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Alymphatic skin islands: A murine model
JOURNAL
OF SURGICAL
MORITZ
RESEARCH
22,643-648 (1977)
Alymphatic
Skin Islands:
M. ZIEGLER, M.D.,
HENRY MAGUIRE,
A Murine
Model1
M.D., AND CLYDE F. BARKER, M.D.
Institute for Cancer Research, Fox Chase, and Department of Surgery, University of Pennsylvania School of Medicine, Children’s Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, Pennsylvania 19104 Submitted for publication November 9, 1976
Immunologically privileged sites provide a tool for the cancer researcher to facilitate favored growth of tumor allografts and xenografts, thereby providing a mechanism to study the effect of radiotherapy, chemotherapy, immunotherapy, or nonspecific factors on such tumor growth [5]. Such sites also have shown favor to the induction of primary tumors [28]. Mice are widely used, genetically well-defined and the least expensive of experimental animals, although heretofore it has not been technically possible to isolate and better define the recognition and effector limbs of their immunologic reflex. Utilizing precise excision of the lymphatic channel carrying panmculus camosus muscle followed by application of the biologic adhesive Eastman 910 (Eastman-Kodak Co., Rochester, N.Y.), it has been possible to adopt a modified rat alymphatic skin island method [26] for use in the smaller mouse. This paper describes the technique involved in preparing a murine alymphatic skin island. Data are also presented answering the question of whether these islands are alymphatic and whether such islands behave as immunologically privileged sites. MATERIALS AND METHODS Animals. Balb/c male and female mice weighing 22-25 g were utilized throughout 1 Supported by U.S. Public Health Service Research Grams CA-08856, CA-06927, CA-13456, RR-05539 and CA-1582202. The technical assistance of D. Evans is acknowledged.
the study and, following experimental procedures, were caged separately. Tumor was harvested from H-2 incompatible donor C57 black male mice. Skin island preparation (Fig. I). Mice were anesthetized with Nembutal, the flank area was clipped, and the skin was cleansed with alcohol. A l.O- to 1.5-cm diameter rectangular island was outlined with ink, and then a 3.0- to 4.0-mm-wide surrounding strip of skin and underlying panniculus carnosus muscle was excised using curved iris scissors. The musculocutaneous defect then widened and the nonfixed central island floated freely. To cover the exudative wound and to stabilize the island the defect was Wed with the methyl-Zcyano acrylate monomer, Eastman 910. After adhesive drying the animals were returned to individual cages to prevent canabalism. Visualization of afferent lymphatics and their draining lymph nodes. Utilizing a 1: 1 dilution of 2% berlin blue and 2% patent blue dye, intradermal injections of O.Ol- to 0.03-cc vol were done via a 30-gauge needle. Injections were made into either normal flank skin or alymphatic islands. After IO-, 30-, and 60-min intervals, dissection was done to expose lymphatic channels and regional axillary and inguinal lymph nodes to inspect them for dye staining. Tumor experiments. B-16 melanoma was maintained in C-57 black male mice by trocar innoculation. The tumor donor was killed by cervical dislocation and the viable tumor was quickly excised and minced with a blade after discarding necrotic portions. Enzymatic digestion for 20 min was done in
643 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0022.4804
644
JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
5 days following island preparation and serial tumor diameter measurements were made until either the mouse died or until all tumor growth had regressed. Statistics. The Mann-Whitney U test was utilized for statistical analysis. RESULTS
FIG. 1. Surgical technique to construct a murine alymphatic island: (1) 15cm rectangle outlined on mouse tlank, (2) full thickness of skin and underlying panniculus camosus muscle excised with iris scissors, (3) alymphatic channel complete and, (4) Eastman 910 applied over wound.
