Ex vivo Pretreatment of Islets with Mitomycin C: Reduction in Immunogenic Potential of Islets by Suppressing Secretion of Multiple Chemotactic Factors

Abstract

Strategies to reduce the immunogenicity of pancreatic islets and to prevent the activation of proinflammatory events are essential for successful islet engraftment. Pretransplant islet culture presents an opportunity for preconditioning to improve outcomes of islet transplantation. We previously demonstrated that ex vivo mitomycin C (MMC) pretreatment and subsequent culture significantly prolonged graft survival. Fully understanding the biological process of pretreatment could result in the development of a protocol to improve the survival of islet grafts. Microarrays were employed to conduct a comprehensive analysis of genes expressed in untreated or MMC-treated rat islets that were subsequently cultured for 3 d. A bioinformatics software was used to identify biological processes that were most affected by MMC pretreatment, and validation studies, including in vivo and in vitro assay, were performed. The gene expression analysis identified significant downregulation of annotated functions associated with cellular movement and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies revealed significantly decreased levels of interleukin 6 (IL-6), monocyte chemoattractant protein 3 (MCP-3), and matrix metallopeptidase 2 (MMP2) in culture supernatants of MMC-treated islets compared with controls. Moreover, we showed the suppression of leukocyte chemotactic activity of MMC-treated islets in vitro. We also showed that MMC-treated islets secreted lower levels of chemoattractants that synergistically reduced the immunogenic potential of islets. Histological and immunohistochemical analyses of the implant site revealed that infiltration of monocytes, CD3-positive T cells, and B cells was decreased in MMC-treated islets. In conclusion, the ex vivo pretreatment of islets with MMC and subsequent culture can reduce the immunogenic potential and prolong the survival of islet grafts by inducing the suppression of multiple leukocyte chemotactic factors.

Keywords: inflammation; islets transplantation; preconditioning; tolerance.

Fig. 1.

Comprehensive interpretation of biological functions in nontreated and mitomycin C (MMC)-treated islets in response to 3-d culture. We used the ingenuity pathway analysis (IPA) algorithm to determine the z-score and the P value to identify significant biological functions associated with MMC-induced engraftment (MMC- and nontreated d3-islets compared with control islets [nontreated d0-islets]). Each category was sized and arranged in order of –log (P value) and colored by the activation z-score (red; upregulation, blue; downregulation). Compared to nontreated islets, many biological functions were downregulated in MMC-treated d3-islets; most significantly downregulated function was cellular movement.

Fig. 2.

Significant alteration of annotated molecular and cellular functions in nontreated islets. We sorted molecular and cellular function annotations according to the z-score as follows: >2 (upregulation) or ≤2 (downregulation). The P value (square dot), which was calculated using the Fisher exact test, indicates the statistical significance of the association between the expression of a set of genes and a biological function (P value ≤ 0.05, −log₁₀ ≥ 1.3). Expression levels of numerous genes involved function such as “cellular movement” were upregulated significantly in nontreated d3-islets during culture period prior to transplantation.

Fig. 3.

Significant alteration of annotated molecular and cellular functions in mitomycin C (MMC)-treated islets. Significant alteration of annotated molecular and cellular functions in MMC-treated islet was evaluated, as described in Fig. 2. A number of downregulated functions were increased over subsequent 3-d culture period in MMC-treated islets, and more than half of the functions, including “cellular movement,” were downregulated in MMC-treated d3-islets.

Fig. 4.

Rat pancreatic islets produce and secrete interleukin 6 (IL-6), monocyte chemoattractant protein 3 (MCP-3), and metallopeptidase 2 (MMP-2). Supernatants from primary cultures of rat islets were analyzed using the enzyme-linked immunosorbent assay (ELISA) Kit. The IL-6, MCP-3, and MMP2 levels in islet culture supernatants were significantly higher in untreated islets than in MMC-treated islets.

Fig. 5.

Mouse monocyte chemotactic activity of supernatants harvested from cultures of MMC-treated or untreated islets. Undiluted supernatants of cultured islets (day 1 or 3) were used in chemotaxis assays of mouse monocytes. The results are expressed as the mean number of migrated monocytes toward the islet culture supernatant per high-power field. (A) The number of monocytes was significantly lower in MMC-treated islets than in untreated islets. (B) Compared with controls, the mean number of migrated monocyte was significantly decreased in islet culture supernatants treated with anti- monocyte chemoattractant protein 3(MCP-3) monoclonal antibody (mAb) or the metallopeptidase 2 (MMP) inhibitor GM6001.

Fig. 6.

Histology of representative Wistar rat islets harvested from C57BL/6 recipient on day 4 or 8. Representative sections of xenografts treated with 10 µg/mL mitomycin C (MMC; A, C, E, and G) or untreated xenografts (B, D, F, and H). Paraffin-embedded sections were reacted with antibodies specific to T cells (CD3), macrophages (F4/80), and B cells (CD45 R).

Fig. 7.

Analysis of metallopeptidase 2 (MMC)-induced immunosuppression of xenografts. Immune cells that infiltrated the xenografts were stained and counted. MMC-treated (circles) and untreated (squares) islet xenografts. The dominant components of the leukocyte population that migrated to the xenograft were CD3-positive T cells and macrophages. The numbers of T cells, macrophages, and B cells significantly decreased in MMC-treated islet xenografts (P < 0.05).

Fig. 8.

Survival of metallopeptidase 2 (MMC)- and untreated islet xenografts (Wistar rat islets to B6 mice). Prolonged survival of MMC-treated d3-islets (solid line) compared with that of untreated d0- and d3-islets (dotted lines; P < 0.01).