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The Journal of Immunology, 2003, 170: 5530-5537.
Copyright © 2003 by The American Association of Immunologists

Absence of Allograft ICAM-1 Attenuates Alloantigen-Specific T Cell Priming, But Not Primed T Cell Trafficking into the Graft, to Mediate Acute Rejection1

Qi-Wei Zhang*, Danielle D. Kish* and Robert L. Fairchild2,*,{dagger},{ddagger}

* Department of Immunology and {dagger} Urological Institute, Cleveland Clinic Foundation, Cleveland, OH 44195; and {ddagger} Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression and function of ICAM-1 are critical components in the initiation and elicitation of many T cell-mediated responses. Whether ICAM-1 expression is required on the T cells or on the APC during T cell priming remains unclear. To address this issue in alloantigen-specific T cell activation, the priming and function of T cells in response to heart allografts from MHC-mismatched wild-type vs ICAM-1-/- donors were tested. Wild-type C57BL/6 (H-2b) heart allografts were rejected by A/J (H-2a) recipients on days 7–9, whereas B6.ICAM-1-/- allografts survived until days 18–23 post-transplant. On day 7 post-transplant, infiltrating macrophages and CD4+ and CD8+ T cells in the ICAM-1-/- allografts were 20–30% those observed in the wild-type allografts. ELISPOT analyses indicated that the number of alloantigen-specific T cells producing IFN-{gamma} from recipients of ICAM-1-deficient grafts was 60% lower than that from recipients of wild-type allografts. On day 16 post-transplant, these numbers did not markedly increase in ICAM-1-deficient allograft recipients. Consistent with the reduced priming of alloreactive T cells, isolated dendritic cells from ICAM-1-/- mice stimulated allogeneic T cell proliferation poorly compared with wild-type dendritic cells. When A/J mice were primed with wild-type dendritic cells and then received wild-type or ICAM-1-deficient heart allografts 3 days later, the primed recipients rejected the wild-type and ICAM-1-/- allografts on days 5–6 post-transplant. These results indicate that optimal priming of alloreactive T cells requires allograft expression of ICAM-1, but, once primed, recipient T cell infiltration into the allograft is independent of graft ICAM-1 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Allogeneic organ transplantation stimulates a potent T cell response that, unless checked by immunosuppression, results in rejection of the allograft. The graft rejection process is initiated by the surgical trauma and ischemia/reperfusion injury imposed on transplanted organs that induce high levels of inflammation in the graft. There are two critical consequences of this tissue inflammation. First, the graft inflammation induces interstitial dendritic cells (DC)3 to emigrate from the graft and traffic to recipient lymphoid tissues draining the graft where the alloantigen-specific T cell response is primed (1, 2, 3). Second, the inflammation stimulates graft endothelium to up-regulate the expression of adhesion molecules and chemokines that direct recipient leukocytes, including alloantigen-primed T cells, into the allograft (4, 5, 6).

Similar to activation of other Ag-specific T cell responses, functional priming of alloantigen-specific T cells requires a complex series of interactions with alloantigen-presenting cells from the graft. These requirements include recognition of allogeneic MHC molecules or allogeneic peptide/self MHC complexes as well as the delivery of costimulatory signals to the T cell. A primary source of costimulation is provided through T cell-expressed CD28 interactions with B7 molecules on the APC (7, 8). Recently, several other costimulatory pairs have been reported that may also be required or may substitute for the absence of CD28-B7-mediated costimulation (9). Optimal activation of T cells in many responses may also require interaction of T cell-expressed LFA-1 with ICAM-1 or ICAM-2 molecules on the APC surface (10, 11, 12, 13, 14).

Acute allograft rejection is mediated by the coordinated infiltration of T cells into the allograft, followed by the expression of T cell effector functions that destroy the graft tissue (15, 16). Alloantigen-primed T cell trafficking to and through the vascular endothelium is mediated by endothelial cell-expressed adhesion molecules and chemokines. Recent studies using neutralizing Abs or genetically modified animals as graft donors or recipients have demonstrated a role for specific chemokines and/or receptors in directing Ag-primed T cells into allografts (17, 18, 19, 20). Similarly, the administration of adhesion molecule-specific Abs to recipients prolonged skin and heart allograft survival (21, 22, 23). Although the use of anti-ICAM-1 mAb was partially effective in extending skin and heart allograft survival, the combination of ICAM-1- and LFA-1-specific Abs was effective in promoting long term graft survival in many experimental models. In addition to their function in cell adhesion, both ICAM-1 and LFA-1 are signaling molecules, and the effects of specific Abs in blocking cellular interactions vs the delivery of negative signaling cannot be clearly distinguished in experiments using such Abs.

