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Disappearance of Langerhans cells and melanocytes after cryopreservation of skin
British Journal ol' Plastk" SurgeO' (1995), 48. 405-409 c~ 19t)5 The British Association of Plastic Surgeons
BRITISH
JOURNAL
OF
•••
PLASTIC
SURGERY
Disappearance of Langerhans cells and melanocytes after cryopreservation of skin S. Abe, T. Fujita and Y. Takami
Deparmwnt o[" Plastic and Reconstructh,e Surge/3,, Sapporo Medical College, Sapporo, Japan SUMMA R Y. Cryopreserved skin allografts have been extensively reported to remain viable for longer periods after grafting, both in the laboratory and in the clinic, than skin stored by other methods. We investigated the immunocytochemical and electron microscopic properties of samples of cryopreserved human skin ( - 196°C) for comparison with fresh samples. In an immunocytochemical study of fresh skin, reagents S-100 and CDIa indicated numerous mesenchymal origin cells in the squamous cell layer, basal layer and dermis; 2B7 identified these cells in the basal layer and PC-10 identified them in the basal and squamous cell layers. In cryopreserved skin, however, few cells reacted to these reagents. An electron microscopic study of the cryopreserved skin showed Langerhans cells (LC); however, these had suffered degeneration, with partial defects of the cell membrane and vacuolation in the cytoplasm. We speculate these effects are responsible for the virtually complete abolition of LC membrane and cytoplasm markers. In summary, we detected few mesenchymal origin cells, melanocytes, Langerhans cells, or S-phase cells, in cryopreserved skin by immunocytochemical methods. Langerhans cells existed but had degenerated. These results indicate that cryopreservation at - 196°C causes degeneration of Langerhans cells, and that is the reason for the prolonged viability of cryopreserved allograft.
theless, there has been relatively little work on biological alteration of epidermal Langerhan cells during and after cryopreservation.
The early and rapid coverage of large cutaneous defects is a primary goal of burn care. The most efficient and successful method for coverage of large burns is still split-thickness cutaneous autografts, despite progress in skin tissue culture technology. We have treated several hundred burn patients including, since 1990, four Russians who were brought from Sakhalin or Kamchatka for emergency treatment.' We were able to apply Japanese cadaver allografts to three of the Russian patients, but it is very difficult to obtain cadaver skin, especially in Japan. These problems underlie the increase in need for human skin and for development of skills in storage against future clinical need. Advances in the field of cryobiology have resulted in the development of practical storage techniques for many cells and tissues, 2 including skin. 'a-"The purpose of this paper is to describe the viability and immunocytochemical properties of cryopreserved human skin as compared with fresh samples. Cryopreserved samples were also studied under the electron microscope. '° It is well known that frozen skin survives longer than fresh skin, once it has " t a k e n " . " - ' ~ It has long been suspected that skin immunogenicity is altered by freezing. '6''7 In addition to k6ratinocytes, epidermis contains several minor cell populations each of which is dendritic in shape. These include Langerhans cells (LC), melanocytes and Merkel cells. Interest in LC was sparked several years ago when researchers discovered that LC were MHC (major histocompatibility complex) class II positive leukocytes which were derived from bone marrow, and were capable of allo-activating T-helper cells, None-
Materials and methods
Residual human split skin was obtained during operations on patients ranging in age from 15 to 55 years. Skin was incubated in 1.4 M glycerin in phosphate Programmed Freezing (HOXAN CRYOCELL AP~
,C I 20 -6'
-40,
"•C/ml•2C/mm
-Z0.
LN2
F 196.
! " ........