a 1:l solution of 0.25% pronase and 0.005% desoxyribonuclease, and the subsequent single cell suspension was washed twice and suspended in Prehn’s modified Eagle’s medium. Exclusion of a 1:lO dilution of 0.5% trypan blue was used to count viable tumor cells. A dose of lo5 viable tumor cells in 0.05 ml of medium was injected intradermally to three experimental groups in the following sites (Fig. 2): (i) intact flank skin in nonoperated controls; (ii) intact flank skin in stress control mice which have had an alymphatic island constructed on their opposite flank; or (iii) directly into the alymphatic island. Animals were innoculated 4 or Alymphottc Island
Operative results. The average operative time for constructing an alymphatic island was 5 min and the procedure was easily learned by several personnel. A total of 259 mice in all experiments underwent this operation and 15 died from 1 to 3 days thereafter, this being a mortality of 5.7%. Such islands often were edematous and the circumferential alymphatic channel remained open for a mean time of 14 days. Dye studies. Dye injections were done in 86 mice. Dye in intact flank skin traveled via cutaneous or deeper lymphatic channels lying in the epimysium of the panniculus carnosus muscle to lymph node chains in the axilla and to the posterolateral node in the inguinal region. Dye injected into alymphatic islands as late as 19 days after surgery failed to stain either the lymphatic channels leaving the island or the regional lymph nodes; however, after such an interval, wound contraction had occurred to such an extent that the alymphatic rectangle around the island
Str&s;,,Wpund
Intact Skin Control
FIG. 2. Recipient transplant groups in tumor allograft experiments: alymphatic island recipients, intact skin stress controls, and intact skin controls.
ZIEGLER, MAGUIRE, AND BARKER: ALYMPHATIC
was obliterated and the lymphatic continuity was restored. Tumor studies (Table 1). The 27 control Balb/c mice receiving B-16 melanoma allografted to flank skin with intact lymphatic channels failed to demonstrate any tumor growth. These results contrast sharply with those seen in 41 recipients of tumor allografts in alymphatic islands. Thirty-five or 85.3% of these mice developed tumors and 25 of those 35 mice (71.4%) went on to die of the melenoma, the mean time of death being 59.5 days. Autopsies done on the dead mice demonstrated diffuse metastatic disease in only three; the remainder apparently died of inanition. In 25 stress control mice, four tumors developed (16%) and two of these animals died after intervals of 106 and 121 days (Table 3). The above data suggest that the specific ablation of the afferent limb of the immunologic reflex, namely, the interrupted lymphatic channels, confirms immunologic privilege to the murine skin islands. That this result is not due to nonspecific surgical stress alone is confirmed by the statistically significant (P < 0.01) smaller tumor incidence seen when alymphatic islands and stress control mice are compared. That such murine islands have an intact efferent limb of the immunologic reflex (Fig. 3) is suggested by the fact that none of 15 Balb/c mice previously rejecting either B-16 melanoma tumor allografts or C57 black skin grafts demonstrated growth of a challenge dosage of melanoma placed into an alymphatic island (Table 2).
SKIN ISLANDS
645
TABLE 1 B-16 MELANOMAPRIMARYALLOGRAFTGROWTH IN Balb/c RECIPIENTS
Recipient groups
Number of mice
Tumor incidence/ total group (percentage)
P"
27
0127(0)
Intact skin controls Alymphatic island Stress wound controls (2Mann-Whitney
41
35/41 (85.3)
25
4/2.5(16.0)
U test.
filter diffusion chamber [23], the heterotopically relocated hamster cheek pouch [2, 61, the vascularized guinea pig skin pedicle [l, 1l] the vascularized rat skin pedicle [8, 223, the rat skeletal muscle bed [4], the rat flank skin island [26, 271, and now the murine flank skin island. All of these sites have a deficient or absent lymphatic drainage, and this interruption of the afferent limb of the immunologic reflex with its inherent impaired central antigenic recognition may account for the prolonged survival of allografts or heterografts transplanted to them [l, 2, 41. More recently immunologic enhancement has been postulated as also playing a role in this prolonged graft survival [16, 251; however, other data suggest that such sites are privileged because the lack of lymphatic drainage diverts antigen through the blood vascular route to the spleen where immunoregulation converts DISCUSSION a tissue destructive to a protective role [13]. Immunologically privileged sites can be To confirm that such immunologic privilege classified into those occurring naturally and provided allografts in these sites is not due those created surgically. The anterior cham- to an efferent blockade all of these models ber of the eye [24], the brain [17,19], and the have been shown to express sensitivity; testes [18] have been designated to be im- that is, a sustained or subsequent allograft munologically privileged, but the natural in the alymphatic site is rapidly lost if site of most experimental usefulness is the the host animal is or has been actively or hamster cheek pouch [2,6]. Various surgical adoptively sensitized to that allograft [ 1,261. techniques have been described to create This paper reports for the tirst time a such sites and these include the Millipore surgically constructable immunologically
646
JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
FIG. 3. Experimental protocol to test the efferent limb of the immunologic reflex with tumor allografts in immunized mice: recipient mice sensitized with tumor or skin graft alloantigens (top), and sensitized recipients challenged with donor strain B-16 melanoma in three recipient sites (bottom).
