecular mechanisms for CD200RL signaling have not been fully elucidated, but CD200R3 has been linked to recruitment of the adaptor protein DAP12, which contains an immune activation or immunoreceptor tyrosine-based activation motif (ITAM) sequence [14]. Recent data suggest that CD200 specifically binds to CD200R and not to the other CD200RL proteins [12]. Thus, the only defined function of CD200 is to transmit an immunosuppressive signal by binding to CD200R (Fig. 1). In this review, CD200R refers specifically to the inhibitory CD200 receptor. In contrast to the rather broad expression pattern of CD200, CD200R expression is restricted to leukocytes (Fig. 1). It has been exclusively detected on macrophages, mast cells, neutrophils, basophils, DCs, B cells and some T cells [10]. Recent studies reveal that CD200R is expressed on both LCs and activated dendritic epidermal T cells (DETCs) in murine epidermis [15].
1.2. CD200 imparts an immunoregulatory signal to CD200R-expressing cells Multiple lines of evidence support a model whereby CD200 ligation of CD200R results in immune regulation of CD200R+ cells, with a downstream effect of attenuating both inflammation and autoimmunity (Fig. 2). Results from studies with CD200-deficient mice attest to the validity of such a model [15,16]. These mice have constitutively activated CD200Rexpressing leukocytes in the CNS, peripheral lymphoid tissues and skin. Although they do not develop overt autoimmunity de novo, CD200-deficient mice display an exaggerated inflammatory response to trauma and an increased susceptibility to induction of autoimmune disease, including experimental autoimmune encephalomyelitis and collageninduced arthritis [16]. Additional evidence in sup-
Fig. 2 CD200 imparts an immunoregulatory signal to CD200R-expressing cells. Various in vivo and in vitro studies have demonstrated that CD200—CD200R-mediated signaling results in an attenuation of inflammatory and/or autoimmune responses through multiple mechanisms. Relevant references are denoted in parentheses.
168 port of the concept that CD200 attenuates inflammatory and immune responses is provided by the recent identification of a viral homologue of CD200 that transmits an inhibitory signal to tissue macrophages and DCs, thereby reducing their ability to prime Tcells [17]. Collectively, these results suggest that CD200-mediated regulation of CD200R+ cells plays a role in attenuating innate inflammatory responses as well as increasing the threshold for the development of both adaptive immune responses and autoimmunity. Evidence for how CD200R ligation results in immune regulation has been elucidated in several in vitro assays utilizing various CD200R-expressing cell types. In a model of mast cell activation, Cherwinski et al. [18] have shown that signaling through CD200R inhibits both human and murine mast cell degranulation in response to FCeRI cross-linking. CD200R engagement also resulted in a potent suppression of TNFa and IL-13 secretion by mast cells [18]. Macrophages are targets of CD200—CD200Rmediated regulation, and Gorczynski [19] has shown that signaling through CD200R in purified splenic macrophages results in increased production of IL6, a cytokine implicated in immune regulation [20]. CD200R ligation on murine DCs results in increased expression of indoleamine-2,3-dioxygenase (IDO) [21], an enzyme that promotes immunosuppression of T cells through tryptophan catabolism [22]. We (Rosenblum, unpublished data) and others [10] have shown that a subset of CD4+ T cells express CD200R. The functional significance of CD200R on CD4+ Tcells is not yet clear. We found that co-culture of CD4+ T cells in vitro with autologous DCs from normal CD200+ mice (as compared to those from CD200deficient mice) resulted in diminished secretion of pro-inflammatory cytokines (TNFa and IFNg) by autoreactive T cells [23].
