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SCID chimera
International Congress Series 1257 (2003) 3 – 9
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Tonsil—a mysterious organ-novel approaches using $ human skin/SCID chimera Noboru Yamanaka *, Yoshikazu Yamamoto, Muneki Hotomi, Masaki Suzumoto, Keiji Fujihara, Kiyonori Kuki Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University, 811-1, Kimiidera, Wakayama 641-8510, Japan Received 22 July 2003; received in revised form 30 July 2003; accepted 25 August 2003
Abstract. The concept that altered antigens in the squamous epithelium of the palatine tonsil may serve as a source of autoantibodies for diseases of the palms and soles, i.e., pustulosis palmaris et plantaris (PPP), has been considered for a long time. To study the role of tonsillar mononuclear cells in the onset of PPP lesion, we developed a xenogeneic transplantation model, human skin/severe combined immunodeficiency (SCID) chimera and reconstituted the chimera with human lymphocytes from PPP patients. D 2003 Published by Elsevier B.V. Keywords: Tonsil; Pustulosis palmaris et plantaris; SCID mouse; Chimera mouse
1. Introduction The human palatine tonsils and the nasopharyngeal tonsil are lymphoepithelial tissues located in strategic areas of the oropharynx and nasopharynx. These immunocompetent tissues represent the defense mechanism of first line against ingested or inhaled foreign pathogens. However, the fundamental immunological roles of tonsils have yet to be addressed. One primary obstacle hindering investigators is the absence of an appropriate animal model for studying functions of tonsils in the mucosal immune system. In this study, we employed a novel animal model, human skin/severe combined immunodeficiency (SCID) mouse chimera, to study immunological roles of tonsils.
$
5th International Symposium on Tonsils and Mucosal Barriers of Upper Airways—President lecture. * Corresponding author. Tel.: +81-73-441-0651; fax: +81-73-448-2434. E-mail address: [email protected] (N. Yamanaka).
0531-5131/ D 2003 Published by Elsevier B.V. doi:10.1016/S0531-5131(03)01545-0
4
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
Pustulosis palmaris et plantaris (PPP) has been considered as one of the typical tonsillar focal infections based on the marked clinical improvement of the skin lesions after tonsillectomy [1 –4]. Despite the accumulation or data showing the clinical efficacy of tonsillectomy for this skin lesion, fundamental etiological and pathophysiological issues have yet to be addressed. In the early stage of PPP, it has been reported that lymphocytes, predominantly CD4+ T lymphocytes, infiltrate the palmar and plantar skins. However, the origin and mechanism of infiltration by these lymphocytes is not clear and there are very few reports on whether tonsillar mononuclear cells react directly with the skin. We have been intrigued by the ability to engraft human cells onto severe combined immunodeficiency (SCID) mice [5], together with the opportunity for long-term graft survival and the ability to adoptively transfer various human immunocompetent cells. In this review, we addressed the existing deficiencies in our understanding of the relationship between tonsils and PPP by using emerging transplantation technology involving SCID mice. 2. Materials and methods 2.1. Engraftment of human tonsillar mononuclear cells (TMC) in human skin/SCID chimera To study the role of TMC in the onset of PPP lesion, we developed a xenogeneic transplantation model [6]. In this model, we transfer full-thickness human skin grafts onto SCID mice and simultaneously inject the patients’ tonsillar mononuclear cells intraperitoneally. 2.1.1. Human skin/SCID mouse chimera In 1975, Krueger et al. [7] grafted lesions of psoriasis vulgaris onto the thorax of nude mice. Grafts were fixed on each nude mouse with Band-aids. These were removed on the
Fig. 1. Scheme of transplantation method of uninvolved skin specimens of PPP in SCID mice.
