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In situ effector mechanisms in rat kidney allograft rejection
CELLULAR
IMMUNOLOGY
In Situ Effector
&,327-336
(1979)
Mechanisms
in Rat Kidney Allograft
Rejection
II. Heterogeneity of the Effector Cells in the Inflammatory vs that in the Spleen of the Recipient Rat EEVA Transplantation
VON WILLEBRAND, Laboratory,
Fourth SF 00290 Received
ANU
SOOTS, AND P. H~YRY
Department of Surgery, Helsinki 29, Finland November
Infiltrate
University
of Helsinki,
27, 1978
We have isolated the infiltrating inflammatory cells from rejecting rat kidney allografts via collagenase-DNase dispersion and lg velocity sedimentation. A representative cell sample, devoid of blood contamination and subclass-specific cell losses, is thus obtained in a functionally viable state. The infiltrating inflammatory cells display an immunologically specific in vitro cytotoxicity to donor-strain lymphoblasts. The peak cytotoxicity takes place in the graft before it takes place in the lymphatic system of the recipient rat. The aUog&t-infiltrating cytotoxic cells display a far wider distribution profile in lg velocity than do the recipient spleen killer cells. The donor-strain lymphoblasts are highly sensitive to the lytic effect of both allograftinfiltrating and spleen killer cells, but the transplant parenchymal cells, consisting mainly of tubular and glomerular cells, are relatively insensitive. The findings suggest heterogeneity of the effector cells in the allograft infiltrate vs that in the spleen, and indicate that different structures of the target organ are differently sensitive to cellular cytotoxicity.
INTRODUCTION In a previous communication (1) we described how rat kidney allografts transplanted across the major AgB barrier may be disaggregated with collagenase and DNase. We also analyzed the distribution and lymphocyte subclasses of the allograft-infiltrating inflammatory cells. The inflammatory infiltrate consisted (in order of magnitude) of mononuclear phagocytes (i.e., monocytes and macrophages), lymphocytes, (T) lymphoblasts, (B) plasmablasts, plasma cells, and relatively few granulocytes. Most of the infiltrating lymphocytes carried neither the T-specific Pta.1.A surface antigen nor expressed the surface Ig, but had a low electrophoretic mobility of non-T cells. In this communication we discuss how we have separated the allograft-infiltrating inflammatory cells from the allograft parenchymal cells via lg velocity sedimentation, and tested the ability of the inflammatory cells and immune spleen cells to kill donor-derived lymphocytic target cells and transplant parenchymal cells in vitro. The results demonstrate a heterogeneity of effector cells in the allograft infiltrate vs that in the spleen, and suggest that the transplant parenchymal cells (consisting in this experiment of mainly tubular cells and glomerular podocytes) are relatively insensitive to short-term cellular cytotoxicity in vitro. 327
0008-8749/79/100327-10$02.00/O Copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
328
VON
WILLEBRAND,
MATERIALS
SOOTS,
AND
HiiYRY
AND METHODS
Rats. The nuclei for the inbred strains DA (AgB-4), A0 (AgB-2), and HO (AgB-5) were obtained from Professor J. L. Gowans, Dunn School of Pathology, Oxford, England. The rats were bred in our own colony. Transplantations. The micromethods described by Lee (2) for rat kidney transplantation were used. The organs were procured and perfused with physiological saline containing 40 II-l/ml heparin (Medica Pharmaceuticals, Helsinki, no preservative added). End to side anastomoses were performed on the recipients’ aorta and vena cava, and the donor ureter was anastomized to the recipient bladder with the aid of a bladder flap. Silk, 9-O to 10-0, was used throughout. Dispersion of the allogrufts. The transplant was throughly perfused, minced with a scalpel to pieces of approximately 0.5 x 0.5 mm in diameter, and immersed into Hepes-buffered RPM1 1640 (Gibco Bio-Cult, Glasgow, Scotland) medium containing collagenase (2.0 mg/ml, Worthington Biochemical Co., Freehold, N.J.) plus DNase (0.2 mg/ml, Sigma Chemical Co., St. Louis, MO.). The material was incubated at 37°C with a magnetic stirrer, until most of the tissue was dissolved. The few remaining clumps were removed by sedimentation and the resulting single-cell suspension was washed twice with RPM1 medium. Preparation of recipient lymphoid cells. Blood mononuclear leukocytes were recovered via density centrifugation of 1: 10 diluted blood over Ficoll (Pharmacia Fine Chemicals, Uppsala, Sweden)-Isopaque (Nyregaard & Co., Oslo, Norway), density 1.081 g/ml. Spleen and lymph node leukocytes were obtained by teasing apart the respective organs, by removing of the clumps via sedimentation, and by lysing the residual red cells with 0.83% ammonium chloride. Separation ofthe infiltrating cellsfrom ullogruftpurenchymul sedimentation. The Ig velocity method fractionates particles,
cells by lg velocity
such as cells, OR the basis of their size. Our application of this method has been described elsewhere (3). In short, the single-cell suspension of allograft suspension was loaded on a 0.75 to 1.5% BSA-PBS gradient and sedimented for 2-4 hr. Ten to 15 fractions of lo-15 ml were drained from the gradient. Cell smears were made from each fraction with a cytocentrifuge, and differential counts were performed after MayGriinwald-Giemsa (MGG) staining. The differential counts permitted the identification of the sites for proper “cuts” in the profile (see Fig. 1). In the fastestsedimenting “parenchymal cell” fractions approximately 90-95% of cells were morphologically kidney parenchymal (i.e., glomerular, tubular, endothelial, etc.) cells, and only approximately 5% were infiltrating cells (mainly macrophages). In the slowest-sedimenting “infiltrating cell” fractions up to 95% of all cells consisted of inflammatory cells of various types (Fig. 2). Target cells and cytotoxicity assay. Concanavalin A blast cells were cultured from donor- or recipient-strain spleen by methods described elsewhere (4). The transplant parenchymul cells were separated from the graft-infiltrating cells via the lg velocity sedimentation method. Prior to the assay all target cell populations were washed, labeled with sodium 51Cr chromate (The Radiochemical Center, Amersham, Great Britain) in conditions described (4), washed three times, and diluted into RPM1 1640 medium containing 5% fetal calf serum. The cytotoxicity assays were performed as described previously (4) in 200 ~1 vol at 37” in a humidified atmosphere of 5% CO, in air by employing 8 hr exposure time and triplicate
