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Possible involvement of T cell co-stimulation in pustulosis palmaris et plantaris via the induction of inducible co-stimulator in chronic focal infections
Journal of Dermatological Science (2008) 50, 197—207
www.intl.elsevierhealth.com/journals/jods
Possible involvement of T cell co-stimulation in pustulosis palmaris et plantaris via the induction of inducible co-stimulator in chronic focal infections Hiroko Sakiyama a, Satomi Kobayashi b,*, Umberto Dianzani c, Hideki Ogiuchi a, Makoto Kawashima b, Takehiko Uchiyama d, Junji Yagi d a
Department of Maxillofacial Surgery, Tokyo Women’s Medical University School of Medicine, Tokyo 162-8666, Japan b Department of Dermatology, Tokyo Women’s Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan c Interdisciplinary Research Center of Autoimmune Disease, Department of Medical Sciences, A. Avogadro University of Eastern Piedmont, Novara, Italy d Department of Microbiology and Immunology, Tokyo Women’s Medical University School of Medicine, Tokyo 162-8666, Japan Received 30 August 2007; received in revised form 25 December 2007; accepted 27 December 2007
KEYWORDS Pustulosis palmaris et plantaris; Focal infection; Co-stimulation; ICOS; B7h
Summary Background: Inducible co-stimulator (ICOS) is a co-stimulatory receptor on activated T cells that provides the signals needed for Th1 and Th2 responses via its interaction with B7h. Chronic focal infections are closely related to pustulosis palmaris et plantaris (PPP), but the involvement of ICOS in PPP has not been clarified. Objective: To investigate the effectiveness of treatments for focal infections on PPP skin lesions and the involvement of ICOS-positive T cells at focal infection sites in the tonsils and in PPP lesional skin. Methods: In patients that had undergone a tonsillectomy or dental treatment, the clinical activities of PPP, both the skin lesions and pustulotic arthro-osteitis were followed for over 2 years. The expressions of ICOS and various other activation
Abbreviations: ICOS, inducible co-stimulator; PPP, pustulosis palmaris et plantaris; APC, antigen presenting cell; CLA, cutaneous lymphocyte-associated antigen; TGF, transforming growth factor; IFN, interferon; TNF, tumor necrosis factor; PBMC, peripheral blood mononuclear cell; hICOSmIg, human ICOS-mouse IgG Fc fusion protein; hCTLA-4hIg, human cytotoxic T lymphocyte-associated antigen 4human IgG Fc fusion protein. * Corresponding author. Tel.: +81 3 3353 8111; fax: +81 3 5269 4769. E-mail address: [email protected] (S. Kobayashi). 0923-1811/$30.00 # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.12.010
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H. Sakiyama et al. markers on T cells were examined in tonsil tissue from both PPP patients and non-PPP patients, and the expression levels in peripheral blood were also evaluated in PPP patients and healthy donors. ICOS-positive T cells and B7h expression in PPP and normal skin were examined immunohistochemically. Results: The above treatments for focal infections led to a dramatic and persistent improvement in the PPP skin lesions and pustulotic arthro-osteitis. The expression of ICOS, but not of other activation markers, was higher in tonsil tissues from PPP patients than in tonsil tissues from non-PPP patients. B7h was upregulated without numerous ICOS-positive T cell infiltrates in the skin lesions. Conclusion: The activation of T cells via ICOS co-stimulation in focal infections likely triggers the skin and skeletal inflammation associated with PPP, resulting in tissue damage. # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Pustulosis palmaris et plantaris (PPP) is a chronic recurrent dermatosis characterized by sterile intraepidermal pustules associated with erythematous scaling on the palm and sole [1]. Some PPP patients have skeletal involvement in intrasternocostoclavicular, manubriosternal, spinal ligament or peripheral joints [2]. Histologically, the primary eruptions are spongiotic vesicles in the lower epidermis that contain mononuclear cells; pustulation of the vesicle rapidly begins after it comes in contact with the stratum corneum [3]. A variable density of T cells infiltrate around the pustules. A number of cytokines have been suggested to play a pivotal role in the pathogenesis of PPP [4—7]. In addition, previous reports have shown elevated levels of immune complexes and anti-keratin antibodies in the sera of patients with PPP [8]. Thus, these lines of evidence suggest that abnormal responses by various types of cells, such as T, B and antigen presenting cells (APCs), may be involved in disease onset. Chronic focal infections in the tonsils and oral regions are well known to be involved in the induction of PPP, since a tonsillectomy [1,9,10] and the treatment of oral chronic infections such as periodontal disease and apical periodontation [11] often improve the skin lesions. A tonsillectomy leads to the reduction of serum levels of IL-8 and IL-6 in PPP patients [4,12], suggesting that inflammatory cytokines are related to the induction of PPP by tonsillar focal infections. Furthermore, Nozawa et al. [6] showed that the number of T cells expressing cutaneous lymphocyte-associated antigen (CLA), a homing receptor related to migration into the skin, in tonsillar mononuclear cells and tonsillar tissues was higher in patients with PPP than in patients without PPP. An in vitro stimulation of tonsillar Tcells with astreptococcal antigens from patients with PPP, but not from those without PPP, enhanced CLA expres-
sion and transforming growth factor (TGF)-b production [6]. Murakata et al. [5] also reported a higher production of cytokines [IL-6, interferon (IFN)-g and tumor necrosis factor (TNF)-a] from tonsillar T cells stimulated with a-streptococci in patients with PPP than in patients without PPP. Thus, these data suggest the following scenario. Chronic focal infection by bacteria like a-streptococci may activate T cells to enhance CLA expression, thereby facilitating the movement of those T cells into the skin, and cytokines produced by these T cells may play a key role in tissue damage. However, the molecular events in the onset of PPP induced by focal infections are not fully understood. Inducible co-stimulator (ICOS) [13] is a member of CD28 family that binds to its ligand, B7h [14], on APCs and provides co-stimulatory signals to augment T cell proliferation and the production of various cytokines [13,15,16]. In contrast to CD28, ICOS is not expressed on naı¨ve T cells, but is induced on T cells after T cell receptor engagement [13] and has important roles in both Th1 and Th2 responses [17—19], and antibody production [20]. B7h mRNA is expressed by lymphoid and non-lymphoid tissues, and is upregulated by TNF-a and LPS [14], suggesting that the interaction between ICOS and B7h may function as an important pathway in peripheral tissue inflammation. We previously reported that ICOS expression is increased on activated T cells in the synovial fluid of patients with rheumatoid arthritis [21]. The production of various cytokines was enhanced in CD4+ T cells derived from synovial fluid after ICOS engagement [21]. The possible involvement of ICOS in several other human immune-mediated diseases, such as systemic lupus erythematosus [22], multiple sclerosis [23], common variable immunodeficiency [24], and X-linked lymphoproliferative disease [25], has also been reported. However, the role of ICOS in chronic inflammatory skin diseases has not yet been elucidated. In the present study, in an attempt to clarify
Case
Age/sex
Diagnosis
History of disease (year)
Puatulotic arthroosteitis
Focal infections
+ +
Tonsil Tonsil Tonsil, dental Tonsil Tonsil Tonsil Tonsil Tonsil Tonsil Tonsil Dental Dental, sinusitis Dental
1 2 3 4 5 6 7 8 9 10 11 12 13
54/M * 53/F 54/F 50/M 53/F*** 35/F 47/M 27/F 52/F 37/M 35/M ** 53/F 68/F
PPP PPP PPP PPP PPP PPP PPP PPP PPP PPP PPP PPP PPP
5 1 3 1 13 0.5 1 1 2.5 3 0.3 1 1
14
31/F
PPP
0.3
15 16 17 18 19 20
35/M 24/F 27/F 30/M 33/M 27/M
Reccurent tonsillitis Reccurent tonsillitis Reccurent tonsillitis Reccurent tonsillitis Sleep apnea syndrome Sleep apnea syndrome
52 6 2 4 52 Unknown
Healthy individuals (n = 16) 24—65 (mean 40.3)/male:femal = 10:6
+
+
Dental
Treatment of focal infections Tonsillectomy Tonsillectomy Tonsillectomy Tonsillectomy Tonsillectomy Tonsillectomy — — Penicillin for 4 months — Tooth extraction Tooth extraction Tooth and dental metal extraction —
