Trichohyalin is a Potential Major Autoantigen in Human Alopecia Areata

Trichohyalin is a Potential Major Autoantigen in Human Alopecia Areata Man Ching Leung,† Chris W. Sutton,‡ David A. Fenton,§ and Desmond J. Tobin* Centre for Skin Sciences, School of Life Sciences, University of Bradford, Bradford, West Yorkshire, United Kingdom BD7 1DP, Institute of Cancer Therapeutics, University of Bradford, Bradford, West Yorkshire, United Kingdom, and St. Thomas’s Hospital, St. John’s Institute of Dermatology, London, United Kingdom Received May 8, 2010

Several lines of evidence support an autoimmune basis for alopecia areata (AA), a common putative autoimmune hair loss disorder. However, definitive support is lacking largely because the identity of hair follicle (HF) autoantigen(s) involved in its pathogenesis remains unknown. Here, we isolated AAreactive HF-specific antigens from normal human scalp anagen HF extracts by immunoprecipitation using serum antibodies from 10 AA patients. Samples were analyzed by LC-MALDI-TOF/TOF mass spectrometry, which indicated strong reactivity to the hair growth phase-specific structural protein trichohyalin in all AA sera. Keratin 16 (K16) was also identified as another potential AA-relevant target HF antigen. Double immunofluorescence studies using AA (and control sera) together with a monoclonal antibody to trichohyalin revealed that AA sera contained immunoreactivity that colocalized with trichohyalin in the growth phase-specific inner root sheath of HF. Furthermore, a partial colocalization of AA serum reactivity with anti-K16 antibody was observed in the outer root sheath of the HF. In summary, this study supports the involvement of an immune response to anagen-specific HFs antigens in AA and specifically suggests that an immune response to trichohyalin and K16 may have a role in the pathogenesis of the enigmatic disorder. Keywords: alopecia areata • autoantigen • trichohyalin • keratin 16 • proteomics • immunoprecipitation • immunofluorescence • LC-MALDI-TOF/TOF

Introduction Alopecia areata (AA) is a reversible immune-mediated disorder with a putative autoimmune basis that is associated with inflammatory but nonscarring hair loss. It has an estimated incidence at 0.1% of the general population at any one time, but a lifetime risk of approximately 1.7%.1 AA is a very unpredictable and heterogeneous disorder and can present as either patchy hair loss,2 loss of 100% scalp hair with none/ some loss of body hair termed alopecia totalis (AT), or 100% loss of scalp and body hair termed alopecia universalis (AU). While AA can be recurring, complete remission is still possible for most patient types,3 especially those affected by patchy disease. Although AA is not life-threatening, the resulting dramatic changes in the patient’s appearance can bring great psychological distress and a decreased quality of life.4 The lower hair follicle (HF) including bulb is reported to be an immunologically privileged site, and some have suggested * To whom correspondence should be addressed. Professor Desmond J. Tobin, Centre for Skin Sciences, School of Life Sciences, University of Bradford, Bradford, West Yorkshire BD7 1DP, United Kingdom. Phone: 01274233585. Fax: 01274-309742. E-mail: [email protected]. † Centre for Skin Sciences, School of Life Sciences, University of Bradford. ‡ Institute of Cancer Therapeutics, University of Bradford, Bradford, West Yorkshire, United Kingdom. § St. Thomas’s Hospital, St. John’s Institute of Dermatology, London, United Kingdom. 10.1021/pr100422u

 2010 American Chemical Society

that the onset of AA may be associated with a collapse of this privilege.5 In this way class I and II MHC antigens, which are normally absent from the proximal anagen HF, are upregulated when immune privilege is lost, enabling the inappropriate presentation of HF autoantigens to the immune system.5,6 It has long been known that AA can target HF when they are in the growth (or anagen) phase of the hair cycle,7 though other pigmented tissues including nail8,9 and eye10,11 may also be involved. As a result anagen HF growth is commonly interrupted at early anagen (anagen III). This coincides with the reconstruction of the HF pigmentary unit and the point when expression of trichohyalin (THH) signifies the formation of inner root sheath (IRS) and the onset of hair cortex keratinization. These AA-targeted HFs may survive for some time under a distressed state and may produce a dystrophic and pathognomic so-called “exclamation-mark” hair, before the HF is prematurely precipitated into the regression phase of the hair cycle (i.e., catagen).7,12 These histopathologic changes are associated with the infiltration of lymphocytes and inflammatory cells into or around the anagen hair bulb13-15 and a role for T cells in AA pathogenesis is supported by hair regrowth in AA models (e.g., C3H/HeJ mice and DEBR model) after depletion of CD4+ and/or CD8+ T cells.16-18 In addition to cell-mediated immune response in AA, circulating antibodies against antigens expressed by anagen Journal of Proteome Research 2010, 9, 5153–5163 5153 Published on Web 07/19/2010