An alternative approach to directly testing the roles of specific signaling proteins in the immune response is using animals with a targeted gene deletion in the protein of interest. Initial studies testing the rejection of vascularized heart allografts from ICAM-1-/- donors indicated no difference in rejection time compared with rejection of grafts from wild-type donors (24). Another line of mice with a targeted deletion in ICAM-1 has been generated and tested in experimental models of inflammation where they have been found to be resistant to septic shock and renal ischemia/reperfusion injury (25, 26). These studies prompted us to investigate the roles of recipient and allograft ICAM-1 during the priming of alloreactive T cells and acute rejection of heart allografts using grafts from the latter ICAM-1 deficient donors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

A/J (H-2a), C57BL/6 (H-2b), and SJL (H-2s) mice were obtained through Dr. C. Reeder at the National Cancer Institute (Frederick, MD). ICAM-1-deficient mice on a C57BL/6 background (C57BL/6-Icam1tm1/cgr) were purchased from The Jackson Laboratory (Bar Harbor, ME). Adult males, 7–12 wk of age, were used throughout this study.

Antibodies

For immunohistological analyses the following Abs were used: GK1.5 (rat anti-mouse CD4) and 53-6.7 (rat anti-mouse CD8{alpha}) purchased from BD PharMingen (San Diego, CA); MOMA-2 (rat anti-mouse macrophage) purchased from BioSource International (Camarillo, CA); and biotinylated polyclonal rabbit anti-rat IgG Ab purchased from DAKO (Carpinteria, CA).

Heterotopic cardiac transplantation

Cardiac transplants were performed using the method of Corry and co-workers (27). Briefly, donor and recipient mice were anesthetized with phenobarbital. Donor hearts were harvested and placed in chilled lactated Ringer’s solution while the recipient was prepared. Total cold ischemic time was always <45 min during recipient preparation. The donor heart was anastomosed to the recipient abdominal aorta and vena cava using microsurgical techniques. Upon completion of the anastomosis and organ perfusion, the heart grafts resumed spontaneous contraction. The strength and quality of cardiac graft impulses were examined each day by palpation of the recipient abdomen. Rejection of cardiac grafts was considered complete by cessation of impulse and was confirmed visually for each graft by laparotomy. In A/J recipients, complete rejection of wild-type C57BL/6 cardiac grafts occurs 8–10 days after transplantation, and isografts function for >300 days.

Histology

For immunohistology, iso- and allogeneic heart grafts were retrieved on days 7 or 16 post-transplant, embedded in OCT compound (Sakura Finetek U.S.A., Torrance, CA), and frozen in liquid nitrogen. Sections (8 µm) were cut and mounted onto slides. Slides were dried overnight, fixed in acetone for 10 min, and air-dried. Slides were immersed in PBS for 10 min and then in 0.03% H2O2 for 10 min to eliminate endogenous peroxidase activity. The slides were then stained for 1 h at room temperature with GK1.5 or 53-6.7 diluted to 5 µg/ml in 0.05% Tris-HCl buffer with 1% BSA or with MOMA-2 diluted 1/5 in the buffer. Control slides were incubated with rat IgG as the primary Ab. After three 5-min washes in PBS, slides were incubated for 20 min at room temperature with biotinylated rabbit anti-rat IgG diluted 1/300 in PBS. After three washes in PBS, slides were incubated with streptavidin-HRP (DAKO) for 20 min at room temperature. The substrate-chromagen solution was prepared by dissolving a 10-mg 3,3'-diaminobenzidine tablet (Sigma-Aldrich, St. Louis, MO) in 15 ml of PBS and adding 12 µl of 30% H2O2 just before use. After three 5-min washes in PBS, the 3,3'-diaminobenzidine solution was applied to each slide and incubated for 3–7 min at room temperature. After a final wash in H2O, slides were counterstained with hematoxylin, rinsed, and immersed in 37 mM NH4OH for 10 s. The slides were dehydrated and viewed under light microscopy, and images were captured using ImagePro Plus (Media Cybernetics, Silver Spring, MD). The numbers of cells staining positively were counted in five random fields from three different tissue sections from three different grafts. The significance between the number of positively staining cells per field in wild-type vs ICAM-1-/- grafts was determined using Mann-Whitney U test.

