Auteur : Dr Laurent MISERY (Faculté de Médecine de SAINT-ETIENNE).
Travail subventionné par l’AAA (subvention 1999) à hauteur de 29 700 F.
Subvention débloquée le 1er août 1999.
Résumé fourni en février 2001.
Compte rendu détaillé fourni le 25 avril 2002.
RESUME (texte intégral)
Diminution de l’expression du CGRP au cours de la pelade.
D.Meyronet, K. Jaber, A. Gentil-Perret, F. Cambazard, L. Misery. Services d’Anatomie Pathologique et de Dermatologie, Hôpital Nord, Saint-Etienne, France.
Les poussées de pelade sont souvent considérées comme pouvant être associées au stress. La dénervation permet la repousse. Le système nerveux cutané paraît donc impliqué dans la physiopathogénie de la pelade.
Chez 15 patients atteints de pelade, nous avons réalisé une étude en cuir chevelu sain et en cuir chevelu malade. Nous avons recherché l’expression de la substance P et du CGRP (calcitonin gene-related peptide), neuromédiateurs co-exprimés par les fibres nerveuses sensitives. Cette étude immunohistochimique a été réalisée sur des coupes congelées. Les anticorps monoclonaux ont été révélés immunoperoxydase.
Nous n’avons pas observé de différences d’expression de la substance P entre le cuir chevelu sain et le cuir chevelu atteint de pelade. En revanche, le CGRP était fortement exprimé sur les cellules de couches basales de l’épiderme et des follicules pileux en cuir chevelu sain alors que sa détection était difficile dans les zones alopéciques.
Cette chute de l’expression du CGRP peut correspondre à une diminution de sa production ou de l’expression de son récepteur. Une autre étude a montré une diminution des taux de CGRP sanguins. Le CGRP est un neuromédiateur vasodilatateur et immuno-suppresseur. On peut donc supposer que la chute de son expression favorise la vasoconstriction et l’action des lymphocytes, qui induisent la formation de plaques de pelade. Les agonistes du CGRP pourraient donc représenter une nouvelle voie thérapeutique. Des travaux sur d’autres neuromédiateurs sont en cours.
COMPTE RENDU DETAILLE (texte intégral)
Inhibition of CGRP expression in alopecia areata
David Meyroneta, Kahena Jaberb, Anne Gentil-Perreta,
Frédéric Cambazardb, Laurent Miseryb.
Departments of Pathologya and Dermatologyb, University Hospital, Saint-Etienne, France.
The course of alopecia areata appears to be associated to stress. Denervation allows hair to grow again. Hence, the nervous system is involved in the pathophysiogeny of this disease.
Fifteen outpatients with alopecia areta have undergone biopsies of healthy scalp and lesions of alopecia areata. We have searched for the expression of substance P, somatostatin and CGRP (calcitonin gene-related peptide), which are neurotransmitters co-expressed by sensitive nervous fibres, as well as the expression of NGF (nerve growth factor) and its receptor. This immunohistochemical study has been performed on frozen slides. Monoclonal antibodies were revealed by immunoperoxidase.
Substance P, somatostatin, NGF and its receptor were expressed in a same manner in healthy scalp and lesions. On the contrary, CGRP was strongly expressed on basal cells of epidermis and hairy folicles in healthy scalp whereas it was almost undetectable in lesions of alopecia areata.
This dramatic inhibition of CGRP expression could be due to a decrease of production or to a diminished expression of CGRP receptor. A recent study reveals a strong decrease on CGRP blood amounts. CGRP is a neurotransmitters with vaodilatator and immunosupressive properties. We suggest the inhibition of CGRP expression in lesions favours vasoconstriction and the effects of lymphocytes, which are known to induce alopecia areata patches. Hence, CGRP agonists could be used to treat this disease.
Key-words : alopecia areata ; neurotransmitter ; CGRP ; substance P ; somatostatin ; NGF
Alopecia areata can be considered as an auto-immune disease. This disorder can be transmitted by T lymphocytes (1). T cytotoxic lymphocytes induce hair folicle cell apoptosis through Fas-Fas ligand system (2). However, auto-antibodies recognizing hair folicles can be detected (3).
Genetic factors are involved in the pathophysiogeny of alopecia areata. Hence, HLA-DQB1*03 is associated with alopecia areata (4). The severity of the disease is linked with a mutation of interleukin 1-receptor antagonist (IL1-RA) gene (5).