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
5
Fig. 2. Scheme of the skin fenestration transplantation method.
seventh day, and 2- to 3-mm biopsies were taken at 2, 4 and 6 weeks after grafting. A drawback of this human skin/nude mouse chimera was that the maintenance and preservation of the grafts were unsatisfactory. In contrast, Sugai et al. [8] and Ohkawa et al. [9] reported that psoriatic lesions were maintained over a long period after transplantation into the skin of SCID mice. To develop the human-TMC/SCID mouse chimera, we employed the skin fenestrating transplantation method (SFTM) reported by Ueda et al. [10]. After approval by the local ethics committee, patients with PPP and non-PPP patients gave written consent to donate blood and plantar skin samples for the study. Clinically uninvolved skin specimens were dissected into pieces of 8 – 10 mm in diameter and subsequently transplanted onto the subcutaneous tissue onto the back of female CB-17 SCID mice with the surface of the epidermis facing upward (human skin/SCID chimera) (Fig. 1). The skin of the SCID mouse covering the human skin was removed 2 weeks after the transplantation (SFTM) (Fig. 2). The preservation of the grafts 3 weeks after the transplantation was more than 80% in our study.
Table 1 Summary of the immunohistochemical analysis of the grafts
CD3 CD4 CD8 LFA-1 ICAM-1 CD20 Hu-IgG Hu-IgM a
Human-TMC/PPP (n = 7)
Human-PBL/PPP (n = 8)
Human-PBL/non-PPP (n = 3)
+
+
F
+
F
25.0 25.0 25.0 25.0 62.5 0 50.0 0
37.5 25.0 37.5 25.0 12.5 0 – –
0 0 0 0 0 0 0 0
100 100 33.3 0 100 0 – –
F a
85.7 85.7 71.4 71.4 100 0 42.9 0
0 14.3 28.6 14.3 0 0 – –
Percentage of cases studied.
14.3 0 0 14.3 0 100 57.1 100
37.5 50.0 37.5 50.0 25.0 100 50.0 100
0 0 66.7 100 0 100 100 100
6
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
2.1.2. Reconstitution of human skin/SCID chimera with human lymphocytes from PPP patients and non-PPP patients TMC and peripheral blood lymphocytes (PBL) were isolated by Ficoll Hypaque separation. TMC and PBL (1 –3 107/mouse) in phosphate-buffered saline were injected intraperitoneally immediately after the skin transplantation. Four weeks after the transplantation, the mice were killed and the transplanted human skins were excised with
Fig. 3. Immunohistochemical staining of grafts from human-TMC/human PPP skin/SCID mice. (a) CD3+ T lymphocytes, (b) CD4+ T lymphocytes, (c) CD20+ B lymphocytes, (d) ICAM-1.
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
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surrounding mouse skin. Tissues were embedded in OCT-compound, snap frozen in liquid nitrogen, and stored at 80 jC until analyzed. 3. Results 3.1. Immunohistochemical analysis of the grafts Cryosections of 3 – 5 Am were mounted on gelatin-coated slides, air-dried, fixed in acetone and were handled for immunoperoxidase staining. The antibodies against CD3, CD4, CD8, CD2O, LFA-l (lymphocyte function-associated antigen-I) intercellular adhesion molecule-I (ICAM-I) and cutaneous lymphocyte-associated antigen (CLA) were utilized. The immunohistochemical analysis of the grafts is summarized in Table 1. 3.1.1. Human-TMC/human PPP skin/SCID mice SCID mice with transplanted skin specimens from patients with PPP were injected with patients’ TMC. In these mice, CD3+ and CD4+ T lymphocytes infiltrated the area around
Fig. 4. Immunohistochemical staining of CLA.
