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*
Department of Pathology, Biology Research Center, Massachusetts General
Hospital/Harvard Medical School, Boston, Massachusetts
Department of Transplantation, Biology Research Center, Massachusetts
General Hospital/Harvard Medical School, Boston, Massachusetts
Department of Pathology, University of Chicago, Chicago,
Illinois
§
Department of Pathology, Nippon Medical School, Tokyo, Japan.
Correspondence to Dr. Robert B. Colvin, Department of Pathology, Massachusetts General Hospital, 55 Fruit Street (WRN 225), Boston, MA 02114; Phone: 617-726-2966; Fax: 617-726-7533; E-mail: colvin{at}helix.mgh.harvard.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In acute renal allograft rejection in humans, infiltration of the interstitium with mononuclear cells, including cytotoxic T cells, is a characteristic feature (6,7,8,9), and the cells show marked activation and/or proliferation (10,11,12). However, cell infiltration is also detected in normally functioning grafts with no obvious clinical evidence of rejection (13,14,15). Graft-infiltrating cells are also seen in the induction of tolerance to allogeneic grafts in animal models (16,17,18,19). The distinguishing features of cell infiltrates in normal functioning or accepting grafts in the development of tolerance are unclear.
We therefore studied the intragraft morphologic features associated with induction and subsequent development of tolerance (which we have termed the "acceptance reaction"), focusing on the activation, proliferation, and apoptosis in graft-infiltrating cells and cell-mediated apoptotic graft injury. Serial monitoring was performed to assess whether (1) the phenotypic characteristics, (2) level of immune activation, (3) proliferation, and (4) extent of apoptosis in graft infiltrating cells or (5) level of apoptotic graft injury was useful in distinguishing graft acceptance from rejection as well as in predicting further development of transplantation tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histologic Examination
Serial open kidney biopsies were taken on postoperative days 4, 7, 8, and
11 for all groups and on days 18, 30, 60, and 100 for the CsA-treated graft
acceptance and syngeneic control groups. For light microscopic examination,
tissue was fixed in 10% buffered formalin and embedded in paraffin.
Hematoxylin and eosin and periodic acid-Schiff stains were performed for
histologic examination. The biopsy samples were diagnosed using National
Institutes of Health-Cooperative Clinical Trials in Transplantation
classification of allograft rejection
(21,22,23).
A standard of the avidin-biotin-peroxidase complex (ABC) technique (9) was used in frozen sections to detect phenotype of infiltrating cells. Primary antibodies included anti-pig monoclonal antibodies MSA4 (anti-swine CD2), BB23-8E6 (anti-swine CD3), 74-12-4 (anti-swine CD4), 76-2-11 (anti-swine CD8), BB6-11C9 (anti-swine CD21; B cells), K231-3B2 (anti-swine interleukin-2 receptor; IL2R), and 74-22-15A (macrophages) (24) and anti-human CD3 polyclonal antibodies (DAKO, Glostrup, Denmark). The anti-human CD3 antibody was confirmed to react with swine pan T cells using swine thymus, lymph nodes, and spleen.
For the detection of proliferating cell nuclear antigen (PCNA), 10% buffered formalin-fixed, paraffin-embedded tissue blocks were used, and sections were stained using ABC technique. To optimize detection of PCNA, microwave treatment (heat for 2 x 5 min in 0.01 M sodium citrate [pH 6.0] in a 750-W microwave oven at full power and then immediately chilling to 4°C) and 1:1000 dilution of PC10 (DAKO), was used (25). Double immunostaining for PCNA and CD3 was performed in formalin-fixed paraffin sections using a two-color staining technique (9). The sections first were stained with PCNA and incubated with alkaline phosphatase-labeled anti-mouse IgG (Vector, Burlingame, CA) with a blue reaction product (Alkaline Phosphatase Substrate Kit III, Vector). Sections then were stained with polyclonal CD3, horseradish peroxidase-labeled anti-goat antibody (DAKO), hydrogen peroxide (H2O2) containing 3,3'-diaminobendizine (DAB; Research Genetics, Hansville, AL), which has a brown reaction product. Controls included substitution of the primary antibody with an irrelevant antibody and use of anti-CD3 before anti-PCNA. Sections of thymus and lymph node were used as positive controls to ensure that the staining procedures were effective.
In histologic sections, fragmented nuclear DNA associated with apoptosis was labeled by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) (26). The 2.5-µm thick sections were deparaffinized and incubated with proteinase K 100 µg/ml for 15 min. After blocking endogenous peroxidase by immersion in distilled water containing 2% H2O2, sections were rinsed in TdT buffer and incubated with TdT 1:25 and biotinylated-dUTP 1:20 in TdT buffer for 60 min at 37°C. The biotinylated nuclei were detected with avidin-peroxidase and H2O2 containing DAB. Double immunostaining with TUNEL and CD3 for the identification of the origin of TUNEL+ cells was performed by immunoalkaline phosphatase using TUNEL, followed by an antibody to CD3, horseradish peroxidase-labeled anti-goat, then incubated with H2O2 containing DAB. Negative controls consisted of omission of dUTP or TdT. The positive control was swine thymus, which shows numerous TUNEL+ thymocytes.