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HFs have been detected in the serum of AA patients. Antibody reactivity to HFs can also be detected in serum of normal control individuals, but these occur in much lower levels.20 The higher titer HF antibodies in AA sera suggest that AA patients have an abnormally increased production of antibodies to antigens expressed on apparently normal anagen HFs.20 Moreover, anti-HF antibodies in AA sera are mainly of the IgG isotype, while those in normal individuals are usually IgM but with some low titer IgG antibodies also. A distinctive feature of AA is that the anti-HF antibody reaction patterns and intensities differ markedly between AA patients. Still, these antibodies most commonly reacted to antigens of molecular weight (MWt) between 44 and 57 kDa,20 while antigens of 62,21 115, 155, 185, 200, and 220 kDa19,20 can also be routinely detected. These antigens appear to be expressed in multiple structures of the HFs, but principally in the outer root sheath (ORS) and IRS, hair bulb matrix and hair shaft.21 Type I HFspecific keratins of 44 and 46 kDa are reported to be targeted by the anti-HF antibodies in human AA and also in the C3H/ HeJ mouse model of AA.22-24 Also, sera of AA patients (but not normal controls) can immunoprecipitate a 44 kDa HF-specific keratin. Therefore, this HF-specific keratin may be an autoantigen targeted in AA.22 Moreover, AA studies in other mammalian species including horses and dogs have also showed very similar pathology to human AA, including peri- and intrafollicular T cell infiltration25 serum antibodies to HF antigens 40-60 kDa25,26 and a 200-220 kDa doublet, the latter which was identified as THH.26,27 The above approach to autoantigen identification is tedious and inefficient, depending as it does on the presence of corresponding monoclonal antibodies. To identify AA-targeting HF antigens using a nonbiased manner, a proteomics approach was taken in this study.

Materials and Methods Chemicals and Antibodies. All chemicals were purchased from Sigma-Aldrich Company Ltd., Dorset, unless otherwise stated. Antibodies used included: Mouse anti-THH AE15 monoclonal antibody (Gift of T. T. Sun from New York University Medical Center, New York, NY); keratin 16 (C-12) (Santa Cruz Biotechnology Inc., Santa Cruz, CA); FITC goat antihuman IgG (whole) (Sigma-Aldrich Company Ltd., Dorset, U.K.); Alexa 594 Donkey antimouse IgG (H+L) antibody (Invitrogen Corporation, Paisley, U.K.); and Alexa 488 Donkey antimouse IgG (H+L) antibody (Invitrogen Corporation, Paisley, U.K.). Serum Samples. Human serum samples (AA patients, identifiers with “AA”) were obtained with informed consent from 10 individuals diagnosed with different AA subtypes [9 F, 1 M; mean age ) 39 y]. Two patients had patchy AA, 5 AU, 2 AT, and 1 with ophiasis pattern. Four patients exhibited some areas of active hair loss while 6 individuals exhibited some areas of hair regrowth. AA can be associated with contemporaneous hair loss and hair regrowth on the same scalp. Normal control sera (identifiers with “N”) were obtained with informed consent from 10 individuals [6 F, 4 M; mean age ) 28 y]. The IgG concentration of each serum was determined by radial immunodiffusion using BINDARID (The Binding Site, Birmingham, U.K.) and was found to be within normal range (8-17 mg/ mL) for all AA and control sera. Hair Follicle Extract. Plucked hair follicles (HF) were obtained from the androgen insensitive parietal scalp of a normal healthy male (age 40 y). Only the follicle portion was 5154