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N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
the papillary dermis, in great numbers, whereas CD20+ B lymphocytes were hardly seen in the grafts (Fig. 3a– c). Additionally, the epidermis overlying the papillary dermis showed focal neo-expression of ICAM-1 (Fig. 3d). The intensity of ICAM-I seemed to be in proportion to T lymphocyte infiltration of the epidermis and the dermis. The distribution of LFA-l-positive cells was almost consistent with that of CD4+ T lymphocytes. In addition, infiltrating CD4 + T lymphocytes were also positive for CLA (Fig. 4). 3.1.2. Human-PBL/human PPP skin/SCID mice SCID mice transplanted with skin specimens from patients with PPP were injected with patients’ PBL. The immunohistology in these mice contrasted well with the mice reconstituted with TMC. Only a few infiltrating CD3+ T lymphocytes were seen around the papillary dermis and the intensity of ICAM-I was much weaker in these mice. 3.1.3. Human-PBL/human non-PPP skin/SCID mice SCID mice with transplanted skin specimens from non-PPP donors were injected with their PBL. The immunohistological study revealed almost no positive staining. 4. Conclusion Although multiple factors are likely to contribute to the pathogenesis of PPP, there is increasing evidence for a central role of T lymphocytes in this disease. We show formation of an infiltrate in grafts derived from clinically uninvolved PPP skin that also comprises CD3 and CD4 expressing T lymphocytes. The ICAM-1 expression observed in our model is restricted to those regions overlying the papillary dermis and is lacking at the tips of the rete ridges. This pattern is thought to be due to a Th-1 cell-mediated response [11]. Moreover, infiltrating lymphocytes exhibited a pronounced exocytosis which was associated with the expression of the cutaneous T lymphocyte-associated antigen, the skinselective homing receptor [12]. Thus, our data clearly demonstrate that this type of xenogeneic transplantation system is valuable to study recruitment of human tonsillar mononuclear cells into human skin. Conclusively, the experimental approach described here, should prove to be very helpful in further studies on the pathogenesis of autoimmune disorders primarily affecting the skin. References [1] G.C. Andrews, G.F. Machacek, Pustular bacterids of the hand and feet, Arch. Dermatol. 32 (1935) 837 – 847. [2] G. Husby, G. Rajka, T.E. Larsen, Immunofluorescence studies in pustulosis palmaris et plantaris, Acta Derm. Venereol. (Stockh.) 53 (1973) 123 – 126. [3] N. Yamanaka, S. Sambe, A. Kataura, Conceptual understanding of pustulosis palmaris et plantaris as an immune complex disease clue to local tonsillar infections, Acta Otolaryngol. (Stockh.) 401 (1983) 68 – 74 (Supplement). [4] N. Yamanaka, F. Shido, A. Kataura, Tonsillectomy-induced changes in anti-keratin antibodies in patients with pustulosis palmaris et plantaris: a clinical correlation, Arch. Otorhinolaryngol. 246 (1989) 109 – 112. [5] G.C. Bosma, R. Custer, M.J. Bosnia, A severe combined immunodeficiency mutant in the mouse, Nature 301 (1983) 527.
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
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[6] Y. Yamamoto, K. Kuki, Y. Hayashi, N. Yamanaka, A study of the pathogenesis of tonsillar focal infectiontransplantation of human tonsillar lymphocytes and human skin into SCID mice, J. Otolaryngol. Jpn. 103 (2000) 796 – 802. [7] G.G. Krueger, D.D. Manning, J. Malouf, B. Orden, Long-term maintenance of psoriatic human skin on congenitally athymic (nude) mice, J. Invest. Dermatol. 64 (1975) 307 – 312. [8] J. Sugai, M. Iizuka, Y. Kawakubo, et al., Some observations after the transplantation of psoriatic skin using the SCID mice. The 8th Japanese Annual Meeting of Psoriasis, Yokohama. [9] T. Ohkawa, T. Fujimura, Y. Hamada, et al., A study on immunohistochemical and expressions of cytokine mRNA in transplanted psoriatic skin in SCID mice. The 8th Japanese Annual Meeting of Psoriasis, Yokohama. [10] K. Ueda, M. Yanagihara, T. Fujita, Ultrastructure of the lesions of psoriasis vulgaris transplanted into the subcutaneous tissue of nude mice by the skin fenestrating transplantation method, J. Dermatol. 24 (1997) 141 – 146. [11] K. Uyemura, M. Yamamura, D.Y. Fivenson, R.L. Modlin, B.J. Nickololl, The cytokine network in psoriasis characterized by a T-helper type I cell mediated response, J. Invest. Dermatol. 101 (1993) 701 – 705. [12] D.Y.M. Leung, M. Gately, A. Trumble, et al., Bacterial superantigens induce T cell expression of the skinselective homing receptor, the cutaneous lymphocyte-associated antigen, via stimulation of interleukin 12 production, Exp. Med. 181 (1995) 747 – 752.