1.3. CD200 and CD200R expression in the skin We first became interested in the biology of CD200 in the skin through in vivo studies examining UVBmediated suppression of contact hypersensitivity. It is well established that UVB can suppress contact hypersensitivity to cutaneously applied hapten [24]. This mechanism is thought to involve UVB-mediated apoptosis of LCs [25]. Earlier, we had reported that CD200 expression was increased on the surface of murine DCs as they underwent apoptosis [23]. Thus, we hypothesized that CD200 may play a role in UVmediated immune suppression in the skin. Strong inhibition of contact hypersensitivity was observed in WT mice exposed to UVB; however, UVB did not suppress contact hypersensitivity in CD200-deficient
M.D. Rosenblum et al. mice [23], suggesting that CD200 plays a role in cutaneous immune responses. To determine whether LCs expressed CD200, epidermal cell suspensions from healthy C57BL/6 mice were examined by multi-color flow cytometry. As expected, LCs constitutively expressed low levels of CD200, but we were surprised to find that a distinct subpopulation (10—20%) of KCs also expressed CD200 [5]. CD200 expression on KCs had not been previously reported. We confirmed that KCs expressed CD200 by performing RT-PCR and flow cytometry on primary human and murine epidermal cultures, as well as human and murine KC cell lines [5]. Both human and murine primary epidermal cell cultures expressed mRNA for CD200; however, human epidermal cells (in contrast to mouse epidermal cells) did not express CD200 glycoprotein on the cell surface. In addition, only one of three murine KC cell lines tested (line 308) expressed cell surface CD200 [5]. Taken together, these findings provided the first evidence that CD200 is expressed in murine epidermis and that expression is limited to LCs and a distinct subset of KCs. To characterize where CD200+ KCs resided within the epidermis, murine skin was examined by both immunohistochemistry and fluorescent scanning confocal microscopy. These studies revealed that CD200 was preferentially expressed on cytokeratin 14-expressing KCs that comprised the ORS of HFs [5]. Very little CD200 was observed on KCs in the interfollicular epidermis. Thus, the subpopulation of KCs that preferentially expressed cell surface CD200 (as detected by flow cytometry) were KCs that localize almost exclusively to HFs. It is tempting to speculate that the HF microenvironment induces cell surface CD200 expression. This could explain why most primary KCs cultured ex vivo did not express cell surface CD200 [5]. Accordingly, the CD200-expressing murine KC cell line may have been derived from CD200+ KCs of the ORS. Cherwinski et al. have characterized CD200R expression in the skin [18]. Using immunofluorescent microscopy, they detected CD200R on the majority of mast cells and on a significant percentage of macrophages, DCs and T cells in both human and murine skin. Recently, we reported that CD200R was expressed on LCs and activated DETCs in mouse epidermis [15]. We also found that LCs from CD200deficient mice were maintained in a heightened state of immune activation in comparison to those of WT mice, suggesting that CD200—CD200R signaling attenuates their basal level of activation [15]. Taken together, these findings indicate that leukocytes in the skin have the potential to be regulated by CD200—CD200R interactions.
CD200, a ‘‘no danger’’ signal for hair follicles
1.4. CD200 suppresses inflammatory/ autoimmune alopecia A functional role for cutaneous CD200 expression was first elucidated using gender-mismatched (male-onto-female) skin grafting. This is a wellcharacterized model of cutaneous inflammation and T cell-mediated adaptive immunity [26,27]. Female mice reject strain-matched (syngeneic) male skin due to minor histocompatibility (H) antigens encoded by the Y chromosome (i.e., H-Y mismatch). Experiments were designed to test whether CD200 influenced the rate of gender-mismatched skin graft rejection. Tail skin from either normal or CD200-deficient male mice was grafted onto the flanks of normal female recipients, and graft size was measured daily (Fig. 3A). Normal male skin was completely rejected at a mean of 59 days, whereas CD200-deficient male skin was completely rejected at a mean of 25 days (Fig. 3A). Thus, CD200 in the skin significantly attenuated graft rejection.
