September 1999
Volume 40, Issue 10
Free
Cornea  |   September 1999
Increase in Orthotopic Murine Corneal Transplantation Rejection Rate with Anterior Synechiae
Author Affiliations
  • Satoru Yamagami
    From the Department of Ophthalmology, Jichi Medical School, Tochigi, Japan.
  • Tadahiko Tsuru
    From the Department of Ophthalmology, Jichi Medical School, Tochigi, Japan.
Investigative Ophthalmology & Visual Science September 1999, Vol.40, 2422-2426. doi:
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Satoru Yamagami, Tadahiko Tsuru; Increase in Orthotopic Murine Corneal Transplantation Rejection Rate with Anterior Synechiae. Invest. Ophthalmol. Vis. Sci. 1999;40(10):2422-2426.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

purpose. To evaluate the immunologic effect of anterior synechiae (AS) in a murine model of corneal transplantation.

methods. Orthotopic penetrating keratoplasty with 12 interrupted sutures was performed on C57BL/6 donor mice and BALB/c recipient mice without AS (AS− group). In contrast to suturing in the AS− group, 3 of the 12 sutures were placed to create AS (AS+ group). The average graft opacity scores and rejection rates of both groups were compared. Cytotoxic T-lymphocyte (CTL) reactions and delayed hypersensitivity (DH) were evaluated 3 weeks after transplantation. Corneal cytokine expression was evaluated.

results. The opacity scores of the AS+ group were consistently greater than those of the AS− group, and the rejection rate of the AS+ group was significantly greater than that of the AS− group (86% versus 54%, P = 0.03). The AS+ group had significantly higher CTL activity compared with the AS− group. There was no significant difference in DH between the two groups. The cytokine expression pattern in the AS+ group became similar to that of the AS− group in which the grafts were rejected.

conclusions. These findings indicate that AS impairs ocular immune privilege by mediating CTL activity, but without intensifying the DH response. Therefore, AS is a critical risk factor in allograft rejection in a murine model of corneal transplantation.

