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1 From the Department of Ophthalmo-Immunology, The Netherlands Ophthalmic Research Institute, Amsterdam; the 2 Department of Ophthalmology, University Hospital Groningen, The Netherlands; the 3 Department of Ophthalmology, Hospital Geral de Santo António, Pôrto, Portugal; the 4 Department of Cell Biology, Free University, Amsterdam, The Netherlands; and the 5 Department of Ophthalmology, University Hospital Dijkzigt, Rotterdam, The Netherlands.
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Abstract |
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METHODS. Transplantations of corneal buttons from PVG rats were performed in AO rats. After surgery, one group received clodronate liposomes subconjunctivally at five time points, and the other group remained untreated. On postoperative day (POD) 3, 7, 12, or 17, rats were killed, the presence of CTLs was investigated at three different anatomic locations, and antibodies against donor antigens were tested.
RESULTS. No significant differences were found between the groups tested 3 and 7
days after surgery. But on POD 12 (the time of onset of rejection in
the untreated group) and on POD 17, the CTL activities detected in the
submandibular lymph nodes (P 
0.008) and the spleen
(P 0.009) were significantly less in the treated
groups compared with the untreated groups. In the untreated groups
complement-independent antibodies were present only on POD 17, whereas
no antibodies were found in the treated rats.
CONCLUSIONS. Local treatment with clodronate liposomes was shown to downregulate local and systemic CTL responses and to prevent the generation of antibodies. Local depletion of macrophages in the initiation phase of the immune response appears to lead to a less vigorous attack on the grafted tissue and therefore to promote graft survival.
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Introduction |
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Histopathologic studies of corneal graft rejection show an influx of not only T lymphocytes but also many macrophages.9 Although the current opinion is that rejection is mediated by T lymphocytes, Van der Veen et al.10 performed a study to determine the contribution of the macrophage to immunologic corneal graft rejection in high-risk rats. Using dichloromethylene diphosphonate (clodronate)–containing liposomes, macrophages could be selectively depleted. Subconjunctival administration of these liposomes after orthotopic corneal transplantation to recipient rats with a history of corneal inflammation, surprisingly resulted in complete graft survival in all treated rats for a follow-up period of 100 days, whereas the control group rejected the grafts between 12 and 17 days after surgery. Therefore, macrophages also seem to play an important role in corneal graft rejection.
It remains to be determined how the clodronate liposomes interfered in the rejection process. With regard to the various functions of macrophages, they can be involved in both the afferent and the efferent arc of the immune response leading to graft rejection. Through processing and presentation of foreign antigen to T lymphocytes they can participate in the afferent arc, but they are also able to play the role of effector cells and destroy the graft.11 12
In this study, we used a different rat strain combination without prior induction of keratitis to validate the clodronate liposome treatment. To unravel the immunologic basis for the absence of graft rejection in clodronate liposome–treated rats we used this strain combination to measure the cytotoxic T lymphocyte (CTL) and antibody responses from postoperative days (PODs) 3 to 17.
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Methods |
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Anesthesia
Donor rats were killed by an intracardial injection of
pentobarbital (0.5 ml; Euthesate; Apharmo, Arnhem, The Netherlands).
Recipients were anesthetized by an intramuscular injection of a mixture
of fluanisone and fentanyl citrate (0.5 ml/kg; Hypnorm; Janssen
Pharmaceutica, Beerse, Belgium) and an intraperitoneal injection of
diazepam (2.5 mg/kg). Oxybuprocaine hydrochloride (0.4%) was used for
topical anesthesia of the cornea.
Orthotopic Corneal Transplantation
Full-thickness corneal transplantations were performed in the
right eye of the recipient rat using a technique previously
described.10
Briefly, before surgery dilation of the iris
was obtained by subcutaneous injection of atropine sulfate (0.15 mg/kg)
and topical administration of atropine 1% sulfate and 10%
phenylephrine hydrochloride. With a 3-mm trephine and curved Vannas
scissors, the corneal button was removed from the donor and recipient
rats. The donor cornea was stored in Eagle’s modified essential medium
with 2% fetal calf serum, 100 IU/ml penicillin and 100 µg/ml
streptomycin (Life Technologies, Breda, The Netherlands) until use.