Quantification of Histologic Findings
Morphometric studies were performed to determine the number of CD3-, CD4-,
CD8-, or CD21-positive cells and macrophages per mm2, as well as
the proportions of graft-infiltrating cells that were PCNA+, IL2R+, or TUNEL+.
All counts and pathologic evaluation were performed on coded slides, without
prior knowledge of the clinical or histologic diagnosis, at 400x, using
an optical grid area of 0.0625 mm2. Counts were expressed as the
numbers of positive cells per mm2 and as the percentage of the
infiltrating mononuclear cells. In addition, the percentage of cortex with
infiltrating cells was estimated in low-power fields (at 40x) in all
fields of the renal cortex. To determine the degree of graft parenchymal cell
injury, we counted the number of TUNEL+ glomerular cells per glomerular cross
section, the number of TUNEL+ endothelial cells in peritubular capillaries per
mm2, the percentage of TUNEL+ tubular epithelial cells, or the
percentage of TUNEL+ arteries in CD3 and TUNEL double-stained sections and
examined the correlation between CD3+ cells and TUNEL+ graft parenchymal
cells. In this study, we identified the cell type of TUNEL+ cells by its
location, morphologic appearance, and CD3 expression; those in which the cell
type could not be determined were excluded (5% or less). More than 40
glomerular cross sections or more than 40 fields of renal cortex were counted
in all biopsy specimens. These results were expressed as the mean ± SD
or SEM, and statistical analysis was performed using the t test.
Cell-Mediated Lympholysis Assay
Cell-mediated lympholysis (CML) assays were performed using peripheral
blood leukocyte (PBL) as described previously
(4). Briefly, lymphocyte
cultures containing 4 x 106 responder and 4 x
106 irradiated (25 G) stimulator PBL in 2 ml of medium were
incubated for 6 d at 37°C in 7.5% CO2 and 100% humidity. Bulk
cultures were harvested, and effector cells were tested on
51Cr-labeled blasts. The tests were run at serially diluted ratios
(100:1, 50:1, 25:1, 12.5:1). After 5.5 h of effector cell incubation with the
5 x 103 specific targets, supernatants were harvested and
51Cr release was determined on a gamma counter (Micromedics,
Huntsville, AL). Maximum lysis was obtained with a 1% solution of the nonionic
detergent NP-40 (BLR, Rockville, MD). Baseline levels were measured as the
rate of spontaneous release of 51Cr from 5 x 103
targets. The data were expressed as percentage of specific lysis:
% specific lysis = {[experimental release (cpm) - spontaneous release (cpm)]/[maximum release (cpm) - spontaneous release (cpm)]} x 100
The results of the control (rejector) and the acceptor groups were expressed as the mean ± SD, and statistical analysis was performed using the t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Graft-Infiltrating Cells in Rejecting Grafts
In control rejecting recipients, a progressive mononuclear cell infiltrate
with cellular proliferation and activation developed and led to irreversible
rejection. By day 7, the mononuclear cell infiltrate extended diffusely in the
interstitium and caused tubulitis, glomerulitis, and endotheliitis in arteries
(Figure 2A). Infiltrating cells
had large nuclei and nucleoli, indicating typical activated lymphocytes
(Figure 2B). CD3+ T cells
diffusely infiltrated the cortex (Figure
2C). The mononuclear cells infiltrated more than 60% of
interstitium in the cortex on day 7 and were composed of a similar number of
CD3+ cells (2500/mm2) and macrophages (2000/mm2)
(Figure 3). CD8+ cells were the
predominant T cells (1500/mm2), with lesser amounts of CD4+ cells
(700/mm2). Occasional CD21+ B cells (90/mm2) were found.
Many graft-infiltrating cells were PCNA+
(Figure 2D). In double staining
with PCNA and CD3, many PCNA+ cells expressed CD3
(Figure 2E), indicating that T
cells were proliferating in the grafts. IL2R+ infiltrating cells were present
diffusely in the cortex (Figure
2F). TUNEL+ apoptotic cells and apoptotic bodies were found in the
infiltrate (Figure 2G), and
double stain with TUNEL and CD3 showed that infiltrating T cells underwent
apoptosis in the grafts (Figure
2H).