privileged site in the mouse. Such alymphatic islands are durable avoiding the easily damaged pedicle grafts, are simple to construct avoiding the need for microsurgical techniques, are readily visible facilitating inspection, and are constructable in large numbers for a single experiment. The construction of these murine alymphatic islands adopts a method previously described for rats [26], modified by the fact that in mice we were unable to separate the epimysium from the underlying belly of the panniculus carnosus muscle. Therefore in mice the entire panniculus muscle with its attached lymphatic channel carrying epimysium is excised. After muscle removal the island became freely mobile, but by using the biologic adhesive which solidifies on contact with tissue water it could be fixed in a central location. TABLE 2 CHALLENGE TUMOR ALLOGRAFT GROWTH IN Balblc RECIPIENTS PREVIOUSLY SENSITIZED BY TUMOR OR SKIN GRAFTS
Recipient group
Number of mice
Tumor incidence/ total group (percentage)
Alymphatic island
15
o/15 (0)
The data in this tumor allograft murine system are also comparable to the data in studies of such islands in rats [26]. The B-16 melanoma tumor allograft transplanted to murine alymphatic islands grows more frequently, faster, and more aggressively than when the tumor is transplanted to flank skin with intact lymphatics. Such favored growth of either tumor allografts or tumor isografts with known tumor specific antigens was also seen in rat alymphatic island recipients [26, 271. Whether the role of lymphatic ablation is impairment of antigen delivery to the regional lymph node or whether it involves impairment of transmission of either primed host lymphocytes or of graft leukocytic passenger cells cannot be determined from our data [3, 9, 211. Surgical stress produces both more frequent “takes” and more aggressive growth of tumor allografts both in rats and mice as well as tumor isografts in rats [26, 271, a fmding consistent with the recently described immune depression seen following surgery [20]. However, that the improved growth of tumor allografts in alymphatic islands is not due to nonspecific factors is indicated statistically by a greater tumor incidence and more aggressive growth pattern when such a graft is transplanted to skin devoid of lymphatic drainage. It was
ZIEGLER,
MAGUIRE,
AND BARKER: TABLE
ALYMPHATIC
SKIN ISLANDS
647
3
FATE OF Balblc MICE WITH GROWINGB-16 MELANOMA
Recipient group
Tumor incidence/ total group (percentage)
Alymphatic island
35/41 (85.3)
Stress controls
Death incidence (percentage)/ mean diameter at death (cm)
4/25 (16.0)
also apparent that when such B-16 melanoma tumors reached a larger critical size the mouse defense mechanism would be overwhelmed and the animal would die; however, if such tumor growth had not progressed as far by the time the alymphatic space around the island had contracted, and therefore lymphatic channel continuity had been restored, or by the time the stress wound had healed, then host factors would result in tumor regression and animal survival. These data lend support to the concept that host antitumor surveillance mechanisms are interfered with when tumors are in alymphatic sites [7, 191. When animals were presensitized by donor antigen growth of challenge doses of tumor allograft transplanted to alymphatic islands, the challenge dose would never grow either in the rat or mouse models conhrming that the efferent limb of the immune reflex was intact to such islands