Figure l--Planning of cooling rate to -70°C in our system. 405
406
British J o u r n a l o f Plastic Surgery
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Figure 2--S-100 immunostaining of the cryopreserved skin and fresh skin. (Above) Most of the positively stained cells in the epidermis are Langerhans cells (LC); in addition there are a small number of melanocytes in the basal layer of the fresh skin. (Below) No positive cells in the cryopreserved skin ( × 340). Figure 3---OKT6 immunostaining of the cryopreserved skin and fresh skin. (Above) Positive cells are observed
Disappearance of Langerhans cells and melanocytes after cryopreservation of skin buffer solution (PBS) (NaCI: 8 g, KCI: 0.2 g, Na.,H PO4:i.15 g, KH._,PO4: 0.2 g in 1000cc distilled water. pH 7.2) for one hour at 4°C, then removed and placed immediately (while still saturated with solution) in a sterile freezing bag (Charter Med. Inc. Blood freezing bag). The bag was laid out fiat in a cassette and the cassette was placed in a computer-programmed freezer, Hoxan CryocelI-AP (Sapporo, Japan), which cooled the samples. When the cooling process had stopped at the final temperature of -70°C, the cassettes were removed and immediately put into liquid nitrogen for storage at - 1 9 6 ° C for 3 weeks (Fig. 1). The temperature of each sample was verified by a thermosensor inserted into each bag. A preliminary experiment was conducted to ensure that the apparatus worked correctly. Immediately prior to examination, the bags were put into a 40°C water bath for rapid thawing. The fresh and cryopreserved skin samples were stained with haematoxylin, eosin and an antidesmoplakin antibody. Desmoplakin is a component of the desmosomes, which form junctions between the epidermal cells and provide much of the resilience and plasticity of normal epidermis..In the immunocytochemical study, the samples were stained with: S-100 antibody (Dako) against S-100, an acid calcium binding protein present in the cytoplasm of a variety of cells including LC and melanocytes; OKT6 (Dako), which reacts with LC and recognises a 49000 dalton cell surface glycoprotein, CDI; 2B7 (Queensland Institute for Medical Research), an antityrosinase antibody which yields a strong colour reaction in the perinuclear region of melanocytes; and PC- 10 (Dako) a monoclonal antibody against all nuclear antigens in S-phase proliferating cells. Electron micrographs of the cryopreserved skin were also taken.
Results
Haematoxylin and eosin stains indicated that the structure of the epidermis in the preserved skin was well retained; there were no visible differences from that of the fresh skin. The desmoplakin antibody showed no differences in staining intensity between the fresh and the cryopreserved skin. S-100 positive cells were observed in the squamous cell layer, basal layer and dermis in the fresh skin. Most of the positively stained cells in the epidermis were Langerhans cells (LC); in addition there was a small number of melanocytes in the basal layer of the fresh skin. No S-100 positive cells were observed in the cryopreserved skin (Fig. 2). OKT6 positive cells were observed in the fresh skin, but were not detected in the cryopreserved skin (Fig. 3). The. population of epidermal melanocytes was
407
assessed using the 2B7 antibody. Melanocytes were observed in the basal layer of the fresh skin but were not seen in the preserved skin (Fig. 4). Cells reacting with PC-10 antibody were often seen in fresh skin, but never in cryopreserved skin (Fig. 5). Epidermal structures such as intercellular bridges were clearly observed in the cryopreserved skin. Degenerated LC were seen in electron micrographs of the cryopreserved skin. This degeneration was manifested by partial defects of the cell membrane and vacuolation in the cytoplasm. The surrounding keratinocytes had no apparent degenerative changes. Highmagnification photomicrographs of the LC showed Birbeck granules (tennis racquet shaped structures) (Fig. 6).
Discussion
Young and Hyatt H reported the results of cryopreserved skin allograft dressing in 1960. Glycerolprotected frozen skin showed a considerable variation in graft retention, ranging from 14 to 52 days, though once the initial "'take" had been accomplished the allografts tended to survive longer. We have also observed programmed frozen allograft on severely burned patients. Studies during the past decade have demonstrated that LC are effective antigen-presenting cells in allogeneic, antigen-specific proliferative and cytotoxic T-cell responses.~S-2° Suspecting that cryopreservation changes LC in some degree for long-term survival of allograft, we examined morphological changes in LC and melanocytes after cryopreservation, by electron microscopy and with the immunocytochemical agents OKT6, S-100 protein, 2B7 and PC-10. We found few positive cells for OKT6, S-100 protein, 2B7 and PC-10 in the preserved specimens. This suggests degeneration of LC, melanocytes and synthetic phase keratinocytes, though it is known that the disappearance of S-100 and OKT6 antigens in LC does not signify a substantial depletion of this cell population. '-'~ Electron micrographs of the preserved skin showed the existence of LC and partial defects in the membranes and vacuolation in the cytoplasm of LC, though the appearance of the keratinocytes was normal. Similar findings have also been reported after laboratory and clinical ultraviolet irradiation of epidermal LCs. "2'23 In vitro, UVB irradiation of epidermal cells induces an impairment of antigen-specific T lymphocyte proliferation in mixed epidermal cell lymphocyte reactions, suggesting the abrogation of the antigen-presenting function of epidermal LC/4 In vivo, UVB irradiation induces skin cancer in mice. These mice have suppressor T cells specific for the UVinduced antigenic determinants in their lymphoid tissue before they develop cancer/s'~6 The unrespon-
in the freshskin. (Below)No positivecells in the cryopreservedskin ( x 340). Figure4--2B7 immunostainingof the cryopreservedskin and fresh skin. (Above)Positivemelanocytesin the basal layerof the freshskin. (Below)No positivecellsin the cryopreservedskin ( x 340). Figure ~-PC-10 immunostainingof the cryopreservedskin and fresh skin. (Above) Positive S-phase of the cell cycleobserved in the basal and squamous cell layersof the fresh skin. (Below)No positivecells in the cryopreservedskin ( x 340).