Mixed lymphocyte reactivity

Priming of alloantigen-specific T cells from heart graft recipients was tested by performing short term MLR assays. The responder spleen cells from recipients on days 5 and 16 post-transplant were treated with Tris-NH4Cl for 2.5 min at room temperature to lyse erythrocytes, washed three times, and resuspended at 3 x 106 cells/ml in complete medium, RPMI (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (Sigma-Aldrich), 2 mM L-glutamine, 5 x 10-5 M 2-ME, 10 mM HEPES, and 20 µg/ml gentamicin, and 100-µl aliquots were delivered in triplicate to the wells of a 96-well, flat-bottom tissue culture plate. Stimulator cells were prepared from the spleens of syngeneic (i.e., A/J), allograft donor (i.e., C57BL/6), and third-party allogeneic SJL mice. The stimulator cells were treated with Tris-NH4Cl, washed three times, and then treated with 50 µg/ml mitomycin C for 30 min at 37°C. After washing, the cells were resuspended in culture medium at 6 x 106 cells/ml, and 100-µl aliquots were delivered to each well in the culture plates. After 56 h, cultures were pulsed with 0.25 µCi of [3H]thymidine, and 16 h later the cultures were harvested onto fiber filter mats, and the amount of 3H incorporation was determined by liquid scintillation counting.

ELISPOT assay

Priming of alloantigen-specific T cells from heart allograft recipients was also tested by enumerating IFN-{gamma}-producing T cells using ELISPOT assays as previously described (28). Briefly, ELISA spot plates (Unifilter350, Polyfiltronics, Rockland, MA) were coated with 2 µg/ml IFN-{gamma}-specific mAb and incubated overnight at 4°C. The plates were blocked with 1% BSA in PBS and then washed four times with PBS. Spleen cell suspensions from graft recipients were prepared on days 5 and 16 post-transplantation and used as responder cells. Spleen cells from A/J, C57BL/6, and SJL mice were prepared and treated with mitomycin C for use as stimulator cells in the assay as described above. Responder and stimulator cells were cultured in serum-free HL-1 medium (BioWhittaker, Walkersville, MD) supplemented with 1 mM L-glutamine. After 24 h of cell culture at 37°C in 5% CO2, cells were removed from the plate by extensive washing with PBS. Biotinylated anti-IFN-{gamma} mAb (2 µg/ml) was added, and the plate was incubated for 6 h at room temperature. The plate was washed three times with PBS/0.05% Tween 20, and streptavidin-conjugated alkaline phosphatase was added to each well. After 2 h at room temperature, the plates were washed with PBS, and nitro blue tetrazolium-5-bromo-4-cloro-3-indolyl substrate (Kirkegaard & Perry, Gaithersburg, MD) was added for the detection of IFN-{gamma}-producing cells. The resulting spots were counted with an ImmunoSpot Series I analyzer (Cellular Technology, Cleveland, OH) that was designed to detect ELISA spots with predetermined criteria for spot size, shape, and colorimetric density.

Preparation of bone marrow-derived DC

Bone marrow cells were flushed from the femurs of wild-type C57BL/6 mice and cultured for 5 days in medium containing 10 ng/ml GM-CSF and 10 ng/ml IL-4 to generate mature DC. After culture, DC were isolated by centrifuging the cells through a 14.5% metrizamide gradient. After washing three times, 2.5 x 106 interface cells were injected i.v. into each A/J mice, and 3 days later the mice were transplanted with wild-type C57BL/6 or ICAM-1-/- heart allografts.