[l, 261. Since it is now possible to isolate the murine afferent and efferent limbs of the immunologic reflex using such islands it will be feasible to dissect mouse immune reactivity to allografts and isografts in a more detailed fashion. This should also permit study of immune reactivity as it applies to tumor therapy. In addition, the skin, because of its rich lymphatic endowment and its ready access, has been a favored site for the induction of delayed-type hypersensitivity with contact allergens [lo, 141 and such alymphatic islands will facilitate the
Regression incidence (percentage)/ mean diameter at regression (cm)
214(50) 1.75
study of this response in the mouse as it has in other species. A clinically more useful application of this model is dependent on whether such murine alymphatic islands will support the growth of human tumor xenografts. Alymphatic areas such as the hamster cheek pouch, brain, or Millipore filter diffusion chambers have been successfully used for growth of human tumor xenografts [S], and once such growing tumors are established either pharmacologic or radiation therapy can be tested against such tumors in their foreign hosts. The mouse alymphatic skin island remains to be tested as a recipient site for tumor xenografts, but should tumors grow there it would provide both a less expensive and more durable model than the xenografting of human tumors to nude mice [12]. REFERENCES Barker, C. F., and Billingham, R. E. The role of afferent lymphatics in the rejection of skin homografts. J. Zxp. Med. 128: 197, 1%8. Barker, C. F., and Billingham, R. E. The lymphatic status of hamster cheek pouch tissue in relation to its properties as a graft and as a graft site. J. Exp. Med. 133: 620, 1971. Barker, C. F. Lymphatics and hemograft reaction and the role of peripheral sensitization. In W. Montogra and R. E. Billingham (Eds.), Advances in Biology of Skin: Vol. 11: Immunology Skin, p. 199. Appleton-Century-Crofts,
and the
New York, 1971. Barker, C. F., and Billingham, R. E. Skeletal muscle as a privileged site for orthotopic skin allografts. J. Exp. Med. 138: 289, 1973.
648
JOURNAL
OF SURGICAL
RESEARCH:
5. Barker, C. F. Union of interrational cancer com-
mission workshop new animal models for chemotherapy of human solid tumors. Personal communication, Budapest, 1974. 6. Billingham, R. E., and Silvers, W. K. Studies on cheek pouch skin homografts in the Syrian hamster. In G. E. Wolstenholme and M. P. Cameron (Eds.), Ciba Foundation Symposium on Transplantation. Churchill, London, 1%2. 7. Burnet, M. Immunological Surveillance. Pergamon, Sydney, 1970. 8. Cho, S. I., Marcus, F. S., and Kountz, S. L. A new model for study of allograft rejection in the rat: use of skin with an intact vascular pedicle. I. Effect of vascularity on allograft survival. Transplantation
13: 496, 1972.
9. Elves, M. W. Migration of small lymphocytes from the skin to the regional lymph nodes. Nature (London) 227: 725, 1970. 10. Frey, J. R., and Wenk, P. Experimental studies on the pathogenesis of contact eczema in the guinea pig. Intern. Arch. Allergy Appl. Immunol. 11: 81, 1957.
11. Futrell, J. W., and Myers, G. H., Jr. Regional lymphatics and cancer immunity. Ann. Surg. 177: 1, 1973. 12. Giovanella, B. C., Stehlin, J. S., and Williams, L. J., Jr. Heterotransplantation of human mahgnant tumors in “nude” thymusless mice. II. Malignant tumors induced by injection of cell cultures derived from human solid tumors. J. Nat. Cancer Inst. USA 52: 921, 1974. 13. Kaplan, H. J., and Streilein, J. W. Do immunologically privileged sites require a functioning spleen. Nature (London) 251: 553, 1974. 14. Landsteiner, K., and Chase, M. W. Studies on the sensitization of animals with simple compounds. VI. Experiments on the sensitization of guinea pigs to poison ivy. J. Exp. Med. 69: 767, 1939. 15. Medawar, P. B. Introduction to transplantation of tissues and organs. Brit. Med. Bull. 122: 360, 1965.