408
British Journal of Plastic Surgery
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Fig. 6 Figure ~-Electromicroscopy of the cryopreserved human skin. (Left) Keratinocytes and degenerated LC. Degeneration of the LC is shown by partial defects of the cell membrane and vacuolation in the cytoplasm. ( x 4000). (Right) High-magnification photomicrograph of LC shows Birbeck granules (tennis racquet shaped structures) [arrow heads). ( x 8000).
siveness is due to an impairment of the epidermal LC antigen-presenting function a n d / o r induction of suppressor T cells. ~r'~s Streilein et al. found that tape-stripping removes almost all LC from the epidermisP They grafted tapestripped skin on a mouse and found that it survived longer than their unstripped controls, though it was ultimately rejected. They concluded that (a) dermal cells which may express M H C class II antigens are sufficient to induce allograft immunity, a n d / o r (b) the very few LC that remain after skin stripping, especially those near the openings of hair follicles and within the follicles, are sufficient to immunise. Thin human skin which was treated by ultraviolet radiation and glucocorticoid incubation showed depression of LC and morphological change which shortened the dendritic processes.:" :]' Samples of such prepared split thin skin was grafted on burned patients and crural ulcers. These allografts showed late rejection (after day 14). These results correspond to the findings of Ray-Keil and Chandler with corneal allograftsP They have reported that a reduction in the incidence of rejection of mouse heterotopic corneal allograft was achieved by in vitro pretreatment of the graft with UVB at dose of 150 mJ/cm 2 and 300 mJ/cm~. :~2UVB irradiation of epidermal sheet prior to epidermal allografting may have clinical applications for allogeneic skin grafting. The behaviour of LC has been examined after skin transplantation and in an organ culture system by Larcen et al. s3 These observations established a direct route for migration of LC from the epidermis into dermis and then out of the skin. In vivo, epidermal LC migrate via the dermis into the lymphatic system and then to the draining nodes. LC are regarded as a peripheral afferent path of the immunological system of the skin. Reducing the scope of this path has enabled prolonged acceptance of allograft. In cryopreserved skin, the mechanism for the observed extreme decrease in LC density is not clear. First, there may have been irreversible damage and an actual decrease in number through cellular death. A
second possibility is that the cells were affected functionally as a result of alterations in membrane surface antigen and enzyme expression. The type of ultrastructural damage appeared to be reversible, although no direct enumeration of LC was attempted by electron microscopy. The observed extreme decrease in LC density with cryopreservation may stem from reversible damage. This would explain the longer survival of frozen skin once it has fused with host skin, as has been reported in many papers. Although LC migrate similarly out of both allografts and autografts, in the case of an allogeneic skin graft, delivery of the allogeneic LC into the recipient's nodes would provide a powerful stimulus for initiation of rejection. The exact mechanism for depression of LC in cryopreserved cadaver split skin is unknown, but cryopreserved skin allografts showed prolonged take. We propose the following hypothesis: LC membrane and cytoplasm are damaged during cryostorage. The resulting impairment in LC function after thawing prevents many of the LC from migrating at full vigour, so that an insufficient number of them arrive at the draining node to cause the host tissue to institute immediately the rejection reaction. The state of a "compromised host" may last several weeks. But the LC are able to repair themselves during that time; as they do so, they migrate into the recipient lymph nodes and the graft is subsequently rejected. Some recent reports support our hypothesis. Taylor et al. studied the cryobiology of human and rat splenic dendritic leucocytes. TM They demonstrated that dendritic leukocytes showed optimal survival after cooling at 0.3 or 1.5°C/min and that survival fell with faster cooling rates. The yield of viable human dendritic leukocytes was less than 10% when cooled at - 2 0 ° C / m i n or faster. In spite of Taylor's negative results with high cooling rates, the present study found high viability preservation at the cooling rate of - 3 0 ° C / m i n . This result is consistent with that of Ingham et al., who employed the same cooling rate with murine skin samples in Me.,SO and glycerol. ]7 They found about 80% of fresh skin viability with optimal concentrations of both reagents and also
Disappearance of L a n g e r h a n s cells and melanocytes after cryopreservation of skin suggested that LC had been impaired by the freezing process. H u m a n a l l o g r a f t s k i n is a n e s s e n t i a l s o u r c e o f b i o l o g i c a l d r e s s i n g in t h e t r e a t m e n t o f b u r n s . W e h a v e s h o w n t h a t g r a f t s f r o m f r o z e n s k i n last l o n g e r t h a n f r e s h s k i n , as t h e r e s u l t o f d e g e n e r a t i o n o f L C . S k i n a l l o g r a f t s w i t h d e c r e a s e d a n t i g e n i c i t y will m a k e a greater contribution to the treatment of burned patients.