169 In addition to the interesting results observed in the gender-mismatched animals, gender-matched skin grafts (which were used as negative controls) yielded a novel finding [5]. Female tail skin from either normal or CD200-deficient mice was grafted onto normal female recipients (Fig. 3A). In this completely gender-matched, syngeneic system, the only known genetic difference between the two groups was presence of CD200 in the normal wildtype grafts and absence of CD200 in the CD200deficient grafts. Both normal and CD200-deficient grafts were accepted long-term with no differences in rejection between the two groups (Fig. 3). However, a pronounced alopecia was observed in the CD200-deficient skin grafts. Early post-grafting (10— 15 days), an exaggerated perifollicular and intrafollicular inflammatory cell infiltrate was observed in CD200-deficient grafts with heightened recruitment of both CD4 and CD8 T cells [5]. This infiltrate persisted long-term (>40 days), eventually leading to complete destruction of HFs within the graft, but
Fig. 3 CD200 inhibits skin graft rejection and HF destruction in syngeneic skin-grafting models. (A) Tail skin from normal (wildtype, WT) or CD200-deficient (CD200 / ) C57BL/6 (B6) male mice was grafted onto the trunks of normal female B6 recipients (gender-mismatched). As controls, gender-matched tail skin from either normal or CD200-deficient female B6 mice was grafted onto the trunks of normal female B6 recipients. *Denotes a significant difference from all other groups ( p < 0.01) by Wilcoxon rank sum test. (B) Representative views of gender-matched tail skin grafts at 80 days post-grafting [5]. Normal (WT) grafts display full density short, fine hairs typical for murine tail skin. CD200-deficient (CD200 / ) skin grafts display scarred, atrophic skin that was largely devoid of hair (photographs reproduced with permission from Blackwell Publishing, Oxford, UK).
170 not rejection of the graft itself (Fig. 3B). In contrast, inflammation in grafts of normal skin completely resolved within 2 weeks post-grafting, and these grafts persisted long-term with hair (Fig. 3B). Interestingly, CD200-deficient grafts remained hairless, as they did not re-grow hair for the remaining life of the animals (>200 days post-grafting for some). In addition, hair was only lost within the CD200-deficient skin graft itself and not elsewhere on the grafted CD200+ normal host. Even when female CD200-deficient skin was grafted onto female CD200-deficient hosts, alopecia was confined to the graft itself [5]. Although alopecia was localized to grafted skin, cells capable of mediating alopecia were systemically distributed and not restricted to the skin graft. Both whole spleen cells [5] and purified T cells (Fig. 4) from ‘‘HF-sensitized’’ donors transferred alopecia to naı¨ve CD200-deficient hosts. These ‘‘HF-sensitized’’ T cells-induced extensive patchy alopecia (Fig. 4). Thus, destruction of HFs and alopecia in CD200-deficient skin grafts was, at least in part, mediated by systemically distributed autoreactive T cells.
1.5. CD200 as a ‘‘no danger’’ signal for hair follicles Mammalian HFs are evolutionarily conserved structures which play a major role in many important biological processes. Perhaps one of the most important functions of HFs is to house epidermal stem cells, which possess the capacity to regenerate not only HFs but the epidermis as well [28]. Given their vital importance, it has been proposed that HFs have evolved mechanisms to evade recognition by the immune system (termed ‘‘immune privilege’’) and thus thwart potential autoimmune responses [29]. A
M.D. Rosenblum et al. breakdown of this privilege can result in immunemediated hair loss, such as that observed in inflammatory alopecias and alopecia areata. The immune system maintains tissue integrity by providing protection from external as well as internal threats (e.g., infectious agents, cellular necrosis, cancer, autoreactivity, etc.). This has led to the concept of a ‘‘danger model’’ of immune recognition [30]. The danger model proposes that the immune system does not differentiate between self and non-self when deciding whether to mount a response, but instead, discerns between that which is dangerous or not dangerous to the host. A danger signal in this context is defined as any signal capable of inciting inflammation. Implicit to this model are the ideas that the immune system can and will respond to self-antigens if these antigens are presented in the context of danger (i.e., strong inflammatory mediators), and conversely, that inflammation must be regulated to avoid autoimmunity. Based on results from CD200-deficient