VOL. 22, NO. 6, JUNE
1977
16. Merriam, J. C., and Tilney, N. L. A humoral component in prolongation of alymphatic skin allografts in rats. Surg. Forum 27: 336, 1976. 17. Murphy, J. B., and Sturm, E. Conditions determining transplantability of tissues in brain. J. Exp. Med. 28: 183, 1923. 18. Russel, P. S., and Monaco, A. P. The Biology of Tissue Transplantation. Little, Brown, Boston, 1964. 19. Schneck, S. A., and Penn, I.De novo brain tumors in renal transplant recipients. Lancet 1: 983, 1971. 20. Slade, M. S., Simmons, R. L., Yunis, E., and Greenberg, L. J. Immunodepression after major surgery in normal patients. Surgery 78: 363, 1975. 21. Strober, S., and Bowans, J. H. The role of lymphocytes in the sensitization of rats to renal homografts. J. Exp. Med. 122: 347, 1965. 22. Tilney, N. L., and Gowans, J. L. The sensitization of rats by allografts transplanted to alymphatic pedicles of skin. J. Exp. Med. 133: 951, 1971. 23. Weaver, J. M., Algire, G. H., and Prehn, R. T. The growth of cells in vivo in diffusion chambers. II. The role of cells in the destruction of hemografts in mice. J. Nat. Cancer. Inst. USA 15: 1737, 1955. 24. Woodruff, M. F., and Woodruff, H. G. The transplantation of normal tissues; with special reference to auto- and homotransplants of thyroid and spleen in the anterior chambers of the eye and subcutaneously in guinea pigs. Philos. Trans. R. Sot. Lond. Ser. B. Biol. Sci. 224: 539, 1950.
25. Wustrack, K. 0.. Gruber, R. P., and Lucas, Z. J. Immunological enhancement of skin allografts in the rat role of vascular and lymphatic reconstitution. Transplantation 19: 156, 1975. 26. Ziegler, M. M., Miller, E. E., and Barker, C. F. Tumor transplants in alymphatic skin islands. Surg. Forum 23: 127, 1972. 27. Ziegler, M. M., Miller, E. E., and Barker, C. F. Regional lymphatics and the mechanism of action of neuraminidase. Surg. Forum 24: 290, 1973. 28. Ziegler, M. J., Lopez, V., and Barker, C. F. Carcinogenesis in an immunologically privileged site. J. Surg. Res. 18: 201, 1975.
OF SURGICAL
MORITZ
RESEARCH
22,643-648 (1977)
Alymphatic
Skin Islands:
M. ZIEGLER, M.D.,
HENRY MAGUIRE,
A Murine
Model1
M.D., AND CLYDE F. BARKER, M.D.
Institute for Cancer Research, Fox Chase, and Department of Surgery, University of Pennsylvania School of Medicine, Children’s Hospital of Philadelphia, 34th Street and Civic Center Boulevard, Philadelphia, Pennsylvania 19104 Submitted for publication November 9, 1976
Immunologically privileged sites provide a tool for the cancer researcher to facilitate favored growth of tumor allografts and xenografts, thereby providing a mechanism to study the effect of radiotherapy, chemotherapy, immunotherapy, or nonspecific factors on such tumor growth [5]. Such sites also have shown favor to the induction of primary tumors [28]. Mice are widely used, genetically well-defined and the least expensive of experimental animals, although heretofore it has not been technically possible to isolate and better define the recognition and effector limbs of their immunologic reflex. Utilizing precise excision of the lymphatic channel carrying panmculus camosus muscle followed by application of the biologic adhesive Eastman 910 (Eastman-Kodak Co., Rochester, N.Y.), it has been possible to adopt a modified rat alymphatic skin island method [26] for use in the smaller mouse. This paper describes the technique involved in preparing a murine alymphatic skin island. Data are also presented answering the question of whether these islands are alymphatic and whether such islands behave as immunologically privileged sites. MATERIALS AND METHODS Animals. Balb/c male and female mice weighing 22-25 g were utilized throughout 1 Supported by U.S. Public Health Service Research Grams CA-08856, CA-06927, CA-13456, RR-05539 and CA-1582202. The technical assistance of D. Evans is acknowledged.