Acknowledgement We gratefully acknowledgc the girl of 2B7 from Dr Peter G. Parsons. Queensland Institute of Medical Research, Australia. This paper was presented at the 18th Annual Scientific Meeting of the Japanese Society of Burn Injury, in Hakone. Kanagawa on 7 May 1992.
18. 19. 20. 21. 22. 23. 24.
References
25.
I. Abe S. Skin ullograft for severe burned patients. Jpn J Med Transplantation Now. 1992: 5: 463-8. 2. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 1984: 247: c125-c142. 3. Lawrence JC. Storage and skin motabolism. Br J Plast Surg 1972; 25: 440-53. 4. Ninnemann JL. Fisher JC, Frank HA. Clinical skin banking: a simplified system for processing, storage and retrieval of human allografts. J Trauma 1978; 18: 723-5. 5. Wachtel T L Ninneman J. Fisher JC, Frank HA, Inancsi W. Viability of frozen allografts. Am J Surg 1979; 138: 783-7. 6. Pegg DE, Taylor MJ. Immunological modification of grafts during storage. In: Calne RY, ed. Transplantation Immunology--Clinical and Experimental. Oxford: University Press. Oxford, 1984: 391-409. 7. May SR, Guttman RM, Wainwright JF. Cryopreservation of skin using an insulated heat sink box stored at -70°C. Cryobiology 1985; 22: 205-14. 8. Fielding GA, Pegg SP. Homograft skin banking--current practices and future trends. Aust N Z J Surg 1988; 58: 153-6. 9. Kearney JN. Wheldon LA, Gowland G. Effects of cryobiological variables on the survival of skin using a defined murine model. Cryobiology 1990; 27: 164-70. 10. Fujita T. Abe S, Takami Y, Kamibayashi Y, Yamaguchi T, Takahashi M. Usefulness of cryopreserved human alloskin. Jpn J Burn lnj 1993: 19: 19-25. I I. Young JM, Hyan GW. Stored skin homografts in extensively burned patients. AMA Arch Surg 1960; 80: 208-13. 12. Lehr HB. Berggren RB, Lotke PA et al. Permanent survival of preserved skin autografts. Surgery 1964; 56: 742-6. 13. Berggren RB, Lehr HB. Clinical use of viable frozen human skin. JAMA 1965: 194: 149-51. 14. Bondoc CC, Burke JF. Clinical experience with viable frozen skin and a frozen skin bank. Ann Surg 1971; 174: 371-82. 15. Baldwin WM 3rd, Cohen N, Hrapchak DB. Prolonged survival of murine skin grafted across a weak histocompatibility barrier as a function of skin grafting technique. Transplantation 1973: 15: 419-22. 16. Taylor M J, London NJM, Thirdborough SM, Lake SP, James RFL. The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing. Cryobiology 1990: 27: 269-78. 17. Ingham E, Matthews JB, Kearney JN. Gowland G. The effects of variation of cryopreservation protocols on the immuno-
26. 27.