Eight stitches were made using a continuous 10-0 monofilament nylon
suture (Alcon, Fort Worth, TX) to secure the donor button into the
recipient graft bed. After surgery, no attempt was made to re-form the
anterior chamber or to remove the suture.
Clinical Evaluation
Grafts were observed for occurrence of graft rejection using an
operating microscope, on each alternate day until POD 14 and once a
week thereafter. Rejection was diagnosed as an opacity score of 3 or
higher in a previously clear graft. Opacity was scored from 0 to 4. A
score of 3 meant that the iris vessels were not visible, but the pupil
margins could be distinguished.9
Corneal transplantations
that were complicated by technical failures were excluded from the
study, as well as grafts that had an opacity score of 3 or higher
during clodronate liposome injections (see experimental design).
Preparation of Clodronate Liposomes
Clodronate liposomes were prepared as has been
described.13
In short, 75 mg phosphatidylcholine and 11 mg
cholesterol were dissolved in chloroform and a thin lipid film was
produced by low-vacuum rotary evaporation. This film was dispersed in
10 ml phosphate-buffered saline (PBS) solution in which 1.8 to 1.9 ml
dichloromethylene diphosphonate (clodronate
[Cl2MDP]; the kind gift of Boehringer Mannheim,
Mannheim, Germany) was dissolved. The suspension was maintained at room
temperature for 2 hours followed by sonication for 3 minutes in a water
bath sonicator. After another 2 hours at room temperature, the
dichloromethylene diphosphonate liposome suspension was centrifuged at
100,000g for 30 minutes to remove free dichloromethylene
diphosphonate. The liposomes were resuspended in 4 ml PBS and stored at
4°C until use.
Experimental Design
Ninety-one transplantations were performed of which five were
excluded: four because of technical failures of the transplantations,
such as extensive synechiae and hyphema, and one because of an
anesthesia problem. Four of the transplantations were autografts. The
allografted animals were divided into two experimental groups: an
untreated and a clodronate liposome–treated group. The latter received
subconjunctival injections of clodronate liposomes immediately after
transplantation and on PODs 2, 4, 6, and 8. At each time point 100 µl
of these liposomes was injected near the limbus, dispensed over four
locations (±25 µl each), to achieve an equal distribution around the
cornea. Previously, it had been demonstrated that during clodronate
liposome injections the graft became slightly more opaque than the
untreated grafts.10
It was found that an opacity score of
three or higher was not acceptable, because the graft will not regain
clarity after POD 8. For this reason, 3 of the 41 clodronate
liposome–treated animals had to be excluded from the study because the
graft rejection time could not be determined. In these rats, failure
was thought to be due to mechanical reasons, such as liposome clotting
or increased pressure around the eyeball. An immunologic cause for
rejection seems improbable so soon after grafting in an avascular
cornea and had never occurred this early in the untreated group.
Experiment 1.
Twenty-one grafts were observed for the appearance of graft rejection
for 100 days. The four autografts in AO recipients were used to test
for the correct transplantation technique and the nine untreated
allografts to determine the rejection time. Eight allogeneic grafts
were treated with clodronate liposomes to determine whether this
treatment also prevents graft rejection in the PVG-AO rat strain
combination.
Experiment 2.
To determine the immunologic basis for the absence of graft rejection
after clodronate liposome treatment, rats were killed at four different
time points. Two time points were chosen before rejection occurred in
the untreated allogeneic group (PODs 3 and 7), one at the onset of
graft rejection in the untreated allogeneic group (POD 12) and
approximately one 5 days after rejection (POD 17). Lymphoid cells were
isolated from three locations to determine the presence of CTLs: the
submandibular lymph nodes (SLNs), the mesenteric lymph nodes (MLNs),
and the spleen. To analyze for the presence of alloantibodies, serum
was obtained at these same time points. All serum samples were
heat-inactivated and stored at -20°C in small aliquots until use.