Stimulatory capacity assay of APC

To directly test the stimulatory capacity of alloantigen-presenting cells from ICAM-1-deficient vs wild-type C57BL/6 mice, DC were isolated from split ear cultures following culture in RPMI for 48 h. Staining and flow cytometric analysis of the isolated cells from wild-type and ICAM-1-/- cultures indicated no difference in expression levels of I-Ab molecules (data not shown). The cells were irradiated (2500 rad) and used as stimulator cells in MLR assays with lymph node cells from C57BL/6 and A/J mice as responder T cells as detailed above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Survival of ICAM-1-deficient heart allografts

To begin to investigate the requirement for ICAM-1 in cardiac allograft rejection, the ability of A/J (H-2a) mice to reject heart allografts from wild-type C57BL/6 (H-2b) vs B6.ICAM-1-/- donors was compared. Consistent with previous studies using this model, wild-type allografts were rejected on days 8–9 post-transplant (Fig. 1a). In contrast, B6.ICAM-1-/- heart allografts were not rejected by A/J recipients until days 18–23 post-transplant. Wild-type C57BL/6 and B6.ICAM-1-/- mice were also used as recipients of A/J heart allografts, and each set of recipients rejected the allografts on days 8–9 post-transplant (Fig. 1b).



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  		FIGURE 1. Delayed rejection of MHC-mismatched cardiac allografts from ICAM-1-/- donors. a, Groups of A/J mice received cardiac allografts from wild-type C57BL/6 (n = 6) or B6.ICAM-1-/- (n = 6) donors. b, Groups (n = 6) of wild-type C57BL/6 and B6.ICAM-1-/- mice received MHC-mismatched cardiac allografts from A/J donors, and A/J mice received heart allografts from B6.ICAM-1-/- donors. Allograft rejection was confirmed visually in all recipients by laparotomy. Wild-type and ICAM-1-/- isografts were maintained >100 days.

 
On day 7 post-transplant, wild-type C57BL/6 and B6.ICAM-1-/- allografts were retrieved from A/J recipients and stained with specific Abs to assess graft infiltration by macrophages and CD4+ and CD8+ T cells. Typical of acute rejection of these allografts, intense macrophage, CD8+ T cell, and modest CD4+ T cell infiltration into wild-type C57BL/6 allografts was observed (Fig. 2A, a–c). In contrast, examination of ICAM-1-deficient allografts indicated low to absent infiltration by macrophages and CD8+ and CD4+ T cells (Fig. 2A, d–f). To support these histological findings, heart sections were examined, and the number of positive cells in at least eight random fields in three different sections using sections from at least five different hearts was counted (Fig. 3). The number of macrophage and CD4+ T cells infiltrating ICAM-1-deficient allografts was 20% that observed in wild-type grafts on day 7 post-transplant, and the number of CD8+ T cells infiltrating ICAM-1-deficient allografts was 30% that observed in wild-type allografts. However, at the time of ICAM-1-/- heart allograft rejection (e.g., day 20 post-transplant) there was no difference in the level of mononuclear cell graft infiltration compared with that in wild-type allografts on day 8 post-transplant (Fig. 2B). These results indicated that optimal T cell-mediated rejection of allogeneic heart grafts is dependent upon graft expression of ICAM-1.



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  		FIGURE 2. Histological analyses of cardiac allografts from wild-type vs ICAM-1-/- donors. Groups of A/J mice received cardiac allografts from wild-type C57BL/6 or B6.ICAM-1-/-donors. A, Wild-type C57BL/6 (a–c) and B6.ICAM-1-/- (d–f) allografts were retrieved on day 7 post-transplant. Frozen sections were prepared and stained with mAb to detect CD4+ T cells (a and d), CD8+ T cells (b and e), and macrophages (c and f). Representative areas of the slides are shown at x200 magnification. Intense macrophage, CD8+, and modest CD4+ T cell infiltration into wild-type C57BL/6 allografts was observed (a–c), whereas ICAM-1-deficient allografts had low to absent infiltration by macrophages, CD8+, and CD4+ T cells (d–f). B, A/J isografts and wild-type C57BL/6 heart allografts were retrieved on day 8 post-transplant, and B6.ICAM-1-/- allografts were retrieved on day 20 post-transplant. The grafts were fixed with formalin, and prepared 8-µm sections were stained with H&E. Magnification, x200.