Study sample Blood
Tonsil
# # # # # N.D. N.D. N.D. # # # # N.D.
# # # # # # N.D. N.D. N.D. N.D. N.D. N.D. N.D.
N.D.
N.D.
N.D. N.D. N.D. N.D. N.D. N.D.
# # # # # #
#
N.D.
Induction of ICOS in chronic focal infections in PPP
Table 1 Samples in the study
(*), (**), (***) see Figs. 1—3 and the text; (#) sample was tested; N.D.: not done.
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200 the involvement of ICOS in the onset of PPP induced by focal infections, we examined ICOS expression on T cells from various sites, including the tonsils, in patients with PPP.
2. Materials and methods 2.1. Patient samples The characteristics of samples from patients and healthy donors are shown in Table 1. After obtaining informed consent, samples were studied from 14 patients with PPP, 4 patients with recurrent tonsillitis, and 2 patients with obstructive sleep apnea syndrome who had been admitted to the Tokyo Women’s Medical University Hospital between 2003 and 2005, as well as from 16 healthy volunteers. Ten of the 14 patients had suspicious tonsillar focal infections according to their clinical histories, 6 patients underwent a tonsillectomy, and 3 of those 6 patients had symptomatic pustulotic arthro-osteitis. Five of the 14 patients suffered from dental focal infections, such as pericoronitis, periodontal disease, or apical periodontation. Three of these 5 patients underwent tooth extraction. Peripheral blood samples were obtained from 9 patients with PPP (men, 4; women, 5) and from 16
H. Sakiyama et al. healthy volunteers. Full-thickness 4-mm punch biopsies were obtained from pustules on the palms or soles of the PPP patients and from normalappearing skin of three healthy donors. All the patients had not received any treatment for at least 1 week prior to biopsy. Samples of the tonsil tissue were obtained from six patients with PPP and from six patients without PPP (four with recurrent tonsillitis and two with obstructive sleep apnea syndrome). The use of human peripheral blood, skin and tonsil tissues from the patients and healthy donors was approved by the ethical review board of Tokyo Women’s Medical University. The age distribution of the non-PPP group was younger than that of the PPP group.
2.2. Estimation of the severity of PPP skin lesions and pustulotic arthro-osteitis Since an accepted clinical method for scoring the severity of PPP eruptions does not exist, we established a scoring system based on a modification of the psoriasis area and severity index (PASI) [26] to estimate the clinical activity of PPP after treatments for focal infections. Briefly, scores (0—4) estimated according to the degree of erythema, scaling, and pustule formation, rather than the erythema, scaling and induration used in the PASI,
Fig. 1 Representative clinical course of PPP demonstrating rapid improvement after tonsillectomy. Sole (upper) and palm (lower) of a 54-year-old Japanese man before (a), 1 week (b) and 3 months (c) after a tonsillectomy. The patient’s profile and clinical course are indicated by the * in Table 1 and Fig. 3A.
Induction of ICOS in chronic focal infections in PPP were summed and further multiplied by the area score (0—6), determined by the percentage of the lesional area on bilateral palms or soles. The resulting scores for bilateral palms or bilateral soles were multiplied by 0.5 and further summed to determine the skin score, ranging from 0 to 72 like the PASI. The degree of pain from arthroosteitis was self-evaluated using a visual analogue scale (VAS; ranging from 0 to 100 mm) by the patients.
2.3. Antibodies and reagents The C398.4A monoclonal antibody specific for ICOS that was used in this study has been described previously [27]. The following conjugated monoclonal antibodies were used: FITC-anti-CD3 (UCHT1; Immunotech-Beckman-Coulter, Villepinte, France), PE-anti-HLA-DR (Immu-357; Immunotech S.A., Cedex, France), PE-anti-CD25 (B1.49.9; Immunotech S. A.), PE-anti-CLA (HECA-452; BD Biosciences Pharmingen, San Jose, CA, USA), PE-streptavidin (BD Biosciences Pharmingen), PC5-anti-CD4 (13B8.2; Immunotech, Marseille, France) and PC5anti-CD8 (B9.11; Immunotech S.A.). Anti-ICOS monoclonal antibody was conjugated with biotin in our laboratory.
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2.6. Flow cytometry analysis To detect the expression of ICOS and various activation molecules on PBMCs and tonsillar mononuclear cells, the cells were stained with several combinations of the appropriate PE-, FITC- and PC5-conjugated monoclonal antibodies and were examined using three-color flow cytometry analysis. Samples of 1 105 viable cells were analyzed with an EPICS flow cytometer (Beckman Coulter, Miami, FL, USA). The statistical significance of differences between any two groups was analyzed using a Student’s t test. The results were considered significant if p < 0.05.
2.4. Human ICOS-mouse IgG Fc fusion protein (hICOSmIg) The Fc portion of mouse IgG2a (CH2 plus CH3 domain) was inserted into the extracellular portion of the mouse ICOS-inserted pEFIII vector. The ICOS portion was then replaced in frame with the corresponding region of human ICOS. pEFIII/human ICOSmouse IgG Fc was transiently transfected into 293T cells using the calcium phosphate method, and the supernatant was collected and purified using a protein G column (Pharmacia Fine Chemicals, Uppsala, Sweden), then dialyzed against PBS. The protein concentrations were measured with Bradford reagent (Bio-Rad, Hercules, CA, USA).
2.5. Preparation of cells Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Conray density gradient centrifugation. Tonsil tissues obtained during surgery were cut into small pieces, and the single-cell suspensions were prepared by squeezing the pieces between two glass slides, filtering them through stainless steel meshes. Dead cells and red blood cells were removed following centrifugation on a Ficoll-Conray density gradient. The cells were washed with PBS two times before use.
Fig. 2 Representative clinical course of PPP after dental treatment. (A) Soles in a 35-year-old Japanese man are shown before (a), 3 months (b) and 1.5 years (c) after the extraction of the involved tooth. (B) A pantomography showing pericoronitis (§) before treatment. The patient’s profile and clinical course are indicated by the ** in Table 1 and Fig. 3A.