The ocular anterior segment is considered to have immunologic privilege. The cornea in particular has several advantages: lack of vessels, a deficiency of antigen-presenting cells in the central cornea, immunosuppressive activity by corneal fibroblasts and the endothelium, and Fas ligand expression that suppresses immunoreaction. 1 Because of these unique immunologic properties, the success rate of human corneal transplantation, compared with other types of vascularized organ transplants, is high in a normal corneal bed. 2  
Allograft rejection of corneal transplants, however, is a critical unresolvable problem, particularly in high-risk patients with vascularized corneas, previously failed corneal grafts, or both. 2 3 Anterior synechiae (AS) of the iris to the corneal endothelium is also a complication that often occurs after corneal transplantation. Two decades ago, the presence of AS was reported as a possible risk factor for rejection in human corneal transplantation. 4 5 6 7 However, whether or not AS is a real risk factor in corneal allografts and how it modulates the anterior segment immune reaction and cellular immunity, including cytokine expression, have not been fully addressed. 
The cytokine network, which regulates the immune reaction, is a critical factor in determining immunologic properties. Cytokines, produced by CD4+ T cells, are divided into T helper (Th)1 (interleukin [IL]-2, interferon-gamma [IFN-γ], and IL-12) and Th2 (IL-4, IL-5, IL-6, and IL-10) groups. These cytokines affect T-cell–mediated immune responses and cross-regulate each other. In corneal allorejection, a predominance of Th1 cytokine expression in cells infiltrating the graft has been reported in murine models. 8 9  
We developed a new AS mice corneal transplantation model and investigated the local and systemic immunologic properties of AS, including cytokine expression. We showed that AS is a risk factor for rejection in corneal transplants and that AS elicits cytotoxic T-lymphocyte (CTL) activity without intensifying delayed hypersensitivity (DH). 
Materials and Methods
Animals
Inbred strains of male BALB/c (H-2d) mice (Clea Japan Co., Tokyo, Japan) were the recipients of the grafts. C57BL/6 (H-2b) and BALB/c mice were the respective donors of the allografts and isografts. The major and minor histocompatibility antigens differ in the BALB/c and C57BL/6 strains. Male mice 8 to 14 weeks of age were used in all experiments. All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Surgical Technique of Orthotopic Corneal Transplantation
Orthotopic penetrating keratoplasty was performed as described previously with some modification. 8 Briefly, corneas of the mydriated recipients were marked with a trephine and excised with microscissors. Donor corneas excised with a 2.0-mm trephine (Inami, Tokyo, Japan) were transplanted to the same size recipient corneas, and 12 interrupted 11-0 nylon sutures (Mani, Tochigi, Japan) were placed without AS (AS− group). To create the AS (AS+ group), the last three sutures were passed through the iris and the cornea in three quadrants. The corneal sutures were removed 8 days after surgery. Grafts with an opacity score of 3+ or greater at suture removal were eliminated from the study because of the development of postoperative complications. When AS was absent in eyes in the AS+ group and vice versa, the eyes were excluded. 
Definition of Rejection and Clinical Observation
The corneal grafts were observed weekly for 8 weeks under an operating microscope. Graft opacity was scored from 0 to 5+ based on the following criteria 10 : 0, clear; 1+, some opacity at the graft margin or superficial edema; 2+, iris vessels readily visible; 3+, some iris vessel discernible; 4+, only pupil margin visible; and 5+, anterior chamber not visible. Corneal grafts were considered to be rejected when the opacity scores were greater than 2+. 
Histologic Evaluation
Some of the grafted mice were killed for histologic study 3 weeks after surgery. The enucleated eyes were fixed in 4% paraformaldehyde and then stained with hematoxylin and eosin for light microscopy. 
Immunohistochemical Study
The biotin-conjugated monoclonal antibodies (mAbs) used for immunostaining were anti-mouse IFN-γ (XMG1.2, 4 μg/ml; PharMingen, San Diego, CA), anti-mouse IL-2 (JES6-5H4, 4 μg/ml; PharMingen), anti-mouse CD4 (H129.19, 4 μl/ml; PharMingen), and anti-mouse CD8a (53-6.7, 4 μg/ml; PharMingen). The immunoperoxidase technique was performed as follows. Frozen specimens (7 μm) were sectioned in a cryostat, then fixed in acetone for 10 minutes, and washed with phosphate-buffered saline (PBS). The mAbs were applied overnight, after which the mAb-labeled sections were exposed for 20 minutes to horseradish peroxidase–labeled streptavidin. The sections were incubated for 1 minute in diaminobenzidine and then stained with Mayer’s hematoxylin for 10 seconds. Biotin-conjugated rat IgG1 and IgG2b (PharMingen) were used for the negative control study. Positive cells were scored on a scale of 0 to 4+ per 100× high power field: +, 1 to 10 cells; ++, 11 to 20 cells; +++, 21 to 30 cells; and ++++, ≥ 31 cells. Counting and grading on the stained slides were limited to the central zone of each graft and host area to avoid the host–graft junction area. The gradings were the averages obtained from three separate compilations of the numbers of positive cells performed during immunohistochemical study. 
Assay for DH
DH responses to alloantigens were determined by measuring ear swelling. B6 splenocytes were irradiated with 30 Gy, resuspended at a concentration of 1 × 106 cells in 10 μl, and injected into the right pinnae. PBS was injected into the left pinnae. Naive mice and mice immunized by subcutaneous injection of 1 × 107 B6 splenocytes into the back, the negative and positive controls, respectively, were used. After 24 hours, ear thickness was measured with a low-pressure micrometer (Mitsutoyo, Tokyo, Japan). DH-dependent ear swelling was calculated according to the following formula:  
\[\mathrm{Specific\ ear\ swelling{=}}\]
(24-hour measurement of right ear 
−0-hour measurement of right ear) 
−(24-hour measurement of left ear 
−0-hour measurement of left ear)×10−3 mm. 
CTL Assay
Recipient mice were killed, and their spleens and draining lymph nodes (LNs) were removed. Cervical LNs from the same side as the corneal graft were harvested as draining LNs. Single-cell suspensions from 5 × 106 splenocytes and 4 × 106 LN cells were restimulated for 3 days, respectively, with irradiated (30 Gy) 5 × 106 and 4 × 106 splenocytes from the donor syngeneic mice. Draining LNs were recovered from four to six mice per group and processed together. Naive mice LNs and splenocytes were the negative controls. Allospecific CTLs were assayed with a 4-hour chromium-51 (51Cr) release assay using donor syngeneic EL-4 (H-2b) tumor target cells. Maximum and spontaneous 51Cr release rates, respectively, were determined by measuring the amount of 51Cr released into the medium by 51Cr-labeled targets incubated with 1% Nonidet P-40 and into the medium without effector cells. The percentage of cytotoxicity was calculated according to the following formula:  