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used for this study and typically contained substantial ORS and most of the anagen bulb. Only rarely was the follicular papilla (FP) also retained after plucking. No non-HF (i.e., epidermis/ dermis) material is present. The HF were solubilized in 6 M urea at pH 8.5 overnight at 4 °C with 5 µL of Protease Inhibitor Cocktail per 1 mL of volume. A small pestle was used to crush the HF and the soluble supernatant (the extract) was collected after centrifuging 3 times (3 min each at 14 385× g). The protein content of the extract was determined using Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA), aliquoted and stored at -80 °C. Immunoprecipitation of Hair Follicle Antigens. Immunoprecipitations of antigens contained within the HF extract were performed individually with each of the 20 human serum samples (300 µg of HF extract and serum volume equivalent to 300 µg of IgG). In addition, immunoprecipitation negative controls included AA serum alone (AA5) without HF extract or HF extract alone without serum. Briefly, Eppendorfs were precoated with PBS/0.65% Tween 20 (pH 7.5). Protein GSepharose, in 20% ethanol, was washed with dH2O before it was added in a 1:1 volume ratio with each serum sample, and incubated for 1 h at room temperature. The supernatants were discarded and the Sepharose beads washed twice with PBS/ 0.65% Tween 20 and then once with 200 mM triethanolamine (pH 8). The beads were then incubated for 1 h at room temperature with 20 mM dimethyl pimelidate dihydrochloride (DMP) in 200 mM triethanolamine (pH 8) which cross-links IgG to the Protein G. The supernatant was discarded and the beads were then incubated for 15 min with 50 mM Tris (pH 7.5), washed twice with 50 mM glycine/0.65% Tween 20 (pH 2.7) and once with washing buffer (25 mM EDTA, 63 mM Trizma base, 10% glycerol (BDH, UK)). The beads were then resuspended in washing buffer and incubated with HF extract (precleared with Protein G-Sepharose for 1 h at room temperature) for 1 h at room temperature or overnight at 4 °C with shaking to allow for IgG-antigen binding. The beads were washed 4 times with washing buffer, and then resuspended in 20 µL of fresh washing buffer and boiled in a water bath for 3 min to elute the antigens. Preparation of Protease Digests. All 23 immunoprecipitates (10 AA samples, 10 normal control samples, 3 negative controls) were processed at the same time. Each immunoprecipitate was lyophilized and resuspended in 9 µL of 8 M urea and then reduced by incubating with 1 µL of 50 mM dithiothreitol for 20 min at 60 °C. Alkylation was carried out (in the dark) by incubating with 1 µL of 0.3 mM iodoacetamide at room temperature for 20 min. The sample was then diluted with 25 µL of 100 mM ammonium bicarbonate to bring the urea concentration down to 2 M. The proteins were digested into peptides by incubating with a 10 ng/µL solution of sequencegrade, modified bovine trypsin (Roche Diagnostics, Mannheim, Germany) at 28 °C for 22 h. Myoglobin was used as a positive control for trypsin digestion and mass spectrometric analysis indicated that trypsin digest was complete. The samples were desalted using ZipTip µ-C18 pipet tips (Millipore Corporation, Massachusetts), lyophilized and resolubilized in 6.5 µL of 10% acetonitrile (ACN) just before loading into the LC. Liquid Chromatography-Matrix-Assisted Laser Desorption/ Ionization-Tandem Time-of-Flight Analysis (LC-MALDI-TOF/ TOF). A Dionex Ultimate 3000HPLC system with a C18, 300 µm × 5 mm, 5 µm diameter, 100 Å PepMap precolumn (25 µL/ min) followed by a C18, 75 µm × 15 cm, 3 µm diameter, 100 Å PepMap separating column (0.3 µL/min) was used (Dionex,

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Human AA Potential Autoantigen: Trichohyalin Table 1. Proteins Identified by MALDI-TOF/TOF with Mascot Scores over 30, Excluding IgGs, False Positives and Those Observed Only in Control or Negative Controls K16/Swiss-Prot accession no. P08779

THH/Swiss-Prot accession no. Q07283

identifier

Mascot score

sequence coverage (%)

peptide matches

Mascot score

sequence coverage (%)

peptide matches

AA1 AA2 AA3 AA4 AA5 AA6 AA7 AA8 AA9 AA10 N1 N2 N3 N4 N5 N6 N7 N8 N9 N10

85 116 30 84 106 48 65 61 55 104 37 67 -

5 12 3 12 18 27 13 16 17 19 7 10 -

9 6 1 4 6 9 4 6 7 10 3 4 -

256 137 47 53 117 79 142 232 73 140 41 46 116 33 165 -

11 6 2 2 6 3 6 6 3 8 2 2 5 1 7 -

18 9 4 3 9 6 10 10 5 13 3 3 9 1 12 -

Table 2. Frequency of Occurrence, Mean Score and SEM for K16 and THH Identified by MALDI-TOF/TOF of the AA and Normal Groups K16

THH p

AA

Normal

Frequency Mean Score SEM Frequency Mean Score SEM

8/10 74 13 4/10 66 12

0.097

p

10/10 128 22 5/10 80 18

0.005*

Sunnyvale). The peptides were separated into 48 fractions using a gradient of solvent A/solvent B 90%/10% to 60%/40% (Solvent A: 2% ACN/0.05% TFA; Solvent B: 80% ACN/0.05% TFA). The fractions were collected automatically every 15 s for 12 min by PROTEINEER fc liquid handler (Bruker Daltonics, Massachusetts), mixed automatically with 0.8 µL R-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics, Massachusetts) and codispensed onto the MTP AnchorChip 800-384 target plate (Bruker Daltonics, Massachusetts). Samples were processed through the LC consecutively to fill the whole target (8 samples on each target). Peptide Calibration Standard (Angiotensin I, Angiotensin II, Substance P, Bombesin, ACTH clip 1-17, ACTH clip 18-39, Somatostatin 28, Bradykinin fragment 1-7 and Renin Substrate Tetradecapeptide porcine; covered mass range 700 Da - 3200 Da, Bruker Daltonics, Massachusetts), was spotted onto the target manually and the matrix codeposited with it. Mass spectrometric analysis of the LC fractions was carried out using a MALDI-TOF/TOF UltraFlex II instrument (Bruker Daltonics, Bremen, Germany) with the reflectron in positive ion mode. A fully automated work flow was performed using WarpLC software (version 1.2), which encompassed data acquisition (FlexControl version 3.0), initially in MS mode to screen for peptide signals (FlexAnalysis version 3.0) between

Figure 1. Detection of K16 in normal human scalp HF section by indirect immunofluorescence using an antibody to K16. CTS, connective tissue sheath; ORS, outer root sheath; CL, companion layer; FP, follicular papilla.