the study and, following experimental procedures, were caged separately. Tumor was harvested from H-2 incompatible donor C57 black male mice. Skin island preparation (Fig. I). Mice were anesthetized with Nembutal, the flank area was clipped, and the skin was cleansed with alcohol. A l.O- to 1.5-cm diameter rectangular island was outlined with ink, and then a 3.0- to 4.0-mm-wide surrounding strip of skin and underlying panniculus carnosus muscle was excised using curved iris scissors. The musculocutaneous defect then widened and the nonfixed central island floated freely. To cover the exudative wound and to stabilize the island the defect was Wed with the methyl-Zcyano acrylate monomer, Eastman 910. After adhesive drying the animals were returned to individual cages to prevent canabalism. Visualization of afferent lymphatics and their draining lymph nodes. Utilizing a 1: 1 dilution of 2% berlin blue and 2% patent blue dye, intradermal injections of O.Ol- to 0.03-cc vol were done via a 30-gauge needle. Injections were made into either normal flank skin or alymphatic islands. After IO-, 30-, and 60-min intervals, dissection was done to expose lymphatic channels and regional axillary and inguinal lymph nodes to inspect them for dye staining. Tumor experiments. B-16 melanoma was maintained in C-57 black male mice by trocar innoculation. The tumor donor was killed by cervical dislocation and the viable tumor was quickly excised and minced with a blade after discarding necrotic portions. Enzymatic digestion for 20 min was done in
643 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0022.4804
644
JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
5 days following island preparation and serial tumor diameter measurements were made until either the mouse died or until all tumor growth had regressed. Statistics. The Mann-Whitney U test was utilized for statistical analysis. RESULTS
FIG. 1. Surgical technique to construct a murine alymphatic island: (1) 15cm rectangle outlined on mouse tlank, (2) full thickness of skin and underlying panniculus camosus muscle excised with iris scissors, (3) alymphatic channel complete and, (4) Eastman 910 applied over wound.
a 1:l solution of 0.25% pronase and 0.005% desoxyribonuclease, and the subsequent single cell suspension was washed twice and suspended in Prehn’s modified Eagle’s medium. Exclusion of a 1:lO dilution of 0.5% trypan blue was used to count viable tumor cells. A dose of lo5 viable tumor cells in 0.05 ml of medium was injected intradermally to three experimental groups in the following sites (Fig. 2): (i) intact flank skin in nonoperated controls; (ii) intact flank skin in stress control mice which have had an alymphatic island constructed on their opposite flank; or (iii) directly into the alymphatic island. Animals were innoculated 4 or Alymphottc Island
Operative results. The average operative time for constructing an alymphatic island was 5 min and the procedure was easily learned by several personnel. A total of 259 mice in all experiments underwent this operation and 15 died from 1 to 3 days thereafter, this being a mortality of 5.7%. Such islands often were edematous and the circumferential alymphatic channel remained open for a mean time of 14 days. Dye studies. Dye injections were done in 86 mice. Dye in intact flank skin traveled via cutaneous or deeper lymphatic channels lying in the epimysium of the panniculus carnosus muscle to lymph node chains in the axilla and to the posterolateral node in the inguinal region. Dye injected into alymphatic islands as late as 19 days after surgery failed to stain either the lymphatic channels leaving the island or the regional lymph nodes; however, after such an interval, wound contraction had occurred to such an extent that the alymphatic rectangle around the island
Str&s;,,Wpund
Intact Skin Control
FIG. 2. Recipient transplant groups in tumor allograft experiments: alymphatic island recipients, intact skin stress controls, and intact skin controls.
