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Effector mechanisms in allograft rejection I. Assembly of “sponge matrix” allografts
CELLULAR
IMMUNOLOGY
26, 160-167 (1976)
Effector Mechanisms in Allograft I. Assembly of “Sponge Matrix”
Rejection
Allografts
P. J. ROBERTS AND P. H;~YRY Transjlantation Laboratory, Fourth Department of Surgery, and Third Department of Pathology, hiversity of Helsinki, SF 00290 Helsinki 29, Fixland Received May l&l976 This communication describes a model that makes it possible to quantitatively recover allograft-infiltrating cells in a functionally viable state. The model is based on the use of an inert spongious matrix tissue into which fibroblasts of strain “A” are grown. Upon transplantation to an allogeneic “B” strain host, graft-directed killer cells infiltrate the sponge. Mere physical compression of the sponge releases virtually all infiltrating cells. More than 9 0 % of the viable cells infiltrating the sponge graft are thus recovered. About 1.5% of the infiltrating cells are blasts, about 3 5 % are lymphocytes, and about 3 0 % are monocytes and macrophages. The rest are predominantly granulocytes. The allograft-infiltrating cells display an immunologically specific cytolytic response to relevant “‘Cr-labeled target cells in vitro. The infiltrating cells are much more efficient killer cells than spleen or draining lymph node cells. The allograft-infiltrating cells are thus functionally intact and are recovered without enzymatic treatment by mere mechanical means. W e therefore consider the sponge matrix model a suitable and reproducible method for studying allograftinfiltrating cells.
INTRODUCTION A prominent histological feature in an allograft undergoing acute rejection is its infiltration by lymphocytes of varying sizes (1, 2). Both T (3) and B (4) lymphocytes have been reported to infiltrate the allograft. In addition to the lymphocytes, macrophages/monocytes are also present in the cellular infiltrates, and the more necrotic areas of the graft usually display an infiltration of polymorphonuclear leukocytes (5). Approximately 6@-80% of the allograft-infiltrating cells are equipped with (idiotypic) receptors directed to the antigens of the graft (6). The significance of finding several cell classes in the graft-infiltrating cell population is unknown, as are the functions of the different cell types in the graft-destructive process. It has recently become possible to identify and fractionate the various subclasses of immunocompetent cells on the basis of their surface markers (7, S), physical (9, lo), or biological (8, 11) properties. Concomitantly, methods have been developed enabling the characterization of the functions of these cells in vitro. These studies have demonstrated that both T lymphocytes (12, 13) and non-T lymphocytes (14) and even monocytes and macrophages (15) can function as killer cells to relevant
allogeneic
target
cells in vitro. 160
Copyright 1976 by Academic Press, Inc. rights o3 reproductionin any form reserved.
All
These in vitro
results
therefore
suggest
SPONGE
MATRIX
ALLOGRAFTS
161
that several different cell-bound effector mechanisms are functioning in allograft destruction in v&o. In order to link these in vitro findings to the functions of allograft-infiltrating cells, it was necessary to quantitatively isolate the infiltrating cells in an intact, functionally viable state. Since the isolation of the infiltrating cells from ordinary skin or organ allografts was not possible without enzymatic treatment (4), we have developed a model for this purpose. The model is based on the use of an inert “spongious matrix” tissue into which fibroblasts of strain “A” are grown. The sponge is transplanted to a strain “B” allogeneic host, and removed at various times thereafter. Specifically cytotoxic cells were found to infiltrate the sponge and mere physical compression of the sponge released virtually all of the infiltrating cells. In this communication we report the assembly of the sponge matrix grafts and the use of these grafts in transplantation studies in the mouse. A preliminary report describing the histological and cytological features of the SMG has been published (16). In forthcoming papers we will describe in detail the physical properties, the surface characteristics, and the receptor specificities of the allograft-infiltrating cells, and link these characteristics to the ability of the various cell populations to perform in vitro target cell lysis. MATERIALS
AND
METHODS
Mice. The CBA/H-T6T6, DBA/Z, and C57BL/6 mouse strains were originally obtained from the Jackson Laboratory, Bar Harbor, Maine, and were bred in our colony. Sponge matrix grafts. As supporting matrix we used viscous cellulose sponge. The sponge was purchased from Kongsfors Fabrikker A/S, Oslo. Prior to use, the sponge was cut into cubical 3- X 3- X 3-mm pieces and washed 10 times in distilled water and 70% ethanol. The sponges were kept immersed in physiological saline in small glass bottles and were sterilized by autoclaving them before use. The sponges were “filled up” with allogeneic cells by placing 5 to 10 pieces of sponge for 6 to 7 days into the peritoneal cavity of the donor strain. During this time the sponge was uniformly infiltrated mainly by fibroblastoid cells plus some lymphocytes and macrophages of the donor (16). Final allotransplantations were performed subcutaneously to the neck of the recipient via a hind-region incision. Usually two pieces of sponge were transplanted simultaneously on both sides of the midline under strictly aseptical conditions. In order to keep several transplanted animals in the same cage, it was necessary to remove hair only at the site of the incision; removal of hair at the site of the transplant resulted in biting of the transplant sites, thus causing infections of the transplants. In all transplantations the animals were under intraperitoneal chloralhydrate (360 mg/kg) anesthesia. During the first week after transplantation, 4 g/liter of oxytetracycline was added to the drinking water. Six to eight days after transplantation the recipients were sacrificed. The sponges, draining (axillary and inguinal) and nondraining (mesenteric) lymph nodes, and the spleens of the recipients were removed. The recipient blood was obtained from the venous eye plexus, and in some cases the thymus and the cell contents of the humeral and femural bone cavities were also harvested. The cell contents of the sponge were recovered to tissue culture medium by compression. Earlier experience