700-4000 Da (300 laser shots per fraction). The system then compiled a nonredundant list of compounds (minimum signal: noise ratio of 7 required, compounds within a window of 5 fractions and 70 ppm were considered indistinguishable, and a mass (70 ppm that occurred in >30% of the fractions was defined as background), generation of data-dependent MS/MS of each compound (900 laser shots each) using LIFT mode (FlexControl) and the processing of spectra to produce fragment mass lists for each peptide (FlexAnalysis). External calibration was performed for each surrounding 4 fractions during MS analysis. Protein identification was performed using the Mascot search engine (http://www.matrixscience.com/) to search MS/MS fragmentation patterns from each peptide against the SwissProt database (version 52.4), comprising 16 356 human protein sequences. Preset database search parameters: taxonomy, Homo sapiens; variable modifications, carbamidomethylation and methionine oxidation; up to 1 trypsin miscleavage; MS tolerance of 100 ppm and MS/MS tolerance of 0.7 Da. The threshold of significance of p < 0.05 was used for Mascot search results and the mean and SEM of each protein’s Mascot scores were calculated for each AA and control groups. MudPIT scoring was applied to the search results and, in general, proteins were only accepted if they had at least two unique peptides. Journal of Proteome Research • Vol. 9, No. 10, 2010 5155

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Figure 2. Detection of THH in normal human scalp HF section by indirect immunofluorescence using the anti-THH antibody AE15. CTS, connective tissue sheath; ORS, outer root sheath; IRS, inner root sheath.

Double Indirect Immunofluorescence. Anagen HF were isolated as described previously28 from face-lift skin samples (females, 62 and 59 years old for THH and K16 experiments respectively), embedded in OCT compound (Tissue-Tek, Sakura Finetek, Torrance, CA) and stored at -80 °C. Sections of 5 µm thick were cut, air-dried for 1 h at room temperature and then fixed in ice-cold acetone for 10 min and rinsed in PBS. Sections were then air-dried, rehydrated in PBS for 5 min. They were incubated first with 10% blocking serum for 2 h at room temperature followed by the first primary antibody (AE15 monoclonal mouse antitrichohyalin antibody, gift of T. T. Sun from New York University Medical Center, New York, NY or antibody to K16 (C-12), Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 18 h at 4 °C. At the end of this incubation period, the sections were washed 3 times for 5 min each in 0.05% Tween 20 in PBS washes and once for 5 min in PBS alone. The sections were then incubated with the first secondary antibody (Alexa 594 donkey antimouse IgG, Invitrogen Corporation, Paisley, U.K.) for 1 h at room temperature. After that, sections were washed and blocked as before followed by incubation with the second primary antibody (patient or control serum) for 2 h at room temperature. The sections were washed as before and then incubated for 1 h in second secondary antibody (FITC goat antihuman IgG, Sigma-Aldrich Company Ltd., Dorset, 5156


U.K.) at room temperature followed by the same sequence of washes. The sections were then coverslipped with mounting medium with 4′,6-diamidino-2-phenylindole (VECTASHIELD, Vector Laboratories, Burlingame, CA) to stain the nuclei. Sections were photodocumented on the same day. All photographs were taken under the same exposure levels. For single immunofluorescence studies, the HF sections were coverslipped after the washes following the first secondary antibody incubation. Negative controls were conducted by replacing primary antibody and secondary antibody with PBS.

Results LC-MALDI-TOF/TOF Identified Trichohyalin (THH) in AA Serum Immunoprecipitated HF Antigen. Several HF proteins were immunoprecipitated by antibodies present in AA and control sera. IgG subunits were identified among these, although these were possibly from the serum component of the immunoprecipitation, indicating less than complete crosslinking of the IgG to the beads. As expected, IgG subunits were not observed in the negative control immunoprecipitations that used HF only. Albumin was also identified in three of the ten AA immunoprecipitates and was also considered a “pull-down contaminant” from immunoprecipitation. Keratins K1, K9 and

Human AA Potential Autoantigen: Trichohyalin

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Figure 3. Co-localization of (a) AA sera antibodies (AA1-AA10) and (b) control sera antibodies (N1-N10) targeted antigens (green fluorescence) with AE15-reactive THH (red fluorescence) and in normal human scalp HF sections by double indirect immunofluorescence analysis. Note the weak reactivity of control sera antibodies with antigens expressed in these normal HF and THH hyaline presence shows little colocalization with these anti-HF antibodies. Magnifications ) ×100.

