Corneal transplantation has restored sight to millions of blind people worldwide, but there have been no major changes in pharmacotherapy for decades since the advent of corticosteroids and most high-risk grafts are still rejected despite maximum anti-inflammatory therapy. Deletion or blockade of molecular mediators of alloimmunity potentially offers the possibility of more precise inhibition with fewer side effects. The deletion or blockade of chemokines has been partially successful in several organ transplantations,16–24,33
but mechanistic studies unraveling chemokine pathways in corneal transplantation have not been performed thus far. The present study demonstrates, for the first time, that targeting the chemokine receptor CCR1 can lead to a significant increase in long-term corneal graft survival.
Our previous work evaluating patterns of chemokine expression in the late phase of corneal transplant rejection identified CCL5/RANTES, CCL3/MIP-1α, CCL4/MIP-1β, CCL2/ MCP-1, and CXCL10/IP-10 mRNA overexpression, though the functional relevance of these findings in the immunopathogenesis of graft rejection was not investigated.11
In this study, we focused on the expression and functional role of specific CC and CXC chemokine receptors implicated in corneal alloimmunity by virtue of their overexpression and examined the effect of selective gene deletion to demonstrate that CCR1 suppression had a significant effect in promoting allograft survival, even beyond that seen with Cy administration.
In light of our previous findings, the current findings may be surprising, since the deletion of CCR1 led to a significantly improved allograft survival, whereas deletion of other chemokine receptors, whose ligands were upregulated after graft rejection did not. Although the results obtained by Yamagami et al.11
were obtained from whole eyes that included both donor and recipient tissue, the current experiments were performed using recipient KO mice. Hancock et al.21
showed elegantly that when CXCL10/IP-10 KO allografts were transplanted into WT recipients, a significantly improved survival rate was demonstrated. In contrast, the survival was not improved when WT allografts were transplanted into CXCL10/ IP-10 KO recipients. Therefore, the KO mice used herein as recipients might demonstrate improved corneal graft survival if used as donors. In addition, there is a high level of promiscuity in the chemokine system. Despite this promiscuity, there are functional differences when the same ligands bind to different receptors. This is, in part, because different chemokine receptors are expressed by different cell types. We show here that in addition to CCR1 KO mice, the CCR5 KO mice recipients (CCR1 and CCR5 share ligands) demonstrated a delayed rejection compared with WT mice. The role of CCR5 and CCL5/ RANTES in recruitment of MHC class II-positive LCs in the corneal epithelium
has been shown by us recently.30
Our current data suggest that while some stromal DCs also express CCR5, they are the only antigen-presenting cell (APC) population that express CCR1, because epithelial LCs do not express this receptor. This finding underscores the importance of intrastromal graft infiltration by APCs in corneal alloimmunity. Moreover, the different expression patterns of CCR1 and CCR5 by the epithelial and stromal DCs could explain the different results obtained between CCR1 KO and CCR5 KO mice in this study. Similarly, previous studies have shown that chemotactic migration of monocytic-derived DCs (like corneal stromal DCs), is abolished with Abs to CCR1, but not with Abs to CCR5.34
The different roles of epithelial LCs and stromal DCs are further confirmed by the fact that, although CCL5/RANTES blockade led to a CCR5-mediated suppression in LC migration after inflammation, CCL5/RANTES blockade alone did not improve corneal graft survival, suggesting that other chemokines are involved in the CCR1-mediated corneal allograft rejection.
Corneal allograft rejection is primarily mediated by CD4+
This study demonstrates the mediation of Th1 responses by CCR1, as CCR1 deletion was associated with a reduced influx of T cells into the cornea, as well as abolished gene expression of Th1 cytokines IL-2 and IFN-γ. IFN-γ, a product primarily of Th1 cells, can induce production of Th1-attracting chemokines, whereas antagonizing Th2-attracting chemokines.38
CXCL10/IP-10 for instance, is induced by IFN-γ and is expressed abundantly in rejected corneal transplants.11
IFN-γ can also synergize with IL-1 and TNF-α to stimulate chemokine production, and as such, demonstration of the critical role of CCR1 in mediating corneal allograft rejection provides indirect confirmation for our previous work implicating IL-1 and TNF-α in alloimmunity.30,32,35,39,40
Leukocyte recruitment into the corneal graft is a major component of the early alloimmune response, as is the case for other tissue grafts. A recent study demonstrated in a renal inflammatory model that CCR1, but not CCR5, is required for leukocyte recruitment.41
Further, Eis et al. demonstrated through the use of the CCR1- antagonist BX471 and CCR5-blocking antibodies that CCL5/RANTES-induced arrest of monocytes and T cells was predominantly mediated by CCR1 but not CCR5.41
In addition, neutrophils have been reported to express CCR1, explaining our observation that CCR1 blockade has a profound effect on neutrophil infiltration into corneal grafts. Earlier studies have shown that neutrophils from CCR1 KO mice are nonresponsive to CCL3/MIP-1α, suggesting that CCR1 may be the dominant CCL3/MIP-1α binding receptor subtype in these cells.