162
ROBERTS
AND
HiYRY
has shown that almost all sponge-infiltrating cells were thus released (16). The cells of the various lymphoid organs were processed for experimentation as described (12). Prior to the experiments the red cells were lysed from all populations with 0.83% ammonium chloride (12). The cells recovered from the various lymphoid organs demonstrated a good (> 95%) cell viability in trypan blue staining; the cells recovered from the sponge were usually contaminated with cell debris and with 35-40s dead cells. In order to remove the dead cells and the cell debris, the sponge-infiltrating cells were centrifuged (5000 rpm/lS min) over a 35% bovine serum albumin (BSA, Sigma Chemical Company, St. Louis, MO.) one-step density gradient. The cell population collected at the interface contained > 90% of the viable cells and no debris (Table 2). Diflerentiul counts. The differential counts were performed from May-Gruenwald-Giemsa (MGG) stained cytocentrifuged cell smears. Cell-mediated cytotoxicity. The method of Brunner et al. (17) was modified for microculture conditions. The target cells were the P-815-X2 mastocytoma line of DBA/2 (obtained from Dr. K. T. Brunner, Swiss Institute for Experimental Cancer Research, Lausanne) and the EL-4 lymphoma line of C57BL/6 (obtained from Dr. Leif C. Andersson, Department of Immunology, University of Uppsala). The target cells were carried in alternate in z&o-in vitro passages in peritoneal cavity and in suspension culture. For cytotoxicity test the target cells were labeled with 51Cr (sodium 51chromate, The Radiochemical Center, Amersham) in conditions described (17). Ten-thousand target cells were plated in 100 ~1 of 5% fetal calf serum containing Eagle’s Minimal Essential Medium per well in round-bottom microtitration plates (Cooks Microtiter System, U.K.). The effector cells were added to the target cells in the same volume by employing at least two effector/ target cell ratios. After a short centrifugation (809 for 3 min), the plates were incubated in a humidified atmosphere of 5% CO, in air for 6-18 hr at 37”C, and the amount of Yr released to the culture medium was used to indicate the rate of target cell damage. The “specific 5*Cr release” was calculated according to the formula (17) : y. Specific release = 100 x
Release in presence of effector cells - Spontaneous Maximal release - Spontaneous release
The spontaneous release never exceeded 10% of the maximum release in these conditions,
(Triton
release ’ X-100)
RESULTS Rediscovery of graft-infiltrating cells. In the first experiment we quantitated the number of cells infiltrating an allogeneic and a syngeneic sponge matrix transplant. Cubical pieces of sponge, sized 3 x 3 x 3 mm, were inserted into the peritoneal cavity of DBA/2 or CBA mice. Seven days later, the sponges were removed and transplanted subcutaneously into the neck of CBA recipients. Each recipient received two transplants, one on either side of the neck. Seven days after the transplantation, the cell contents of the sponges were released by compression. Cytocentrifuged cell smears were made for differential counts, and the rest of the cells were used for further purification in one-step BSA density centrifugation.
SPONGE
MATRIX
163
ALLOGRAFTS
TABLE
1
Cellular Infiltrates in Allogeneic and Syngeneic Sponge Matrix Grafts and in Control Sponges 7 Days after Subcutaneous Transplantation to the Neck Cells per graft X lo6 5
Type of graft
Allogeneic graft Syngeneic graft Sponge along
Blasts
Lymphocytes
Granulocytes
Monocytes/ macrophages
Total
0.94 f 0.41 0.0 0.0
3.12 f 1.08 0.056 f 0.019 0.068 f 0.028
1.59 f 0.36 0.461 f 0.248 0.151 f 0.040
1.40 f 0.55 0.283 f 0.020 0.031 f 0.016
7.04 f 1.24 0.80 f 0.23 0.25 f 0.08
a The infiltrating cells were released by compression, differential cell counts of viable cells were performed from MGG-stained cytocentrifuged cell smears, and the actual numbers of different types of cells per a 3- X 3- X 3-mm graft were calculated on the basis of viable cell counts. Mean f SD from triplicate determinations.
After releasing the cell contents, practically no cells were retained in the matrix as described earlier (16) and verified by histological examinations of the sponges. The released cells consisted predominantly of white blood cells and macrophages ; the sticky fibroblasts died and distinguished during the releasing procedure (16). Actual numbers of the different types of released cells per a 9-mm3 sponge graft were assessed on the basis of differential counts and of viable cell numbers. As shown in Table 1, 10 times more inflammatory cells were released from an allograft than from a syngeneic graft, and the number of inflammatory cells infiltrating the control sponges was less than l/20 of that infiltrating the allografts. The blast cells and lymphocytes are the most prominent cell elements in the allograft-infiltrating population (Table 1). In average, 4 x lo6 lymphocytic cells were recovered from a single allograft as compared to only 0.06 x lo6 obtained from a single syngeneic graft. The number of monocytes and macrophages was approximately five times higher in allografts as compared to syngeneic grafts. Approximately 20% of the allograft-infiltrating cells were granulocytes. Granulocytes were also prominent, present in the cellular infiltrates of syngeneic grafts and control sponges, although in somewhat smaller quantities. Since the granulocytic response against viscous cellulose sponges is especially prominent in the mouse (and man) but nearly missing in the rat (J. Ahonen, unpublished), it is difficult to evaluate their significance in the cellular infiltrates. TABLE Separation of Viable Graft-Infiltrating Treatment
2
Cells by BSA Density Centrifugation
Dead/alive”
Cells per graft X lo6 b Allograft
Syngeneic graft
Sponge alone
No treatment
D L
1.7 f 1.0 7.1 f 2.9
0.47 f 0.2 1.2 f 0.2
0.25 f 0.2 0.6 f 0.5
After BSA-centrifugation
D L
0.2 f 0.3 6.3 f 2.9
0.0 1.1 f 0.2
0.0 0.4 i 0.2
0 D = dead; L = living cells in trypan blue staining. b Cells released per a 3- X 3- X 3-mm graft 7 days after grafting. Triplicate f SD.