K10 were also identified and these common contaminants found in skin and dandruff.29 Table 1 shows proteins identified with Mascot scores over 30,30 excluding IgGs, false positives and proteins that were also observed in the controls. The frequencies of each antibody-targeted HF protein were assessed (Table 2) and used for comparison between the AA group and control group. Given that all AA and control immunoprecipitate samples were prepared, processed and analyzed in the same controlled way, the Mascot scores for the same target protein recognized by antibodies in the different serum samples could be used at least semiquantitatively. The Mean scores (and their standard error of mean; SEM) for each protein in the AA group and control group were calculated and compared, and their significance evaluated using paired two-tail Student’s t test (Table 2). All 10 AA sera generated trichohyalin (THH) Mascot scores of over 47, while it was only detected in 20% of the control samples. Moreover, the difference of average scores between AA/control serum groups was found to be statistically significant (p < 0.05%). The frequency of immune reactivity to keratin 16 (K16) in sera of patients with AA was twice as great in AA (60% with Mascot score over 60) as compared to the control group (30% with Mascot score over 60), although failing to reach statistical significance (p < 0.097). Both THH and K16 were not detected in the negative controls. Colocalization of Serum Antibody Reactivity with THH and K16 Expression in Normal Human HF. Double indirect immunofluorescence was carried out with AA or control sera

on frozen sections of normal anagen scalp HFs to determine whether there was any colocalization of binding of these serum antibodies with monoclonal antibodies to THH and K16. The anti-K16 antibody reacted with the ORS and companion layer (CL) keratinocytes (Figure 1),31 whereas the anti-THH antibody (AE15) bound to all three layers of the IRS (Figure 2).32 When comparing the anti-HF reactivities of AA serum antibodies and control serum antibodies (Figure 3), antibodies present in AA sera exhibited much higher reactivity (see more intense green fluorescence signals), indicating a higher titer than antibodies present in control sera. While immunoreactivities to other HF components were less common, some AA sera (e.g., AA5) showed very intense binding to the precortex. AA5 serum also contained antibodies that reacted with the hair bulb matrix; the proliferatively active component of the growing HF. This heterogeneous pattern of immunoreactivity to HF components was similar to previous reports.21 The region of greatest antigen colocalization exhibited by AA serum antibody and AE15 antibody (as evidenced by the overlap of red and green fluorescence to give orange/yellow fluorescence) was the proximal IRS (Figures 3 and 5). Within this compartment, greater immunoreactivity was to external Henle’s layer, with less reactivity to the most internal Huxley’s layer and IRS cuticle. There was also partial colocalization of AA serum antibodies and K16 monoclonal antibody binding in the ORS and this was more frequent and more intensive in Journal of Proteome Research • Vol. 9, No. 10, 2010 5157

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Figure 4. Detection of colocalization of (a) AA sera antibodies (AA1-AA10) and (b) normal control sera antibodies (N1-N10) (green fluorescence) with anti-K16 antibody (red fluorescence) in normal human scalp HF sections by double indirect immunofluorescence analysis. Magnifications ) ×100.

AA sera-probed sections than control sera-probed sections (Figures 4 and 6). Correlation between Mascot Score (THH) and Serum Donor Age. The relationship between THH Mascot scores and the age of serum donors was investigated. No correlation was found in either AA and control groups (Figure 7).

Discussion The most significant impediment to progress in understanding the etiology and pathogenesis of AA, the main pathological form of hair loss, has been our failure to identify the HF autoantigen(s) targeted in this disorder. Several attempts have previously been made to identify AA-relevant antigens in HF, including pigment-cell associated proteins that were capable of activating T cells that could transmit AA in SCID mice.33 We have previously shown the binding of human AA serum antibodies to the hair specific 44/46 kDa keratins by Western Blotting22 and immunoprecipitation of THH by AA sera in canine and equine AA.26,27 In addition, we used a cDNA library (of murine anagen skin) approach to determine target antigens, but with limited success. To attempt to move this field forward, we adopted here an approach combining immunoprecipitation and the capability of MALDI mass spectrometry in protein identification to investigate AA target antigens. Using this approach THH was identified as a target HFspecific antigen in all 10 of the immunoprecipitates of AA sera. This protein was much less commonly detected by antibodies 5158