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2.7. Immunohistological staining Three cryostat sections from each skin tissue sample were air-dried and fixed in acetone. The sections were then incubated overnight at 4 8C with primary anti-ICOS monoclonal antibodies to stain ICOS-positive cells, or hICOSmIg to stain B7h expressing cells followed by secondary and third reactions using the Omnitag kit (Lipshow/Immunon, Pittsburg, PA, USA), according to the manufacturer’s instructions. AEC was used as the chromogen, and the sections were counterstained with hematoxylin. Skin samples from four donors with PPP and three healthy individuals were examined. Representative staining profiles are shown.
3. Results 3.1. Treatment of chronic focal infections resulted in an improvement of the skin lesions As reported previously by other groups [4,9,10,12], the patients with PPP showed dramatic and rapid improvements in their skin lesions after undergoing a tonsillectomy. As shown in Fig. 1a, multiple small pustules with erythema and scaling disseminated over bilateral palms and soles were visible prior to the tonsillectomy. One week after the tonsillectomy (Fig. 1b), a substantial reduction in the pustules and erythema was seen on the soles, and the improvements on the soles and palms were even
H. Sakiyama et al. more evident at 3 months after the tonsillectomy (Fig. 1c). No recurrences occurred during a 2-year follow up period (Fig. 3A). Out of six patients, one patient (*** in Table 1 and Fig. 3A) with a long clinical history did not show any apparent improvement in her skin lesions after undergoing a tonsillectomy. The treatment of dental focal infections was also effective for improving of PPP skin lesions. As shown in Fig. 2A, tooth extraction in a patient with pericoronitis (Fig. 2B) led to a marked reduction in pustules, erythema and scaling, although the course of the improvements was more gradual (Fig. 2Ab and c). Other patients who underwent a tonsillectomy or who received treatment for dental focal infections of periodontal disease and apical periodontation also showed rapid or gradual improvements in their skin lesions, respectively (Fig. 3A). In contrast, patients who received only topical steroid and narrowband UVB irradiation did not exhibit any changes in their clinical scores for skin lesion severity (Fig. 3A). Thus, the treatment of focal infections led to a long-lasting recovery from the PPP eruptions. In addition, arthro-osteitis also improved after tonsillectomy (Fig. 3B).
3.2. Comparison of the expressions of ICOS and various activation markers in tonsillar CD4+ T cells between the patients with PPP and those without PPP Since the above results indicated a close involvement between the tonsils and PPP eruptions, we assumed that the activation states of the cells in the tonsils
Fig. 3 Changes in clinical extent of PPP lesions after treatments for focal infections. (A) Patients underwent a tonsillectomy (^–), dental treatments for periodontal disease or apical periodontation (^- - -), or treatment only with topical steroid and narrowband UVB irradiation (nbUVB) (^—). The activities of the PPP lesions were scored according to a method described in Section 2. *, **, ***; see Figs. 1 and 2 and the text. (B) VAS according to the pain of pustulotic arthroosteitis recorded by three patients who underwent tonsillectomies.
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Fig. 4 ICOS expression on CD4+ T cells is significantly higher in tonsil tissues from PPP patients. (A) Representative flow cytometric profile of ICOS expression on CD4+ T cells from PPP and non-PPP patients. Numbers show the percentages of ICOS-positive cells in CD4+ cells. (B) Percentages of ICOS-, CD25-, HLA-DR- and CLA-expressing T cells in CD4+ T cells from PPP patients (^) and from non-PPP patients (recurrent tonsillitis (&); obstructive sleep apnea syndrome (&)). Data are shown as the mean S.E.M.
might be different between patients with PPP and those without PPP. To investigate this matter, the cell surface expressions of various molecules were examined. As shown in Fig. 4A and B, the percentage of ICOS-expressing CD4+ T cells from PPP patients was significantly higher than that from non-PPP patients. The patient whose ICOS expression was the lowest among the six patients was also the patient who did not show any clinical improvement after undergoing a tonsillectomy, as described above (Fig. 3A). The numbers of CD25 or HLA-DR-positive CD4+ T cells were similar between the two groups (Fig. 4B). Regarding the expression of CLA, two of the six PPP patients exhibited a much higher percentage
in CD4+ T cells, but a significant difference was not observed between the two groups (Fig. 4B). The CD8+ T cells from the two groups did not exhibit any significant differences in the expressions of the above-mentioned molecules (data not shown). These results indicate that the tonsillar CD4+ T cells from PPP patients contained more activated and/or effector T cells preferentially expressing ICOS, but not CD25, HLA-DR or CLA, than those from non-PPP patients. The expression of various surface markers on tonsillar lymphocytes is known to change with aging. However, the surface marker expression levels that we evaluated were similar in the PPP and nonPPP groups in the present study with the exception of
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Fig. 5 CD4+ Tcells are not activated in peripheral blood from PPP patients. (A) Representative flow cytometric profile of ICOS expression on CD4+ T cells from PPP patients and healthy donors (control). Numbers show the percentages of ICOSpositive cells in CD4+ cells. (B) The percentages of ICOS-, CD25-, HLA-DR- and CLA-expressing T cells among CD4+ T cells from PPP patients (^) and healthy donors (&). Data are shown as the mean S.E.M.
ICOS, indicating that the expression of ICOS was specifically higher in the tonsils of PPP patients. Additionally, the ICOS, CD25, HLA-DR and CLA expression levels on CD4+ Tcells in PBMCs from PPP patients were similar to those on cells from non-PPP patients (Fig. 5A and B).
3.3. ICOS and B7h expressions in the skin lesions of PPP patients Other reports have suggested that tonsillar CLA+ T cells may migrate into the skin of PPP patients, resulting in tissue damage [6]. Therefore, ICOS+ T cells may also migrate in the same way and may play a
role in the development of the disease by interacting with its ligand B7h and enhancing the production of various cytokines. To examine this possibility, we analyzed ICOS- and B7h-expressing cells in PPP skin lesions and skin from healthy donors using immunohistological staining. ICOS+ T cells were rarely observed in the control skin, and only a few ICOSexpressing T cells had infiltrated in the PPP skin (Fig. 6A). In contrast, B7h, which is known to be induced by proinflammatory cytokines like IFN-g and TNF-a [14], was strikingly expressed in PPP skin. As shown in Fig. 6B, B7h was expressed in the epidermis directly under the pustules, not only on scattered inflammatory cells, but also on cells adhering to
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Fig. 6 Immunohistochemical staining for ICOS and B7h in PPP skin. ICOS-expressing T cells (A) and B7h expression (B) in normal skin and PPP lesional skin were examined by immunohistochemical staining, as described in Section 2. In (A) arrows indicate ICOS-expressing T cells, in (B), B7h expression on inflammatory cells in the epidermis (*) and on cells adhering to the basal membrane zone (**).
the basal membrane zone, however, the identity of these positive cells was not clear. In control skin, we could not find any B7h expression (Fig. 6B). Thus, an interaction between ICOS and B7h may play some role in pathogenesis, but the striking difference between the expression of ICOS and B7h suggests that inflammation may be elicited by some mechanism other than an ICOS-B7h interaction in PPP skin.