determinations
164
ROBERTS
AND
TABLE
H;CYRY
3
Effect of BSA Density Centrifugation on the Cytolytic Infiltrating and Spleen Killer Cells Source of cells
Treatment
Fraction recovered
Effect of Graft-
Percentage viablea cells
Percentage specific Wr releaseb 5O:l
25:l
Graft
None BSA BSA
Interphase Bottom
60 90 4
28 59 0
25 58 0
Spleen
None BSA BSA
Interphase Bottom
80 9.5 5
20 23 0
9 13 0
a Trypan blue staining. b Transplants: DBA/2 - SMA to CBA; target cell P-815 (DBA/2), taneous release at most 12% of maximal.
assay time 10 hr. Spon-
Approximately 2040% of the cells released from an allograft or from a syngeneic graft were dead in the trypan blue uptake test (Tables 2, 3) ; the released cells were also contaminated with substantial amounts of cell debris. In the preliminary experiments (16), it had also become apparent that the dead cells and TABLE
4
Lysis of Relevant Allogeneic Target Cells by Graft-Infiltrating Cells and by Cells Recovered from Various Lymphoid Organs Type of graft
Allograftc
Days after transplantation
Percentage specific 6’Cr release” Graftb Draining lymph node
Nondraining lymph node
Spleen
Blood
Bone marrow
6
so:1 2.5:1
51 19
18 8
2 1
22 10
2 2
3 0
7
so:1 25:l
61 35
24 12
0 1
29 12
0 1
1 0
8
so:1 25:l
78 50
28 15
0 1
34 18
0 0
so:1 2.5:1
60 2.5
15 10
0
38 20
-1
so:1 25:1
1 0
0 0
-1
2 0
-1
0
2
1 0
-1 0
1 1
9 Syngeneic graft*
Effector/ target cell ratio
6 8
so:1 25:1
-1
-1
Q Effector cells from the listed lymphoid organs. Target cell P-815 of DBA/Z. 14 hr. Background release at most 140/, of maximal. b Purified over 35% BSA density centrifugation. c DBA/Z graft to CBA. * CBA graft to CBA.
-1 1
0
0 0
1
0 1
0 2
1 0
Exposure time
SPONGE
MATRIX
TABLE Specificity Graft recipient
Graft donor
of Target Source of effector cells
165
ALLOGRAFTS
5 Cell Lysis Effector/ target cell ratio
Percentage specific Vr release” P-815 (DBA/2
EL-4 (C57BL/6J
CBA
DBA/2
Graft b
so:1 25:l
68 50
7 3
CBA
C57BL/6
Graft
so:1 25:l
3 2
39 22
CBA
CBA
Graft
so:1 25:1
0 -2
-1 -1
DBA/2
CBA
Graft
5O:l 25:l
1 0
3 2
CBA
DBA/2
Spleen
so:1 25:l
28 11
4 0
CBA
C57BL/6
Spleen
so:1 25:l
3 2
41 21
CBA
CBA
Spleen
so:1 25:l
-1
so:1 25:l
-1
DBA/Z
CBA
Spleen
a Target cells P-81.5 (DBA/2) or EL-4 (C57BL/6). most 10% of maximal. b Purified over 3.5y0 BSA density centrifugation.
Exposure
0 0
time 8 hr, background
-2 -3 0 2 release at
cell debris partially inhibited cytolysis of relevant target cells. The viable cells were therefore purified by a one-step density centrifugation over 35% BSA. As seen in Table 2, the percentage of viable cells recovered in the interphase was of the order of 88-92% of the input of viable cells. The cells recovered from the interphase were essentially free of dead cells and cell debris. In vitro target cell lysis by the allograft-infiltrating cells. DBA/Z sponge grafts were transplanted to the neck of CBA mice as above, and the transplants were removed 7 days later. Spleen cells of the graft-recipient mice were equally removed, and processed for experiments. Both types of cells were centrifuged over the 35% BSA gradient and were tested for in vitro cytotoxicity to P-815 target cells of DBA/Z origin. As demonstrated in Table 3, both the graft-infiltrating cells and the spleen cells were cytotoxic to relevant allogeneic target cells. Removal of the dead cells and the cell debris increased the cytotoxicity in the graft-infiltrating population but had only a negligible effect on the cytotoxicity of immune spleen cells (already demonstrating a good cell viability). The (dead) cells recovered from the bottom of the gradient were not cytotoxic to relevant allogeneic target cells. Lysis of relevant target cells by graft-infiltrating cells and by cells deriving from various lymphoid organs. DBA/Z or CBA sponge grafts were transplanted to the neck of CBA mice as above, and the grafts were removed 6, 7, 8, and 9 days after the transplantation. The recipients were sacrificed at the time of the removal of
166
ROBERTS
AND
HiiYRY
the graft and their various lymphoid “organs” were removed at the same time. The axillary and inguinal lymph nodes were considered as “draining" lymph nodes and the mesenteric nodes as “nondraining” nodes. The cells from the various organs were tested for in vitro cytotoxicity against relevant allogeneic target cells. As demonstrated in Table 4, the cells recovered from the allograft were most efficient in performing relevant target cell lysis. The cells recovered from the draining lymph nodes and the spleen were also good, though less efficient than those recovered from the graft. No cytotoxicity could be demonstrated by cells recovered from blood, from nondraining lymph nodes, from bone marrow, or from thymus (controls). The cells recovered from the syngeneic graft were incapable of performing target cell lysis. Specificity of target cell lysis by graft-infiltrating cells. Finally, we examined whether the lysis of target cells by graft-infiltrating cells was immunologically specific. CBA mice were transplanted with CBA, DBA/2, or C57BL/6 sponge transplants. Seven days after transplantation, the graft-infiltrating cells and the spleen cells of the recipient mice were recovered and tested for cytotoxicity to relevant and irrelevant allogeneic target cells (P-815 of DBA/2 and EL-4 of C57BL/6 origin). Table 5 gives the