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Graft-Infiltrating Cells in Accepted Grafts
In syngeneic control graft, only a few infiltrating cells and CD3+ cells
were evident during the experimental periods. Despite stable renal function in
animals that received CsA, a cellular infiltrate began on day 4 and reached a
maximum on days 8 to 18 (Figure
3). However, the cell infiltrate spontaneously resolved by day
100. On days 8 to 18, the patchy mononuclear cell infiltrate with CD3+, CD8+,
and CD4+ cells was seen in perivascular areas; tubulitis and glomerulitis were
less prominent and endotheliitis was absent (Figures
4, A and B, and
5). The peak concentration of
CD3+ (1200/mm2), CD8+ (800/mm2), and CD4+
(300/mm2) cells and macrophages (1000/mm2) were
significantly lower in acceptance reactions than in the rejecting grafts, but
the proportion of each subset was similar in both groups
(Figure 3). The number of CD21+
B cells, which peaked on day 30 (60/mm2), was not significantly
different from rejecting grafts. Fewer PCNA+, PCNA+ CD3+, or IL2R+ graft
infiltrating cells were seen in accepting grafts (Figures
5D and
6), and the peak % of PCNA+ and
IL2R+ cells on day 8 (13.3 and 11.0%, respectively) was significantly lower
than for the rejecting group (24.3 and 21.2%, respectively; P <
0.01; Figure 7). A similar
fraction of the peak TUNEL+ graft-infiltrating cells was observed in both
rejecting and accepting grafts (4.9 and 4.3%, respectively; Figures
7 and
8).
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After reaching a peak on day 18, the mononuclear cell, T-cell, and macrophage infiltrate began to decrease by day 30 and further decreased by day 100 (Figures 3 and 4). The number of cells labeling for PCNA+ and IL2R+ decreased earlier (starting on day 11) and more rapidly than the infiltrate cell decrease (Figures 6 and 7). Apoptosis of graft-infiltrating cells continued at nearly peak levels for up to 60 d, then gradually decreased by day 100 (Figures 7 and 8). In double staining with TUNEL and CD3 (Figure 8D, inset), TUNEL+ cells expressed CD3, indicating that some of the infiltrating T cells undergo apoptosis in the grafts.
Cell-Mediated Apoptotic Graft Injury
In syngeneic graft, only a few CD3+ infiltrating cells and TUNEL+ apoptotic
graft parenchymal cells were evident during experimental periods. In rejecting
grafts by day 7, many CD3+ cells and TUNEL+ apoptotic cells were seen in the
lesions in the arterial intima, glomeruli, tubules, and peritubular
capillaries (Figure 9, A through
D). Occasional TUNEL+ cells were adjacent to infiltrating CD3+
cells.
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In accepting grafts, CD3+ cells were less prominent and fewer TUNEL+ apoptotic cells were found in tubular, glomerular, and peritubular capillary lesions at days 8 to 18; no TUNEL+ cells were identified in arterial endothelium (Figure 9, E through H). The peak number of TUNEL+ tubular epithelial cells (0.95%), glomerular cells (1.0 cells/glomerular cross section), and peritubular capillary endothelial cells (17 cells/mm2) in accepting graft was significantly less than in rejecting grafts (2.6%, 1.9 cells/glomerular cross section and 36 cells/mm2, respectively; P < 0.01; Figure 10). TUNEL+ cells in tubules, glomeruli, and peritubular capillaries diminished by day 60, and very few TUNEL+ cells remained in these sites at day 100.
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In antidonor and third-party type class I CML assays, rejectors without CsA treatment maintained strong cytotoxic T-cell (CTL) responses against donor type targets aftr kidney transplant (Figure 11). In contrast, acceptors developed specific unresponsiveness to the donor class I antigens by postoperative day 30, and this was maintained until day 100, although they had strong CTL responses against third-party type targets. Positive in vitro CTL reactivity correlated with TUNEL+ donor parenchymal cells in the grafts (Figures 10 and 11).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells composed the largest leukocyte population in the rejecting and the accepting grafts. The proportions of CD3+, CD8+, and CD4+ cells and macrophages were similar in all time points in accepting and rejecting grafts, confirming that T-cell subsets had neither prognostic nor diagnostic significance. In rejection in the present study, the interstitial mononuclear cells, CD3+, CD8+, and CD4+ T cells, and macrophage infiltrate doubled in extent and intensity compared with the accepting grafts. Although the intensity or extent of the infiltrate has no or only negligible predictive value for graft prognosis in clinical renal transplantation under antirejection therapy, these may be important in distinguishing between acceptance and rejection reactions in tolerance induction protocols.
Recent reports demonstrated that the analysis of cell activation and proliferation of graft infiltrating cells could be useful in differentiating between rejection and other causes of graft dysfunction (10,11,12). In the present study, the peak percentage of PCNA+ and IL2R+ cells in the graft infiltrate were significantly less in accepting grafts compared with rejecting grafts, suggesting that these analyses are also useful in the diagnosis of acceptance reaction. Moreover, our results suggest that a limitation of T-cell proliferation and activation occurs at the time of first exposure to alloantigens in the induction of tolerance and supports the hypothesis that the development of tolerance depends on a relative deficit of T-cell help in the presence of alloantigens (5,27).