Experiment 3.
To exclude the possibility that local administration of clodronate
liposomes suppresses the whole immune system, five AO rats were
immunized intraperitoneally with PVG lymphocytes. Three of them were
treated with subconjunctival injections of clodronate liposomes on the
day of immunization and 2, 4, 6, and 8 days later; the other two rats
remained untreated. Ten days after immunization, rats were killed, and
the presence of CTLs and antibodies directed toward PVG lymphocytes was
tested in a chromium-release assay and an immunofluorescence assay.
Cell-Mediated Cytotoxicity Assay
The specific cytotoxic activity of lymphoid cells derived from
grafted rats was determined by a chromium 51 release assay. Cells
isolated from the SLNs, MLNs, and spleen were measured separately.
Single-cell suspensions were prepared by gently teasing the tissue
through a nylon-mesh strainer (70-µm Cell Strainer; Falcon, Becton
Dickinson, Franklin Lakes, NJ). The cell suspensions were washed twice
and resuspended in culture medium (RPMI 1640 containing 2.0 mM
L-glutamine, 10 mM HEPES buffer [Life Technologies], 100
IU/ml penicillin, 100 µg/ml streptomycin, 10% heat-inactivated fetal
calf serum [Life Technologies], and 3 x
10-5 M 2-mercaptoethanol [Sigma, St. Louis,
MO]) at a concentration of 5 x 106
cells/ml. Thymocytes derived from a young naive PVG rat were prepared
as described and irradiated (30 Gy) with a cesium 137 source. These
irradiated stimulator cells were adjusted to a cell concentration of
5 x 106 cell/ml; 0.5 ml of the suspension
was mixed with 1 ml effector cells in 24-well trays (Costar, Cambridge,
MA), total volume 1.5 ml; and cells were incubated at 37°C in a
humidified 5% CO2 atmosphere. After 5 days the
effector cells from six identical wells were pooled, and cell viability
was determined by trypan blue exclusion. Cells were centrifuged at
200g for 10 minutes and resuspended in culture medium at the
following concentrations: 10 x 106/ml;
5 x 106/ml; 2.5 x
106/ml and 1.25 x
106/ml.
SLNs and MLNs from a naive PVG rat served as target cells. Target cells
were cultured for 5 days in culture medium at a concentration of 5 x 106 cells/ml, and after 2 days in culture
concanavalin A (ConA 2 µg/ml; Life Technologies) was added. On the
day of the assay, target cells were washed and labeled with 100 µCi
Na251CrO4
(Amersham, Little Chalfont, UK) at 37°C for 1 hour. Labeled cells
were washed three times with culture medium, incubated for 30 minutes
in 20 ml culture medium to remove spontaneously released
51Cr, centrifuged (10 minutes, 200g)
and resuspended at a concentration of 2 x
105 cells/ml. Fifty microliters of labeled target
cells (104 cells) were plated into wells of
round-bottomed 96-well microtiter plates (Costar). One hundred
microliters of effector cells in different concentrations was added in
triplicate to the target cells. Effector-to-target ratios ranged from
100:1 to 12.5:1. Plates were centrifuged at 100g for 3
minutes, followed by a 4-hour incubation at 37°C in a humidified 5%
CO2 atmosphere. After 4 hours, the plates were
centrifuged for 10 minutes at 500g and 100 µl supernatant
was removed from each well for counting of radioactivity in a
spectrometer (Auto-gamma; Packard, Meriden, CT). The cytotoxicity of
each sample was determined as follows:
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Assay of Alloantibodies in Serum
The presence of specific antibodies against PVG cells was tested
by indirect immunofluorescence assay. PVG lymphocytes were isolated and
cultured for 3 days in the presence of ConA (2 µg/ml) to increase
their size and enhance their major histocompatibility complex (MHC)
expression. After 3 days, the cells were washed and fixed with 2%
paraformaldehyde. Subsequently, these fixed cells were washed twice in
washing medium (PBS, 1% bovine serum albumin [Sigma], and 0.2%
sodium azide), centrifuged for 10 minutes at 200g, and
resuspended at 5 x 106 cells/ml. Ten
microliters serum was added to 90 µl of these cells. After an
incubation for 1 hour, cells were washed and incubated for another hour
at 4°C with a 1:50 dilution of rabbit anti-rat Ig antibody labeled
with fluorescein isothiocyanate (Rara-Ig-FITC; Nordic, Tilburg, The
Netherlands). Cells were washed once, 1 drop of Evans blue was added to
the pellet, and cells were washed again. Finally, they were suspended
in PBS-glycerol, a drop of Vectashield (Vector; Burlingame, CA) was
added, and they were mounted on a glass slide with a coverslip. The
cells were scored for the presence of immunofluorescence-positive cells
by a masked observer, and the percentage of positive cells was
calculated. In each assay, sera from grafted rats and samples from a
pool of naive AO rats or from a pool of AO rats immunized against PVG
antigens were included.