result of one such experiment. Target lysis was immunologically specific. In any one of the tested strain combinations, highest lytic effect was always recorded against the relevant target cell, whereas irrelevant target cells were not significantly lysed. The lytic effect of the graft-infiltrating cells was again stronger than that of spleen cells, although equal target specificity was displayed by the spleen cells. DISCUSSION Very little information is currently available on the type and functions of cells infiltrating allografts during rejection. This has been partially due to the extreme difficulties met in the isolation of the graft-infiltrating cells from conventional organ allografts. Strom, Carpenter, and their co-workers have isolated infiltrating cells from human renal allografts (4) and from rat heart allografts (18). The infiltrating cells were obtained by mechanical means followed by enzymatic treatment of the tissue, and the purification of the infiltrating cells also included two gradient centrifugations over Ficoll-Isopaque. The cell yield was extremely low, and possibly is not representative, since selective cell losses are expected during the Ficoll-Isopaque centrifugations. The aim of this communication is to describe a model that enables quantitative isolation of allograft-infiltrating cells. The model is based on the use of a spongious matrix tissue into which cells of strain “A” are grown. Upon transplantation to an allogeneic strain “B” recipient, the sponge induces a vigorous alloimmune response. The cells infiltrating the sponge graft are released simply by compression and practically all the infiltrating cells are thus obtained (16). When one-step BSAcentrifugation is used as the only proliferation step for viable cells, more than 90% of the viable graft-infiltrating cells are recovered with no detectable losses of killer cells. Any enzymatic treatment required for the liberation of the allograft-infiltrating cells may harm the surface structures used for their classification (19). Equally, if the receptor for antigen expressed on the cell surface is not a product of the cell itself (11, 14), the loss of this receptor during the release procedure will also
SPONGE
MATRIX
ALLOGRAFTS
167
hamper the analysis of the function of this kind of cell. The fact that no enzymatic treatment is used in the recovery of the infiltrating cells thus guarantees that the receptor structures on the cells are intact. We have documented this in the rat system by direct visualization of the idiotype of the receptor molecule by employing FITC-conjugated anti-idiotype sera (6). The classification of immunocompetent cells is primarily based on the use of various antigenic or receptor structures on the cell surface. These methods have been tested and found reliable also in the classification of cells infiltrating a sponge matrix allograft (unpublished). The sponge matrix allograft model may primarily mimic a nonvascularized organ allograft, such as skin graft in the mouse. This conclusion may be drawn from our results where specific killer cells were demonstrated in the regional lymph nodes but not in nonrelated nodes. It is also noteworthy that at the time when specific killer cells can be demonstrated in the graft, in draining lymph nodes, and in the spleen, no killer activity is evident in the blood nor in the rest of the lymphatic system. This result coincides with that of Tilney et al. (18). In conclusion, we consider the sponge matrix allograft method a simple and and reliable method for the study of allograft-infiltrating cells in laboratory animals. ACKNOWLEDGMENTS The authors are indebted to Mrs. Hilkka Sokura for performing the differential counts. The work was financed by grants from the Sigrid Juselius Foundation and Finska LikaresUskapet, Helsinki, and by NIH-NC1 Contract No. NOl-CB-64032.
REFERENCES 1. 2. 3. 4.
Medawar, P. B., J. Anat. Medawar, P. B., J. Anat. Balch, C. M., Wilson, C. Strom, T. B., Tilney, N. 1257, 1975. 5. Simonsen, M., Buemann, Microbial.
Stand.
78, 176, 1944. 79, 157, 1945.
B., Lee, S., and Feldman, J. D., J. Ezp. Med. 138, 1584, 1973. L., Carpenter, C. B., and Busch, G. J., New Engl. J. Med. 292, A., Gammeltaft, F., Jensen, F., and Jorgensen, K., Acta Pathol.
32, 1, 1953.
6. Binz, H., Wigzell, H., and HPyry, P., Nature (London) 259, 401, 1976. Rev. 6, 52, 1971. 8. Wigzell, H., and Andersson, B., Annu. Rev. Microbial. 25, 291, 1971. 9. Hiyry, P., and Andersson, L. C., Stand. J. Immunol., in press. 10. Shortman, K., Anna. Rev. Biophys. Bioeng. 1, 93, 1972. 11. Wigzell, H., and Hiyry, P., Curr. Top. Ma’crobiol. Immunol. 67, 1, 1974. 12. Hiyry, P., Andersson, L. C., Nordling, S., and Virolainen, M., Transplant. Rev. 12, 91, 1972. 13. Cerottini, J., and Brunner, T., Adv. Immunol. 18, 67, 1974. 14. Trinchieri, G., DeMarchi, M., Mayer, W., Savi, M., and Ceppellini, R., Transplant. Proc. 5, 1631, 1973. 1.5. Evans, R., and Alexander, P., Nature (London) 228, 620, 1970. 16. Roberts, P. J., and Hsyry, P., Transplantation 21, 437, 1976. 17. Brunner, K. T., Mauel, J., Rudolf, H., and Chapuis, B., Immunology 18, 501, 1970. 18. Tilney, N. L., Strom, T. B., MacPherson, S. G., and Carpenter, C. B., Transplantation 20, 323, 1975. 19. Mijller, G. (Ed.), Transplant. Rev. 16, 1, 1973. 7. Raff, M. C., Transplalzt.