www.ics-elsevier.com
Tonsil—a mysterious organ-novel approaches using $ human skin/SCID chimera Noboru Yamanaka *, Yoshikazu Yamamoto, Muneki Hotomi, Masaki Suzumoto, Keiji Fujihara, Kiyonori Kuki Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical University, 811-1, Kimiidera, Wakayama 641-8510, Japan Received 22 July 2003; received in revised form 30 July 2003; accepted 25 August 2003
Abstract. The concept that altered antigens in the squamous epithelium of the palatine tonsil may serve as a source of autoantibodies for diseases of the palms and soles, i.e., pustulosis palmaris et plantaris (PPP), has been considered for a long time. To study the role of tonsillar mononuclear cells in the onset of PPP lesion, we developed a xenogeneic transplantation model, human skin/severe combined immunodeficiency (SCID) chimera and reconstituted the chimera with human lymphocytes from PPP patients. D 2003 Published by Elsevier B.V. Keywords: Tonsil; Pustulosis palmaris et plantaris; SCID mouse; Chimera mouse
1. Introduction The human palatine tonsils and the nasopharyngeal tonsil are lymphoepithelial tissues located in strategic areas of the oropharynx and nasopharynx. These immunocompetent tissues represent the defense mechanism of first line against ingested or inhaled foreign pathogens. However, the fundamental immunological roles of tonsils have yet to be addressed. One primary obstacle hindering investigators is the absence of an appropriate animal model for studying functions of tonsils in the mucosal immune system. In this study, we employed a novel animal model, human skin/severe combined immunodeficiency (SCID) mouse chimera, to study immunological roles of tonsils.
$
5th International Symposium on Tonsils and Mucosal Barriers of Upper Airways—President lecture. * Corresponding author. Tel.: +81-73-441-0651; fax: +81-73-448-2434. E-mail address: [email protected] (N. Yamanaka).
0531-5131/ D 2003 Published by Elsevier B.V. doi:10.1016/S0531-5131(03)01545-0
4
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
Pustulosis palmaris et plantaris (PPP) has been considered as one of the typical tonsillar focal infections based on the marked clinical improvement of the skin lesions after tonsillectomy [1 –4]. Despite the accumulation or data showing the clinical efficacy of tonsillectomy for this skin lesion, fundamental etiological and pathophysiological issues have yet to be addressed. In the early stage of PPP, it has been reported that lymphocytes, predominantly CD4+ T lymphocytes, infiltrate the palmar and plantar skins. However, the origin and mechanism of infiltration by these lymphocytes is not clear and there are very few reports on whether tonsillar mononuclear cells react directly with the skin. We have been intrigued by the ability to engraft human cells onto severe combined immunodeficiency (SCID) mice [5], together with the opportunity for long-term graft survival and the ability to adoptively transfer various human immunocompetent cells. In this review, we addressed the existing deficiencies in our understanding of the relationship between tonsils and PPP by using emerging transplantation technology involving SCID mice. 2. Materials and methods 2.1. Engraftment of human tonsillar mononuclear cells (TMC) in human skin/SCID chimera To study the role of TMC in the onset of PPP lesion, we developed a xenogeneic transplantation model [6]. In this model, we transfer full-thickness human skin grafts onto SCID mice and simultaneously inject the patients’ tonsillar mononuclear cells intraperitoneally. 2.1.1. Human skin/SCID mouse chimera In 1975, Krueger et al. [7] grafted lesions of psoriasis vulgaris onto the thorax of nude mice. Grafts were fixed on each nude mouse with Band-aids. These were removed on the
Fig. 1. Scheme of transplantation method of uninvolved skin specimens of PPP in SCID mice.