 


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  		FIGURE 3. Mononuclear cell infiltration into cardiac allografts from wild-type vs ICAM-1-/- donors. Groups of A/J mice received cardiac allografts from wild-type C57BL/6 or B6.ICAM-1-/- donors, and the allografts were retrieved on day 7 post-transplant. Frozen sections were prepared and stained with specific Abs to detect macrophages and CD4+ and CD8+ T cells. The slides were viewed at x200 magnification, and the number of positively staining cells was counted in eight random fields of three different frozen sections from five different grafts. The numbers of graft-infiltrating CD4+ ({blacksquare}), CD8+ (), and macrophages ({square}) were significantly less (**, p < 0.0001; *, p < 0.0002; by Mann-Whitney U test) for each cell population in grafts from B6.ICAM-1-/- compared with cell infiltration into wild-type allografts.

 
Alloantigen priming of graft recipient T cells

The prolonged survival of ICAM-1-deficient heart allografts could be due to delayed priming of alloantigen-specific T cells by graft alloantigen-presenting cells and/or to the inability of primed T cells to infiltrate the allograft in the absence of ICAM-1. The priming of alloreactive T cells in A/J recipients of ICAM-1-deficient and wild-type heart allografts was examined 5 days after transplantation by performing short-term MLR assays. Splenocytes from A/J recipients of wild-type C57BL/6 allografts had high proliferative reactivity to C57BL/6 stimulator cells compared with the proliferative responses of splenic T cells from A/J recipients of ICAM-1-deficient and naive A/J mice (Fig. 4). Proliferative responses to the third-party SJL (H-2s) allogeneic stimulator cells were essentially equivalent to responses to syngeneic stimulator cells for each of the three groups of responder cells.



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  		FIGURE 4. Decreased alloantigen-specific T cell priming in recipients of ICAM-1-/- heart allografts. A/J mice received cardiac allografts from wild-type C57BL/6 or B6.ICAM-1-/- donors. On day 5 post-transplant, splenic T cells from allograft recipients and from naive A/J mice were prepared, and 3 x 105 cells were cultured with 6 x 105 mitomycin C-treated spleen cells from A/J ({square}), C57BL/6 (), or SJL ({blacksquare}) mice. After 48 h cultures were pulsed with 1 µCi of [3H]thymidine, and 16 h later the cultures were harvested onto fiber filter mats, and the amount of 3H incorporation was tested by liquid scintillation. The data are presented as the mean 3H incorporation ± SD and are representative of two individual experiments.

 
To further investigate the priming of alloreactive T cells in response to wild-type and ICAM-1-deficient heart allografts, the frequency of alloantigen-specific T cell development to IFN-{gamma}-producing cells in the recipients was tested by ELISPOT assay. On days 5 and 16 post-transplant, recipient cell suspensions were prepared and cultured with mitomycin C-treated spleen cells from A/J, C57BL/6, or SJL mice on anti-IFN-{gamma} mAb-coated wells. After 24 h of culture, the cells were removed, and the assay was developed to enumerate the number of IFN-{gamma}-producing cells. On day 5 post-transplant (2–3 days before completion of wild-type allograft rejection), the number of alloantigen-specific T cells primed to produce IFN-{gamma} in recipients of ICAM-1-deficient heart allografts was almost 3-fold less than the number in recipients of wild-type allografts (Fig. 5a). IFN-{gamma}-producing cells were not observed in cultures with syngeneic A/J splenocytes as stimulator cells, and the number in cultures with SJL spleen cell stimulators was <35 in all groups of responder T cells tested. On day 16 post-transplant, 3–4 days before completion of ICAM-1-deficient allograft rejection, the number of alloantigen-specific T cells primed to produce IFN-{gamma} was almost equivalent in recipients of ICAM-1-deficient and wild-type allografts (Fig. 5b). It should be noted, however, that the increase in numbers of IFN-{gamma}-producing T cells in recipients of ICAM-1-deficient grafts from day 5 to day 16 post-transplant was modest.



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  		FIGURE 5. Decreased development of alloantigen-specific T cells producing IFN-{gamma} in recipients of ICAM-1-/- cardiac allografts. A/J mice received cardiac allografts from wild-type C57BL/6 () or B6.ICAM-1-/- ({blacksquare}) donors. On days 5 (a) and 16 (b) post-transplant, recipient and naive A/J ({square}) spleen cell suspensions were prepared, and 2 x 105-cell aliquots were cultured with 6 x 105 mitomycin C-treated spleen cells from A/J, C57BL/6, or SJL mice on anti-IFN-{gamma}-coated wells. Two days later the cells were removed, and the assay was developed to determine the number of IFN-{gamma}-producing cells by ELISPOT assay. Detected spots were counted by computer-assisted image analysis and are expressed as the mean value of duplicate wells. No spots were observed for A/J stimulator cells, and the number of spots in cultures with SJL stimulator cells was <35 in all responder groups tested. The results are representative of two individual experiments.