EFFECTOR
MECHANISMS
IN RAT KIDNEY
2
4
6
FRACTION
ALLOGRAFT
8
REJECTION
II
329
10
NUMBER
FIG. 1. Velocity sedimentation (Ig) profile of collagenase-DNase dispersate of a rat kidney allograft (a). Section (b) gives the relative distribution of allograf? parenchymal cells (open triangles) and host inflammatory infiltrate (broken line) within the profile. When the profile is cut between fractions 5 and 6, more than 95% of the inflammatory infiltrate is recovered in the fractions on the right, whereas more than 95% of the allograft parenchymal cells is recovered in the fractions on the left. The parenchymal cell fractions (shaded area) can be further purified from the contaminating inflammatory monocytes and macrophages with adherence to plastic.
determinations. The triplicate values fell consistently within 5% of the mean. The specific cytotoxicity was expressed according to the formula: Sp Wr
release =
release with effector cells - spontaneous release x 100. maximal release - spontaneous release
Regardless of target cell type, the spontaneous 14% of the maximal release (Triton X-100).
release was consistently
below
RESULTS Separation Cells
of the Allograft-Injiltrating
Cells from the Transplant
Parenchymal
A0 allografts were transplanted into DA rats, removed 6 days later, and disaggregated with collagenase and DNase. The cells were loaded on a linear OS1.5% bovine serum albumin/PBS gradient. After 3 hr of separation at lg, the gradient was divided into 12 or more fractions. Cytological cell smears were made from each fraction with a cytocentrifuge and stained with MGG. One such fractionation experiment is illustrated in Fig. 1. The kidney parenchymal, i.e., glomerular and tubular cells, on the average larger than the infiltrating cells and usually in small clumps, sedimented faster than did the infiltrating cells. By performing a proper “cut” in the profile, determined by differential analysis of cytocentrifuged preparations and located in Fig. 1 between fractions 5 and 6,
330
VON WILLEBRAND,
SOOTS, AND HAYRY
FIG. 2. (A) A microphotograph of the allograft inflammatory infiltrate recovered by lg velocity sedimentation displaying blast cells, monocytes, macrophages, and lymphocytes. (B) Immune spleen cells. (C) Transplant parenchymal cehs obtained via Ig velocity sedimentation and adherence of the contaminating host cells to plastic. Note that the cells consist nearly exclusively of large tubular cells, small podocytes, and very few endothelial cells.
a population consisting of more than 95% infiltrating cells was obtained. Furthermore, due to differences in cell size, the infiltrating cells were also fractionated into distinct, though overlapping cell populations. Most of the monocytes and macrophages were recovered in the immediate fractions after the parenchymal cells, the bulk of the blast cells were obtained in the intermediary fractions, and small lymphocytes, were obtained in the last fractions of the profiie. The transplant parenchymal cell fraction was contaminated with a sizable pro-
EFFECTOR
MECHANISMS
IN RAT KIDNEY
ALLOGRAFT
REJECTION
II
331
portion of host cells, mainly monocytes and macrophages. Short-term adherence to plastic in vitro, however, depleted the parenchymal cell fraction from most of the host cells (and endothelial cells of the transplant), and the resulting population consisted of more than 80% transplant parenchymal cells, mainly tubular cells and glomerular podocytes (Fig. 2). Cytotoxic Activity of Allagraft-lnjiltrating Injlammatory Recovered from the Recipient Lymphoid System
Cells, and Effector Cells
A0 recipients were transplanted with DA kidney allografts and the grafts were recovered on Day 6 after the transplantation. The allograft-infiltrating cells were released with collagenase plus DNase, and purified via Ig velocity sedimentation. The infiltrating cells, the recipient spleen, blood, and (irrelevant) lymph node cells, were tested for in vitro cytotoxicity to donor- or recipient-strain-derived Con A blast cells in the 6-hr short-term 51Cr release assay. The result is displayed in Fig. 3. Both the allograft-infiltrating cells and the white cells recovered from the recipient lymphoid organs displayed a distinct dose-dependent cytotoxicity to the relevant target cell in vitro. The recipient-strain blasts (not shown) were not lysed. The highest cytotoxic activity was seen in the allograft infiltrate, followed by spleen, blood, and the (irrelevant) lymph node cell populations. Another experiment was designed to investigate the sequence of appearance of cytotoxic cells into the allograft and into the recipient lymphoid system. We also wanted to investigate the specificity of the lysis at different time periods after the transplantation, and the relative sensitivity of donor-derived lymphocytic target cells vs that of the transplant parenchymal cells to the cytotoxic effect by the in situ effector cells, and by the effector cells recovered from the central lymphatic system of the recipient rat. A0 rats were transplanted with DA kidney allografts, the grafts were removed on Days 3-16 after the transplantation, and the recipients were exterminated. The different effector cell populations were recovered and tested for in vitro cytotoxicity to donor-strain Con A lymphoblasts, to recipient-strain lymphoblasts,
104
50:)
1OO:l
EFFECTOR
5O:l TARGET
x)0:1
RATIO
FIG. 3. Cytotoxic activity of allograft-infiltrating cells (closed circles), spleen cells (closed squares), irrelevant lymph node cells (open circles), and blood leukocytes (closed triangles) to donor-strain Con A lymphoblasts on Day 6 after transplantation of a DA kidney allograft to an A0 recipient.