in control sera. Similarly, immunoreactivity to K16, which is commonly associated with THH,34 was identified in most AA sera immunoprecipitates but in a minority of control sera. All immunoprecipitates were randomized and were prepared, treated and analyzed under the same conditions and in the closest possible temporal succession to avoid fluctuations in instrument performances. By carefully controlling these experimental conditions, the MALDI-TOF/TOF Mascot scores were used as a semiquantitative measure of relative level of immunoprecipitated HF proteins. Given that a higher quantity of a particular HF protein in the immunoprecipitates will result in a more favorable signal-to-noise ratio in the resulting spectra, there is a corresponding higher probability that this protein will be identified with a higher Mascot score, than would be the case if a particular protein were present at a lower quantity. Therefore, Mascot scores awarded for a particular positively identified protein in different immunoprecipitates can be compared to reflect the relative quantity of this protein, and so can refer to the level of immune-reactivity to the target antigen, inferring an association of this to AA. For the immunoprecipitates containing trichohyalin (THH), the THH Mascot scores were very significantly greater for AA patients than among the normal control individuals (p ) 0.005). This suggests that there was a relatively much greater amount of THH antigen present, on average, in all 10 of the AA immunoprecipitates than in the THH-positive control immunoprecipitates. There is likely to be more than one way to


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Figure 5. Detection of Colocalization of (a) AA1 and (b) AA3 serum antibodies with anti-THH antibody AE15. He, Henle’s layer.

interpret this finding as the higher amount of THH protein present in AA immunoprecipitates can reflect either (1) a higher titer of anti-THH IgG antibodies in serum of AA patients than in control serum or (2) that the relative amount of THH-reactive IgG is higher in AA than in control sera. Either or both of these possibilities are likely, because the starting amount of normal HF extract and IgG used in all immunoprecipitations was similar. It is important to note that both AA and control serum IgG are reactive against THH present in extracts of normal HF. The identification of THH as an immuno-dominant antigen in AA is of particular interest physiologically, as THH is a critical structural protein present in the anagen-specific IRS of normal HF.35,36 A disturbance of THH by an inappropriate immune response is likely to negatively affect the efficient progression of anagen. The successful identification of THH-reactive IgG antibodies by immunoprecipitation supports the view that these antibodies bind to a relevant conformational epitope of THH, as the HF extracts were not denatured during the protein solubilization, unlike in Western Blotting analyses in which protein epitopes are denatured including during the SDS-PAGE separation. The above results also indicate that the normal control sera which were used to generate these immunoprecipitates had either very little IgG to THH, or had IgG with very low avidity to THH. Previously, we have reported that antibody reactivity

to HFs is not a feature of unrelated autoimmune skin diseases (e.g, pemphigous, bullus pemphigoid) or inflammatory disease of the HFs, such as lichen planoilaris.22 This would suggest a degree of specificity for AA, and AA remains the only hair loss disorder for which antibodies to HFs have been reported. Indeed, antibodies to THH have not been reported in any other cutaneous disorder to date. When comparing the mean Mascot scores of K16 identified in the 8 AA serum samples (74) and in the 4 control serum samples (66), the difference was only p ) 0.097. However, the higher mean score and 2-fold more frequent incidence for the AA patient group could be interpreted as the presence of higher amounts of anti-K16 IgG or higher avidity of anti-K16 IgG in AA sera compared to the normal control group. While interpreting data from AA patients and normal healthy controls, it is useful to consider the clinical status of both groups of serum donors. The average age of serum donor was approximately one decade higher in the AA group than in the normal control group, and while most AA patients were female, 40% of the control group were male. While autoimmune disease is more common in females, no gender bias has been shown in AA.1 Also, the level of natural autoantibodies is reported to increase with age.37,38 Thus, the level of autoantibody in the somewhat older AA group may be expected to be higher than in the normal control group, although these may not necessarily be related to antigens expressed by normal HF. However, Journal of Proteome Research • Vol. 9, No. 10, 2010 5159

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Figure 6. Detection of colocalization of (a) AA1 and (b) AA3 serum antibodies with anti-K16 antibody. ORS, outer root sheath.

we found no correlation between the THH Mascot score and serum donor age. This suggests that the higher THH Mascot score among AA patients than the normal control group is not related to their higher average age. It is unlikely that a decade difference in mean age would explain these findings, as there was no apparent trend when individual titers were compared in the two serum groups. The literature suggests that the impact of age on antibody production is not clear. For example, antielastin antibodies are reported to be much higher in normal individuals of 18-20 years of age compared with those over this age, and this is even more pronounced in individuals with atherosclerosis.39 This issue would benefit however from an analysis of a larger cohort of AA patients. Another factor affecting the relative levels of HF-reactive IgG in sera of AA patients is their disease status at the time of serum collection. While all AA patients had long-standing disease, some were experiencing hair regrowth and hair loss either sequentially or contemporaneously. Moreover, the half-life of 5160