skin grafting experiments and an emerging body of evidence on the immunosuppressive role of CD200—CD200R interactions, we propose a model in which HF-associated expression of CD200 imparts a ‘‘no danger’’ signal to the immune system. In essence, we postulate that CD200 signaling acts to both inhibit the initiation and attenuate the propagation of HF-associated inflammation. In doing so, CD200 may also play a role in suppressing HF-specific autoimmunity. Under quiescent or steady-state conditions, CD200 expression by KCs of the ORS imparts an immunoregulatory signal to CD200R+ leukocytes resident within the epidermis. This continuous signaling acts to maintain CD200R+ cells in a low or basal state of immune activation, thereby raising the threshold required to initiate an inflammatory response. Upon
Fig. 4 Tcells from a ‘‘HF-sensitized’’ B6 donor-induced alopecia in a syngeneic CD200-deficient host. Two tail skin grafts from female CD200-deficient B6 mice were placed onto a normal female WT B6 recipient 8 months apart. One year after the second graft, spleen and lymph nodes were collected, and Thy1+ Tcells were isolated by immunomagnetic separation. One day after lethal irradiation (10 Gy) of the female CD200-deficient B6 host, 5 106 enriched T cells (70% Thy1+; 38% CD4+; 30% CD8+; 17% B cells) were injected i.v. along with 10 106 normal bone marrow (BM) cells. Alopecia began to appear in the recipient within 2 weeks after injection. The animal is shown shortly before death on day +61 posttransplant.
CD200, a ‘‘no danger’’ signal for hair follicles initiation of inflammation, we speculate that CD200—CD200R signaling acts to attenuate inflammatory cell activity surrounding HFs, sparing them from excessive damage and subsequent autoimmune attack. In the syngeneic skin grafting model, we propose that epidermal and dermal CD200R+ leukocytes (e.g., LCs, dermal DCs and mast cells) are maintained at a heightened state of immune activation in CD200-deficient skin, skewing the balance of immune homeostasis towards ‘‘danger’’ (Fig. 5). This is consistent with the observation that CD200R+ cells, including LCs, display a more activated phenotype in various tissues of CD200-deficient mice [15,16]. Inflammation that is induced by the surgical procedure of skin grafting may lead to further dysregulation of CD200R+ cells resulting in increased production of pro-inflammatory mediators and a heightened recruitment of inflammatory cells. This is similar to the exaggerated inflammatory response observed when the CNS is traumatized in CD200-deficient mice [16]. In the absence of CD200 expression, HF-associated KCs have a diminished capacity to regulate multiple inflammatory cell types that have migrated into follicular epithelium, and thus perifollicular and intrafollicular inflammation persists. We speculate that a small number of HF-specific autoreactive T cells are among the peripheral T cells recruited to sites of inflammation, presumably as a consequence of trauma-induced chemokine release. In the absence of direct CD200—CD200R-mediated regulation and in the context of a heightened or dysregulated inflammatory milieu, presentation of HF-associated autoantigens may result in activation of these T cells. Consequently, they proliferate and attack HF epithelial cells that express the cognate antigen. Of note, healthy young CD200-deficient mice do not show any obvious abnormalities in skin, hair or hair follicles. Older mice appear to have slightly thinner hair, but hair density has not been quantified. After plucking, anagen phase hair growth appears to proceed normally in young animals, but older mice, especially males, show patches of alopecia. Transient alopecia has been seen around the bite wounds inflicted on males that were housed with aggressive non-littermates. Anecdotally, we have seen extensive dermatitis of unknown etiology in a small number of older CD200-deficient mice. To date, we have been able to reproducibly induce alopecia only by skin grafting and not by exogenous administration of inflammatory agents or cutaneous irritants. Destruction of HFs did not extend beyond the borders of the CD200-deficient skin grafts on either normal [Ref. [5] and Fig. 3B] or CD200-deficient
171 hosts (unpublished data). The fact that alopecia did not develop outside the skin graft suggests that other regulatory pathways may also contribute to controlling HF-associated autoimmunity. In our model, such regulatory mechanisms were apparently overwhelmed in the CD200-deficient host by systemic injection of relatively large numbers of lymphocytes [5] or enriched T cells (Fig. 4) containing autoreactive T cells.