 
The results from MLR and ELISPOT assays indicated suboptimal priming of alloantigen-specific T cells in recipients of ICAM-1-deficient allografts. To directly test the stimulatory capacity of APC from ICAM-1-deficient vs wild-type C57BL/6 mice, mature DC were isolated from 48-h split ear cultures. The cells were collected, irradiated (2500 rad), and used as stimulator cells in long term MLR assays with naive responder T cells from C57BL/6 and A/J mice. Although equivalent levels of I-Ab expression were observed, the DC isolated from ICAM-1-deficient mice were 8- to 10-fold less stimulatory in activating allogeneic A/J T cell proliferation than DC from wild-type mice (Fig. 6). It should be noted, however, that the proliferative responses of syngeneic T cells to ICAM-1-/- DC were 8- to 10-fold lower than the responses to wild-type, ICAM-1+/+ DC, and the stimulation indexes of allogeneic T cell proliferation to wild-type and ICAM-1 DC were similar.



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  		FIGURE 6. Decreased ability of DC from ICAM-1-/- vs wild-type C57BL/6 mice to stimulate allogeneic T cell proliferation. Split ears from wild-type C57BL/6 (a) and ICAM-1-/- (b) mice were cultured for 48 h. The cells that migrated out of the ears were collected, irradiated (2500 rad), and used as stimulator cells in mixed lymphocyte assays with lymph node cells from syngeneic, C57BL/6 ({diamond}) and allogeneic A/J ({square}) mice as responder T cells (5 x 105/well). After 72 h cultures were pulsed with 1 µCi [3H]thymidine, and 16 h later the cultures were harvested onto fiber filter mats, and the amount of 3H incorporation was tested by liquid scintillation. Numbers in parentheses indicate the stimulation index for each data point. Note the log difference in the scales for the alloreactive T cell proliferation stimulated by wild-type (a) and ICAM-1-/- (b) DC.

 
Primed recipient T cells do not require allograft expression of ICAM-1 to infiltrate the graft

The results of the previous experiments indicated the decreased stimulatory capacity of allogeneic DC that is consistent with the delayed T cell priming and rejection observed when B6.ICAM-1-/- heart allografts were transplanted to A/J recipients. The ability of graft alloantigen-primed T cells to infiltrate ICAM-1-deficient heart allografts remained unclear. To test alloantigen-primed T cell infiltration into ICAM-1-deficient allografts, A/J mice were primed with bone marrow-derived DC from wild-type C57BL/6 mice and 3 days later received a heart allograft from either a wild-type C57BL/6 or a B6.ICAM-1-/- donor. In contrast to unprimed A/J recipients of ICAM-1-deficient heart allografts, the allogeneic DC-primed A/J mice rejected the ICAM-1-deficient heart allografts at the same time as the wild-type allografts were rejected, days 5–8 post-transplant (Fig. 7a). Furthermore, similar levels of mononuclear cell infiltration into the allografts were observed at the time of rejection (day 5 post-transplant; Fig. 7b). These results indicate that once primed, alloreactive T cells do not require allograft expression of ICAM-1 for infiltration into the graft parenchyma and acute rejection.



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  		FIGURE 7. Primed alloreactive T cells infiltrate ICAM-1-deficient and wild-type heart allografts with equivalent efficiency. a, A/J mice were primed with 2.5 x 106 bone marrow-derived DC from wild-type C57BL/6 mice and 3 days later received a heart allograft from either a wild-type C57BL/6 (n = 5) or a B6.ICAM-1-/- (n = 5) donor. Allograft rejection was confirmed visually in all recipients by laparotomy. b, On the day of rejection allografts were retrieved, and formalin-fixed sections were prepared and stained with H&E. Magnification, x200.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As a major participant in cellular interactions and trafficking, adhesion molecules have been an attractive target for therapeutic strategies to attenuate inflammatory processes. Several approaches have been used to inhibit the appearance or function of specific adhesion molecules to test the effects on cellular infiltration and survival of allografts in experimental models. These approaches have included treatment with specific Abs and antisense oligonucleotides (21, 23, 29). Studies by Isobe and co-workers (21) demonstrated that treatment with mAb to both ICAM-1 and LFA-1 resulted in long term survival of MHC-mismatched cardiac allografts and the induction of donor-specific tolerance. Treatment with either mAb alone had modest effects on attenuating T cell priming to donor alloantigens and prolonging allograft survival. Subsequent studies by other investigators have used treatment with anti-ICAM and anti-LFA-1 mAb to prolong the survival of many different tissue allografts, including heart, skin, and pancreatic islets (22, 23, 30). The poor efficacy of treatment with anti-ICAM-1 mAb alone observed in these studies may be due to the ability of LFA-1 on recipient T cells and other leukocytes to bind to alternative ligands, such as ICAM-2 expressed on DC and vascular endothelial cells.