IgG in blood is approximately 1 month40 and so circulating levels of IgG will fluctuate according to immuno-stimulation. Therefore, it is difficult to conclude firmly whether the most disease relevant IgG antibodies were present in the AA patient serum at the time of collection. Using this proteomics approach, we did not detect antigens related to melanocytes and some of the HF-associated keratins, as previously reported.33 It is possible that the level and nature of antigens associated with melanocytes is reduced in plucked HF extracts as used in this study compared to studies of whole and intact HFs. A similar case can be made for the absence of potential AA target antigens associated with the connective tissue sheath and follicular papilla, which would not be present in plucked HF extracts. Previously we reported using Western Blotting,20 the presence of multiple immunoreactive bands for most AA sera when probing HF antigens separated by 1D SDSPAGE and electroblotted. To what extent these bands were AA specific was not clear from those earlier studies, and these


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Figure 7. Relationship between THH Mascot score and age and gender of (a) all donors, (b) AA patients, and (c) normal control donors.

could include some proteins excluded from study here (i.e., not unique to AA). The number of proteins identified was fewer than expected in a study of this type, possibly because the very low amount of proteins present in the immunoprecipitates were outside the limits of detection for the instrument. One should also be cautious in interpreting that THH and K16 are immunodominant HF antigens associated with AA, although, the fact that THH was observed in all 10 of the AA sera does suggest that THH may be one of the major AA-associated-HF antigens. Other potential AA-relevant antigens may be present in the immunoprecipitates, although they were not identified using the assays conducted here. The identification of THH as an AA-associated antigen raises the question of how disruption of THH could affect hair growth in AA. THH is a critical structural protein of the IRS in that it is required for the alignment of keratins in this growth-specific layer of the follicle.34 Indeed, THH is one of the earliest differentiation antigens to be expressed during the anagen or growth phase.41 Disruption in hyalinisation and development of these layers would have a serious effect on the growth of the hair shaft and the HF as a whole. In this study colocalization with THH was found most prominently in the proximal IRS where THH is likely still in its granular form before it leaves the granule and cross-links with other proteins in the IRS.32,34 Therefore, it is possible that the anti-THH IgG in AA sera are binding the granular form of the THH during IRS differentiation, where its impact on hair growth may be even greater, and less to the THH on the surface of the granule or cross-linked THH. Even if these THH-reactive antibodies do not have pathogenic potential themselves in AA, they may as in the case of other diseases like diabetes mellitus, share epitopes with effector T lymphocytes. Disruption of IRS structure integrity is a feature of other hair follicle disorders (cathepsin L (CTSL) deficiency).42 The current study also reports that most AA sera

contain prominent IgG-immunoreactivity to a second major target HF-associated antigen, K16, which exhibited at least partial colocalization of binding with a commercial anti-K16 monoclonal antibody on haired human scalp hair tissue. Similar colocalization of binding was rare with normal control serum antibodies. Expression of the K16 keratin can be found in ORS keratinocytes, a cell population that is both capable of proliferation and partial differentiation and is a very common site for AA antibody binding.4 Thus, an immune-mediated disruption of the ORS could cause hair cycle effects and possibly a premature induction of HF regression or catagens a feature of AA pathobiology. Just why anti-HF antibodies are generated in AA is unclear, and it remains to be determined whether these antibodies predate the onset of clinical hair loss in humans (as they may do in the Dundee Experimental Bald Rat (DEBR) model43). Stimulation of the immune system to generate anti-HF IgG antibodies may occur after an inappropriate presentation of normal HF antigens to the immune system.5,44,45 A perplexing aspect of autoimmunity and autoantibody elicitation is the identification of the events involved in the initiation of the response. Exogenous triggers may be involved (e.g., microbial antigen cross-reactivity) that can initiate some autoimmune responses. Alternatively, there is significant evidence for drugand chemical-induced autoimmunity. It is also possible that epitope spreading can increase the range of antigenic sites recognized by antibodies in the circulation of patients with AA. Whether THH is a secondary antigen (e.g., in response to tissue damage), rather than a unique immuno-dominant epitope, is not yet clear. In any event, secondary antigens may still be involved in disease progression in other autoimmune conditions.46 The targeting of the antihair follicle immune response to THH in AA is particularly interesting, as this is one of the first follicular differentiation proteins to be expressed in earliest Journal of Proteome Research • Vol. 9, No. 10, 2010 5161

research articles anagen (growth) of the hair growth cycle and is at that stage relatively exposed in terms of cell distances from a vascularized dermal sheath. The results presented in this current study support an involvement of THH and K16 as target antigens in AA. It will be useful now to determine whether these IgG antibodies have functional effects on the growing HF.