Assay of Complement-Dependent Alloantibody in Serum
To test the specific cytotoxic activity of the serum samples a
chromium release assay was used. The Cr 51-labeled target cells were
prepared as described. Fifty microliters of these cells (5 x
106 cells/ml) was plated into wells of a
round-bottomed 96-well plate and incubated at 37°C for 60 minutes
with 50 µl serum dilution (1:10, 1:20, 1:40, and 1:80) obtained from
graft-recipient rats. Subsequently, 50 µl fresh normal rat serum was
added, as a source of complement, and the incubation continued for 60
minutes. The plates were then centrifuged at 500g for 10
minutes, and 100 µl of the supernatant was removed and counted in a
gamma counter. The percentage of specific release of serum samples was
determined as described. In each assay, a serum sample from a pool of
naive AO rats and a serum sample from a pool of AO rats that had been
immunized against PVG antigens were tested as a standard.
Statistical Analysis
The graft survival curves were compared using the log rank test.
Statistical analysis of the cytotoxic and immunofluorescence assays was
performed using the Mann–Whitney test. The two groups were compared at
the effector-to-target ratio of 100 to 1 and serum dilution of 1:10.
The statistical analyses were corrected for multiple comparisons;
P < 0.03 was considered statistically significant.
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Results |
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CTL Activity against Donor Antigens in Recipients of Corneal
Allografts
In experiment 2, at four time points after orthotopic
allotransplantation, untreated AO rats and clodronate liposome–treated
AO rats were killed to determine the presence of CTLs against donor PVG
cells. Cells were isolated from three locations to determine
differences in local and systemic CTL activity between the two groups.
Local CTL activity was determined by taking cells derived from the SLNs
(Fig. 2)
. Comparing the two treatment groups at the effector-to-target ratio of
100 to 1, no difference was noted on PODs 3 and 7. But at time of the
onset of graft rejection in the untreated group, POD 12 and 5 days
later, a significant difference was noted (P = 0.006
and P = 0.008, respectively). Far higher CTL activity
was found in the untreated rats than in the clodronate
liposome–treated rats.
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Effect of Subconjunctival Administration of Clodronate Liposomes on
Immune Responses to Intraperitoneally Injected Allogeneic
Lymphocytes
After intraperitoneal injections of PVG lymphocytes, high CTL
responses were detected in SLNs and MLNs in untreated and clodronate
liposome–treated rats (Table 2)
. Also, the generation of complement-dependent antibodies was not
downregulated by subconjunctival injections of clodronate liposomes in
systemically immunized rats (Table 3) .
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Discussion |
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The present study determined the influence of macrophage depletion on the presence of CTLs and antibodies early after transplantation. In the first week after transplantation, no significant difference was found between the two treatment groups, but when the onset of graft rejection occurred in the untreated rats (on POD 12) and on POD 17, the CTL activity detected locally and systemically was significantly less in the treated group. Generation of antibodies also seemed to be impaired in macrophage-depleted rats.