332
VON WILLEBRAND,
SOOTS, AND HAYRY
to the transplant parenchymal cells recovered by the lg velocity sedimentation, and to the (control) graft-infiltrating cells themselves. The results of the short-term 6-hr 51Cr release assay are displayed in Fig. 4. The peak cytotoxic activity was seen in the graft already on Day 6 after the transplantation, whereafter it declined. The peak cytotoxic activities in the central lymphatic organs of the recipient rat, in the spleen, blood and lymph nodes, were observed later, at the time when the cytotoxic activity in the graft had declined. The lytic effect was specific, as donor-strain Con A lymphoblasts but not recipient-strain lymphoblasts were lysed by any one type of effector cell. In contrast to donor-derived lymphocytic target cells, the transplant parenchymal cells obtained by lg velocity sedimentation, consisting of more than 80% kidney tubular and glomerular cells, were highly insensitive to lysis. Velocity Sedimentation Distribution of Allograft-Infiltrating Cells
and Spleen Effector
A0 recipients were transplanted with DA kidney allografts, and the transplants were removed 6 days later. The allografts were dispersed with collagenase plus DNase, and the recipient spleen cells were treated with the same enzymes under
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DAYSAFTERTRANSPLANTATIGN FIG. 4. Time-course profile of the in virro cytotoxicity of allograft-infiltrating inflammatory cells and the recipient spleen, blood, and (nonregional) lymph node cells to donor-strain (relevant) and recipientstrain (irrelevant) Con A lymphoblasts, to the 1s isolated transplant parenchymal cells, and to (control) graft-infiltrating inflammatory cells themselves.
EFFECTOR
MECHANISMS
IN RAT
KIDNEY
ALLOGRAFT
REJECTION
II
333
similar conditions. Both cell populations were fractionated in Ig velocity sedimentation. Differential cell counts were performed from each fraction, and the cytotoxic activity of the fractionated cells was tested, fraction by fraction, to donor-strain Con A-induced lymphoblastoid target cells. As the parenchymal cells were rather insensitive to short-term cellular cytotoxicity, we believed that they would not interfere with the assay, but also fractions containing parenchymal cells (l-3) were included in the test. One of three experiments with similar results is displayed in Figs. 5 and 6. As seen in sections (b) and (c) of Fig. 5, the lg velocity method rather efficiently separated lymphocytes, blast cells, and the mononuclear phagocytes, monocytes, and macrophages. A considerable overlapping between the adjacent fractions was, however, evident. When the various fractions were tested for in vitro cytotoxicity to relevant blast target cells (Fig. 6), all cytotoxicity in the spleen was present in two or at most three fractions only. In contrast, in the allograft infiltrate high cytotoxic activities, equal to or higher than those in the input population, were observed in at least five or six different fractions. DISCUSSION We have previously demonstrated that the collagenase-DNase method does not affect the subclass-specific surface markers of lymphoid cells (5). Neither does the treatment invalidate such functional responses of lymphoid or monocytic GRAFT
SPLEEN
248810
2 FRACTION
4
a
a to
NUMBER
FIG. 5. Velocity sedimentation (lg) profiles of the allograft dispersate and the recipient spleen. (a) Total cell distribution profile. (b) Distribution of the inflammatory cells (broken line), transplant parenchymal cells (open triangles), and granulocytes (X) within the profile. (c) Distribution of mononuclear phagocytes (broken line), macrophages (closed squares), and monocytes (open squares). (d) Distribution of lymphocytes (closed dots), plasmablasts and plasma cells (open circles), lymphoblasts (closed circles), and lymphocytic cells in general (broken line).