IMMUNOLOGY
26, 160-167 (1976)
Effector Mechanisms in Allograft I. Assembly of “Sponge Matrix”
Rejection
Allografts
P. J. ROBERTS AND P. H;~YRY Transjlantation Laboratory, Fourth Department of Surgery, and Third Department of Pathology, hiversity of Helsinki, SF 00290 Helsinki 29, Fixland Received May l&l976 This communication describes a model that makes it possible to quantitatively recover allograft-infiltrating cells in a functionally viable state. The model is based on the use of an inert spongious matrix tissue into which fibroblasts of strain “A” are grown. Upon transplantation to an allogeneic “B” strain host, graft-directed killer cells infiltrate the sponge. Mere physical compression of the sponge releases virtually all infiltrating cells. More than 9 0 % of the viable cells infiltrating the sponge graft are thus recovered. About 1.5% of the infiltrating cells are blasts, about 3 5 % are lymphocytes, and about 3 0 % are monocytes and macrophages. The rest are predominantly granulocytes. The allograft-infiltrating cells display an immunologically specific cytolytic response to relevant “‘Cr-labeled target cells in vitro. The infiltrating cells are much more efficient killer cells than spleen or draining lymph node cells. The allograft-infiltrating cells are thus functionally intact and are recovered without enzymatic treatment by mere mechanical means. W e therefore consider the sponge matrix model a suitable and reproducible method for studying allograftinfiltrating cells.
INTRODUCTION A prominent histological feature in an allograft undergoing acute rejection is its infiltration by lymphocytes of varying sizes (1, 2). Both T (3) and B (4) lymphocytes have been reported to infiltrate the allograft. In addition to the lymphocytes, macrophages/monocytes are also present in the cellular infiltrates, and the more necrotic areas of the graft usually display an infiltration of polymorphonuclear leukocytes (5). Approximately 6@-80% of the allograft-infiltrating cells are equipped with (idiotypic) receptors directed to the antigens of the graft (6). The significance of finding several cell classes in the graft-infiltrating cell population is unknown, as are the functions of the different cell types in the graft-destructive process. It has recently become possible to identify and fractionate the various subclasses of immunocompetent cells on the basis of their surface markers (7, S), physical (9, lo), or biological (8, 11) properties. Concomitantly, methods have been developed enabling the characterization of the functions of these cells in vitro. These studies have demonstrated that both T lymphocytes (12, 13) and non-T lymphocytes (14) and even monocytes and macrophages (15) can function as killer cells to relevant
allogeneic
target
cells in vitro. 160
Copyright 1976 by Academic Press, Inc. rights o3 reproductionin any form reserved.
All
These in vitro
results
therefore
suggest
SPONGE
MATRIX
ALLOGRAFTS
161
that several different cell-bound effector mechanisms are functioning in allograft destruction in v&o. In order to link these in vitro findings to the functions of allograft-infiltrating cells, it was necessary to quantitatively isolate the infiltrating cells in an intact, functionally viable state. Since the isolation of the infiltrating cells from ordinary skin or organ allografts was not possible without enzymatic treatment (4), we have developed a model for this purpose. The model is based on the use of an inert “spongious matrix” tissue into which fibroblasts of strain “A” are grown. The sponge is transplanted to a strain “B” allogeneic host, and removed at various times thereafter. Specifically cytotoxic cells were found to infiltrate the sponge and mere physical compression of the sponge released virtually all of the infiltrating cells. In this communication we report the assembly of the sponge matrix grafts and the use of these grafts in transplantation studies in the mouse. A preliminary report describing the histological and cytological features of the SMG has been published (16). In forthcoming papers we will describe in detail the physical properties, the surface characteristics, and the receptor specificities of the allograft-infiltrating cells, and link these characteristics to the ability of the various cell populations to perform in vitro target cell lysis. MATERIALS
AND
METHODS
Mice. The CBA/H-T6T6, DBA/Z, and C57BL/6 mouse strains were originally obtained from the Jackson Laboratory, Bar Harbor, Maine, and were bred in our colony. Sponge matrix grafts. As supporting matrix we used viscous cellulose sponge. The sponge was purchased from Kongsfors Fabrikker A/S, Oslo. Prior to use, the sponge was cut into cubical 3- X 3- X 3-mm pieces and washed 10 times in distilled water and 70% ethanol. The sponges were kept immersed in physiological saline in small glass bottles and were sterilized by autoclaving them before use. The sponges were “filled up” with allogeneic cells by placing 5 to 10 pieces of sponge for 6 to 7 days into the peritoneal cavity of the donor strain. During this time the sponge was uniformly infiltrated mainly by fibroblastoid cells plus some lymphocytes and macrophages of the donor (16). Final allotransplantations were performed subcutaneously to the neck of the recipient via a hind-region incision. Usually two pieces of sponge were transplanted simultaneously on both sides of the midline under strictly aseptical conditions. In order to keep several transplanted animals in the same cage, it was necessary to remove hair only at the site of the incision; removal of hair at the site of the transplant resulted in biting of the transplant sites, thus causing infections of the transplants. In all transplantations the animals were under intraperitoneal chloralhydrate (360 mg/kg) anesthesia. During the first week after transplantation, 4 g/liter of oxytetracycline was added to the drinking water. Six to eight days after transplantation the recipients were sacrificed. The sponges, draining (axillary and inguinal) and nondraining (mesenteric) lymph nodes, and the spleens of the recipients were removed. The recipient blood was obtained from the venous eye plexus, and in some cases the thymus and the cell contents of the humeral and femural bone cavities were also harvested. The cell contents of the sponge were recovered to tissue culture medium by compression. Earlier experience