ZIEGLER, MAGUIRE, AND BARKER: ALYMPHATIC
was obliterated and the lymphatic continuity was restored. Tumor studies (Table 1). The 27 control Balb/c mice receiving B-16 melanoma allografted to flank skin with intact lymphatic channels failed to demonstrate any tumor growth. These results contrast sharply with those seen in 41 recipients of tumor allografts in alymphatic islands. Thirty-five or 85.3% of these mice developed tumors and 25 of those 35 mice (71.4%) went on to die of the melenoma, the mean time of death being 59.5 days. Autopsies done on the dead mice demonstrated diffuse metastatic disease in only three; the remainder apparently died of inanition. In 25 stress control mice, four tumors developed (16%) and two of these animals died after intervals of 106 and 121 days (Table 3). The above data suggest that the specific ablation of the afferent limb of the immunologic reflex, namely, the interrupted lymphatic channels, confirms immunologic privilege to the murine skin islands. That this result is not due to nonspecific surgical stress alone is confirmed by the statistically significant (P < 0.01) smaller tumor incidence seen when alymphatic islands and stress control mice are compared. That such murine islands have an intact efferent limb of the immunologic reflex (Fig. 3) is suggested by the fact that none of 15 Balb/c mice previously rejecting either B-16 melanoma tumor allografts or C57 black skin grafts demonstrated growth of a challenge dosage of melanoma placed into an alymphatic island (Table 2).
SKIN ISLANDS
645
TABLE 1 B-16 MELANOMAPRIMARYALLOGRAFTGROWTH IN Balb/c RECIPIENTS
Recipient groups
Number of mice
Tumor incidence/ total group (percentage)
P"
27
0127(0)
Intact skin controls Alymphatic island Stress wound controls (2Mann-Whitney
41
35/41 (85.3)
25
4/2.5(16.0)
U test.
filter diffusion chamber [23], the heterotopically relocated hamster cheek pouch [2, 61, the vascularized guinea pig skin pedicle [l, 1l] the vascularized rat skin pedicle [8, 223, the rat skeletal muscle bed [4], the rat flank skin island [26, 271, and now the murine flank skin island. All of these sites have a deficient or absent lymphatic drainage, and this interruption of the afferent limb of the immunologic reflex with its inherent impaired central antigenic recognition may account for the prolonged survival of allografts or heterografts transplanted to them [l, 2, 41. More recently immunologic enhancement has been postulated as also playing a role in this prolonged graft survival [16, 251; however, other data suggest that such sites are privileged because the lack of lymphatic drainage diverts antigen through the blood vascular route to the spleen where immunoregulation converts DISCUSSION a tissue destructive to a protective role [13]. Immunologically privileged sites can be To confirm that such immunologic privilege classified into those occurring naturally and provided allografts in these sites is not due those created surgically. The anterior cham- to an efferent blockade all of these models ber of the eye [24], the brain [17,19], and the have been shown to express sensitivity; testes [18] have been designated to be im- that is, a sustained or subsequent allograft munologically privileged, but the natural in the alymphatic site is rapidly lost if site of most experimental usefulness is the the host animal is or has been actively or hamster cheek pouch [2,6]. Various surgical adoptively sensitized to that allograft [ 1,261. techniques have been described to create This paper reports for the tirst time a such sites and these include the Millipore surgically constructable immunologically
646
JOURNAL OF SURGICAL RESEARCH: VOL. 22, NO. 6, JUNE 1977
FIG. 3. Experimental protocol to test the efferent limb of the immunologic reflex with tumor allografts in immunized mice: recipient mice sensitized with tumor or skin graft alloantigens (top), and sensitized recipients challenged with donor strain B-16 melanoma in three recipient sites (bottom).