2. Future directions The findings and models discussed in this review raise a number of questions: How does CD200— CD200R signaling act to maintain immune homeostasis in skin? Mast cells are early sentinels of danger in the skin and serve as potent initiators of inflammation upon activation. CD200 inhibits degranulation and cytokine secretion by CD200R+ mast cells, and others have suggested that CD200— CD200R signaling may establish the activation threshold (‘‘danger level’’) for mast cells [18]. CD200-mediated immunosuppression in the skin may involve LCs and IDO production. As previously mentioned, IDO is an enzyme that regulates immune responses in various tissues [22]. Human LCs can be induced to produce IDO, and IDO secretion has been suggested as a mechanism by which LCs maintain epidermal immune homeostasis [31]. Interestingly, CD200—CD200R signaling is a potent stimulus for IDO expression in murine splenic DCs [21]. Constitutive CD200 signaling may promote IDO expression in LCs, and in doing so, promote an immunoregulatory environment within the epidermis. In addition, DETCs help to maintain skin homeostasis and are involved in wound healing [32]. We recently reported that expression of CD200R was increased on freshly isolated DETCs following activation in vitro and that CD200-binding inhibited proliferation and cytokine secretion of a CD200R+ DETC cell line (7—17) in vitro [15]. Thus, CD200—CD200R signaling may regulate immune homeostasis in murine skin, at least in part, by regulating DETC activity. What are the antigens recognized by HF-specific Tcells? This is a question under intense investigation by several groups, as these antigens have yet to be fully defined. Although HF autoantigens could be dominant self-antigens, it is more likely that they are among the poorly processed and poorly presented cryptic self-antigens that are recognized by self-reactive T cells only under inflammatory conditions [33]. Both dominant and cryptic selfantigens are recognized by autoreactive T cells that escape negative selection in the thymus. These T cells are recruited to sites of inflammation and must
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Fig. 5 Proposed model of CD200—CD200R signaling as a ‘‘no danger’’ signal for murine HFs. In the absence of inflammatory signals (i.e., steady-state), CD200+ KCs located in the ORS of HFs interact with CD200R+ leukocytes (e.g., LCs and DETCs). This acts as a constitutive inhibitory or ‘‘no danger’’ signal, maintaining these cells in a quiescent, non-activated state. CD200—CD200R signaling may also act to attenuate low-grade perifollicular inflammation, sparing the HF and associated stem cells from excessive tissue damage. In the absence of CD200 (i.e., CD200-deficient mice as indicated with a red X), CD200R+ leukocytes are in a heightened state of activation ( ), thereby lowering the threshold required to incite an inflammatory response. Trauma to the skin (e.g., transient ischemia and surgery associated with skin grafting) results in activation and/or dysregulation of CD200R+ leukocytes ( ) and chemokine-mediated recruitment of inflammatory cells ( ). Cells of the pro-inflammatory infiltrate express CD200R, and thus, are susceptible to CD200-mediated immunoregulation. In the absence of CD200, there is diminished attenuation of the heightened inflammatory response ( ), resulting in persistent perifollicular and intrafollicular inflammation. Either the heightened level of inflammation or the prolonged duration of inflammation (or both) is sufficient to break peripheral T cell tolerance leading to the recruitment and activation of autoreactive HF-specific T cells ( ). These T cells cause further destruction of HFs within the inflammatory milieu and mediate HF destruction when transferred into vulnerable CD200-deficient hosts as shown in Fig. 4.