LFA-1 and ICAM-1 not only participate in cell adhesion and trafficking, but also are signaling molecules, and the binding of specific Abs can have multiple effects on the function of these molecules and the cells expressing them. The use of mice with a targeted deletion in genes encoding adhesion molecules, including integrins, selectins, and members of the Ig superfamily, have provided valuable tools for testing the roles of these molecules in immune responses. Schowengerdt and co-workers (24) generated and used mice with a targeted deletion in ICAM as either recipients or donors of MHC-mismatched heart grafts. The absence of ICAM-1 in either the recipient or the graft did not prolong allograft survival beyond that observed in the wild-type animals. Similar results were observed in the current study when B6.ICAM-1-/- mice were transplanted with ICAM-1+/+ A/J heart allografts, indicating that ICAM-1 is not required by recipient alloreactive T cells to reject the allograft. However, the absence of ICAM-1 in the heart allograft prolonged survival up to 2 wk beyond survival of wild-type allografts; this is in direct contrast to the results of the previous study. Associated with the prolonged survival of ICAM-1-deficient heart allografts was a marked attenuation of alloantigen-specific T cell priming in the graft recipients. This priming was tested by both mixed lymphocyte reactivity as well as ELISPOT assay, where the number of direct alloantigen-reactive T cells producing IFN-{gamma} in recipients of ICAM-deficient allografts were approximately one-third that observed in recipients of wild-type allografts. At the time of wild-type heart allograft rejection there was also a marked decrease in CD4+ and CD8+ T cell and macrophage infiltration into the ICAM-1-deficient heart allografts. One obvious difference between the previous and current studies is the source of the ICAM-1-/- mice as well as the use of C3H (H-2k) mice as recipients of the ICAM-1-deficient heart allografts in the previous study. Decreased cellular infiltration and tissue pathology have also been observed in a renal ischemia/reperfusion injury model using the mice studied in the current report (26).

Although the time of ICAM-1-/- heart allograft rejection was delayed compared with that in wild-type grafts, the intensity of mononuclear cell infiltration into the ICAM-1-deficient allografts and the histopathology of the allograft were similar to those observed in wild-type heart allografts at the time of rejection. However, cellular infiltration into ICAM-1-/- allografts at the time of rejection did not correlate with a substantial rise in the number of alloantigen-primed T cells producing IFN-{gamma} in the recipient spleen. In heart allograft recipients treated with both anti-ICAM-1 and anti-LFA-1 mAb to inhibit rejection, there is evidence that the donor reactive T cell compartment develops to a type 2 cytokine-producing (i.e., Th2) phenotype, and these T cells may implement the donor-specific tolerance observed in this model (31). Although it is possible that other mechanisms may mediate the delayed rejection of the ICAM-1-deficient heart allografts, the grafts did not exhibit the intense infiltration with eosinophils that has been observed during rejection mediated by Th2 cells observed in other heart allograft models (32, 33). In light of the histological analyses of ICAM-1-deficient heart grafts at the time of rejection, we favor the interpretation that the attenuated priming of alloantigen-specific T cells in these recipients continues at a pace that eventually directs a sufficient number of primed T cells into the graft to mediate rejection. The expression of the IFN-{gamma}-induced chemokine Mig during the progression of this rejection (Q. Zhang, unpublished observations) suggests that at least a portion of the IFN-{gamma}-producing alloantigen-primed T cells observed in the ELISPOT assays are a component of the cellular infiltrate in the ICAM-1-/- allografts at the time of rejection.