4. Discussion PPP is closely related to chronic focal infections [1,9—12]. The present study showed the effectiveness of a tonsillectomy and dental treatment for the
recovery of clinical disease activity, with persistent improvements in the skin lesions and skeletal diseases observed (Fig. 3). As mentioned in Section 1, a previous report [6] showed that the number of tonsillar CLA+ T cells was higher in patients with PPP than in those without PPP. In the skin lesions, numerous CLA+ Tcells were found around pustules in the epidermis and papillary dermis, but such cells were rarely found in normal skin. Thus, CLA+ T cells in focal infections may migrate into the skin, produce proinflammatory cytokines, and cause tissue damage. Consistent with this hypothesis, Yamanaka et al. [28] injected human tonsillar mononuclear cells or PBMCs from patients with PPP into SCID mice grafted with human PPP skin and found a large
206 number of tonsillar mononuclear cell infiltrates than PBMCs around the papillary dermis of the PPP skin. However, in the present study, tonsillar CD4+ Tcells from only two of the six patients with PPP showed a higher positive percentage of CLA expression, but no significant difference was seen overall between the PPP and non-PPP patients (Fig. 4B). Thus, CLA expression in patients with PPP in the present study was not consistent with the results of the previous report. This discrepancy might be explained by the severity of the focal infections. Although a clinical scoring method enabling comparison with previous reports in not available, the severity of the focal infections may have been lower among our samples, except for in the two above-mentioned patients. Our findings, however, cannot rule out the possibility that CLA+ T cells in chronic focal infections may be involved in the pathogenesis of PPP. CLA+ T cells may trigger disease onset or may exacerbate the severity of the skin lesions during the clinical course of the patients. More intensive studies regarding the relationship between the focal induction of CLA and the clinical scores of PPP eruptions are needed. In the present study, tonsillar CD4+ T cells in patients with PPP expressed significantly higher levels of ICOS than those measured in patients without PPP (Fig. 4A and B). The interaction of ICOS with B7h on tonsillar APCs, the expression of which was clearly induced on infiltrated cells in the epithelium of the tonsillar crypt in PPP patients (data not shown), appears to enhance the production of various cytokines, like IL-4, IL-5, IL-10, IFN-g and TNF-a, in T cells [13,15,16]. On the contrary, we could detect only a few ICOS+ Tcells in our representative PPP skin samples (Fig. 6A), thus, ICOS-B7h-interactions probably did not occur in the skin lesions of the majority of our patients. Our immunohistological staining protocol itself worked well, as numerous ICOS+ Tcells were detected in psoriatic skin from patients with psoriasis vulgaris (data not shown), which is closely related to the inflammatory infiltration of activated T cells and APCs [29]. As shown by the obvious improvement in skeletal symptoms, in addition to the improvement in the skin lesions in all three patients with arthroosteitis after tonsillectomy (Fig. 3B), proinflammatory cytokines may travel via the blood stream to peripheral tissues, accumulate in the skin and ligament, and induce inflammation, resulting in tissue damage. Supporting this possibility, B7h expression, which can be induced by IFN-g and TNF-a [14], was upregulated in representative skin samples from our patients (Fig. 6B). At the same time, ICOS+ T cells at focal infections may contribute to the production of antibodies, including anti-keratin antibodies [8], that are then responsible for skin inflammation. However, T cells activated at the site of focal infections
H. Sakiyama et al. may still migrate and lead to tissue damage in different tissues, as mentioned above. Obviously, future studies are needed to clarify these possibilities. In either circumstance, T cell activation caused by chronic focal infections likely triggered the eruption of PPP. Therefore, the manipulation of T cell activation is expected to regulate the deterioration. A fusion protein composed of the extracellular region of human cytotoxic T lymphocyte-associated antigen 4 and human IgG Fc portion (hCTLA-4hIg) was reported to be effective in suppressing the clinical disease activity of patients with psoriasis vulgaris [30] by blockading T cell co-stimulation via CD28. The present study found that ICOS was significantly induced in chronic focal infections. Therefore, the blockade of ICOS co-stimulation by the administration of hICOS-hIg is expected to be a therapeutic approach to alleviating activated Tcells selectively during chronic focal infections with milder side effects than other therapies, like the administration of cyclosporine A or hCTLA-4hIg–— which inhibit all T cell functions. In conclusion, T cells co-stimulated by ICOS molecules in chronic focal infections may induce the skin lesions by supplying proinflammatory cytokines, such as IFN-g and TNF-a; these cytokines further promote the production of various cytokines and chemokines from other cell types, like monocytes in blood and macrophages, dendritic cells and Langerhans cells in the skin, resulting in substantial inflammation and tissue damage. The induction of CLA expression in focal infections was not constantly higher, suggesting that CLA may not have an important role in the maintenance of the steady state in skin lesions. Based upon our present results, we propose that the blockade of ICOS co-stimulation may effectively inhibit the progression of PPP.
Acknowledgements We thank M. Sakuma, N. Kodama and H. Yagi for their technical assistance, Drs. T. Yoshihara and T. Kaneko for the generous supply of the surgical tonsil tissues, and Dr. Y. Arimura for his technical advice regarding hICOSmIg. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (Japan), the International Research and Educational Institute for Integrated Medical Sciences, Tokyo Women’s Medical University which is supported by the Program for Promoting the Establishment of Strategic Research Centers, Special Coordination Funds for Promoting Science and Technology, from the Ministry of Education, Culture, Sports, Science and Technology
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(Japan), and the Associazione Italiana Ricerca sul Cancro (Milan, Italy).