334
VON WILLEBRAND,
SOOTS, AND HiiYRY
SPLEEN
GRAFT *’
t
Ba
I\
I
Cb
2
4
6
8
10
FRACTION
2
4-6
8
10
NUMBER
FIG. 6. Cytotoxic activity of allograft-infiltrating cells and recipient spleen cells to donor-derived Con A blast cells. The cytotoxicities in the respective input populations are given on the right and left sides of the graph.
cells as, e.g., conduction of lysis of syngeneic tumor cells in vitro in rodents (6) or in man (7), proliferation in vitro in response to mitogenic stimuli (unpublished), or conduction of the so-called “natural killer” activity to an appropriate target cell (unpublished). As it has also been documented that the inflammatory cells released after collagenase-DNase dispersion from a well-perfused rat allograft are devoid of blood contamination (I), and as the differential distribution of the released inflammatory cells is identical to imprint preparations of the very same allografts (1)) we conclude that this enzymatic method provides a representative sample of the allograft-infiltrating cells in a functionally viable state. The lg sedimentation method employed to fractionate the transplant parenchyma1 cells from the allograft-infiltrating cells gives an essentially pure population of infiltrating cells with a minimal (approximately 5%) cell loss. As seen in the profile of Fig. 5, the lost population consists predominantly of large monocytes and macrophages of the highest sedimentation mobility. Two observations have emerged from this study which are potentially important in understanding the modus operandi of allograft rejection. First, the distribution profile of the graft-infiltrating killer cells in lg velocity sedimentation is wider than the profile of spleen killer cells, and, second, different donor-derived target structures display unequal sensitivity to cell-mediated cytotoxicity in short-term 6-hr in vitro cytotoxicity assay.
EFFECTOR
MECHANISMS
IN RAT
KIDNEY
ALLOGRAFT
REJECTION
II
335
The Ig velocity sedimentation method separates also the various cell populations of inflammatory cells into distinct though overlapping fractions (6). Andersson (6) has previously demonstrated that the allograft-directed cytotoxic cells in the recipients’ spleen are shortly after immunization exclusively T cells of relatively high sedimentation velocity. The results of our analysis coincide with the earlier observations. On Day 6 after kidney transplantation, all cytotoxic activity in the recipient spleen was recovered in two fractions only. As removal of monocytes strongly increases the spleen cell cytotoxicity (unpublished), the result favors that T blasts and large lymphocytes are responsible for the cytotoxicity in the spleen. In the graft, on the other hand, the cytotoxic profile was far wider and covered (over the input values) at least six fractions over the profile. The fastest-sedimenting fractions with distinct cytotoxic activity contained predominantly inflammatory monocytes and macrophages, and the intermediary fractions with distinct cytotoxicity were dominated by blast cells, while some of the slowest-sedimenting distinctly cytotoxic fractions contained nearly exclusively small lymphocytes only. Previous evidence has indicated (9) that removal of macrophages and monocytes from the inflammatory infiltrate does not increase the cytotoxic activity. We therefore conclude that, as with sponge matrix allografts (9), several cell types within the inflammatory infiltrate display cytotoxic activity toward donor-derived target cells in vitro. Earlier experience with sponge matrix allografts (10) has indicated that both phagocytic and nonphagocytic allograft-infiltrating inflammatory cells carry idiotypic receptors directed to the major locus antigens of the transplant donor. As only T cells are able to function as killer cells by virtue of an antigen-specific receptor synthetized by the cell itself (1 l), the non-T lymphocytic and phagocytic cells displaying specific lytic activity in the inflammatory infiltrate are most likely armed from other sources. Some of these arming molecules may be T cell derived (12). Also, antibody may render the primarily noncommitted cells specifically cytotoxic (13). Although the amount of (IgG) alloantibody is extremely low in the recipient serum 6 days after transplantation (14), the in situ plasmablasts and antibody-secreting plasma cells, demonstrated to reside in the inflammatory infiltrate (1), may provide the minute amounts of antibody necessary for the arming of the primarily noncommitted killer cells. Different target cells displayed large differences in their sensitivity to cellmediated cytotoxicity in the short-term in vitro cytotoxicity assay. Thus, e.g., donor-derived blast cells were highly sensitive targets. These types of cells are nearly exclusively used in the cytotoxicity experiments intended to document the allograft rejection in vitro. However, allograft parenchymal cells purified from the rejecting allograft via lg velocity sedimentation (followed by adherence of the contaminating mononuclear phagocytes to plastic), were nearly insensitive in the assay. In our experiment these cells consisted essentially of tubular cells and glomerular podocytes only. Although it is improper to make far-reaching conclusions from this observation only, the result seems to indicate that different organelles of the transplant are unequally sensitive to the immune attack, and/or are destroyed via different effector mechanisms. This suggestion is in accordance with a recent finding by Parthenais (unpublished) which demonstrates that muscle cells of rejecting heart allografts are destroyed more readily by ADCG-type mechanisms, whereas the heart endothelial cells are destroyed by cytotoxic T lymphocytes.