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409
genicity of allogeneic skin grafts. Cryobiology 1993: 30: 443-58. Katz SI, Tamaki K, Sachs DH. Epidermal Langerhans cells are derived from cells originating in the bone marrow. Natiare 1979; 282: 324-6. Stingl G, Tamaki K, Katz SI. Origin and function of epidermal Langerhans cells, lmmunol Rev 1980; 53: 149-74. Katz St, Cooper KD, lijima M, Tsuchida T. The role of Langerhans cells in antigen presentation. J Invest Dermatol 1985: 85: 96s-98s. Modlin RL, Rowden G, Taylor CR, Rea TH. Comparison ofS100 and OKT6 antisera in human skin. J Invest Dermatol 1984; 83: 206-9. Aberer W, Schuler G, Stingl G, H6nigsmann H. Wolff K. Ultraviolet light depletes surface markers of Langerhans cells. J Invest Dermatol 1981; 76: 202-10. Friedmann PS. Disappearance of epidermal Langerhans cells during PUVA therapy. Br J Dermatol 1981: 105: 219-21. Stingl G, Gazze-Stingl LA, Aberer W, Wolff K. Antigen presentation by murine epidermal Langerhans cells and its alteration by ultraviolet B light. J lmmunol 1981; 127: 1707-13. Kripke ML. Antigenicity of murine skin tumors induced by ultraviolet light. J Natl Cancer Inst 1974; 53: 1333-6. Fisher MS, Kripke ML. Further studies on the tumor-specific suppressor cells induced by ultraviolet radiation. J lmmunol 1978; 121: 1139-44. Toews GB, Bergstresser PB. Streilein JW. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol 1980; 124: 445-53. Sauder DN, Tamaki K, Moshell AN, Fujiwara H, Katz SI. Induction of tolerance to topically applied TNCB using TNP-conjugated ultraviolet light irradiated epidermal cells. J Immunol 1981: 127: 261-5. Streilein JW, Lonsberry LW, Bergstresser PR. Depletion of epidermal Langerhans cells and la im,nunogenicity from tape-stripped mouse skin. J Exp Med 1982; 155: 863-71. Alsbjorn B. A depression technique of the epidermal Langerhans cells in cadaver split skin. Scand J Plast Reconstr Surg 1984: 18: 61-4. Alsbjorn B. In search of an ideal skin substitute. Scand J Plast Reconstr Surg 1984: 18: 127-33. Ray-Keil L, Chandler JW. Reduction in the incidence of rejection of heterotropic murine corneal transplants by pretreatment with ultraviolet radiation. Transplantation 1986; 42: 403-6. Larsen CP, Steinman RM, Witmer-Pack M, Hankins DF, Morris PJ, Austyn JM. Migration and maturation of Langerhans cells in skin transplants and explants. J Exp Med 1990; 172: 1483-93.
The Authors Seishu Abe, MD, Associate Professor and Head of Department of Plastic and Reconstructive Surgery Tatsuya Fujita MD, Staff surgeon Yoshihiro Takami, MD, Staff Surgeon Department of Plastic and Reconstructive Surgery, Sapporo Medical University, South 1, West 16, Chuo-ku, Sapporo, Japan. Correspondence to Dr S. Abe Paper received 19 September 1994. Accepted 6 March 1995, after revision.
BRITISH
JOURNAL
OF
•••
PLASTIC
SURGERY
Disappearance of Langerhans cells and melanocytes after cryopreservation of skin S. Abe, T. Fujita and Y. Takami
Deparmwnt o[" Plastic and Reconstructh,e Surge/3,, Sapporo Medical College, Sapporo, Japan SUMMA R Y. Cryopreserved skin allografts have been extensively reported to remain viable for longer periods after grafting, both in the laboratory and in the clinic, than skin stored by other methods. We investigated the immunocytochemical and electron microscopic properties of samples of cryopreserved human skin ( - 196°C) for comparison with fresh samples. In an immunocytochemical study of fresh skin, reagents S-100 and CDIa indicated numerous mesenchymal origin cells in the squamous cell layer, basal layer and dermis; 2B7 identified these cells in the basal layer and PC-10 identified them in the basal and squamous cell layers. In cryopreserved skin, however, few cells reacted to these reagents. An electron microscopic study of the cryopreserved skin showed Langerhans cells (LC); however, these had suffered degeneration, with partial defects of the cell membrane and vacuolation in the cytoplasm. We speculate these effects are responsible for the virtually complete abolition of LC membrane and cytoplasm markers. In summary, we detected few mesenchymal origin cells, melanocytes, Langerhans cells, or S-phase cells, in cryopreserved skin by immunocytochemical methods. Langerhans cells existed but had degenerated. These results indicate that cryopreservation at - 196°C causes degeneration of Langerhans cells, and that is the reason for the prolonged viability of cryopreserved allograft.
theless, there has been relatively little work on biological alteration of epidermal Langerhan cells during and after cryopreservation.