\[\mathrm{\%\ Specific\ }^{\mathrm{51}}\mathrm{Cr\ release{=}(experimental\ release\ cpm}\]
−spontaneous release cpm) 
÷(maximum release cpm 
−spontaneous release cpm)×100. 
Statistical Analysis
The Mann–Whitney U test was used to compare the opacity scores and the DH, and the Student’s t-test was used to compare the CTLs. Graft rejection rates were compared using Fisher’s exact method. P < 0.05 was considered significant. 
Results
Clinical Course
The isografts with AS (n = 10) remained clear during the postoperative observation period (data not shown). All the allografts with or without AS became clear before and after suture removal. Figures 1 A and 1B show anterior segment photographs with or without AS on postoperative day 10. Unlike the round pupil in the AS− group, the iris in the AS+ group was connected to the cornea. In the AS+ group, mild vascular invasion of the cornea was observed around the iris synechiae. Figure 2 shows the average postoperative corneal graft opacity scores of both groups. In the AS− group, 50% (14/28) of the corneal grafts had stromal opacity with edema and were considered rejected (AS−Rej group), whereas 50% of the corneal grafts remained transparent 3 weeks after surgery (AS−Acc group). However, 81% (17/21) of the corneas in the AS+ group had graft opacity scores higher than 2+. The mean opacity scores of the AS+ group were consistently higher than those of the AS− group throughout the observation period. At the last observation (8 weeks), the recipient grafts had opacity scores of 3+ or more in 54% (15/28) of the AS− group and 86% (18/21) in the AS+ group (P = 0.03). 
Histologic Evaluation
Many mononuclear cells infiltrated both the host and the graft, and the corneal grafts were edematous in both the AS+ and AS−Rej groups 3 weeks after transplantation. In the AS+ group in which the grafts were successful, many mononuclear cells were observed at the site of AS. In contrast, some infiltrating cells were present, but graft edema was not evident in the AS−Acc group (data not shown). 
Immunohistochemical Study of the Cornea
Table 1 shows the average number of cytokine-positive, CD4+ and CD8+ cells in the host and graft observed by microscopy 3 weeks after surgery. In the AS+Rej and AS−Rej groups, there were numerous CD4+ and CD8+ cells in both the host and the graft of the cornea, with more CD8+ cells in the graft detected in the AS+Rej group. Th1 cytokine–positive cells were present in the host and the graft in the AS−Acc group, but there were fewer cells than in the AS+Rej, AS+Acc, and AS−Rej groups. 
DH Assay
In the AS− group, DH responses to the donor alloantigens correlated well with the determination of rejection or acceptance of the grafts during the third week after surgery (Fig. 3) . There was a significant difference between the AS−Rej and AS−Acc groups (P < 0.01). Some mice in the AS+ group in which the corneal grafts were rejected, however, had lower DH responses to donor alloantigens. No significant differences in DH responses to donor alloantigens were found between the AS− (both the rejected and accepted groups) and AS+ groups. 
CTL Assay
Allospecific CTL responses during the third week are shown in Figure 4 . Splenocytes of the AS+ group had greater CTL responses compared with the AS− group (effector/target ratio; 50, 100, P < 0.05) and the normal control groups (all effector/target ratios, P < 0.05) (Fig. 4A) . There were no significant differences between the AS− Acc and AS+Rej groups (data not shown) or between the AS− and normal control groups in the splenocytes. CTL activity of the draining LNs in the AS+ group was higher than in the AS−Rej and AS−Acc groups. (Fig. 4B) . Data are representative of two experiments. 
Discussion
In this strain combination of a murine corneal transplantation model, the disparity in minor histocompatibility, rather than the major histocompatibility complex (MHC) alloantigen, is a greater barrier to allograft acceptance. 11 Moreover, DH responses, rather than CTL activities, were closely associated with the incidence of allograft rejection. 12 These unique characteristics, distinct from other vascularized organ transplants, may be correlated with immune privilege and are the reasons why further immunologic mechanisms must be determined. 
We analyzed immunologic properties 3 weeks after transplantation, the critical point in determining the fate of a corneal graft 10 and for clarifying dynamic cellular immunity. 12 Our findings clearly show that the presence of AS increased the rejection rate in the murine corneal transplantation model and that enhanced rejection was mediated by CTL activity. Allorejection in corneal transplantation is Th1 cytokine predominant; Th2 cytokines do not have a major role in allograft rejection. 8 9 In the present study, the corneal Th1 cytokine expression pattern in the AS+ group became the same as that of the rejected group without AS, indicating that AS increases the probability of having the same cytokine expression pattern as in the AS−Rej group in the cornea. Although both the DH response and CTL activity are mediated by Th1 cytokines, 13 these cytokines in the AS+ group were associated with upregulation of CTL activity rather than the DH response, as indicated by the low DH response in some of the mice with AS in which the graft was rejected. 
It is unclear why AS, which adds another pathway for presenting alloantigens to recipient T cells, enhances CTL activity alone. The cornea forms the anterior border of the ocular anterior chamber, and antigens may be introduced only through the corneal limbus and aqueous humor in normal eyes. Contact with a donor cornea by recipient iris vessels in the AS+ group, however, may have increased the chance of allorecognition by MHC class Ι–restricted CD8+ lymphocytes and elicited CTL activity by using donor-derived MHC classΙ antigen expression on the corneal graft. The greater CD8+ T-cell infiltration in the graft supports the enhancement of CTL activity in the AS+ group. Moreover, compared with the clear graft found in most of the AS− group 2 weeks postoperatively, the high opacity score of the grafts in the AS+ group may indicate that the vascular-rich iris induces allorecognition, which leads to shortening of the time from transplantation to rejection. 
A similar high-risk model of corneal transplantation, a prevascularization model of the graft bed, has been reported. 14 Interestingly, this model enhanced both CTL in the draining LNs and DH responses 2 weeks after grafting. 14 15 The actual mechanisms by which this prevascularized model and our AS model show different cell-mediated immune responses are not known. In the prevascularized model, allorecognition through preexisting vessels around the corneal limbus may accelerate cell-mediated immunity, both the CTL and DH responses, and easily override ocular immune privilege. 
In conclusion, AS is a critical risk factor for allograft rejection in murine corneal transplantation because it intensifies CTL activity, not the DH response. Corneal cytokine expression patterns in the AS+ group became similar to those in the rejected allograft group without AS. 
 