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References [1] Christophers E, Mrowietz U. Pustular eruptions of palms and soles. In: Freedberg IM, Eisen AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI, editors. Fitzpatrick’s dermatology in general medicine. New York: McGraw-Hill; 2003. p. 628—32. [2] Sonozaki H, Mitsui H, Miyanaga Y, Okitsu K, Igarashi M, Hayashi Y, et al. Clinical features of 53 cases with pustulotic arthro-osteitis. Annal Rheum Dis 1981;40:547—53. [3] Uehara M, Ofuji S. The morphogenesis of pustulosis palmaris et plantaris. Arch Dermatol 1974;109:518—20. [4] Nakamura T, Oishi M, Johno M, Ono T, Honda M. Serum levels of interleukin 6 in patients with pustulosis palmaris et plantaris. J Dermatol 1993;20:763—6. [5] Murakata H, Harabuchi Y, Kataura A. Increased interleukin6, interferon-g and tumour necrosis factor-a production by tonsillar mononuclear cells stimulated with alpha-streptococci in patients with pustulosis palmaris et plantaris. Acta Otolaryngol (Stockh) 1999;119:384—91. [6] Nozawa H, Kishibe K, Takahara M, Harabuchi Y. Expression of cutaneous lymphocyte-associated antigen (CLA) in tonsillar T-cells and its induction by in vitro stimulation with alphastreptococci in patients with pustulosis palmaris et plantaris (PPP). Clin Immunol 2005;116:42—53. [7] Ozawa M, Terui T, Tagami H. Localization of IL-8 and complement components in lesional skin of psoriasis vulgaris and pustulosis palmaris et plantaris. Dermatology 2005;211:249— 55. [8] Yamanaka N, Shido F, Kataura A. Tonsillectomy-induced changes in anti-keratin antibodies in patients with pustulosis palmaris et plantaris: a clinical correlation. Arch Otorhinolaryngol 1989;246:109—12. [9] Andrews GC, Machacek GF. Pustular bacterids of the hands and feet. Arch Dermatol Syphilol 1935;32:837—47. [10] Kataura A, Tsubota H. Clinical analyses of focus tonsil and related diseases in Japan. Acta Otolaryngol (Stockh) Suppl 1996;523:161—4. [11] Yamamoto Y, Hashimoto A, Togashi K, Takatsuka J, Ito A, Shimura H, et al. Effects of treatment for dental focal infection on pustulosis palmaris et plantaris. Jpn J Dermatol 2001;111:821—6 [in Japanese]. [12] Yamamoto T, Katayama I, Nishioka K. Restricted usage of the T-cell receptor Vb repertoire in tonsillitis in association with palmoplantar pustulosis. Acta Derm Venereol (Stockh) 1998;78:161—3. [13] Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, Anagnostopoulos I, et al. ICOS is an inducible T-cell costimulator structurally and functionally related to CD28. Nature 1999;397:263—6. [14] Swallow MM, Wallin JJ, Sha WC. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNFa. Immunity 1999;11:423—32.
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Possible involvement of T cell co-stimulation in pustulosis palmaris et plantaris via the induction of inducible co-stimulator in chronic focal infections Hiroko Sakiyama a, Satomi Kobayashi b,*, Umberto Dianzani c, Hideki Ogiuchi a, Makoto Kawashima b, Takehiko Uchiyama d, Junji Yagi d a
Department of Maxillofacial Surgery, Tokyo Women’s Medical University School of Medicine, Tokyo 162-8666, Japan b Department of Dermatology, Tokyo Women’s Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan c Interdisciplinary Research Center of Autoimmune Disease, Department of Medical Sciences, A. Avogadro University of Eastern Piedmont, Novara, Italy d Department of Microbiology and Immunology, Tokyo Women’s Medical University School of Medicine, Tokyo 162-8666, Japan Received 30 August 2007; received in revised form 25 December 2007; accepted 27 December 2007
KEYWORDS Pustulosis palmaris et plantaris; Focal infection; Co-stimulation; ICOS; B7h
Summary Background: Inducible co-stimulator (ICOS) is a co-stimulatory receptor on activated T cells that provides the signals needed for Th1 and Th2 responses via its interaction with B7h. Chronic focal infections are closely related to pustulosis palmaris et plantaris (PPP), but the involvement of ICOS in PPP has not been clarified. Objective: To investigate the effectiveness of treatments for focal infections on PPP skin lesions and the involvement of ICOS-positive T cells at focal infection sites in the tonsils and in PPP lesional skin. Methods: In patients that had undergone a tonsillectomy or dental treatment, the clinical activities of PPP, both the skin lesions and pustulotic arthro-osteitis were followed for over 2 years. The expressions of ICOS and various other activation
Abbreviations: ICOS, inducible co-stimulator; PPP, pustulosis palmaris et plantaris; APC, antigen presenting cell; CLA, cutaneous lymphocyte-associated antigen; TGF, transforming growth factor; IFN, interferon; TNF, tumor necrosis factor; PBMC, peripheral blood mononuclear cell; hICOSmIg, human ICOS-mouse IgG Fc fusion protein; hCTLA-4hIg, human cytotoxic T lymphocyte-associated antigen 4human IgG Fc fusion protein. * Corresponding author. Tel.: +81 3 3353 8111; fax: +81 3 5269 4769. E-mail address: [email protected] (S. Kobayashi). 0923-1811/$30.00 # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.12.010
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H. Sakiyama et al. markers on T cells were examined in tonsil tissue from both PPP patients and non-PPP patients, and the expression levels in peripheral blood were also evaluated in PPP patients and healthy donors. ICOS-positive T cells and B7h expression in PPP and normal skin were examined immunohistochemically. Results: The above treatments for focal infections led to a dramatic and persistent improvement in the PPP skin lesions and pustulotic arthro-osteitis. The expression of ICOS, but not of other activation markers, was higher in tonsil tissues from PPP patients than in tonsil tissues from non-PPP patients. B7h was upregulated without numerous ICOS-positive T cell infiltrates in the skin lesions. Conclusion: The activation of T cells via ICOS co-stimulation in focal infections likely triggers the skin and skeletal inflammation associated with PPP, resulting in tissue damage. # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Pustulosis palmaris et plantaris (PPP) is a chronic recurrent dermatosis characterized by sterile intraepidermal pustules associated with erythematous scaling on the palm and sole [1]. Some PPP patients have skeletal involvement in intrasternocostoclavicular, manubriosternal, spinal ligament or peripheral joints [2]. Histologically, the primary eruptions are spongiotic vesicles in the lower epidermis that contain mononuclear cells; pustulation of the vesicle rapidly begins after it comes in contact with the stratum corneum [3]. A variable density of T cells infiltrate around the pustules. A number of cytokines have been suggested to play a pivotal role in the pathogenesis of PPP [4—7]. In addition, previous reports have shown elevated levels of immune complexes and anti-keratin antibodies in the sera of patients with PPP [8]. Thus, these lines of evidence suggest that abnormal responses by various types of cells, such as T, B and antigen presenting cells (APCs), may be involved in disease onset. Chronic focal infections in the tonsils and oral regions are well known to be involved in the induction of PPP, since a tonsillectomy [1,9,10] and the treatment of oral chronic infections such as periodontal disease and apical periodontation [11] often improve the skin lesions. A tonsillectomy leads to the reduction of serum levels of IL-8 and IL-6 in PPP patients [4,12], suggesting that inflammatory cytokines are related to the induction of PPP by tonsillar focal infections. Furthermore, Nozawa et al. [6] showed that the number of T cells expressing cutaneous lymphocyte-associated antigen (CLA), a homing receptor related to migration into the skin, in tonsillar mononuclear cells and tonsillar tissues was higher in patients with PPP than in patients without PPP. An in vitro stimulation of tonsillar Tcells with astreptococcal antigens from patients with PPP, but not from those without PPP, enhanced CLA expres-