The early and rapid coverage of large cutaneous defects is a primary goal of burn care. The most efficient and successful method for coverage of large burns is still split-thickness cutaneous autografts, despite progress in skin tissue culture technology. We have treated several hundred burn patients including, since 1990, four Russians who were brought from Sakhalin or Kamchatka for emergency treatment.' We were able to apply Japanese cadaver allografts to three of the Russian patients, but it is very difficult to obtain cadaver skin, especially in Japan. These problems underlie the increase in need for human skin and for development of skills in storage against future clinical need. Advances in the field of cryobiology have resulted in the development of practical storage techniques for many cells and tissues, 2 including skin. 'a-"The purpose of this paper is to describe the viability and immunocytochemical properties of cryopreserved human skin as compared with fresh samples. Cryopreserved samples were also studied under the electron microscope. '° It is well known that frozen skin survives longer than fresh skin, once it has " t a k e n " . " - ' ~ It has long been suspected that skin immunogenicity is altered by freezing. '6''7 In addition to k6ratinocytes, epidermis contains several minor cell populations each of which is dendritic in shape. These include Langerhans cells (LC), melanocytes and Merkel cells. Interest in LC was sparked several years ago when researchers discovered that LC were MHC (major histocompatibility complex) class II positive leukocytes which were derived from bone marrow, and were capable of allo-activating T-helper cells, None-
Materials and methods
Residual human split skin was obtained during operations on patients ranging in age from 15 to 55 years. Skin was incubated in 1.4 M glycerin in phosphate Programmed Freezing (HOXAN CRYOCELL AP~
,C I 20 -6'
-40,
"•C/ml•2C/mm
-Z0.
LN2
F 196.
! " ........
Figure l--Planning of cooling rate to -70°C in our system. 405
406
British J o u r n a l o f Plastic Surgery
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Figure 2--S-100 immunostaining of the cryopreserved skin and fresh skin. (Above) Most of the positively stained cells in the epidermis are Langerhans cells (LC); in addition there are a small number of melanocytes in the basal layer of the fresh skin. (Below) No positive cells in the cryopreserved skin ( × 340). Figure 3---OKT6 immunostaining of the cryopreserved skin and fresh skin. (Above) Positive cells are observed
Disappearance of Langerhans cells and melanocytes after cryopreservation of skin buffer solution (PBS) (NaCI: 8 g, KCI: 0.2 g, Na.,H PO4:i.15 g, KH._,PO4: 0.2 g in 1000cc distilled water. pH 7.2) for one hour at 4°C, then removed and placed immediately (while still saturated with solution) in a sterile freezing bag (Charter Med. Inc. Blood freezing bag). The bag was laid out fiat in a cassette and the cassette was placed in a computer-programmed freezer, Hoxan CryocelI-AP (Sapporo, Japan), which cooled the samples. When the cooling process had stopped at the final temperature of -70°C, the cassettes were removed and immediately put into liquid nitrogen for storage at - 1 9 6 ° C for 3 weeks (Fig. 1). The temperature of each sample was verified by a thermosensor inserted into each bag. A preliminary experiment was conducted to ensure that the apparatus worked correctly. Immediately prior to examination, the bags were put into a 40°C water bath for rapid thawing. The fresh and cryopreserved skin samples were stained with haematoxylin, eosin and an antidesmoplakin antibody. Desmoplakin is a component of the desmosomes, which form junctions between the epidermal cells and provide much of the resilience and plasticity of normal epidermis..In the immunocytochemical study, the samples were stained with: S-100 antibody (Dako) against S-100, an acid calcium binding protein present in the cytoplasm of a variety of cells including LC and melanocytes; OKT6 (Dako), which reacts with LC and recognises a 49000 dalton cell surface glycoprotein, CDI; 2B7 (Queensland Institute for Medical Research), an antityrosinase antibody which yields a strong colour reaction in the perinuclear region of melanocytes; and PC- 10 (Dako) a monoclonal antibody against all nuclear antigens in S-phase proliferating cells. Electron micrographs of the cryopreserved skin were also taken.