162
ROBERTS
AND
HiYRY
has shown that almost all sponge-infiltrating cells were thus released (16). The cells of the various lymphoid organs were processed for experimentation as described (12). Prior to the experiments the red cells were lysed from all populations with 0.83% ammonium chloride (12). The cells recovered from the various lymphoid organs demonstrated a good (> 95%) cell viability in trypan blue staining; the cells recovered from the sponge were usually contaminated with cell debris and with 35-40s dead cells. In order to remove the dead cells and the cell debris, the sponge-infiltrating cells were centrifuged (5000 rpm/lS min) over a 35% bovine serum albumin (BSA, Sigma Chemical Company, St. Louis, MO.) one-step density gradient. The cell population collected at the interface contained > 90% of the viable cells and no debris (Table 2). Diflerentiul counts. The differential counts were performed from May-Gruenwald-Giemsa (MGG) stained cytocentrifuged cell smears. Cell-mediated cytotoxicity. The method of Brunner et al. (17) was modified for microculture conditions. The target cells were the P-815-X2 mastocytoma line of DBA/2 (obtained from Dr. K. T. Brunner, Swiss Institute for Experimental Cancer Research, Lausanne) and the EL-4 lymphoma line of C57BL/6 (obtained from Dr. Leif C. Andersson, Department of Immunology, University of Uppsala). The target cells were carried in alternate in z&o-in vitro passages in peritoneal cavity and in suspension culture. For cytotoxicity test the target cells were labeled with 51Cr (sodium 51chromate, The Radiochemical Center, Amersham) in conditions described (17). Ten-thousand target cells were plated in 100 ~1 of 5% fetal calf serum containing Eagle’s Minimal Essential Medium per well in round-bottom microtitration plates (Cooks Microtiter System, U.K.). The effector cells were added to the target cells in the same volume by employing at least two effector/ target cell ratios. After a short centrifugation (809 for 3 min), the plates were incubated in a humidified atmosphere of 5% CO, in air for 6-18 hr at 37”C, and the amount of Yr released to the culture medium was used to indicate the rate of target cell damage. The “specific 5*Cr release” was calculated according to the formula (17) : y. Specific release = 100 x
Release in presence of effector cells - Spontaneous Maximal release - Spontaneous release
The spontaneous release never exceeded 10% of the maximum release in these conditions,
(Triton
release ’ X-100)
RESULTS Rediscovery of graft-infiltrating cells. In the first experiment we quantitated the number of cells infiltrating an allogeneic and a syngeneic sponge matrix transplant. Cubical pieces of sponge, sized 3 x 3 x 3 mm, were inserted into the peritoneal cavity of DBA/2 or CBA mice. Seven days later, the sponges were removed and transplanted subcutaneously into the neck of CBA recipients. Each recipient received two transplants, one on either side of the neck. Seven days after the transplantation, the cell contents of the sponges were released by compression. Cytocentrifuged cell smears were made for differential counts, and the rest of the cells were used for further purification in one-step BSA density centrifugation.
SPONGE
MATRIX
163
ALLOGRAFTS
TABLE
1
Cellular Infiltrates in Allogeneic and Syngeneic Sponge Matrix Grafts and in Control Sponges 7 Days after Subcutaneous Transplantation to the Neck Cells per graft X lo6 5
Type of graft
Allogeneic graft Syngeneic graft Sponge along
Blasts
Lymphocytes
Granulocytes
Monocytes/ macrophages
Total
0.94 f 0.41 0.0 0.0
3.12 f 1.08 0.056 f 0.019 0.068 f 0.028
1.59 f 0.36 0.461 f 0.248 0.151 f 0.040
1.40 f 0.55 0.283 f 0.020 0.031 f 0.016
7.04 f 1.24 0.80 f 0.23 0.25 f 0.08
a The infiltrating cells were released by compression, differential cell counts of viable cells were performed from MGG-stained cytocentrifuged cell smears, and the actual numbers of different types of cells per a 3- X 3- X 3-mm graft were calculated on the basis of viable cell counts. Mean f SD from triplicate determinations.
After releasing the cell contents, practically no cells were retained in the matrix as described earlier (16) and verified by histological examinations of the sponges. The released cells consisted predominantly of white blood cells and macrophages ; the sticky fibroblasts died and distinguished during the releasing procedure (16). Actual numbers of the different types of released cells per a 9-mm3 sponge graft were assessed on the basis of differential counts and of viable cell numbers. As shown in Table 1, 10 times more inflammatory cells were released from an allograft than from a syngeneic graft, and the number of inflammatory cells infiltrating the control sponges was less than l/20 of that infiltrating the allografts. The blast cells and lymphocytes are the most prominent cell elements in the allograft-infiltrating population (Table 1). In average, 4 x lo6 lymphocytic cells were recovered from a single allograft as compared to only 0.06 x lo6 obtained from a single syngeneic graft. The number of monocytes and macrophages was approximately five times higher in allografts as compared to syngeneic grafts. Approximately 20% of the allograft-infiltrating cells were granulocytes. Granulocytes were also prominent, present in the cellular infiltrates of syngeneic grafts and control sponges, although in somewhat smaller quantities. Since the granulocytic response against viscous cellulose sponges is especially prominent in the mouse (and man) but nearly missing in the rat (J. Ahonen, unpublished), it is difficult to evaluate their significance in the cellular infiltrates. TABLE Separation of Viable Graft-Infiltrating Treatment
2
Cells by BSA Density Centrifugation
Dead/alive”
Cells per graft X lo6 b Allograft
Syngeneic graft
Sponge alone
No treatment
D L
1.7 f 1.0 7.1 f 2.9
0.47 f 0.2 1.2 f 0.2
0.25 f 0.2 0.6 f 0.5
After BSA-centrifugation
D L
0.2 f 0.3 6.3 f 2.9
0.0 1.1 f 0.2
0.0 0.4 i 0.2
0 D = dead; L = living cells in trypan blue staining. b Cells released per a 3- X 3- X 3-mm graft 7 days after grafting. Triplicate f SD.