privileged site in the mouse. Such alymphatic islands are durable avoiding the easily damaged pedicle grafts, are simple to construct avoiding the need for microsurgical techniques, are readily visible facilitating inspection, and are constructable in large numbers for a single experiment. The construction of these murine alymphatic islands adopts a method previously described for rats [26], modified by the fact that in mice we were unable to separate the epimysium from the underlying belly of the panniculus carnosus muscle. Therefore in mice the entire panniculus muscle with its attached lymphatic channel carrying epimysium is excised. After muscle removal the island became freely mobile, but by using the biologic adhesive which solidifies on contact with tissue water it could be fixed in a central location. TABLE 2 CHALLENGE TUMOR ALLOGRAFT GROWTH IN Balblc RECIPIENTS PREVIOUSLY SENSITIZED BY TUMOR OR SKIN GRAFTS
Recipient group
Number of mice
Tumor incidence/ total group (percentage)
Alymphatic island
15
o/15 (0)
The data in this tumor allograft murine system are also comparable to the data in studies of such islands in rats [26]. The B-16 melanoma tumor allograft transplanted to murine alymphatic islands grows more frequently, faster, and more aggressively than when the tumor is transplanted to flank skin with intact lymphatics. Such favored growth of either tumor allografts or tumor isografts with known tumor specific antigens was also seen in rat alymphatic island recipients [26, 271. Whether the role of lymphatic ablation is impairment of antigen delivery to the regional lymph node or whether it involves impairment of transmission of either primed host lymphocytes or of graft leukocytic passenger cells cannot be determined from our data [3, 9, 211. Surgical stress produces both more frequent “takes” and more aggressive growth of tumor allografts both in rats and mice as well as tumor isografts in rats [26, 271, a fmding consistent with the recently described immune depression seen following surgery [20]. However, that the improved growth of tumor allografts in alymphatic islands is not due to nonspecific factors is indicated statistically by a greater tumor incidence and more aggressive growth pattern when such a graft is transplanted to skin devoid of lymphatic drainage. It was
ZIEGLER,
MAGUIRE,
AND BARKER: TABLE
ALYMPHATIC
SKIN ISLANDS
647
3
FATE OF Balblc MICE WITH GROWINGB-16 MELANOMA
Recipient group
Tumor incidence/ total group (percentage)
Alymphatic island
35/41 (85.3)
Stress controls
Death incidence (percentage)/ mean diameter at death (cm)
4/25 (16.0)
also apparent that when such B-16 melanoma tumors reached a larger critical size the mouse defense mechanism would be overwhelmed and the animal would die; however, if such tumor growth had not progressed as far by the time the alymphatic space around the island had contracted, and therefore lymphatic channel continuity had been restored, or by the time the stress wound had healed, then host factors would result in tumor regression and animal survival. These data lend support to the concept that host antitumor surveillance mechanisms are interfered with when tumors are in alymphatic sites [7, 191. When animals were presensitized by donor antigen growth of challenge doses of tumor allograft transplanted to alymphatic islands, the challenge dose would never grow either in the rat or mouse models conhrming that the efferent limb of the immune reflex was intact to such islands
[l, 261. Since it is now possible to isolate the murine afferent and efferent limbs of the immunologic reflex using such islands it will be feasible to dissect mouse immune reactivity to allografts and isografts in a more detailed fashion. This should also permit study of immune reactivity as it applies to tumor therapy. In addition, the skin, because of its rich lymphatic endowment and its ready access, has been a favored site for the induction of delayed-type hypersensitivity with contact allergens [lo, 141 and such alymphatic islands will facilitate the
Regression incidence (percentage)/ mean diameter at regression (cm)
214(50) 1.75
study of this response in the mouse as it has in other species. A clinically more useful application of this model is dependent on whether such murine alymphatic islands will support the growth of human tumor xenografts. Alymphatic areas such as the hamster cheek pouch, brain, or Millipore filter diffusion chambers have been successfully used for growth of human tumor xenografts [S], and once such growing tumors are established either pharmacologic or radiation therapy can be tested against such tumors in their foreign hosts. The mouse alymphatic skin island remains to be tested as a recipient site for tumor xenografts, but should tumors grow there it would provide both a less expensive and more durable model than the xenografting of human tumors to nude mice [12]. REFERENCES Barker, C. F., and Billingham, R. E. The role of afferent lymphatics in the rejection of skin homografts. J. Zxp. Med. 128: 197, 1%8. Barker, C. F., and Billingham, R. E. The lymphatic status of hamster cheek pouch tissue in relation to its properties as a graft and as a graft site. J. Exp. Med. 133: 620, 1971. Barker, C. F. Lymphatics and hemograft reaction and the role of peripheral sensitization. In W. Montogra and R. E. Billingham (Eds.), Advances in Biology of Skin: Vol. 11: Immunology Skin, p. 199. Appleton-Century-Crofts,
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