The pathophysiogeny of alopecia areata is very complex (6,7). This disease could be also considered as a psychosomatic disease (8). Stress seems to be able to induce its occurrence (9). Most of the patients are suffering from pychiatric disorders (10). In summary, psychism appears to be implied in the genesis of alopecia areata, like in the genesis of numerous auto-immune diseases (11).
The close relationship between skin, immunity and the nervous system has led to define the neuro-immuno-cutaneaous system (NICS) (12). Indeed, skin cells and immune cells express receptors for neurotransmitters on their surface, which induce cell activation when linked to these neurotransmitters. The main cutaneous neurotransmitters are substance P, somatostatin and calcitonin gene-related peptide (CGRP). Hair follicles possess a very dense innervation, which is able to induce hair cycle (13). Neurotransmitters modulate skin immunity and hair growth. In the course of dermatoses, there is an inbalance between the expression of receptors and the cutaneous amounts of neurotransmitters. Nerve growth factor (NGF) is an important actor of the NICS. It controls nerve growth, neurotransmitter expression (14) and hair growth (15).
The nervous system is probably strongly involved in alopecia areata lesions since the denervation can induce hair growth in affected skin territories (16). In this study, our aim was to determine if there is any modification of NICS in alopecia areata lesions compared with normal skin.
Material and methods
The study was approved by the ethics commitee of Rhône-Alpes/Loire. All patients gave a written consent. All patients suffered from alopecia areata but no other dermatological disease. They did not have any treatment for alopecia areata since one month. They did not have enteral or parenteral treatment for any reason. They did not apply anything other than shampoo on their scalp. They were 18 years of age or older.
Punch skin biopsies (4mm) were performed on the scalp of 15 patients with alopecia areata. For each patient, two biopsies were obtained : one from healthy scalp and the other one from the border of a lesion of alopecia areata. All specimens were immediatly frozen in isopentane at – 180 °C then preserved at – 80° C. Seven-micron thick cryostat sections were air dried overnight at room temperature and fixed for 10 minutes in cold acetone before use.
Sections were stained with hematoxylin-eosin-safran (HES) and periodic acid shiff (PAS). We assessed on HES stained sections the presence or the absence of hair follicle, the intensity of the inflammatory reaction in dermis, around hair follicle and in the hair itself. On PAS stained sections, we checked the absence of infectious agent as a differential diagnosis of alopecia areata. The diagnosis of healthy scalp biopsy was confirmed when hair and hair follicles were visible and histologigally normal, with no surrounding inflammatory reaction. The diagnosis of alopecia areata was assessed by hair or hair follicle destruction associated with an infiltrate of lymphocytes.
Serial sections from frozen biopsies were cut and stained using a three-step immunoperoxidase technique as described by Mason and Sammons (17). Specific monoclonal antibodies were used. They were directed against CGRP (clone C8198, dilution 1/2000, Sigma Chemical Co, St Louis, Mo.), Substance P (clone S1542, dilution 1/1000, Sigma), somatostatin (clone MAB354, 1/100, Chemicon International, Temecula, California), NGF (clone 25623.1, dilution 1/12, Sigma) and NGFR (clone MAB365, dilution 1/25, Chemicon). Five micron thick cryostat sections were dried overnight at 4° C and fixed 10 minutes in cold acetone before use. After rehydratation in TRIS buffer, tissue sections were incubated for 60 minutes (overnight at 4° C for NGFR) with primary monoclonal antibodies diluted in Tris buffer. Sections were sequentially incubated for 30 min with biotinilated species specific antiimmunoglobulin antibodies and for 30 min with Vectastain reagent (Vectastain Elite Universal, Vector Laboratories, Burlingam, California).
The colour reaction product was developed using Graham and Karnovsky method (18). Peroxydase activity was revealed using 3.3′-diamineobenzidine (Sigma). A light counter-coloration was obtained by staining the section with Harri’s hematoxylin for 50 seconds. Negative controls were set up for each immunstaining procedure omitting the primary antibody.