CD200, a ‘‘no danger’’ signal for hair follicles be suppressed to prevent autoimmunity. Our model of CD200-deficient skin grafting provides a system from which HF-specific T cells might be cloned and the antigens they recognize identified. Is CD200 expressed on human HFs? Although extensive characterization has yet to be reported, Cherwinski et al. [18] provide data to suggest that CD200 is expressed on human HF epithelium. Using immunofluorescent microscopy, they show that HFs in normal human skin express varying levels of CD200. This finding, taken together with our data, prompts examination of CD200 expression in chronic human cutaneous disease states, such as scarring and non-scarring alopecias. Do epidermal stem cells express CD200? The fact that KCs in the ORS of HFs show high levels of CD200 expression coupled with the fact that murine epidermal stem cells reside within the ORS [28], begs the question as to whether these cells express CD200. We found that HFs did not re-grow in CD200-deficient skin grafts after autoimmune destruction. This suggests that epidermal stem cells may have been destroyed (or significantly reduced) in the absence of CD200—CD200R signaling. HF stem cells may be protected by the CD200-expressing KCs that comprise the ORS. Alternatively, they may express CD200 themselves. This latter idea is supported by a preliminary report showing that human HF stem cells express high levels of cell surface CD200 (Dr. Manabu Ohyama, Dermatology Branch, National Cancer Institute, USA and Keio University School of Medicine, Tokyo, Japan).1
3. Conclusion Despite ongoing active investigation, much is still to be learned regarding the mechanisms responsible for establishing and maintaining immune homeostasis in the skin. The CD200:CD200R signaling pathway represents a novel mechanism by which cells of cutaneous tissues suppress inflammation, and thus, preserve vital structure and function. Little is known about the physiological role of the structurally related CD200R-like homologues except that their ligation by receptor-specific antibodies triggers degranulation and cytokine secretion in basophils and mast cells [14]. It is possible that the natural ligands for these receptors may be components of pathogens. We propose that the CD200receptor family is important for maintaining the balance between ‘‘danger’’ and ‘‘no danger’’ signals 1
Oral presentation and personal communication on May 6, 2005 at the 66th Annual Meeting of the Society for Investigative Dermatology, St. Louis, MO, USA.
173 in the skin. Further elucidation of the CD200:CD200R and related signaling pathways may have considerable impact on our understanding of both the pathophysiology and potential treatment of clinical dermatologic disease.
Acknowledgements This work was supported by United States Public Health Service Grant HL079792 from the National Heart, Lung and Blood Institute (Bethesda, MD, USA), the Midwest Athletes Against Childhood Cancer Fund (Milwaukee, WI, USA) and the Research Affairs Committee of the Medical College of Wisconsin (Milwaukee, WI, USA). The authors are grateful to Mr. Aaron Konkol (Milwaukee, WI) for preparation of the figures.
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Robert L. Truitt received his BA and PhD degrees in microbiology from Southern Illinois University, Carbondale, Illinois, USA, in 1968 and 1973, respectively. A former scholar of the Leukemia Society of America, he has been at the Medical College of Wisconsin, Milwaukee, Wisconsin, USA, since 1984 and is currently a professor in the Department of Pediatrics and a member of the Children’s Research Institute. He also is associate director for basic science research in the Cancer Center of the Medical College of Wisconsin. He is a member of the Board of Directors of the American Society for Blood and Marrow Transplantation and serves as an associate editor for the Society’s journal Biology of Blood and Marrow Transplantation. His research interests include the biology of hematopoietic stem cell transplantation and adoptive immunotherapy as well as cellular and molecular pathways of immune regulation. He is internationally known for his research on allogeneic bone marrow transplantation and animal models of graftversus-host and graft-versus-leukemia reactions.
Michael D. Rosenblum received his BS degree in cell biology with honors from the University of Western Ontario, Canada, in 1997. He is a member of the Medical Scientist Training Program at the Medical College of Wisconsin (MCW), Milwaukee, Wisconsin, USA. He received a PhD degree in microbiology in 2004 and was recognized with the Outstanding Doctoral Dissertation Award. He is nearing completion of requirements for the MD degree at MCW, while working part-time as a post-doctoral fellow within the Department of Pediatrics. His research interests include mechanisms of immune regulation, immune tolerance and cutaneous immunology.