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
5
Fig. 2. Scheme of the skin fenestration transplantation method.
seventh day, and 2- to 3-mm biopsies were taken at 2, 4 and 6 weeks after grafting. A drawback of this human skin/nude mouse chimera was that the maintenance and preservation of the grafts were unsatisfactory. In contrast, Sugai et al. [8] and Ohkawa et al. [9] reported that psoriatic lesions were maintained over a long period after transplantation into the skin of SCID mice. To develop the human-TMC/SCID mouse chimera, we employed the skin fenestrating transplantation method (SFTM) reported by Ueda et al. [10]. After approval by the local ethics committee, patients with PPP and non-PPP patients gave written consent to donate blood and plantar skin samples for the study. Clinically uninvolved skin specimens were dissected into pieces of 8 – 10 mm in diameter and subsequently transplanted onto the subcutaneous tissue onto the back of female CB-17 SCID mice with the surface of the epidermis facing upward (human skin/SCID chimera) (Fig. 1). The skin of the SCID mouse covering the human skin was removed 2 weeks after the transplantation (SFTM) (Fig. 2). The preservation of the grafts 3 weeks after the transplantation was more than 80% in our study.
Table 1 Summary of the immunohistochemical analysis of the grafts
CD3 CD4 CD8 LFA-1 ICAM-1 CD20 Hu-IgG Hu-IgM a
Human-TMC/PPP (n = 7)
Human-PBL/PPP (n = 8)
Human-PBL/non-PPP (n = 3)
+
+
F
+
F
25.0 25.0 25.0 25.0 62.5 0 50.0 0
37.5 25.0 37.5 25.0 12.5 0 – –
0 0 0 0 0 0 0 0
100 100 33.3 0 100 0 – –
F a
85.7 85.7 71.4 71.4 100 0 42.9 0
0 14.3 28.6 14.3 0 0 – –
Percentage of cases studied.
14.3 0 0 14.3 0 100 57.1 100
37.5 50.0 37.5 50.0 25.0 100 50.0 100
0 0 66.7 100 0 100 100 100
6
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
2.1.2. Reconstitution of human skin/SCID chimera with human lymphocytes from PPP patients and non-PPP patients TMC and peripheral blood lymphocytes (PBL) were isolated by Ficoll Hypaque separation. TMC and PBL (1 –3 107/mouse) in phosphate-buffered saline were injected intraperitoneally immediately after the skin transplantation. Four weeks after the transplantation, the mice were killed and the transplanted human skins were excised with
Fig. 3. Immunohistochemical staining of grafts from human-TMC/human PPP skin/SCID mice. (a) CD3+ T lymphocytes, (b) CD4+ T lymphocytes, (c) CD20+ B lymphocytes, (d) ICAM-1.
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
7
surrounding mouse skin. Tissues were embedded in OCT-compound, snap frozen in liquid nitrogen, and stored at 80 jC until analyzed. 3. Results 3.1. Immunohistochemical analysis of the grafts Cryosections of 3 – 5 Am were mounted on gelatin-coated slides, air-dried, fixed in acetone and were handled for immunoperoxidase staining. The antibodies against CD3, CD4, CD8, CD2O, LFA-l (lymphocyte function-associated antigen-I) intercellular adhesion molecule-I (ICAM-I) and cutaneous lymphocyte-associated antigen (CLA) were utilized. The immunohistochemical analysis of the grafts is summarized in Table 1. 3.1.1. Human-TMC/human PPP skin/SCID mice SCID mice with transplanted skin specimens from patients with PPP were injected with patients’ TMC. In these mice, CD3+ and CD4+ T lymphocytes infiltrated the area around
Fig. 4. Immunohistochemical staining of CLA.