Results
Haematoxylin and eosin stains indicated that the structure of the epidermis in the preserved skin was well retained; there were no visible differences from that of the fresh skin. The desmoplakin antibody showed no differences in staining intensity between the fresh and the cryopreserved skin. S-100 positive cells were observed in the squamous cell layer, basal layer and dermis in the fresh skin. Most of the positively stained cells in the epidermis were Langerhans cells (LC); in addition there was a small number of melanocytes in the basal layer of the fresh skin. No S-100 positive cells were observed in the cryopreserved skin (Fig. 2). OKT6 positive cells were observed in the fresh skin, but were not detected in the cryopreserved skin (Fig. 3). The. population of epidermal melanocytes was
407
assessed using the 2B7 antibody. Melanocytes were observed in the basal layer of the fresh skin but were not seen in the preserved skin (Fig. 4). Cells reacting with PC-10 antibody were often seen in fresh skin, but never in cryopreserved skin (Fig. 5). Epidermal structures such as intercellular bridges were clearly observed in the cryopreserved skin. Degenerated LC were seen in electron micrographs of the cryopreserved skin. This degeneration was manifested by partial defects of the cell membrane and vacuolation in the cytoplasm. The surrounding keratinocytes had no apparent degenerative changes. Highmagnification photomicrographs of the LC showed Birbeck granules (tennis racquet shaped structures) (Fig. 6).
Discussion
Young and Hyatt H reported the results of cryopreserved skin allograft dressing in 1960. Glycerolprotected frozen skin showed a considerable variation in graft retention, ranging from 14 to 52 days, though once the initial "'take" had been accomplished the allografts tended to survive longer. We have also observed programmed frozen allograft on severely burned patients. Studies during the past decade have demonstrated that LC are effective antigen-presenting cells in allogeneic, antigen-specific proliferative and cytotoxic T-cell responses.~S-2° Suspecting that cryopreservation changes LC in some degree for long-term survival of allograft, we examined morphological changes in LC and melanocytes after cryopreservation, by electron microscopy and with the immunocytochemical agents OKT6, S-100 protein, 2B7 and PC-10. We found few positive cells for OKT6, S-100 protein, 2B7 and PC-10 in the preserved specimens. This suggests degeneration of LC, melanocytes and synthetic phase keratinocytes, though it is known that the disappearance of S-100 and OKT6 antigens in LC does not signify a substantial depletion of this cell population. '-'~ Electron micrographs of the preserved skin showed the existence of LC and partial defects in the membranes and vacuolation in the cytoplasm of LC, though the appearance of the keratinocytes was normal. Similar findings have also been reported after laboratory and clinical ultraviolet irradiation of epidermal LCs. "2'23 In vitro, UVB irradiation of epidermal cells induces an impairment of antigen-specific T lymphocyte proliferation in mixed epidermal cell lymphocyte reactions, suggesting the abrogation of the antigen-presenting function of epidermal LC/4 In vivo, UVB irradiation induces skin cancer in mice. These mice have suppressor T cells specific for the UVinduced antigenic determinants in their lymphoid tissue before they develop cancer/s'~6 The unrespon-
in the freshskin. (Below)No positivecells in the cryopreservedskin ( x 340). Figure4--2B7 immunostainingof the cryopreservedskin and fresh skin. (Above)Positivemelanocytesin the basal layerof the freshskin. (Below)No positivecellsin the cryopreservedskin ( x 340). Figure ~-PC-10 immunostainingof the cryopreservedskin and fresh skin. (Above) Positive S-phase of the cell cycleobserved in the basal and squamous cell layersof the fresh skin. (Below)No positivecells in the cryopreservedskin ( x 340).
408
British Journal of Plastic Surgery
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Fig. 6 Figure ~-Electromicroscopy of the cryopreserved human skin. (Left) Keratinocytes and degenerated LC. Degeneration of the LC is shown by partial defects of the cell membrane and vacuolation in the cytoplasm. ( x 4000). (Right) High-magnification photomicrograph of LC shows Birbeck granules (tennis racquet shaped structures) [arrow heads). ( x 8000).