336
VON WILLEBRAND,
SOOTS, AND HAYRY
ACKNOWLEDGMENTS The editing and typing of the manuscript by Ms. Leena Saraste are gratefully acknowledged. The research was supported by grants from the Sigrid Juselius Foundation and the National Medical Research Council, Helsinki, Finland, and by NIH-NC1 Contract No. (38-64032.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
v. Willebrand, E., and Hayry, P., Cell. Immunol., in press, 1979. Lee, S., Surgery 61, 771, 1967. Hayry, P., and Andersson, L. C., Stand. J. Immunol. S(Suppl.), 31, 1976. H&yry, P., Andersson, L. C., Nordling, S., and Virolainen, M., Trenspfonr. Rev. 12, 91, X972. v. Willebrand, E., and Hayry, P., Cell. Immunol. 41, 358, 1978. Haskill, S., Radov, L., and Hayry, P., In “Contemporary Topics in Immunobiology” (N. L. Warner and M. D. Cooper, Eds.), Vol. 8, p. 107. Plenum, New York, 1978. Totterman, T. H., Hlyry, P., Saksela, E., Timonen, T., and Eklund, B., Eur. J. Immunol. 8, 872, 1978. Andersson, L. C., Stand. .I. Immunol. 2, 75, 1973. Roberts, P. J., and Hiiyry, P., Cell Immunol. 30, 236, 1977. Binz, H., Wigzell, H., and Hlyry, P., Nature (London) 259, 401, 1976. Wigzell, H., and Hiiyry, P., In “Current Topics in Microbiology and Immunology” (W. Arber et al., Eds.), Vol. 67, p. 1. Springer-Verlag, New York/Berlin, 197.5. Evans, R., Grant, C. K., Cox, H., Steel, K., and Alexander, P., J. Exp. Med. 136, 1318, 1972. Trinchieri, G., DeMarchi, M., Mayer, W., Savi, M., and Ceppellini, R., Transplant. hoc. 5, 1631, 1973. Klein, J., Livnat, S., Hauptfeld, V., Jerabek, J., and Weissman, I., Eur. J. Immunol. 4,41, 1974.
IMMUNOLOGY
In Situ Effector
&,327-336
(1979)
Mechanisms
in Rat Kidney Allograft
Rejection
II. Heterogeneity of the Effector Cells in the Inflammatory vs that in the Spleen of the Recipient Rat EEVA Transplantation
VON WILLEBRAND, Laboratory,
Fourth SF 00290 Received
ANU
SOOTS, AND P. H~YRY
Department of Surgery, Helsinki 29, Finland November
Infiltrate
University
of Helsinki,
27, 1978
We have isolated the infiltrating inflammatory cells from rejecting rat kidney allografts via collagenase-DNase dispersion and lg velocity sedimentation. A representative cell sample, devoid of blood contamination and subclass-specific cell losses, is thus obtained in a functionally viable state. The infiltrating inflammatory cells display an immunologically specific in vitro cytotoxicity to donor-strain lymphoblasts. The peak cytotoxicity takes place in the graft before it takes place in the lymphatic system of the recipient rat. The aUog&t-infiltrating cytotoxic cells display a far wider distribution profile in lg velocity than do the recipient spleen killer cells. The donor-strain lymphoblasts are highly sensitive to the lytic effect of both allograftinfiltrating and spleen killer cells, but the transplant parenchymal cells, consisting mainly of tubular and glomerular cells, are relatively insensitive. The findings suggest heterogeneity of the effector cells in the allograft infiltrate vs that in the spleen, and indicate that different structures of the target organ are differently sensitive to cellular cytotoxicity.
INTRODUCTION In a previous communication (1) we described how rat kidney allografts transplanted across the major AgB barrier may be disaggregated with collagenase and DNase. We also analyzed the distribution and lymphocyte subclasses of the allograft-infiltrating inflammatory cells. The inflammatory infiltrate consisted (in order of magnitude) of mononuclear phagocytes (i.e., monocytes and macrophages), lymphocytes, (T) lymphoblasts, (B) plasmablasts, plasma cells, and relatively few granulocytes. Most of the infiltrating lymphocytes carried neither the T-specific Pta.1.A surface antigen nor expressed the surface Ig, but had a low electrophoretic mobility of non-T cells. In this communication we discuss how we have separated the allograft-infiltrating inflammatory cells from the allograft parenchymal cells via lg velocity sedimentation, and tested the ability of the inflammatory cells and immune spleen cells to kill donor-derived lymphocytic target cells and transplant parenchymal cells in vitro. The results demonstrate a heterogeneity of effector cells in the allograft infiltrate vs that in the spleen, and suggest that the transplant parenchymal cells (consisting in this experiment of mainly tubular cells and glomerular podocytes) are relatively insensitive to short-term cellular cytotoxicity in vitro. 327
0008-8749/79/100327-10$02.00/O Copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
328
VON
WILLEBRAND,
MATERIALS
SOOTS,
AND
HiiYRY
AND METHODS