The process of antigen presentation is an indispensable step in the corneal alloimmune response, and our knowledge regarding the presence and importance of corneal APCs has evolved over the past years.42,43
Here, we confirm our previous findings that CCR5, but not CCR1, is expressed on CD11c+
LCs during inflammation.32
Further, we noted that stromal
DCs (that are uniformly CD11b+
) are able to express both CCR1 and -5 during inflammation, and hence are the only APCs that express CCR1 in the cornea. CCR1 and -5 are responsible for the recruitment of immature DCs in a variety of inflamed tissues, through the chemokines CCL3/MIP-1α, CCL5/RANTES, and CCL7/MCP-3. Once activated, DCs secrete high levels of CCL3/MIP-1α (not made constitutively by resident corneal cells)11,44
that lead to additional recruitment of inflammatory cells. This is a likely explanation for the significant effect of CCR1 (but not CCR5) blockade on allograft infiltration and rejection, indicating the important role of stromal DCs in the alloimmune response. In addition, given the critical role of APCs in initiating the alloimmune response, the decreased recruitment of DCs observed in the absence of CCR1 inhibits the initiation and sustained Ag sampling, thereby decreasing or delaying the priming of effector T cells. The role of CCR1, but not CCR5, in regulating the interaction of monocytic-derived DCs and T cells has been demonstrated in the past.34
It is important to address the potential limitations in this study. First, we primarily extracted RNA from whole-eye homogenates for analysis of chemokine receptor mRNA to prevent the problems faced with the very small quantities of RNA extracted from the murine cornea. The use of only corneal RNA would have translated into a significant increase in the number of animals used. We had faced similar problems in our previous studies where we confirmed that the expression of specific chemokine mRNA after allograft rejection that was reflected in the whole-eye data was similar to data in the corneal microenvironment.11
Therefore, although the RPA analysis of whole eyes has the disadvantage of not limiting the assay to the cornea alone, it has the distinct advantage of also assaying chemokine species expressed by other (noncorneal) structures in the anterior segment, such as the vascularized tissues in and around the iris root that also materially contribute to leukocyte infiltration into the anterior eye structures, including the cornea and anterior chamber after corneal transplantation.
Second, although the deletion of CCR1 clearly modulated corneal graft rejection in CCR1 gene-targeted recipient mice, some mice still rejected their corneal grafts. Although leukocyte recruitment into a corneal graft is a major component of the alloimmune response, corneal allograft rejection is controlled by CD4+ T cells. We demonstrated here that corneal allograft survival can be prolonged through a substantial decrease in the number of infiltrating T cells in CCR1 KO recipients compared with WT recipients. However, the fact that some CCR1 KO recipients still rejected their allografts, demonstrates that CCR1-mediated mechanisms, despite their significance, alone cannot entirely account for the alloimmune response. Finally, although we used CCR1 KO recipients in this study to analyze the role of CCR1, we have not used an inhibitor/antagonist of CCR1 in WT recipients to confirm these results, and therefore we cannot comment on alternate (e.g., compensatory) pathways that may exist in the CCR1 KO mice. However, notwithstanding this limitation, it is important to emphasize that these compensatory pathways are typically more problematic in a setting of “false-negative” data with KO animals, in which they can compensate for the lack of a gene through changed expression of other gene products, rather than in this case in which specific gene deletion led to a significant change in outcome.
In summary, this study is the first to identify the role of a critical chemokine receptor in corneal alloimmunity. Targeting CCR1 alone or its ligands may prove to be an effective strategy to suppress the rejection of corneal transplants, thus reducing the risk of the myriad toxic side effects of alternative nonspecific immunosuppressive drugs.