siveness is due to an impairment of the epidermal LC antigen-presenting function a n d / o r induction of suppressor T cells. ~r'~s Streilein et al. found that tape-stripping removes almost all LC from the epidermisP They grafted tapestripped skin on a mouse and found that it survived longer than their unstripped controls, though it was ultimately rejected. They concluded that (a) dermal cells which may express M H C class II antigens are sufficient to induce allograft immunity, a n d / o r (b) the very few LC that remain after skin stripping, especially those near the openings of hair follicles and within the follicles, are sufficient to immunise. Thin human skin which was treated by ultraviolet radiation and glucocorticoid incubation showed depression of LC and morphological change which shortened the dendritic processes.:" :]' Samples of such prepared split thin skin was grafted on burned patients and crural ulcers. These allografts showed late rejection (after day 14). These results correspond to the findings of Ray-Keil and Chandler with corneal allograftsP They have reported that a reduction in the incidence of rejection of mouse heterotopic corneal allograft was achieved by in vitro pretreatment of the graft with UVB at dose of 150 mJ/cm 2 and 300 mJ/cm~. :~2UVB irradiation of epidermal sheet prior to epidermal allografting may have clinical applications for allogeneic skin grafting. The behaviour of LC has been examined after skin transplantation and in an organ culture system by Larcen et al. s3 These observations established a direct route for migration of LC from the epidermis into dermis and then out of the skin. In vivo, epidermal LC migrate via the dermis into the lymphatic system and then to the draining nodes. LC are regarded as a peripheral afferent path of the immunological system of the skin. Reducing the scope of this path has enabled prolonged acceptance of allograft. In cryopreserved skin, the mechanism for the observed extreme decrease in LC density is not clear. First, there may have been irreversible damage and an actual decrease in number through cellular death. A
second possibility is that the cells were affected functionally as a result of alterations in membrane surface antigen and enzyme expression. The type of ultrastructural damage appeared to be reversible, although no direct enumeration of LC was attempted by electron microscopy. The observed extreme decrease in LC density with cryopreservation may stem from reversible damage. This would explain the longer survival of frozen skin once it has fused with host skin, as has been reported in many papers. Although LC migrate similarly out of both allografts and autografts, in the case of an allogeneic skin graft, delivery of the allogeneic LC into the recipient's nodes would provide a powerful stimulus for initiation of rejection. The exact mechanism for depression of LC in cryopreserved cadaver split skin is unknown, but cryopreserved skin allografts showed prolonged take. We propose the following hypothesis: LC membrane and cytoplasm are damaged during cryostorage. The resulting impairment in LC function after thawing prevents many of the LC from migrating at full vigour, so that an insufficient number of them arrive at the draining node to cause the host tissue to institute immediately the rejection reaction. The state of a "compromised host" may last several weeks. But the LC are able to repair themselves during that time; as they do so, they migrate into the recipient lymph nodes and the graft is subsequently rejected. Some recent reports support our hypothesis. Taylor et al. studied the cryobiology of human and rat splenic dendritic leucocytes. TM They demonstrated that dendritic leukocytes showed optimal survival after cooling at 0.3 or 1.5°C/min and that survival fell with faster cooling rates. The yield of viable human dendritic leukocytes was less than 10% when cooled at - 2 0 ° C / m i n or faster. In spite of Taylor's negative results with high cooling rates, the present study found high viability preservation at the cooling rate of - 3 0 ° C / m i n . This result is consistent with that of Ingham et al., who employed the same cooling rate with murine skin samples in Me.,SO and glycerol. ]7 They found about 80% of fresh skin viability with optimal concentrations of both reagents and also
Disappearance of L a n g e r h a n s cells and melanocytes after cryopreservation of skin suggested that LC had been impaired by the freezing process. H u m a n a l l o g r a f t s k i n is a n e s s e n t i a l s o u r c e o f b i o l o g i c a l d r e s s i n g in t h e t r e a t m e n t o f b u r n s . W e h a v e s h o w n t h a t g r a f t s f r o m f r o z e n s k i n last l o n g e r t h a n f r e s h s k i n , as t h e r e s u l t o f d e g e n e r a t i o n o f L C . S k i n a l l o g r a f t s w i t h d e c r e a s e d a n t i g e n i c i t y will m a k e a greater contribution to the treatment of burned patients.
Acknowledgement We gratefully acknowledgc the girl of 2B7 from Dr Peter G. Parsons. Queensland Institute of Medical Research, Australia. This paper was presented at the 18th Annual Scientific Meeting of the Japanese Society of Burn Injury, in Hakone. Kanagawa on 7 May 1992.
18. 19. 20. 21. 22. 23. 24.
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The Authors Seishu Abe, MD, Associate Professor and Head of Department of Plastic and Reconstructive Surgery Tatsuya Fujita MD, Staff surgeon Yoshihiro Takami, MD, Staff Surgeon Department of Plastic and Reconstructive Surgery, Sapporo Medical University, South 1, West 16, Chuo-ku, Sapporo, Japan. Correspondence to Dr S. Abe Paper received 19 September 1994. Accepted 6 March 1995, after revision.