Rats. The nuclei for the inbred strains DA (AgB-4), A0 (AgB-2), and HO (AgB-5) were obtained from Professor J. L. Gowans, Dunn School of Pathology, Oxford, England. The rats were bred in our own colony. Transplantations. The micromethods described by Lee (2) for rat kidney transplantation were used. The organs were procured and perfused with physiological saline containing 40 II-l/ml heparin (Medica Pharmaceuticals, Helsinki, no preservative added). End to side anastomoses were performed on the recipients’ aorta and vena cava, and the donor ureter was anastomized to the recipient bladder with the aid of a bladder flap. Silk, 9-O to 10-0, was used throughout. Dispersion of the allogrufts. The transplant was throughly perfused, minced with a scalpel to pieces of approximately 0.5 x 0.5 mm in diameter, and immersed into Hepes-buffered RPM1 1640 (Gibco Bio-Cult, Glasgow, Scotland) medium containing collagenase (2.0 mg/ml, Worthington Biochemical Co., Freehold, N.J.) plus DNase (0.2 mg/ml, Sigma Chemical Co., St. Louis, MO.). The material was incubated at 37°C with a magnetic stirrer, until most of the tissue was dissolved. The few remaining clumps were removed by sedimentation and the resulting single-cell suspension was washed twice with RPM1 medium. Preparation of recipient lymphoid cells. Blood mononuclear leukocytes were recovered via density centrifugation of 1: 10 diluted blood over Ficoll (Pharmacia Fine Chemicals, Uppsala, Sweden)-Isopaque (Nyregaard & Co., Oslo, Norway), density 1.081 g/ml. Spleen and lymph node leukocytes were obtained by teasing apart the respective organs, by removing of the clumps via sedimentation, and by lysing the residual red cells with 0.83% ammonium chloride. Separation ofthe infiltrating cellsfrom ullogruftpurenchymul sedimentation. The Ig velocity method fractionates particles,
cells by lg velocity
such as cells, OR the basis of their size. Our application of this method has been described elsewhere (3). In short, the single-cell suspension of allograft suspension was loaded on a 0.75 to 1.5% BSA-PBS gradient and sedimented for 2-4 hr. Ten to 15 fractions of lo-15 ml were drained from the gradient. Cell smears were made from each fraction with a cytocentrifuge, and differential counts were performed after MayGriinwald-Giemsa (MGG) staining. The differential counts permitted the identification of the sites for proper “cuts” in the profile (see Fig. 1). In the fastestsedimenting “parenchymal cell” fractions approximately 90-95% of cells were morphologically kidney parenchymal (i.e., glomerular, tubular, endothelial, etc.) cells, and only approximately 5% were infiltrating cells (mainly macrophages). In the slowest-sedimenting “infiltrating cell” fractions up to 95% of all cells consisted of inflammatory cells of various types (Fig. 2). Target cells and cytotoxicity assay. Concanavalin A blast cells were cultured from donor- or recipient-strain spleen by methods described elsewhere (4). The transplant parenchymul cells were separated from the graft-infiltrating cells via the lg velocity sedimentation method. Prior to the assay all target cell populations were washed, labeled with sodium 51Cr chromate (The Radiochemical Center, Amersham, Great Britain) in conditions described (4), washed three times, and diluted into RPM1 1640 medium containing 5% fetal calf serum. The cytotoxicity assays were performed as described previously (4) in 200 ~1 vol at 37” in a humidified atmosphere of 5% CO, in air by employing 8 hr exposure time and triplicate
EFFECTOR
MECHANISMS
IN RAT KIDNEY
2
4
6
FRACTION
ALLOGRAFT
8
REJECTION
II
329
10
NUMBER
FIG. 1. Velocity sedimentation (Ig) profile of collagenase-DNase dispersate of a rat kidney allograft (a). Section (b) gives the relative distribution of allograf? parenchymal cells (open triangles) and host inflammatory infiltrate (broken line) within the profile. When the profile is cut between fractions 5 and 6, more than 95% of the inflammatory infiltrate is recovered in the fractions on the right, whereas more than 95% of the allograft parenchymal cells is recovered in the fractions on the left. The parenchymal cell fractions (shaded area) can be further purified from the contaminating inflammatory monocytes and macrophages with adherence to plastic.
determinations. The triplicate values fell consistently within 5% of the mean. The specific cytotoxicity was expressed according to the formula: Sp Wr
release =
release with effector cells - spontaneous release x 100. maximal release - spontaneous release
Regardless of target cell type, the spontaneous 14% of the maximal release (Triton X-100).
release was consistently
below
RESULTS Separation Cells
of the Allograft-Injiltrating
Cells from the Transplant
Parenchymal
A0 allografts were transplanted into DA rats, removed 6 days later, and disaggregated with collagenase and DNase. The cells were loaded on a linear OS1.5% bovine serum albumin/PBS gradient. After 3 hr of separation at lg, the gradient was divided into 12 or more fractions. Cytological cell smears were made from each fraction with a cytocentrifuge and stained with MGG. One such fractionation experiment is illustrated in Fig. 1. The kidney parenchymal, i.e., glomerular and tubular cells, on the average larger than the infiltrating cells and usually in small clumps, sedimented faster than did the infiltrating cells. By performing a proper “cut” in the profile, determined by differential analysis of cytocentrifuged preparations and located in Fig. 1 between fractions 5 and 6,
330
VON WILLEBRAND,
SOOTS, AND HAYRY
FIG. 2. (A) A microphotograph of the allograft inflammatory infiltrate recovered by lg velocity sedimentation displaying blast cells, monocytes, macrophages, and lymphocytes. (B) Immune spleen cells. (C) Transplant parenchymal cehs obtained via Ig velocity sedimentation and adherence of the contaminating host cells to plastic. Note that the cells consist nearly exclusively of large tubular cells, small podocytes, and very few endothelial cells.