Figure 1.
 
Photographs show the absence or presence of anterior synechiae (AS) on day 10 after transplantation. (A) A round pupil without AS. (B) Two areas of AS (arrows). A portion of the AS has been removed.
Figure 1.
 
Photographs show the absence or presence of anterior synechiae (AS) on day 10 after transplantation. (A) A round pupil without AS. (B) Two areas of AS (arrows). A portion of the AS has been removed.
Figure 2.
 
Average graft opacity scores of mice with and without anterior synechiae (AS) that underwent engraftment. Scores of the AS+ group are higher than those of the AS− group. Significant statistical differences between the AS+ and AS− groups are shown for opacity scores at 2, 3, 6, 7, and 8 weeks. The presence of AS increased the opacity score of the corneal grafts.
Figure 2.
 
Average graft opacity scores of mice with and without anterior synechiae (AS) that underwent engraftment. Scores of the AS+ group are higher than those of the AS− group. Significant statistical differences between the AS+ and AS− groups are shown for opacity scores at 2, 3, 6, 7, and 8 weeks. The presence of AS increased the opacity score of the corneal grafts.
Table 1.
 
Immunohistochemical Study of Corneal Grafts 3 Weeks after Surgery
Table 1.
 
Immunohistochemical Study of Corneal Grafts 3 Weeks after Surgery
Group AS+Rej AS+Acc AS−Rej AS−Acc
Host Graft Host Graft Host Graft Host Graft
CD4+ +++ ++++ ++ +++ +++ ++++ ++ ++
CD8+ ++ +++ ++ ++ ++ ++ + +
IFN-γ +++ ++++ ++ ++ +++ ++++ + +
IL-2 +++ ++++ ++ +++ +++ ++++ ++ ++
Figure 3.
 