In our model, graft rejection seemed to correlate with increase of CTL activity in the local lymph nodes and the spleen on POD 12 and 17. Although corneal graft rejection is possible in the absence of CD8+ CTLs,14 15 it has been shown that rejection can be accompanied by high CTL responses in local lymph nodes or spleen.16 17 18 These CTL activities are detected by an in vitro assay. How these specific CTLs directed toward donor antigens are induced remains speculative, because corneal grafts are thought to be free of antigen-presenting cells. Therefore, the direct route seems improbable. The generation of CTL activity probably occurs mostly through the indirect pathway. It has been shown that preculturing is mandatory to measure CTL responses in these rats.18
Van der Veen et al.18 tested the same locations for presence of cytotoxic activity toward donor antigens in macrophage-depleted rats after the onset of rejection had occurred. Although no difference was detected in local CTL activity, macrophage depletion downregulated CTL activity in the MLNs as well as the spleen. The results, obtained from both local and more remote lymph nodes, are in slight contrast with findings in the current study. Also the difference found between the two groups was more pronounced in the current study. A possible explanation for these findings may be the use of a different rat strain combination and/or the induction of keratitis before transplantation by van der Veen et al. The eight clodronate liposome–treated allogeneic grafts performed in our experiment 1 demonstrated that administration of clodronate liposomes is also effective in prolonging graft survival in this rat strain combination for at least 100 days. Although the CTL response was more vigorous in these untreated rats, it was still downregulated and graft rejection prevented by macrophage depletion. Early rejection after small bowel allotransplantation is also characterized by a massive influx of macrophages.19 In this type of solid graft rejection the macrophages seem to be mandatory as well, because intraperitoneal injection of clodronate liposomes prolonged graft survival.20 Although the precise role of the macrophage in the graft rejection process is unknown, their presence is required for inducing donor-directed cytotoxicity responses by intestinal epithelial cells.21
Another effect of the clodronate liposomes was inhibition of antibody production. Our model showed that in untreated rats, antibodies against donor antigens were detectable 5 days after the onset of graft rejection using an immunofluorescence assay. Unfortunately, it was not possible technically to perform an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, but because the results of the chromium-release assay show that these antibodies were not complement dependent, these results indirectly suggest that they may be cell-dependent cytotoxic antibodies. Van der Veen et al.18 also showed that macrophage depletion impairs generation of complement-dependent antibodies after corneal transplantation. Lung allotransplantation leads to production of both types of antibodies. Depleting donor macrophages before transplantation by intratracheal instillation of clodronate liposomes does not prolong graft survival, but the antibodies involved, both in ADCC and complement-dependent cytotoxicity are significantly decreased.22 Also the presence of these antibodies is found in chronic rejection of cardiac allografts and is related to activation of macrophages.23
As mentioned earlier, corticosteroids have been found to be very effective in preventing graft rejection, but aside from the fact that not all rejections can be inhibited, this drug also has many side effects. In the attempt to find new therapeutic regimens, an important goal is the absence of systemic side effects. A major concern about the mechanism by which clodronate liposomes work could be that macrophage depletion was accomplished not only locally but also systemically. Experiment 3 showed that subconjunctival injection of clodronate-containing liposomes, using the same procedure as was used after corneal transplantation, did not interfere with the cellular and humoral immune response in rats injected intraperitoneally with allogeneic lymphocytes. Probably only the conjunctival macrophages are depleted with this procedure. Van Klink et al.2 showed that in Chinese hamsters, repeated subconjunctival injections with clodronate liposomes led to depletion of acid phosphatase–positive macrophages in the conjunctiva. By contrast, the number of acid-phosphatase–positive cells in the corneal epithelium, stroma, ciliary body, and iris was not affected.24 An explanation for this could be that redistribution of liposomes after local injection is hindered by the anatomic features of the cornea.25
Another point for concern could be that liposome treatment affects not only the macrophage population but also the dendritic cell population. No experimental studies have been published so far in which the number of dendritic cells or the function of the dendritic cell population is measured after subconjunctival injections of clodronate liposomes. It has been shown, however, that after intravenous injections of clodronate liposomes the number and distribution of dendritic cells in the spleen is not affected. Also the antigen-presenting cell activity of splenocytes and lymph node cells, measured by in vitro primary CTL responses to ovalbumin or mixed lymphocyte reactions to allogeneic antigens, were comparable in clodronate liposome–treated and untreated mice.26 27 After intraperitoneal injections with clodronate liposomes, these mice showed similar flow cytometry analysis data for dendritic cells as the control PBS-liposome treated group.28
Using the corneas from the rats used in this study, Torres et
al.29
determined the effect of administration of local
clodronate liposome on cytokine mRNA expression within the cornea.