a population consisting of more than 95% infiltrating cells was obtained. Furthermore, due to differences in cell size, the infiltrating cells were also fractionated into distinct, though overlapping cell populations. Most of the monocytes and macrophages were recovered in the immediate fractions after the parenchymal cells, the bulk of the blast cells were obtained in the intermediary fractions, and small lymphocytes, were obtained in the last fractions of the profiie. The transplant parenchymal cell fraction was contaminated with a sizable pro-
EFFECTOR
MECHANISMS
IN RAT KIDNEY
ALLOGRAFT
REJECTION
II
331
portion of host cells, mainly monocytes and macrophages. Short-term adherence to plastic in vitro, however, depleted the parenchymal cell fraction from most of the host cells (and endothelial cells of the transplant), and the resulting population consisted of more than 80% transplant parenchymal cells, mainly tubular cells and glomerular podocytes (Fig. 2). Cytotoxic Activity of Allagraft-lnjiltrating Injlammatory Recovered from the Recipient Lymphoid System
Cells, and Effector Cells
A0 recipients were transplanted with DA kidney allografts and the grafts were recovered on Day 6 after the transplantation. The allograft-infiltrating cells were released with collagenase plus DNase, and purified via Ig velocity sedimentation. The infiltrating cells, the recipient spleen, blood, and (irrelevant) lymph node cells, were tested for in vitro cytotoxicity to donor- or recipient-strain-derived Con A blast cells in the 6-hr short-term 51Cr release assay. The result is displayed in Fig. 3. Both the allograft-infiltrating cells and the white cells recovered from the recipient lymphoid organs displayed a distinct dose-dependent cytotoxicity to the relevant target cell in vitro. The recipient-strain blasts (not shown) were not lysed. The highest cytotoxic activity was seen in the allograft infiltrate, followed by spleen, blood, and the (irrelevant) lymph node cell populations. Another experiment was designed to investigate the sequence of appearance of cytotoxic cells into the allograft and into the recipient lymphoid system. We also wanted to investigate the specificity of the lysis at different time periods after the transplantation, and the relative sensitivity of donor-derived lymphocytic target cells vs that of the transplant parenchymal cells to the cytotoxic effect by the in situ effector cells, and by the effector cells recovered from the central lymphatic system of the recipient rat. A0 rats were transplanted with DA kidney allografts, the grafts were removed on Days 3-16 after the transplantation, and the recipients were exterminated. The different effector cell populations were recovered and tested for in vitro cytotoxicity to donor-strain Con A lymphoblasts, to recipient-strain lymphoblasts,
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332
VON WILLEBRAND,
SOOTS, AND HAYRY
to the transplant parenchymal cells recovered by the lg velocity sedimentation, and to the (control) graft-infiltrating cells themselves. The results of the short-term 6-hr 51Cr release assay are displayed in Fig. 4. The peak cytotoxic activity was seen in the graft already on Day 6 after the transplantation, whereafter it declined. The peak cytotoxic activities in the central lymphatic organs of the recipient rat, in the spleen, blood and lymph nodes, were observed later, at the time when the cytotoxic activity in the graft had declined. The lytic effect was specific, as donor-strain Con A lymphoblasts but not recipient-strain lymphoblasts were lysed by any one type of effector cell. In contrast to donor-derived lymphocytic target cells, the transplant parenchymal cells obtained by lg velocity sedimentation, consisting of more than 80% kidney tubular and glomerular cells, were highly insensitive to lysis. Velocity Sedimentation Distribution of Allograft-Infiltrating Cells
and Spleen Effector
A0 recipients were transplanted with DA kidney allografts, and the transplants were removed 6 days later. The allografts were dispersed with collagenase plus DNase, and the recipient spleen cells were treated with the same enzymes under
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similar conditions. Both cell populations were fractionated in Ig velocity sedimentation. Differential cell counts were performed from each fraction, and the cytotoxic activity of the fractionated cells was tested, fraction by fraction, to donor-strain Con A-induced lymphoblastoid target cells. As the parenchymal cells were rather insensitive to short-term cellular cytotoxicity, we believed that they would not interfere with the assay, but also fractions containing parenchymal cells (l-3) were included in the test. One of three experiments with similar results is displayed in Figs. 5 and 6. As seen in sections (b) and (c) of Fig. 5, the lg velocity method rather efficiently separated lymphocytes, blast cells, and the mononuclear phagocytes, monocytes, and macrophages. A considerable overlapping between the adjacent fractions was, however, evident. When the various fractions were tested for in vitro cytotoxicity to relevant blast target cells (Fig. 6), all cytotoxicity in the spleen was present in two or at most three fractions only. In contrast, in the allograft infiltrate high cytotoxic activities, equal to or higher than those in the input population, were observed in at least five or six different fractions. DISCUSSION We have previously demonstrated that the collagenase-DNase method does not affect the subclass-specific surface markers of lymphoid cells (5). Neither does the treatment invalidate such functional responses of lymphoid or monocytic GRAFT
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334
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cells as, e.g., conduction of lysis of syngeneic tumor cells in vitro in rodents (6) or in man (7), proliferation in vitro in response to mitogenic stimuli (unpublished), or conduction of the so-called “natural killer” activity to an appropriate target cell (unpublished). As it has also been documented that the inflammatory cells released after collagenase-DNase dispersion from a well-perfused rat allograft are devoid of blood contamination (I), and as the differential distribution of the released inflammatory cells is identical to imprint preparations of the very same allografts (1)) we conclude that this enzymatic method provides a representative sample of the allograft-infiltrating cells in a functionally viable state. The lg sedimentation method employed to fractionate the transplant parenchyma1 cells from the allograft-infiltrating cells gives an essentially pure population of infiltrating cells with a minimal (approximately 5%) cell loss. As seen in the profile of Fig. 5, the lost population consists predominantly of large monocytes and macrophages of the highest sedimentation mobility. Two observations have emerged from this study which are potentially important in understanding the modus operandi of allograft rejection. First, the distribution profile of the graft-infiltrating killer cells in lg velocity sedimentation is wider than the profile of spleen killer cells, and, second, different donor-derived target structures display unequal sensitivity to cell-mediated cytotoxicity in short-term 6-hr in vitro cytotoxicity assay.
