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Chemokines regulate the recruitment of leukocytes to sites of inflammation and may therefore play an important role in lymphocyte trafficking between draining lymph nodes and pancreatic islet tissue allografts. The intragraft expression of alpha- and beta-chemokine mRNA during the rejection of BALB/c proislet (fetal precursor islet tissue) allografts in CBA/H mice was assessed quantitatively and semiquantitatively by RT-PCR analyses. Allograft rejection was associated with the strongly enhanced (from day 4) and prolonged expression (up to day 10) of the alpha-chemokine IP-10 and enhanced intragraft mRNA expression of the beta-chemokines MCP-1, MIP-lalpha, MIP-1beta, RANTES, and eotaxin. Peak transcript expression was identified at day 4 (IP-10, MCP-1), day 5 (eotaxin), day 6 (MIP-1alpha, MIP-1beta), and day 14 (RANTES). To examine the role of beta-chemokine receptors in allograft rejection, additional allografts to CCR2-/- , CCR5-/-, and wild-type CCR+/+ mice were analyzed by histology, immunohistochemistry, and morphometry. In CCR5-/- mice, the intragraft recruitment of T cells and macrophages was slower and allograft destruction was delayed; in CCR2-/- mice, the initial entry of macrophages was retarded but graft survival was not prolonged. These findings suggest that IP-10 regulates the initial influx of T cells into proislet allografts, MCP-1/CCR2 signaling controls initial macrophage entry, and the MIP-1alpha, MIP-1beta, and RANTES/CCR5 pathway contributes to the rejection response by subsequently amplifying the recruitment of T cell subpopulations required for graft destruction.

The mechanism by which inflammatory cells are recruited to pig islet tissue (proislet) xenografts was investigated by examining the intragraft mRNA expression of murine alpha- and beta-chemokines in CBA/H mice from days 3 to 10 post-transplant. Xenograft rejection was associated with early intragraft transcript expression for monocyte chemotactic protein-1 (MCP-1) (3 to 5 days), IP-10 (3 to 4 days) and macrophage inflammatory protein-1alpha (MIP-1alpha) (3 to 5 days) and subsequent expression of eotaxin (days 4 to 10), MIP-1beta (days 4 and 5) and regulated on activation, normal T cell expressed and secreted (RANTES) (days 4 to 6) mRNA. This pattern was consistent with the early recruitment of macrophages (MCP-1, MIP-1alpha), the influx of CD4 T cells (MCP-1, MIP-1alpha, MIP-1beta, IP-10 and RANTES) and the characteristic infiltrate of eosinophils (eotaxin and RANTES) associated with islet xenograft rejection. Inhibition of beta-chemokine signaling in CCR2-/- mice (which lack the major co-receptor for MCP-1) resulted in retarded macrophage and CD4 T cell recruitment, enhanced eosinophil influx and a minor delay in rejection, compared with wildtype mice; there was little effect on leukocyte infiltration in xenografts harvested from CCR5-/- mice (lacking the co-receptor for MIP-1alpha, MIP-1beta and RANTES). The impeded migration of leukocytes into xenografts in CCR2-/- hosts was associated with delayed intragraft expression of MCP-1 and RANTES mRNA; absence of MCP-1/CCR2-mediated signaling led to enhanced intragraft expression of MCP-1, MIP-1alpha and MIP-1beta mRNA. These findings suggest that MCP-1 plays an important role in regulating macrophage and CD4 T cell infiltration to xenograft sites via the CCR2 signaling pathway. Additional treatment of xenografted CCR2-/- transplant recipients with anti-interleukin-(IL)-4 and anti-IL-5 mAbs further delayed xenograft rejection demonstrating the potential for combined antirejection strategies in facilitating pig islet xenotransplantation.