sion and transforming growth factor (TGF)-b production [6]. Murakata et al. [5] also reported a higher production of cytokines [IL-6, interferon (IFN)-g and tumor necrosis factor (TNF)-a] from tonsillar T cells stimulated with a-streptococci in patients with PPP than in patients without PPP. Thus, these data suggest the following scenario. Chronic focal infection by bacteria like a-streptococci may activate T cells to enhance CLA expression, thereby facilitating the movement of those T cells into the skin, and cytokines produced by these T cells may play a key role in tissue damage. However, the molecular events in the onset of PPP induced by focal infections are not fully understood. Inducible co-stimulator (ICOS) [13] is a member of CD28 family that binds to its ligand, B7h [14], on APCs and provides co-stimulatory signals to augment T cell proliferation and the production of various cytokines [13,15,16]. In contrast to CD28, ICOS is not expressed on naı¨ve T cells, but is induced on T cells after T cell receptor engagement [13] and has important roles in both Th1 and Th2 responses [17—19], and antibody production [20]. B7h mRNA is expressed by lymphoid and non-lymphoid tissues, and is upregulated by TNF-a and LPS [14], suggesting that the interaction between ICOS and B7h may function as an important pathway in peripheral tissue inflammation. We previously reported that ICOS expression is increased on activated T cells in the synovial fluid of patients with rheumatoid arthritis [21]. The production of various cytokines was enhanced in CD4+ T cells derived from synovial fluid after ICOS engagement [21]. The possible involvement of ICOS in several other human immune-mediated diseases, such as systemic lupus erythematosus [22], multiple sclerosis [23], common variable immunodeficiency [24], and X-linked lymphoproliferative disease [25], has also been reported. However, the role of ICOS in chronic inflammatory skin diseases has not yet been elucidated. In the present study, in an attempt to clarify
Case
Age/sex
Diagnosis
History of disease (year)
Puatulotic arthroosteitis
Focal infections
+ +
Tonsil Tonsil Tonsil, dental Tonsil Tonsil Tonsil Tonsil Tonsil Tonsil Tonsil Dental Dental, sinusitis Dental
1 2 3 4 5 6 7 8 9 10 11 12 13
54/M * 53/F 54/F 50/M 53/F*** 35/F 47/M 27/F 52/F 37/M 35/M ** 53/F 68/F
PPP PPP PPP PPP PPP PPP PPP PPP PPP PPP PPP PPP PPP
5 1 3 1 13 0.5 1 1 2.5 3 0.3 1 1
14
31/F
PPP
0.3
15 16 17 18 19 20
35/M 24/F 27/F 30/M 33/M 27/M
Reccurent tonsillitis Reccurent tonsillitis Reccurent tonsillitis Reccurent tonsillitis Sleep apnea syndrome Sleep apnea syndrome
52 6 2 4 52 Unknown
Healthy individuals (n = 16) 24—65 (mean 40.3)/male:femal = 10:6
+
+
Dental
Treatment of focal infections Tonsillectomy Tonsillectomy Tonsillectomy Tonsillectomy Tonsillectomy Tonsillectomy — — Penicillin for 4 months — Tooth extraction Tooth extraction Tooth and dental metal extraction —
Study sample Blood
Tonsil
# # # # # N.D. N.D. N.D. # # # # N.D.
# # # # # # N.D. N.D. N.D. N.D. N.D. N.D. N.D.
N.D.
N.D.
N.D. N.D. N.D. N.D. N.D. N.D.
# # # # # #
#
N.D.
Induction of ICOS in chronic focal infections in PPP
Table 1 Samples in the study
(*), (**), (***) see Figs. 1—3 and the text; (#) sample was tested; N.D.: not done.
199
200 the involvement of ICOS in the onset of PPP induced by focal infections, we examined ICOS expression on T cells from various sites, including the tonsils, in patients with PPP.
2. Materials and methods 2.1. Patient samples The characteristics of samples from patients and healthy donors are shown in Table 1. After obtaining informed consent, samples were studied from 14 patients with PPP, 4 patients with recurrent tonsillitis, and 2 patients with obstructive sleep apnea syndrome who had been admitted to the Tokyo Women’s Medical University Hospital between 2003 and 2005, as well as from 16 healthy volunteers. Ten of the 14 patients had suspicious tonsillar focal infections according to their clinical histories, 6 patients underwent a tonsillectomy, and 3 of those 6 patients had symptomatic pustulotic arthro-osteitis. Five of the 14 patients suffered from dental focal infections, such as pericoronitis, periodontal disease, or apical periodontation. Three of these 5 patients underwent tooth extraction. Peripheral blood samples were obtained from 9 patients with PPP (men, 4; women, 5) and from 16
H. Sakiyama et al. healthy volunteers. Full-thickness 4-mm punch biopsies were obtained from pustules on the palms or soles of the PPP patients and from normalappearing skin of three healthy donors. All the patients had not received any treatment for at least 1 week prior to biopsy. Samples of the tonsil tissue were obtained from six patients with PPP and from six patients without PPP (four with recurrent tonsillitis and two with obstructive sleep apnea syndrome). The use of human peripheral blood, skin and tonsil tissues from the patients and healthy donors was approved by the ethical review board of Tokyo Women’s Medical University. The age distribution of the non-PPP group was younger than that of the PPP group.
2.2. Estimation of the severity of PPP skin lesions and pustulotic arthro-osteitis Since an accepted clinical method for scoring the severity of PPP eruptions does not exist, we established a scoring system based on a modification of the psoriasis area and severity index (PASI) [26] to estimate the clinical activity of PPP after treatments for focal infections. Briefly, scores (0—4) estimated according to the degree of erythema, scaling, and pustule formation, rather than the erythema, scaling and induration used in the PASI,
Fig. 1 Representative clinical course of PPP demonstrating rapid improvement after tonsillectomy. Sole (upper) and palm (lower) of a 54-year-old Japanese man before (a), 1 week (b) and 3 months (c) after a tonsillectomy. The patient’s profile and clinical course are indicated by the * in Table 1 and Fig. 3A.
Induction of ICOS in chronic focal infections in PPP were summed and further multiplied by the area score (0—6), determined by the percentage of the lesional area on bilateral palms or soles. The resulting scores for bilateral palms or bilateral soles were multiplied by 0.5 and further summed to determine the skin score, ranging from 0 to 72 like the PASI. The degree of pain from arthroosteitis was self-evaluated using a visual analogue scale (VAS; ranging from 0 to 100 mm) by the patients.
2.3. Antibodies and reagents The C398.4A monoclonal antibody specific for ICOS that was used in this study has been described previously [27]. The following conjugated monoclonal antibodies were used: FITC-anti-CD3 (UCHT1; Immunotech-Beckman-Coulter, Villepinte, France), PE-anti-HLA-DR (Immu-357; Immunotech S.A., Cedex, France), PE-anti-CD25 (B1.49.9; Immunotech S. A.), PE-anti-CLA (HECA-452; BD Biosciences Pharmingen, San Jose, CA, USA), PE-streptavidin (BD Biosciences Pharmingen), PC5-anti-CD4 (13B8.2; Immunotech, Marseille, France) and PC5anti-CD8 (B9.11; Immunotech S.A.). Anti-ICOS monoclonal antibody was conjugated with biotin in our laboratory.
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2.6. Flow cytometry analysis To detect the expression of ICOS and various activation molecules on PBMCs and tonsillar mononuclear cells, the cells were stained with several combinations of the appropriate PE-, FITC- and PC5-conjugated monoclonal antibodies and were examined using three-color flow cytometry analysis. Samples of 1 105 viable cells were analyzed with an EPICS flow cytometer (Beckman Coulter, Miami, FL, USA). The statistical significance of differences between any two groups was analyzed using a Student’s t test. The results were considered significant if p < 0.05.