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N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
the papillary dermis, in great numbers, whereas CD20+ B lymphocytes were hardly seen in the grafts (Fig. 3a– c). Additionally, the epidermis overlying the papillary dermis showed focal neo-expression of ICAM-1 (Fig. 3d). The intensity of ICAM-I seemed to be in proportion to T lymphocyte infiltration of the epidermis and the dermis. The distribution of LFA-l-positive cells was almost consistent with that of CD4+ T lymphocytes. In addition, infiltrating CD4 + T lymphocytes were also positive for CLA (Fig. 4). 3.1.2. Human-PBL/human PPP skin/SCID mice SCID mice transplanted with skin specimens from patients with PPP were injected with patients’ PBL. The immunohistology in these mice contrasted well with the mice reconstituted with TMC. Only a few infiltrating CD3+ T lymphocytes were seen around the papillary dermis and the intensity of ICAM-I was much weaker in these mice. 3.1.3. Human-PBL/human non-PPP skin/SCID mice SCID mice with transplanted skin specimens from non-PPP donors were injected with their PBL. The immunohistological study revealed almost no positive staining. 4. Conclusion Although multiple factors are likely to contribute to the pathogenesis of PPP, there is increasing evidence for a central role of T lymphocytes in this disease. We show formation of an infiltrate in grafts derived from clinically uninvolved PPP skin that also comprises CD3 and CD4 expressing T lymphocytes. The ICAM-1 expression observed in our model is restricted to those regions overlying the papillary dermis and is lacking at the tips of the rete ridges. This pattern is thought to be due to a Th-1 cell-mediated response [11]. Moreover, infiltrating lymphocytes exhibited a pronounced exocytosis which was associated with the expression of the cutaneous T lymphocyte-associated antigen, the skinselective homing receptor [12]. Thus, our data clearly demonstrate that this type of xenogeneic transplantation system is valuable to study recruitment of human tonsillar mononuclear cells into human skin. Conclusively, the experimental approach described here, should prove to be very helpful in further studies on the pathogenesis of autoimmune disorders primarily affecting the skin. References [1] G.C. Andrews, G.F. Machacek, Pustular bacterids of the hand and feet, Arch. Dermatol. 32 (1935) 837 – 847. [2] G. Husby, G. Rajka, T.E. Larsen, Immunofluorescence studies in pustulosis palmaris et plantaris, Acta Derm. Venereol. (Stockh.) 53 (1973) 123 – 126. [3] N. Yamanaka, S. Sambe, A. Kataura, Conceptual understanding of pustulosis palmaris et plantaris as an immune complex disease clue to local tonsillar infections, Acta Otolaryngol. (Stockh.) 401 (1983) 68 – 74 (Supplement). [4] N. Yamanaka, F. Shido, A. Kataura, Tonsillectomy-induced changes in anti-keratin antibodies in patients with pustulosis palmaris et plantaris: a clinical correlation, Arch. Otorhinolaryngol. 246 (1989) 109 – 112. [5] G.C. Bosma, R. Custer, M.J. Bosnia, A severe combined immunodeficiency mutant in the mouse, Nature 301 (1983) 527.
N. Yamanaka et al. / International Congress Series 1257 (2003) 3–9
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[6] Y. Yamamoto, K. Kuki, Y. Hayashi, N. Yamanaka, A study of the pathogenesis of tonsillar focal infectiontransplantation of human tonsillar lymphocytes and human skin into SCID mice, J. Otolaryngol. Jpn. 103 (2000) 796 – 802. [7] G.G. Krueger, D.D. Manning, J. Malouf, B. Orden, Long-term maintenance of psoriatic human skin on congenitally athymic (nude) mice, J. Invest. Dermatol. 64 (1975) 307 – 312. [8] J. Sugai, M. Iizuka, Y. Kawakubo, et al., Some observations after the transplantation of psoriatic skin using the SCID mice. The 8th Japanese Annual Meeting of Psoriasis, Yokohama. [9] T. Ohkawa, T. Fujimura, Y. Hamada, et al., A study on immunohistochemical and expressions of cytokine mRNA in transplanted psoriatic skin in SCID mice. The 8th Japanese Annual Meeting of Psoriasis, Yokohama. [10] K. Ueda, M. Yanagihara, T. Fujita, Ultrastructure of the lesions of psoriasis vulgaris transplanted into the subcutaneous tissue of nude mice by the skin fenestrating transplantation method, J. Dermatol. 24 (1997) 141 – 146. [11] K. Uyemura, M. Yamamura, D.Y. Fivenson, R.L. Modlin, B.J. Nickololl, The cytokine network in psoriasis characterized by a T-helper type I cell mediated response, J. Invest. Dermatol. 101 (1993) 701 – 705. [12] D.Y.M. Leung, M. Gately, A. Trumble, et al., Bacterial superantigens induce T cell expression of the skinselective homing receptor, the cutaneous lymphocyte-associated antigen, via stimulation of interleukin 12 production, Exp. Med. 181 (1995) 747 – 752.