determinations
164
ROBERTS
AND
TABLE
H;CYRY
3
Effect of BSA Density Centrifugation on the Cytolytic Infiltrating and Spleen Killer Cells Source of cells
Treatment
Fraction recovered
Effect of Graft-
Percentage viablea cells
Percentage specific Wr releaseb 5O:l
25:l
Graft
None BSA BSA
Interphase Bottom
60 90 4
28 59 0
25 58 0
Spleen
None BSA BSA
Interphase Bottom
80 9.5 5
20 23 0
9 13 0
a Trypan blue staining. b Transplants: DBA/2 - SMA to CBA; target cell P-815 (DBA/2), taneous release at most 12% of maximal.
assay time 10 hr. Spon-
Approximately 2040% of the cells released from an allograft or from a syngeneic graft were dead in the trypan blue uptake test (Tables 2, 3) ; the released cells were also contaminated with substantial amounts of cell debris. In the preliminary experiments (16), it had also become apparent that the dead cells and TABLE
4
Lysis of Relevant Allogeneic Target Cells by Graft-Infiltrating Cells and by Cells Recovered from Various Lymphoid Organs Type of graft
Allograftc
Days after transplantation
Percentage specific 6’Cr release” Graftb Draining lymph node
Nondraining lymph node
Spleen
Blood
Bone marrow
6
so:1 2.5:1
51 19
18 8
2 1
22 10
2 2
3 0
7
so:1 25:l
61 35
24 12
0 1
29 12
0 1
1 0
8
so:1 25:l
78 50
28 15
0 1
34 18
0 0
so:1 2.5:1
60 2.5
15 10
0
38 20
-1
so:1 25:1
1 0
0 0
-1
2 0
-1
0
2
1 0
-1 0
1 1
9 Syngeneic graft*
Effector/ target cell ratio
6 8
so:1 25:1
-1
-1
Q Effector cells from the listed lymphoid organs. Target cell P-815 of DBA/Z. 14 hr. Background release at most 140/, of maximal. b Purified over 35% BSA density centrifugation. c DBA/Z graft to CBA. * CBA graft to CBA.
-1 1
0
0 0
1
0 1
0 2
1 0
Exposure time
SPONGE
MATRIX
TABLE Specificity Graft recipient
Graft donor
of Target Source of effector cells
165
ALLOGRAFTS
5 Cell Lysis Effector/ target cell ratio
Percentage specific Vr release” P-815 (DBA/2
EL-4 (C57BL/6J
CBA
DBA/2
Graft b
so:1 25:l
68 50
7 3
CBA
C57BL/6
Graft
so:1 25:l
3 2
39 22
CBA
CBA
Graft
so:1 25:1
0 -2
-1 -1
DBA/2
CBA
Graft
5O:l 25:l
1 0
3 2
CBA
DBA/2
Spleen
so:1 25:l
28 11
4 0
CBA
C57BL/6
Spleen
so:1 25:l
3 2
41 21
CBA
CBA
Spleen
so:1 25:l
-1
so:1 25:l
-1
DBA/Z
CBA
Spleen
a Target cells P-81.5 (DBA/2) or EL-4 (C57BL/6). most 10% of maximal. b Purified over 3.5y0 BSA density centrifugation.
Exposure
0 0
time 8 hr, background
-2 -3 0 2 release at
cell debris partially inhibited cytolysis of relevant target cells. The viable cells were therefore purified by a one-step density centrifugation over 35% BSA. As seen in Table 2, the percentage of viable cells recovered in the interphase was of the order of 88-92% of the input of viable cells. The cells recovered from the interphase were essentially free of dead cells and cell debris. In vitro target cell lysis by the allograft-infiltrating cells. DBA/Z sponge grafts were transplanted to the neck of CBA mice as above, and the transplants were removed 7 days later. Spleen cells of the graft-recipient mice were equally removed, and processed for experiments. Both types of cells were centrifuged over the 35% BSA gradient and were tested for in vitro cytotoxicity to P-815 target cells of DBA/Z origin. As demonstrated in Table 3, both the graft-infiltrating cells and the spleen cells were cytotoxic to relevant allogeneic target cells. Removal of the dead cells and the cell debris increased the cytotoxicity in the graft-infiltrating population but had only a negligible effect on the cytotoxicity of immune spleen cells (already demonstrating a good cell viability). The (dead) cells recovered from the bottom of the gradient were not cytotoxic to relevant allogeneic target cells. Lysis of relevant target cells by graft-infiltrating cells and by cells deriving from various lymphoid organs. DBA/Z or CBA sponge grafts were transplanted to the neck of CBA mice as above, and the grafts were removed 6, 7, 8, and 9 days after the transplantation. The recipients were sacrificed at the time of the removal of
166
ROBERTS
AND
HiiYRY
the graft and their various lymphoid “organs” were removed at the same time. The axillary and inguinal lymph nodes were considered as “draining" lymph nodes and the mesenteric nodes as “nondraining” nodes. The cells from the various organs were tested for in vitro cytotoxicity against relevant allogeneic target cells. As demonstrated in Table 4, the cells recovered from the allograft were most efficient in performing relevant target cell lysis. The cells recovered from the draining lymph nodes and the spleen were also good, though less efficient than those recovered from the graft. No cytotoxicity could be demonstrated by cells recovered from blood, from nondraining lymph nodes, from bone marrow, or from thymus (controls). The cells recovered from the syngeneic graft were incapable of performing target cell lysis. Specificity of target cell lysis by graft-infiltrating cells. Finally, we examined whether the lysis of target cells by graft-infiltrating cells was immunologically specific. CBA mice were transplanted with CBA, DBA/2, or C57BL/6 sponge transplants. Seven days after transplantation, the graft-infiltrating cells and the spleen cells of the recipient mice were recovered and tested for cytotoxicity to relevant and irrelevant allogeneic target cells (P-815 of DBA/2 and EL-4 of C57BL/6 origin). Table 5 gives the