The expression levels of each neurotransmitter were evaluated using a semiquantitative grading system based on a three points scale representing the proportion of positive epidermis cells in each case. The grades were defined as follow : 0, no staining ; 1, less of 20 % of epidermis cells staining ; 2, staining of more than 20 % of epidermis cells. For lymphocytes infiltrate, the grades were defined as follows ; 0, no infiltrate ; 1, slight infiltration ; 2, moderate or heavy infiltration.
Hair follicles were partially or totally destroyed in all lesions of alopecia areata and an inflammatory infiltrate consisting mainly of lymphocytes and some macrophages was observed. When totally destroyed, hair follicles were replaced by a dense infiltrate or a sclerosis. In apparently normal scalp, a partial destruction of hair folicles associated with the presence of an infiltrate was observed in only 3 cases and lymphocytes around hair follicles were often observed.
In normal scalp as well as in normal skin, CGRP staining was detected on nerve fibers and on the basal and supra-basal layers of epidermis (Fig. 1) and hair follicles (Fig.2). In the lesions, the expression of CGRP was diminished in these locations in 10 cases (Fig.3 and 4). In 3 cases, there was no difference but the histological appearence of healthy and involved scalp was also not significantly different. CGRP was more expressed in lesions than in healthy scalp in 2 cases.
Substance P expression was found on nerve endings and in the whole epidermis (mainly supra-basal layers) and haire follicles. This expression was weakly diminished in alopecia areata lesions in 6 patients, not modified in 7 cases ans enhanced in 2 patients.
Somatostatin immunostainings were noted on nerve endings and in the epidermis on basal layers and more weakly on supra-basal layers but not in hair follicles. No significant difference was observed between scalp suffering from alopecia areata or not.
NGF was present on nerves and basal cells of epidermis and hair follicles and its expression was not significantly different in healthy and unhealthy scalp.
NGF receptor was found to be expressed in epidermis in 7 patients and in hair follicle in only one. Identifical data were obtained about NGF receptor expression in lesions and in healthy skin.
Whe have shown that CGRP expression is decreased in alopecia areata lesions. A previous study indicated that CGRP immunoreactivity is increased in nerves around eccrine glands of alopecia areata patients (19). A previous work found decreased CGRP blood amounts in these patients (20). These three studies suggest that CGRP is involved in the pathophysiogeny of alopecia areata. Hence, if many neurotransmitters and neuronal growth factors modulate hair growth, only CGRP appears to be involved in alopecia areata.
CGRP is known to be one of the most powerful vosdilatators (21) and a deficiency could induce a vasoconstriction, like in Raynaud’s phenomenon for example. A vasoconstriction could favor hair loss. Laser-Doppler flowmetry was used to study microcirculation of the scalp. Results indicated that patients with alopecia areata have lower basal blood flow and greater vasodilatation following intradermal CGRP injection than control subjects. A vascular hyper-reactivity to vasodilatatory substances such as neuropeptides, probably because of the lack of these substances, was hypothesized (22).
Another hypothesis is that CGRP deficiency might annihilate a CGRP-promoted tolerance to a hapten or antigen responsible for the auto-immune destruction of hairs in alopecia areata. Indeed, CGRP is able to induce an hapten-specific tolerance (23). The therapeutic effects of UV on alopecia areata could be due to the restoration of CGRP cutaneaous amounts through its release by cutaneous nerve endings in response to UV (24). CGRP could act on both T cells (25) and Langerhans cells (26), directly through CGRP receptors (25) or by means of interleukin 10 (23).
Hence, the role of CGRP deficiency in alopecia areata appears to be relate to vascular and immune properties of this neurotransmitter. Nonetheless, the mechanisms of CGRP decrease in alopecia areata remains unknown. We suggest that CGRP deficiency could be induced by CD10 (or neutral endopeptidase or NEP). CD10 is a membrane-bound peptidase, which cleaves many peptides including CGRP, thereby limiting their bilogical actions (27). In alopecia areata, affected hair follicles express strongly this enzyme (28). In hair follicles at the margins of areas of hair loss, like in our study, CD10 staining was oberved in the inner portion of the outer root sheath and in some of the outermost cells the outer root sheath. The expression is stronger in the center of areas of hair loss.
CGRP coul be used for the treatment of alopecia areata but, as a neurotransmitter, CGRP could be very rapidly metabolized. Agonists with long half-life might be more interesting.
Acknowledgements : We thank the Association Alopecia Areata for his financial support.
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