2.4. Human ICOS-mouse IgG Fc fusion protein (hICOSmIg) The Fc portion of mouse IgG2a (CH2 plus CH3 domain) was inserted into the extracellular portion of the mouse ICOS-inserted pEFIII vector. The ICOS portion was then replaced in frame with the corresponding region of human ICOS. pEFIII/human ICOSmouse IgG Fc was transiently transfected into 293T cells using the calcium phosphate method, and the supernatant was collected and purified using a protein G column (Pharmacia Fine Chemicals, Uppsala, Sweden), then dialyzed against PBS. The protein concentrations were measured with Bradford reagent (Bio-Rad, Hercules, CA, USA).
2.5. Preparation of cells Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Conray density gradient centrifugation. Tonsil tissues obtained during surgery were cut into small pieces, and the single-cell suspensions were prepared by squeezing the pieces between two glass slides, filtering them through stainless steel meshes. Dead cells and red blood cells were removed following centrifugation on a Ficoll-Conray density gradient. The cells were washed with PBS two times before use.
Fig. 2 Representative clinical course of PPP after dental treatment. (A) Soles in a 35-year-old Japanese man are shown before (a), 3 months (b) and 1.5 years (c) after the extraction of the involved tooth. (B) A pantomography showing pericoronitis (§) before treatment. The patient’s profile and clinical course are indicated by the ** in Table 1 and Fig. 3A.
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2.7. Immunohistological staining Three cryostat sections from each skin tissue sample were air-dried and fixed in acetone. The sections were then incubated overnight at 4 8C with primary anti-ICOS monoclonal antibodies to stain ICOS-positive cells, or hICOSmIg to stain B7h expressing cells followed by secondary and third reactions using the Omnitag kit (Lipshow/Immunon, Pittsburg, PA, USA), according to the manufacturer’s instructions. AEC was used as the chromogen, and the sections were counterstained with hematoxylin. Skin samples from four donors with PPP and three healthy individuals were examined. Representative staining profiles are shown.
3. Results 3.1. Treatment of chronic focal infections resulted in an improvement of the skin lesions As reported previously by other groups [4,9,10,12], the patients with PPP showed dramatic and rapid improvements in their skin lesions after undergoing a tonsillectomy. As shown in Fig. 1a, multiple small pustules with erythema and scaling disseminated over bilateral palms and soles were visible prior to the tonsillectomy. One week after the tonsillectomy (Fig. 1b), a substantial reduction in the pustules and erythema was seen on the soles, and the improvements on the soles and palms were even
H. Sakiyama et al. more evident at 3 months after the tonsillectomy (Fig. 1c). No recurrences occurred during a 2-year follow up period (Fig. 3A). Out of six patients, one patient (*** in Table 1 and Fig. 3A) with a long clinical history did not show any apparent improvement in her skin lesions after undergoing a tonsillectomy. The treatment of dental focal infections was also effective for improving of PPP skin lesions. As shown in Fig. 2A, tooth extraction in a patient with pericoronitis (Fig. 2B) led to a marked reduction in pustules, erythema and scaling, although the course of the improvements was more gradual (Fig. 2Ab and c). Other patients who underwent a tonsillectomy or who received treatment for dental focal infections of periodontal disease and apical periodontation also showed rapid or gradual improvements in their skin lesions, respectively (Fig. 3A). In contrast, patients who received only topical steroid and narrowband UVB irradiation did not exhibit any changes in their clinical scores for skin lesion severity (Fig. 3A). Thus, the treatment of focal infections led to a long-lasting recovery from the PPP eruptions. In addition, arthro-osteitis also improved after tonsillectomy (Fig. 3B).
3.2. Comparison of the expressions of ICOS and various activation markers in tonsillar CD4+ T cells between the patients with PPP and those without PPP Since the above results indicated a close involvement between the tonsils and PPP eruptions, we assumed that the activation states of the cells in the tonsils
Fig. 3 Changes in clinical extent of PPP lesions after treatments for focal infections. (A) Patients underwent a tonsillectomy (^–), dental treatments for periodontal disease or apical periodontation (^- - -), or treatment only with topical steroid and narrowband UVB irradiation (nbUVB) (^—). The activities of the PPP lesions were scored according to a method described in Section 2. *, **, ***; see Figs. 1 and 2 and the text. (B) VAS according to the pain of pustulotic arthroosteitis recorded by three patients who underwent tonsillectomies.
Induction of ICOS in chronic focal infections in PPP
203
Fig. 4 ICOS expression on CD4+ T cells is significantly higher in tonsil tissues from PPP patients. (A) Representative flow cytometric profile of ICOS expression on CD4+ T cells from PPP and non-PPP patients. Numbers show the percentages of ICOS-positive cells in CD4+ cells. (B) Percentages of ICOS-, CD25-, HLA-DR- and CLA-expressing T cells in CD4+ T cells from PPP patients (^) and from non-PPP patients (recurrent tonsillitis (&); obstructive sleep apnea syndrome (&)). Data are shown as the mean S.E.M.
might be different between patients with PPP and those without PPP. To investigate this matter, the cell surface expressions of various molecules were examined. As shown in Fig. 4A and B, the percentage of ICOS-expressing CD4+ T cells from PPP patients was significantly higher than that from non-PPP patients. The patient whose ICOS expression was the lowest among the six patients was also the patient who did not show any clinical improvement after undergoing a tonsillectomy, as described above (Fig. 3A). The numbers of CD25 or HLA-DR-positive CD4+ T cells were similar between the two groups (Fig. 4B). Regarding the expression of CLA, two of the six PPP patients exhibited a much higher percentage
in CD4+ T cells, but a significant difference was not observed between the two groups (Fig. 4B). The CD8+ T cells from the two groups did not exhibit any significant differences in the expressions of the above-mentioned molecules (data not shown). These results indicate that the tonsillar CD4+ T cells from PPP patients contained more activated and/or effector T cells preferentially expressing ICOS, but not CD25, HLA-DR or CLA, than those from non-PPP patients. The expression of various surface markers on tonsillar lymphocytes is known to change with aging. However, the surface marker expression levels that we evaluated were similar in the PPP and nonPPP groups in the present study with the exception of
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H. Sakiyama et al.
Fig. 5 CD4+ Tcells are not activated in peripheral blood from PPP patients. (A) Representative flow cytometric profile of ICOS expression on CD4+ T cells from PPP patients and healthy donors (control). Numbers show the percentages of ICOSpositive cells in CD4+ cells. (B) The percentages of ICOS-, CD25-, HLA-DR- and CLA-expressing T cells among CD4+ T cells from PPP patients (^) and healthy donors (&). Data are shown as the mean S.E.M.
ICOS, indicating that the expression of ICOS was specifically higher in the tonsils of PPP patients. Additionally, the ICOS, CD25, HLA-DR and CLA expression levels on CD4+ Tcells in PBMCs from PPP patients were similar to those on cells from non-PPP patients (Fig. 5A and B).