Delayed hypersensitivity responses in the anterior synechiae AS+ and AS− groups 3 weeks after surgery. Clinical scores of 3, 4, and 5 indicate rejection of the grafts. All the operations were performed on 2 consecutive days. Mice immunized with donor syngeneic splenocytes and naive mice were the positive and negative controls, respectively. Ear thickness was measured 24 hours after ear challenge with donor-syngeneic, irradiated splenocytes. The correlation between the clinical opacity score and ear swelling shows a significant difference between the AS−Rej and the AS−Acc groups (P < 0.01). Some mice in the AS+ group had low DH responses together with clinical scores of 3, 4, and 5 that indicate rejection of the grafts. There is no significant difference between the AS+ and AS− groups.
Figure 3.
 
Delayed hypersensitivity responses in the anterior synechiae AS+ and AS− groups 3 weeks after surgery. Clinical scores of 3, 4, and 5 indicate rejection of the grafts. All the operations were performed on 2 consecutive days. Mice immunized with donor syngeneic splenocytes and naive mice were the positive and negative controls, respectively. Ear thickness was measured 24 hours after ear challenge with donor-syngeneic, irradiated splenocytes. The correlation between the clinical opacity score and ear swelling shows a significant difference between the AS−Rej and the AS−Acc groups (P < 0.01). Some mice in the AS+ group had low DH responses together with clinical scores of 3, 4, and 5 that indicate rejection of the grafts. There is no significant difference between the AS+ and AS− groups.
Figure 4.
 
Cytotoxic T-lymphocyte (CTL) responses 3 weeks after transplantation. Effector cells derived from BALB/c mice (H-2d) engrafted with a corneal from C57BL/6 (H-2b) (B6) mice were stimulated for 3 days with B6-irradiated splenocytes. A 4-hour 51Cr release assay with EL-4 (H-2b) as the target cells at the indicated effector:target ratios was performed. Mice immunized with a 1 × 107 subcutaneous injection of B6 splenocytes and naive mice were the positive and negative controls, respectively. Results are given as the mean percentage of specific lysis (error bars, ±SEM). Spontaneous release was less than 10% of the maximum release. (A) Splenocytes used as the effector cells. The anterior synechiae (AS) group shows significantly greater cytotoxicity against the donor syngeneic alloantigen than do the mice without AS (effector:target ratios: 50, 100, P < 0.05) and normal control mice (all effector:target ratios, P < 0.05). There is no significant difference between AS−Rej and AS−Acc groups (data not shown). Ten mice constituted the AS− group; four to six mice constituted the other groups. One of two representative data sets is shown. (B) Draining lymph nodes (DLNs) used as effector cells with a group of four to six mice. LNs of mice with AS (AS+) and mice without AS in which the grafts were rejected (AS−Rej) have greater CTL activity than mice without AS in which the grafts were successful (AS−Acc). One of two representative data sets is shown.
Figure 4.
 