The development of tolerance in this model is thymic dependent and therefore involves central mechanisms (28). However, peripheral tolerance mechanisms may also be essential. During the first few weeks after transplant in this model, renal grafts showed the expression of relatively elevated levels of IL-10 in tolerant animals compared with those with rejection (19). The infiltrate contains immunoregulatory cells that are important in the adaptation of the host to the graft in the maintenance of peripheral tolerance (29). Several experimental models of tolerance have shown that Th2-derived cytokines are postulated to contribute to tolerance induction and allograft acceptance by promoting clonal anergy or deletion of alloreactive T lymphocytes (30,31). Thus, intragraft events may be a cause as well as an effect of the induction of tolerance.
It is notable that infiltrating T cells undergo apoptosis that continues at nearly peak levels for up to 60 d in the absence of any exogenous immunosuppressive drug therapy. The prolonged in situ apoptosis may be an important mechanism of containing or eliminating donor reactive cells. Recent studies of rat experimental models of allergic encephalomyelitis (32) and tubulointerstitial nephritis (33) showed that apoptosis is the mechanism that leads to elimination of infiltrating leukocytes during resolution of inflammation. Fas/Fas ligand (Fas-L)-triggered apoptosis may be crucial in this process, as suggested by the marked accumulation of activated lymphocytes in mice deficient in Fas(lpr/lpr) or Fas-L (gld) (34). It is postulated that high-level Fas-L expression on the graft organs promotes the induction of tolerance, probably as a result of the apoptotic deletion of graft-invading cells by Fas/Fas-L interaction (35). In addition, activation-induced T-cell death via Fas/Fas-L pathway, alloantigen-induced T-cell death, and absence of a costimulatory signal during primary activation-induced T-cell death may contribute to graft-infiltrating cell apoptosis (34,35,36). In mouse liver or combined liver/small bowel transplantation, apoptosis of CD8+ T cells is connected with the process of specific tolerance induction (37). In the present study, during the time that apoptosis of infiltrating cells occurred, donor reactive CTL in the PBL decreased. This led us to hypothesize that the effector CTL in grafts undergo apoptosis. Recently, evidence showed that apoptosis of infiltrating CTL in liver grafts occurs during the development of tolerance (38). Although the mechanism is unknown, it seems that in situ apoptosis may regulate the number of infiltrating cells and cell-mediated antigraft activity in the development of graft acceptance.
T-cell—mediated cytotoxicity probably plays an important role in acute allograft rejection by lysis and apoptosis of MHC-incompatible cells (34,39,40). In human renal allografts, apoptosis of graft parenchymal cells occurs during rejection (9,41,42). Our study demonstrated that although CD3+ cells infiltrated the tubules, glomeruli, and peritubular capillaries, only rare TUNEL+ apoptotic graft cells were seen in these sites in accepting grafts, arguing that the infiltrate in accepting grafts was less active in mediating target cell injury. Thereafter, there was a parallel loss of anti—donor-specific CTL reactivity in the PBL and loss of parenchymal cell apoptosis in the grafts, suggesting that a spontaneously remitting immune reaction to donor is important for tolerance to develop.
In this study, we demonstrated that a type of allograft reaction, which precedes long-term graft acceptance, has many similarities with the rejection reaction. However, graft-infiltrating cells in the acceptance reaction are characterized by minimal proliferation, minimal activation, and low frequency of cell-mediated graft cell injury and abundant T-cell apoptosis. The graft acceptance reaction is also characterized by progressively diminishing infiltrating cell proliferation and activation, resolving cell-mediated target cell injury, and persistent apoptosis of infiltrating cells. Analysis of proliferation, activation, and apoptosis in allografts may be useful to determine the status of the donor-reactive immune response and may have practical value in distinguishing the acceptance reaction from rejection. These intragraft events may be relevant in the study of protocol biopsies in humans, and the distinction of subclinical rejection from harmless infiltrate is therapeutically important.
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Acknowledgments |
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,
n = 4) and the accepting ([UNK], n = 4) grafts. In the
control rejector group, plasma Cr levels increase at around day 6, and all
kidneys develop irreversible rejection by day 11. In the acceptor group and
syngenic group, stable Cr levels continue during the experimental periods.
) (A). These infiltrating
cells include numerous proliferating (
) and activated lymphocytes (B),
CD3+ T cells (C), PCNA+ proliferating cells (
, n = 4) and acceptors (,
n = 4) have strong cytotoxic T-cell responses against third-party
target type antigen. Rejectors (