In the macrophage-depleted rats, decreased expression of interleukin
(IL)-1ß, IL-2, IL-4, IL-6, interferon (IFN)-
, and tumor necrosis
factor (TNF)-ß/lymphotoxin (LT) mRNA was found. Treatment
with clodronate liposomes after corneal grafting thus resulted in
decreased local mRNA expression of several macrophage-derived cytokines
and T-cell derived cytokines and reduced levels of CTLs and antibodies.
The mechanism by which the clodronate liposomes induce immunosuppression remains highly speculative. From the fact that clodronate liposomes interfere with only the macrophage population, several hypotheses can be proposed. Because no effector mechanisms, such as CTLs and antibody production, are present, it could be concluded that there is an interruption in the afferent arc of the immune response leading to graft rejection. Macrophages can display a variety of functions that can be immunosuppressive or stimulatory. They are able to present antigen, but less so than dendritic cells or Langerhans’ cells.27 Our hypothesis is that macrophages interact with dendritic cells, and the antigen-presenting function of this latter cell is therefore enhanced. Interaction of lung macrophages with dendritic cells to present antigenic peptides has been reported. Depending on the ratio of interstitial macrophages to dendritic cells, the dendritic cell function is either augmented or inhibited.30 Alternatively, macrophages can mediate with the influx of T cells through upregulation of adhesion molecules or production of cytokines by the macrophage population, T cells are attracted to enter the inflammatory site. Fox et al.26 showed that macrophage depletion after xenografting in nonobese diabetic mice leads to reduced graft infiltration by T lymphocytes and eosinophils without directly affecting the function of the T cells or the dendritic cells. Van der Veen et al.18 also showed an decreased influx of immunocompetent cells into the graft of clodronate-treated rats after corneal allotransplantation and the expression of intercellular adhesion molecule (ICAM)-1 and leukocyte function–associated antigen (LFA)-1 is significantly reduced in these clodronate liposome–treated rats compared with untreated rats.18 31
At this moment a study is in progress to determine the delayed-type hypersensitivity responses of clodronate-treated animals after orthotopic corneal allotransplantation. Results of this study will resolve the question of whether antigen presentation has occurred and the effector phase of rejection has started in these clodronate liposome–treated animals.
Our study shows that after corneal allotransplantation in untreated rats, high CTL responses are detected locally and systemically, as well as complement-independent antibodies at time of rejection. We conclude that macrophage depletion, through repeated subconjunctival injections of clodronate liposomes, downregulates these high CTL responses and impairs antibody formation.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 5, 1999; revised January 11, 2000; accepted January 31, 2000.
Commercial relationships policy: N.
Corresponding author: Tanja P. A. M. Slegers, University Hospital Groningen, Department of Ophthalmology, PO Box 30001, 9700 RB Groningen, The Netherlands. t.slegers{at}ohk.azg.nl
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, T helper 1/T helper 2 cytokines, IgG subclasses, and pathology in a rat model of lung transplantation J Immunol 159,4084-4093[Abstract]
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