Cytotoxic T-lymphocyte (CTL) responses 3 weeks after transplantation. Effector cells derived from BALB/c mice (H-2d) engrafted with a corneal from C57BL/6 (H-2b) (B6) mice were stimulated for 3 days with B6-irradiated splenocytes. A 4-hour 51Cr release assay with EL-4 (H-2b) as the target cells at the indicated effector:target ratios was performed. Mice immunized with a 1 × 107 subcutaneous injection of B6 splenocytes and naive mice were the positive and negative controls, respectively. Results are given as the mean percentage of specific lysis (error bars, ±SEM). Spontaneous release was less than 10% of the maximum release. (A) Splenocytes used as the effector cells. The anterior synechiae (AS) group shows significantly greater cytotoxicity against the donor syngeneic alloantigen than do the mice without AS (effector:target ratios: 50, 100, P < 0.05) and normal control mice (all effector:target ratios, P < 0.05). There is no significant difference between AS−Rej and AS−Acc groups (data not shown). Ten mice constituted the AS− group; four to six mice constituted the other groups. One of two representative data sets is shown. (B) Draining lymph nodes (DLNs) used as effector cells with a group of four to six mice. LNs of mice with AS (AS+) and mice without AS in which the grafts were rejected (AS−Rej) have greater CTL activity than mice without AS in which the grafts were successful (AS−Acc). One of two representative data sets is shown.
Streilein JW, Ksander BR, Taylor AW. Immune deviation in relation to ocular immune privilege. J Immunol. 1997;158:3557–3560. [PubMed]
Price FW, Whitson WE, Marks RG. Graft survival in four common groups of patients undergoing penetrating keratoplasty. Ophthalmology. 1991;98:322–328. [CrossRef] [PubMed]
Williams KA, Roder D, Esterman A, Muehlberg SM, Coster DJ. Factors predictive of corneal graft survival. Ophthalmology. 1992;99:403–414. [CrossRef] [PubMed]
Bourne WM. Reduction of endothelial cell loss during phakic penetrating keratoplasty. Am J Ophthalmol. 1980;89:787–790. [CrossRef] [PubMed]
Cherry PMH, Pashby RC, Tadros ML, et al. An analysis of corneal transplantation: 1-graft clarity. Ann Ophthalmol. 1979;11:461–469. [PubMed]
Tragakis MP, Brown SI. The significance of anterior synechiae after corneal transplantation. Am J Ophthalmol. 1972;74:532–533. [CrossRef] [PubMed]
Smolin G, Biswell R. Corneal graft rejection associated with anterior iris adhesion: case report. Ann Ophthalmol. 1978;10:1603–1604. [PubMed]
Yamagami S, Kawashima H, Endo H, et al. Cytokine profiles of aqueous humor and graft in orthotopic mouse corneal transplantation. Transplantation. 1998;66:1504–1510. [CrossRef] [PubMed]
Sano Y, Osawa H, Sotozono C, Kinoshita S. Cytokine expression during orthotopic corneal allograft rejection in mice. Invest Ophthalmol Vis Sci. 1998;39:1953–1957. [PubMed]
Yamagami S, Kawashima H, Tsuru T, et al. Role of Fas-Fas ligand interactions in the immunorejection of allogeneic mouse corneal transplantation. Transplantation. 1997;27:1107–1111.
Sonoda Y, Streilein JY. Orthotopic corneal transplantation in mice: evidence that the immunogenetic rules of rejection do not apply. Transplantation. 1992;54:694–703. [CrossRef] [PubMed]
Joo CK, Pepose JS, Stuart PM. T-cell mediated responses in a murine model of orthotopic corneal transplantation. Invest Ophthalmol Vis Sci. 1995;36:1530–1540. [PubMed]
Mosmann DA, Coffman RL. Heterogeneity of cytokine secretion patterns and functions of helper T cells. Adv Immunol. 1989;46:111–147. [PubMed]
Sano Y, Ksander BR, Streilein JW. Fate of orthotopic corneal allografts in eyes that cannot support anterior chamber-associated immune deviation induction. Invest Ophthalmol Vis Sci. 1995;36:2176–2185. [PubMed]
Ksander BR, Sano Y, Streilein JW. Role of donor-specific cytotoxic T cells in rejection of corneal allografts in normal and high-risk eyes. Transplant Immunol. 1996;4:49–51. [CrossRef]
Figure 1.
 
Photographs show the absence or presence of anterior synechiae (AS) on day 10 after transplantation. (A) A round pupil without AS. (B) Two areas of AS (arrows). A portion of the AS has been removed.
Figure 1.
 
Photographs show the absence or presence of anterior synechiae (AS) on day 10 after transplantation. (A) A round pupil without AS. (B) Two areas of AS (arrows). A portion of the AS has been removed.
Figure 2.
 
Average graft opacity scores of mice with and without anterior synechiae (AS) that underwent engraftment. Scores of the AS+ group are higher than those of the AS− group. Significant statistical differences between the AS+ and AS− groups are shown for opacity scores at 2, 3, 6, 7, and 8 weeks. The presence of AS increased the opacity score of the corneal grafts.
Figure 2.
 
Average graft opacity scores of mice with and without anterior synechiae (AS) that underwent engraftment. Scores of the AS+ group are higher than those of the AS− group. Significant statistical differences between the AS+ and AS− groups are shown for opacity scores at 2, 3, 6, 7, and 8 weeks. The presence of AS increased the opacity score of the corneal grafts.
Figure 3.
 
Delayed hypersensitivity responses in the anterior synechiae AS+ and AS− groups 3 weeks after surgery. Clinical scores of 3, 4, and 5 indicate rejection of the grafts. All the operations were performed on 2 consecutive days. Mice immunized with donor syngeneic splenocytes and naive mice were the positive and negative controls, respectively. Ear thickness was measured 24 hours after ear challenge with donor-syngeneic, irradiated splenocytes. The correlation between the clinical opacity score and ear swelling shows a significant difference between the AS−Rej and the AS−Acc groups (P < 0.01). Some mice in the AS+ group had low DH responses together with clinical scores of 3, 4, and 5 that indicate rejection of the grafts. There is no significant difference between the AS+ and AS− groups.
Figure 3.
 