In addition to mediating cell adhesion, ICAM-1/LFA-1 interactions provide costimulatory signals to T cells during Ag priming (10, 11, 13, 34, 35). A series of in vitro studies has demonstrated that ICAM-1 engagement of LFA-1 provides costimulatory signals for optimal activation of CD8+ T cells and in some cases may substitute for B7.1-mediated costimulation (12, 36, 37). Costimulation of CD4+ T cells by ICAM-1 during TCR agonism has also been reported (14). The results of the current report indicate the necessity of ICAM-1 expression on allograft donor cells for optimal priming of alloantigen-specific T cells. The induction of recipient T cell responses to ICAM-1-deficient allografts was considerably lower than that observed in response to wild-type grafts. The low infiltration into the ICAM-1-deficient allografts by recipient CD4+ and CD8+ T cells indicates that reduced priming of both alloreactive CD4+ and CD8+ T cells is induced by ICAM-1-/- DC from the allograft. The decreased levels of T cell priming in recipients of ICAM-1-/- heart allografts is not likely to be due to defective APC (i.e., DC) trafficking from the heart allograft into the recipient spleen draining the graft. First, direct alloantigen-specific T cell priming was observed at low levels in recipients of the ICAM-1-/- allografts and was maintained at that level at least up to the time of graft rejection. Second, similar numbers of class II MHC+/CD11c+ DC emigrated from split ear cultures when tissues from ICAM-1-deficient and wild-type mice were compared, and these cells expressed equivalent levels of I-Ab (D. Kish, data not shown). Third, the use of isolated DC from the split ear cultures indicated a substantial decrease in the ability of ICAM-1-deficient DC to stimulate the proliferation of allogeneic T cells compared with DC from the ears of wild-type mice. Finally, there was no difference in the time of rejection of full thickness trunk skin allografts from wild-type C57BL/6 and B6.ICAM-1-/- donors by A/J recipients (R. Fairchild, unpublished observations). It is worth noting that these skin grafts contain many more DC than the heart allografts, which is likely to make the skin allograft much more immunogenic than the heart. The reduced number of graft DC may underlie the attenuated priming of alloreactive T cells in recipients of ICAM-1-/- heart allografts, whereas the greater number of DC in the skin allografts may allow recipient alloreactive T cells to overcome the lack of ICAM-1-mediated signals during alloantigen-specific T cell priming.

The absence of ICAM-1 at the time of priming may also influence the ability of T cells to traffic to tissue sites of inflammation. Recent studies by Camacho and co-workers (14) have demonstrated that OVA/I-Ad-specific CD4+ T cells were clearly activated in the presence or the absence of ICAM-1 in vitro, but were unable to infiltrate pancreatic islets expressing OVA, suggesting that priming in the absence of ICAM-1 may alter the function of T cells. In the current studies, once the T cells were primed by the ICAM-1-deficient allografts there was no apparent change in T cell function, as the histopathology of the rejecting ICAM-1-/- allografts was similar to that observed in the wild-type cells. Furthermore, once the alloreactive T cells were primed, there was no problem in trafficking to and infiltrating the ICAM-1-deficient allografts to mediate rejection. Other ligands for LFA-1, including ICAM-2, also mediate the adhesion of activated lymphocytes to endothelial cells (38, 39, 40). The ability of alloantigen-primed T cells to infiltrate ICAM-1-deficient heart allografts suggests that alternative ligands for LFA-1, such as ICAM-2 expressed on the vascular endothelium of the graft, may facilitate the arrest and infiltration of T cells into the graft. Collectively, these studies suggest that the prolonged survival of ICAM-1-deficient heart allografts is due at least in part to the defective priming of alloantigen-reactive T cells by graft APC and not to the defective trafficking of the primed recipient T cells into the ICAM-1-deficient allograft.


   Acknowledgments
 
We thank Peter Heeger and the Keith Bishop laboratory for helpful suggestions, and the staff of the Cleveland Clinic Foundation BRU for excellent care of the animals used in this study.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant RO1AI40459. Back

2 Address correspondence and reprint requests to Dr. Robert L. Fairchild, NB3-79, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-0001. E-mail address: fairchr{at}ccf.org Back

3 Abbreviation used in this paper: DC, dendritic cell. Back

Received for publication December 30, 2002. Accepted for publication March 31, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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