Acknowledgment. We acknowledge support from the School of Life Sciences, University Bradford, PhD studentship fund, and the willingness of alopecia areata patients and volunteers to donate serum. References (1) Safavi, K. H.; Muller, S. A.; Suman, V. J.; Moshell, A. N.; Melton, L. J., 3rd. Incidence of alopecia areata in Olmsted County, Minnesota, 1975 through 1989. Mayo Clin. Proc. 1995, 70 (7), 628– 633. (2) Shapiro, J.; Madani, S. Alopecia areata: diagnosis and management. Int. J. Dermatol. 1999, 38 (S1), 19–24. (3) Price, V. H. Alopecia areata - clinical aspects. J. Invest. Dermatol. 1991, 96 (Supplement 5s), S68–S68. (4) Tobin, D. J.; Bystryn, J. C. Immunology of alopecia areata. In Hair and Hair Disorders: Research, Pathology and Management; Camacho, F. M., Randall, V. A., Price, V., Eds.; Martin Dunitz Ltd: London, 2000; pp 187-201. (5) Paus, R.; Ito, N.; Takigawa, M.; Ito, T. The hair follicle and immune privilege. J. Investig. Dermatol. Symp. Proc. 2003, 8 (2), 88–194. (6) Hamm, H.; Klemmer, S.; Kreuzer, I.; Steijlen, P. M.; Happle, R.; Bro¨cker, E.-B. HLA-DR and HLA-DQ antigen expression of anagen and telogen hair bulbs in long-standing alopecia areata. Arch. Dermatol. Res. 1988, 280 (3), 179–181. (7) Messenger, A. G.; Slater, D. N.; Bleehen, S. S. Alopecia areata alterations in the hair growth cycle and correlation with the follicular pathology. Br. J. Dermatol. 1986, 114 (3), 337–347. (8) Kasumagic-Halilovic, E.; Prohic, A. Nail changes in alopecia areata: frequency and clinical presentation. J. Eur. Acad. Dermatol. Venereol. 2009, 23 (2), 240–241. (9) Perrin, C.; Michiels, J. F.; Pisani, A.; Ortonne, J. P. Anatomic distribution of melanocytes in normal nail unit: an immunohistochemical investigation. Am. J. Dermatopathol., 1997, 19 (5), 462– 467. (10) Pandhi, D.; Singal, A.; Gupta, R.; Das, G. Ocular alterations in patients of alopecia areata. J. Dermatol. 2009, 36 (5), 262–268. (11) Recupero, S. M.; Abdolrahimzadeh, S.; De Dominicis, M.; Mollo, R.; Carboni, I.; Rota, L.; Calvieri, S. Ocular alterations in alopecia areata. Eye (London, U.K.) 1999, 13 (Pt 5), 643–646. (12) Jackson, D.; Church, R. E.; Ebling, F. J. Alopecia areata hairs scanning electron microscope study. Br. J. Dermatol. 1971, 85 (3), 242–246. (13) Perret, C.; Wiesnermenzel, L.; Happle, R. Immunohistochemical analysis of T cell subsets in the peribulbar and intrabulbar infiltrates of alopecia areata. Acta Derm. Venereol. 1984, 64 (1), 26–30. (14) Ranki, A.; Kianto, U.; Kanerva, L.; Tolvanen, E.; Johansson, E. Immunohistochemical and electron microscopic characterization of the cellular infiltrate in alopecia (areata, totalis, and universalis). J. Invest. Dermatol. 1984, 83 (1), 7–11. (15) Van Scott, E. J. Morphologic changes in pilosebaceous units and anagen hairs in alopecia areata. J. Invest. Dermatol. 1958, 31 (1), 35–43. (16) Carroll, J. M.; McElwee, K. J.; King, L. E.; Byrne, M. C.; Sundberg, J. P. Gene array profiling and immunomodulation studies define a cell-mediated immune response underlying the pathogenesis of alopecia areata in a mouse model and humans. J. Invest. Dermatol. 2002, 119 (2), 392–402. (17) McElwee, K. J.; Spiers, E. M.; Oliver, R. F. In vivo depletion of CD8+ T cells restores hair growth in the DEBR model for alopecia areata. Br. J. Dermatol. 1996, 135 (2), 211–217. (18) McElwee, K. J.; Spiers, E. M.; Oliver, R. F. Partial restoration of hair growth in the DEBR model for alopecia areata after in vivo depletion of CD4+ T cells. Br. J. Dermatol. 1999, 140 (3), 432–437. (19) Tobin, D. J.; Orentreich, N.; Bystryn, J. C. Autoantibodies to hair follicles in normal individuals. Arch. Dermatol. 1994, 130 (3), 395– 396. (20) Tobin, D. J.; Orentreich, N.; Fenton, D. A. Bystryn J. C.Antibodies to hair follicles in alopecia areata. J. Invest. Dermatol. 1994, 102 (5), 721–724.

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Journal of Proteome Research • Vol. 9, No. 10, 2010 5163

Trichohyalin is a Potential Major Autoantigen in Human Alopecia Areata

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