Delayed hypersensitivity responses in the anterior synechiae AS+ and AS− groups 3 weeks after surgery. Clinical scores of 3, 4, and 5 indicate rejection of the grafts. All the operations were performed on 2 consecutive days. Mice immunized with donor syngeneic splenocytes and naive mice were the positive and negative controls, respectively. Ear thickness was measured 24 hours after ear challenge with donor-syngeneic, irradiated splenocytes. The correlation between the clinical opacity score and ear swelling shows a significant difference between the AS−Rej and the AS−Acc groups (P < 0.01). Some mice in the AS+ group had low DH responses together with clinical scores of 3, 4, and 5 that indicate rejection of the grafts. There is no significant difference between the AS+ and AS− groups.
Figure 4.
 
Cytotoxic T-lymphocyte (CTL) responses 3 weeks after transplantation. Effector cells derived from BALB/c mice (H-2d) engrafted with a corneal from C57BL/6 (H-2b) (B6) mice were stimulated for 3 days with B6-irradiated splenocytes. A 4-hour 51Cr release assay with EL-4 (H-2b) as the target cells at the indicated effector:target ratios was performed. Mice immunized with a 1 × 107 subcutaneous injection of B6 splenocytes and naive mice were the positive and negative controls, respectively. Results are given as the mean percentage of specific lysis (error bars, ±SEM). Spontaneous release was less than 10% of the maximum release. (A) Splenocytes used as the effector cells. The anterior synechiae (AS) group shows significantly greater cytotoxicity against the donor syngeneic alloantigen than do the mice without AS (effector:target ratios: 50, 100, P < 0.05) and normal control mice (all effector:target ratios, P < 0.05). There is no significant difference between AS−Rej and AS−Acc groups (data not shown). Ten mice constituted the AS− group; four to six mice constituted the other groups. One of two representative data sets is shown. (B) Draining lymph nodes (DLNs) used as effector cells with a group of four to six mice. LNs of mice with AS (AS+) and mice without AS in which the grafts were rejected (AS−Rej) have greater CTL activity than mice without AS in which the grafts were successful (AS−Acc). One of two representative data sets is shown.
Figure 4.
 
Cytotoxic T-lymphocyte (CTL) responses 3 weeks after transplantation. Effector cells derived from BALB/c mice (H-2d) engrafted with a corneal from C57BL/6 (H-2b) (B6) mice were stimulated for 3 days with B6-irradiated splenocytes. A 4-hour 51Cr release assay with EL-4 (H-2b) as the target cells at the indicated effector:target ratios was performed. Mice immunized with a 1 × 107 subcutaneous injection of B6 splenocytes and naive mice were the positive and negative controls, respectively. Results are given as the mean percentage of specific lysis (error bars, ±SEM). Spontaneous release was less than 10% of the maximum release. (A) Splenocytes used as the effector cells. The anterior synechiae (AS) group shows significantly greater cytotoxicity against the donor syngeneic alloantigen than do the mice without AS (effector:target ratios: 50, 100, P < 0.05) and normal control mice (all effector:target ratios, P < 0.05). There is no significant difference between AS−Rej and AS−Acc groups (data not shown). Ten mice constituted the AS− group; four to six mice constituted the other groups. One of two representative data sets is shown. (B) Draining lymph nodes (DLNs) used as effector cells with a group of four to six mice. LNs of mice with AS (AS+) and mice without AS in which the grafts were rejected (AS−Rej) have greater CTL activity than mice without AS in which the grafts were successful (AS−Acc). One of two representative data sets is shown.
Table 1.
 
Immunohistochemical Study of Corneal Grafts 3 Weeks after Surgery
Table 1.
 
Immunohistochemical Study of Corneal Grafts 3 Weeks after Surgery
Group AS+Rej AS+Acc AS−Rej AS−Acc
Host Graft Host Graft Host Graft Host Graft
CD4+ +++ ++++ ++ +++ +++ ++++ ++ ++
CD8+ ++ +++ ++ ++ ++ ++ + +
IFN-γ +++ ++++ ++ ++ +++ ++++ + +
IL-2 +++ ++++ ++ +++ +++ ++++ ++ ++
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×