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The Ig velocity sedimentation method separates also the various cell populations of inflammatory cells into distinct though overlapping fractions (6). Andersson (6) has previously demonstrated that the allograft-directed cytotoxic cells in the recipients’ spleen are shortly after immunization exclusively T cells of relatively high sedimentation velocity. The results of our analysis coincide with the earlier observations. On Day 6 after kidney transplantation, all cytotoxic activity in the recipient spleen was recovered in two fractions only. As removal of monocytes strongly increases the spleen cell cytotoxicity (unpublished), the result favors that T blasts and large lymphocytes are responsible for the cytotoxicity in the spleen. In the graft, on the other hand, the cytotoxic profile was far wider and covered (over the input values) at least six fractions over the profile. The fastest-sedimenting fractions with distinct cytotoxic activity contained predominantly inflammatory monocytes and macrophages, and the intermediary fractions with distinct cytotoxicity were dominated by blast cells, while some of the slowest-sedimenting distinctly cytotoxic fractions contained nearly exclusively small lymphocytes only. Previous evidence has indicated (9) that removal of macrophages and monocytes from the inflammatory infiltrate does not increase the cytotoxic activity. We therefore conclude that, as with sponge matrix allografts (9), several cell types within the inflammatory infiltrate display cytotoxic activity toward donor-derived target cells in vitro. Earlier experience with sponge matrix allografts (10) has indicated that both phagocytic and nonphagocytic allograft-infiltrating inflammatory cells carry idiotypic receptors directed to the major locus antigens of the transplant donor. As only T cells are able to function as killer cells by virtue of an antigen-specific receptor synthetized by the cell itself (1 l), the non-T lymphocytic and phagocytic cells displaying specific lytic activity in the inflammatory infiltrate are most likely armed from other sources. Some of these arming molecules may be T cell derived (12). Also, antibody may render the primarily noncommitted cells specifically cytotoxic (13). Although the amount of (IgG) alloantibody is extremely low in the recipient serum 6 days after transplantation (14), the in situ plasmablasts and antibody-secreting plasma cells, demonstrated to reside in the inflammatory infiltrate (1), may provide the minute amounts of antibody necessary for the arming of the primarily noncommitted killer cells. Different target cells displayed large differences in their sensitivity to cellmediated cytotoxicity in the short-term in vitro cytotoxicity assay. Thus, e.g., donor-derived blast cells were highly sensitive targets. These types of cells are nearly exclusively used in the cytotoxicity experiments intended to document the allograft rejection in vitro. However, allograft parenchymal cells purified from the rejecting allograft via lg velocity sedimentation (followed by adherence of the contaminating mononuclear phagocytes to plastic), were nearly insensitive in the assay. In our experiment these cells consisted essentially of tubular cells and glomerular podocytes only. Although it is improper to make far-reaching conclusions from this observation only, the result seems to indicate that different organelles of the transplant are unequally sensitive to the immune attack, and/or are destroyed via different effector mechanisms. This suggestion is in accordance with a recent finding by Parthenais (unpublished) which demonstrates that muscle cells of rejecting heart allografts are destroyed more readily by ADCG-type mechanisms, whereas the heart endothelial cells are destroyed by cytotoxic T lymphocytes.
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ACKNOWLEDGMENTS The editing and typing of the manuscript by Ms. Leena Saraste are gratefully acknowledged. The research was supported by grants from the Sigrid Juselius Foundation and the National Medical Research Council, Helsinki, Finland, and by NIH-NC1 Contract No. (38-64032.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
v. Willebrand, E., and Hayry, P., Cell. Immunol., in press, 1979. Lee, S., Surgery 61, 771, 1967. Hayry, P., and Andersson, L. C., Stand. J. Immunol. S(Suppl.), 31, 1976. H&yry, P., Andersson, L. C., Nordling, S., and Virolainen, M., Trenspfonr. Rev. 12, 91, X972. v. Willebrand, E., and Hayry, P., Cell. Immunol. 41, 358, 1978. Haskill, S., Radov, L., and Hayry, P., In “Contemporary Topics in Immunobiology” (N. L. Warner and M. D. Cooper, Eds.), Vol. 8, p. 107. Plenum, New York, 1978. Totterman, T. H., Hlyry, P., Saksela, E., Timonen, T., and Eklund, B., Eur. J. Immunol. 8, 872, 1978. Andersson, L. C., Stand. .I. Immunol. 2, 75, 1973. Roberts, P. J., and Hiiyry, P., Cell Immunol. 30, 236, 1977. Binz, H., Wigzell, H., and Hlyry, P., Nature (London) 259, 401, 1976. Wigzell, H., and Hiiyry, P., In “Current Topics in Microbiology and Immunology” (W. Arber et al., Eds.), Vol. 67, p. 1. Springer-Verlag, New York/Berlin, 197.5. Evans, R., Grant, C. K., Cox, H., Steel, K., and Alexander, P., J. Exp. Med. 136, 1318, 1972. Trinchieri, G., DeMarchi, M., Mayer, W., Savi, M., and Ceppellini, R., Transplant. hoc. 5, 1631, 1973. Klein, J., Livnat, S., Hauptfeld, V., Jerabek, J., and Weissman, I., Eur. J. Immunol. 4,41, 1974.