result of one such experiment. Target lysis was immunologically specific. In any one of the tested strain combinations, highest lytic effect was always recorded against the relevant target cell, whereas irrelevant target cells were not significantly lysed. The lytic effect of the graft-infiltrating cells was again stronger than that of spleen cells, although equal target specificity was displayed by the spleen cells. DISCUSSION Very little information is currently available on the type and functions of cells infiltrating allografts during rejection. This has been partially due to the extreme difficulties met in the isolation of the graft-infiltrating cells from conventional organ allografts. Strom, Carpenter, and their co-workers have isolated infiltrating cells from human renal allografts (4) and from rat heart allografts (18). The infiltrating cells were obtained by mechanical means followed by enzymatic treatment of the tissue, and the purification of the infiltrating cells also included two gradient centrifugations over Ficoll-Isopaque. The cell yield was extremely low, and possibly is not representative, since selective cell losses are expected during the Ficoll-Isopaque centrifugations. The aim of this communication is to describe a model that enables quantitative isolation of allograft-infiltrating cells. The model is based on the use of a spongious matrix tissue into which cells of strain “A” are grown. Upon transplantation to an allogeneic strain “B” recipient, the sponge induces a vigorous alloimmune response. The cells infiltrating the sponge graft are released simply by compression and practically all the infiltrating cells are thus obtained (16). When one-step BSAcentrifugation is used as the only proliferation step for viable cells, more than 90% of the viable graft-infiltrating cells are recovered with no detectable losses of killer cells. Any enzymatic treatment required for the liberation of the allograft-infiltrating cells may harm the surface structures used for their classification (19). Equally, if the receptor for antigen expressed on the cell surface is not a product of the cell itself (11, 14), the loss of this receptor during the release procedure will also
SPONGE
MATRIX
ALLOGRAFTS
167
hamper the analysis of the function of this kind of cell. The fact that no enzymatic treatment is used in the recovery of the infiltrating cells thus guarantees that the receptor structures on the cells are intact. We have documented this in the rat system by direct visualization of the idiotype of the receptor molecule by employing FITC-conjugated anti-idiotype sera (6). The classification of immunocompetent cells is primarily based on the use of various antigenic or receptor structures on the cell surface. These methods have been tested and found reliable also in the classification of cells infiltrating a sponge matrix allograft (unpublished). The sponge matrix allograft model may primarily mimic a nonvascularized organ allograft, such as skin graft in the mouse. This conclusion may be drawn from our results where specific killer cells were demonstrated in the regional lymph nodes but not in nonrelated nodes. It is also noteworthy that at the time when specific killer cells can be demonstrated in the graft, in draining lymph nodes, and in the spleen, no killer activity is evident in the blood nor in the rest of the lymphatic system. This result coincides with that of Tilney et al. (18). In conclusion, we consider the sponge matrix allograft method a simple and and reliable method for the study of allograft-infiltrating cells in laboratory animals. ACKNOWLEDGMENTS The authors are indebted to Mrs. Hilkka Sokura for performing the differential counts. The work was financed by grants from the Sigrid Juselius Foundation and Finska LikaresUskapet, Helsinki, and by NIH-NC1 Contract No. NOl-CB-64032.
REFERENCES 1. 2. 3. 4.
Medawar, P. B., J. Anat. Medawar, P. B., J. Anat. Balch, C. M., Wilson, C. Strom, T. B., Tilney, N. 1257, 1975. 5. Simonsen, M., Buemann, Microbial.
Stand.
78, 176, 1944. 79, 157, 1945.
B., Lee, S., and Feldman, J. D., J. Ezp. Med. 138, 1584, 1973. L., Carpenter, C. B., and Busch, G. J., New Engl. J. Med. 292, A., Gammeltaft, F., Jensen, F., and Jorgensen, K., Acta Pathol.
32, 1, 1953.
6. Binz, H., Wigzell, H., and HPyry, P., Nature (London) 259, 401, 1976. Rev. 6, 52, 1971. 8. Wigzell, H., and Andersson, B., Annu. Rev. Microbial. 25, 291, 1971. 9. Hiyry, P., and Andersson, L. C., Stand. J. Immunol., in press. 10. Shortman, K., Anna. Rev. Biophys. Bioeng. 1, 93, 1972. 11. Wigzell, H., and Hiyry, P., Curr. Top. Ma’crobiol. Immunol. 67, 1, 1974. 12. Hiyry, P., Andersson, L. C., Nordling, S., and Virolainen, M., Transplant. Rev. 12, 91, 1972. 13. Cerottini, J., and Brunner, T., Adv. Immunol. 18, 67, 1974. 14. Trinchieri, G., DeMarchi, M., Mayer, W., Savi, M., and Ceppellini, R., Transplant. Proc. 5, 1631, 1973. 1.5. Evans, R., and Alexander, P., Nature (London) 228, 620, 1970. 16. Roberts, P. J., and Hsyry, P., Transplantation 21, 437, 1976. 17. Brunner, K. T., Mauel, J., Rudolf, H., and Chapuis, B., Immunology 18, 501, 1970. 18. Tilney, N. L., Strom, T. B., MacPherson, S. G., and Carpenter, C. B., Transplantation 20, 323, 1975. 19. Mijller, G. (Ed.), Transplant. Rev. 16, 1, 1973. 7. Raff, M. C., Transplalzt.