3.3. ICOS and B7h expressions in the skin lesions of PPP patients Other reports have suggested that tonsillar CLA+ T cells may migrate into the skin of PPP patients, resulting in tissue damage [6]. Therefore, ICOS+ T cells may also migrate in the same way and may play a
role in the development of the disease by interacting with its ligand B7h and enhancing the production of various cytokines. To examine this possibility, we analyzed ICOS- and B7h-expressing cells in PPP skin lesions and skin from healthy donors using immunohistological staining. ICOS+ T cells were rarely observed in the control skin, and only a few ICOSexpressing T cells had infiltrated in the PPP skin (Fig. 6A). In contrast, B7h, which is known to be induced by proinflammatory cytokines like IFN-g and TNF-a [14], was strikingly expressed in PPP skin. As shown in Fig. 6B, B7h was expressed in the epidermis directly under the pustules, not only on scattered inflammatory cells, but also on cells adhering to
Induction of ICOS in chronic focal infections in PPP
205
Fig. 6 Immunohistochemical staining for ICOS and B7h in PPP skin. ICOS-expressing T cells (A) and B7h expression (B) in normal skin and PPP lesional skin were examined by immunohistochemical staining, as described in Section 2. In (A) arrows indicate ICOS-expressing T cells, in (B), B7h expression on inflammatory cells in the epidermis (*) and on cells adhering to the basal membrane zone (**).
the basal membrane zone, however, the identity of these positive cells was not clear. In control skin, we could not find any B7h expression (Fig. 6B). Thus, an interaction between ICOS and B7h may play some role in pathogenesis, but the striking difference between the expression of ICOS and B7h suggests that inflammation may be elicited by some mechanism other than an ICOS-B7h interaction in PPP skin.
4. Discussion PPP is closely related to chronic focal infections [1,9—12]. The present study showed the effectiveness of a tonsillectomy and dental treatment for the
recovery of clinical disease activity, with persistent improvements in the skin lesions and skeletal diseases observed (Fig. 3). As mentioned in Section 1, a previous report [6] showed that the number of tonsillar CLA+ T cells was higher in patients with PPP than in those without PPP. In the skin lesions, numerous CLA+ Tcells were found around pustules in the epidermis and papillary dermis, but such cells were rarely found in normal skin. Thus, CLA+ T cells in focal infections may migrate into the skin, produce proinflammatory cytokines, and cause tissue damage. Consistent with this hypothesis, Yamanaka et al. [28] injected human tonsillar mononuclear cells or PBMCs from patients with PPP into SCID mice grafted with human PPP skin and found a large
206 number of tonsillar mononuclear cell infiltrates than PBMCs around the papillary dermis of the PPP skin. However, in the present study, tonsillar CD4+ Tcells from only two of the six patients with PPP showed a higher positive percentage of CLA expression, but no significant difference was seen overall between the PPP and non-PPP patients (Fig. 4B). Thus, CLA expression in patients with PPP in the present study was not consistent with the results of the previous report. This discrepancy might be explained by the severity of the focal infections. Although a clinical scoring method enabling comparison with previous reports in not available, the severity of the focal infections may have been lower among our samples, except for in the two above-mentioned patients. Our findings, however, cannot rule out the possibility that CLA+ T cells in chronic focal infections may be involved in the pathogenesis of PPP. CLA+ T cells may trigger disease onset or may exacerbate the severity of the skin lesions during the clinical course of the patients. More intensive studies regarding the relationship between the focal induction of CLA and the clinical scores of PPP eruptions are needed. In the present study, tonsillar CD4+ T cells in patients with PPP expressed significantly higher levels of ICOS than those measured in patients without PPP (Fig. 4A and B). The interaction of ICOS with B7h on tonsillar APCs, the expression of which was clearly induced on infiltrated cells in the epithelium of the tonsillar crypt in PPP patients (data not shown), appears to enhance the production of various cytokines, like IL-4, IL-5, IL-10, IFN-g and TNF-a, in T cells [13,15,16]. On the contrary, we could detect only a few ICOS+ Tcells in our representative PPP skin samples (Fig. 6A), thus, ICOS-B7h-interactions probably did not occur in the skin lesions of the majority of our patients. Our immunohistological staining protocol itself worked well, as numerous ICOS+ Tcells were detected in psoriatic skin from patients with psoriasis vulgaris (data not shown), which is closely related to the inflammatory infiltration of activated T cells and APCs [29]. As shown by the obvious improvement in skeletal symptoms, in addition to the improvement in the skin lesions in all three patients with arthroosteitis after tonsillectomy (Fig. 3B), proinflammatory cytokines may travel via the blood stream to peripheral tissues, accumulate in the skin and ligament, and induce inflammation, resulting in tissue damage. Supporting this possibility, B7h expression, which can be induced by IFN-g and TNF-a [14], was upregulated in representative skin samples from our patients (Fig. 6B). At the same time, ICOS+ T cells at focal infections may contribute to the production of antibodies, including anti-keratin antibodies [8], that are then responsible for skin inflammation. However, T cells activated at the site of focal infections
H. Sakiyama et al. may still migrate and lead to tissue damage in different tissues, as mentioned above. Obviously, future studies are needed to clarify these possibilities. In either circumstance, T cell activation caused by chronic focal infections likely triggered the eruption of PPP. Therefore, the manipulation of T cell activation is expected to regulate the deterioration. A fusion protein composed of the extracellular region of human cytotoxic T lymphocyte-associated antigen 4 and human IgG Fc portion (hCTLA-4hIg) was reported to be effective in suppressing the clinical disease activity of patients with psoriasis vulgaris [30] by blockading T cell co-stimulation via CD28. The present study found that ICOS was significantly induced in chronic focal infections. Therefore, the blockade of ICOS co-stimulation by the administration of hICOS-hIg is expected to be a therapeutic approach to alleviating activated Tcells selectively during chronic focal infections with milder side effects than other therapies, like the administration of cyclosporine A or hCTLA-4hIg–— which inhibit all T cell functions. In conclusion, T cells co-stimulated by ICOS molecules in chronic focal infections may induce the skin lesions by supplying proinflammatory cytokines, such as IFN-g and TNF-a; these cytokines further promote the production of various cytokines and chemokines from other cell types, like monocytes in blood and macrophages, dendritic cells and Langerhans cells in the skin, resulting in substantial inflammation and tissue damage. The induction of CLA expression in focal infections was not constantly higher, suggesting that CLA may not have an important role in the maintenance of the steady state in skin lesions. Based upon our present results, we propose that the blockade of ICOS co-stimulation may effectively inhibit the progression of PPP.
Acknowledgements We thank M. Sakuma, N. Kodama and H. Yagi for their technical assistance, Drs. T. Yoshihara and T. Kaneko for the generous supply of the surgical tonsil tissues, and Dr. Y. Arimura for his technical advice regarding hICOSmIg. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (Japan), the International Research and Educational Institute for Integrated Medical Sciences, Tokyo Women’s Medical University which is supported by the Program for Promoting the Establishment of Strategic Research Centers, Special Coordination Funds for Promoting Science and Technology, from the Ministry of Education, Culture, Sports, Science and Technology
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(Japan), and the Associazione Italiana Ricerca sul Cancro (Milan, Italy).
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