Ganciclovir transiently attenuates murine cytomegalovirus-associated renal allograft inflammation

Abstract

Background: Prophylactic ganciclovir (GCV) is used in high-risk renal transplant patients to prevent acute cytomegalovirus (CMV) disease, but its impact on inflammation within the allograft itself remains undefined.

Methods: To study the effect of GCV prophylaxis on allograft inflammation, murine CMV (MCMV)-infected allografts were analyzed in a murine donor positive/recipient negative allogeneic renal transplantation model by flow cytometry and immunofluorescent staining.

Results: By flow cytometry, CD45+ leukocyte infiltrates were more abundant in MCMV-infected allografts at 14 days posttransplant compared with uninfected grafts (P<0.01) and decreased in the presence of GCV (P<0.05). CD11c+ dendritic cells, Gr-1+ myeloid cells, CD204+ macrophages, and CD49b+ natural killer cells were reduced in GCV-treated allografts compared with MCMV-infected grafts without GCV treatment (P<0.05). However, GCV failed to reduce these cell types to levels found in MCMV-uninfected allografts. By day 7 after cessation of GCV prophylaxis, dendritic cells, macrophages, and natural killer cells increased in number and became statistically indistinguishable from numbers of cells found in MCMV-infected allografts without GCV. GCV treatment did not affect the numbers of CD4+, CD8+, or CD19+/B220+ lymphocytes infiltrating the allografts. Infiltrates were confirmed histologically by immunofluorescent staining for CD3+ and CD11b+ cells.

Conclusions: In this model, MCMV-infected allografts developed significantly greater innate and adaptive leukocytic infiltrates compared with uninfected grafts. GCV attenuated the MCMV-associated innate leukocyte infiltrates in infected allografts but not the lymphocytic infiltrates. The attenuated innate response was limited to the period of GCV prophylaxis.

FIGURE 1.

Ganciclovir (GCV) treatment terminates murine cytomegalovirus (MCMV) replication and is associated with reduction of CD45+ leukocyte infiltrates into the allograft. (A) DNA was extracted from whole blood of CMV-infected animals with and without GCV treatment at 14 days posttransplant, quantitative DNA PCR performed using primers, probe and standards specific for MCMV immediate-early 1, and results depicted as copies per mL blood. MCMV DNA was consistently detectable in blood from CMV-infected transplant recipients without GCV (CMV, closed squares) but was not detectable (nd) in animals treated with GCV (GCV). (B) CD45+ leukocytes were quantitated by flow cytometry from allografts of uninfected (Mock, open squares), CMV-infected (CMV, closed squares), and CMV-infected, GCV-treated (GCV, hatched squares) animals, and depicted as cells per organ. CMV-infected allografts showed greater CD45+ infiltrates compared with Mock allografts (P<0.01). CD45+ leukocytes in the GCV allografts were decreased compared with CMV allografts (P<0.05) but were still greater (P<0.01) than numbers found in Mock allografts.

FIGURE 2.

Innate inflammatory infiltrates are induced in murine cytomegalovirus (MCMV)-infected allografts and reduced with ganciclovir (GCV) treatment. (A) Cellular infiltrates from allografts of uninfected (Mock, open squares), CMV-infected (CMV, closed squares), and CMV infected, GCV-treated (GCV, hatched squares) animals were stained for CD45+/CD11b+ cells, evaluated by flow cytometry, and depicted as cells per organ. CD11b+ infiltrates were greater in the CMV grafts compared with Mock grafts (P<0.01) and were reduced in GCV grafts compared with CMV grafts (P<0.01). CD11b+ infiltrates were greater in the GCV grafts compared with Mock (P<0.05). (B) Allo-grafts were analyzed for CD11b+/CD11c+, CD11b+/Gr-1+, CD45+/CD204+, and CD3-/CD49b+ cells by flow cytometry (cells per organ). CMV grafts had greater infiltrates of all innate subsets compared with Mock grafts (P<0.01 for all except CD49b+, P<0.02). GCV grafts had lower numbers of all innate subsets compared with CMV grafts (P<0.05 for all except CD204+, P<0.02). GCV grafts contained greater numbers of CD11c+ and Gr-1+ cells (P<0.05) as well as CD204+ and CD49b+ cells (P<0.01) compared with Mock grafts.

FIGURE 3.

Adaptive inflammatory infiltrates are induced in murine cytomegalovirus (MCMV)-infected allo-grafts but are not affected by ganciclovir (GCV) treatment. (A) Cells from uninfected (Mock, open squares), CMV-infected (CMV, closed squares), and CMV-infected, GCV-treated (GCV, hatched squares) allografts were stained for CD45+/CD8+ and CD45+/CD4+ surface markers, evaluated by flow cytometry and shown as cells per organ. CMV grafts showed induction of CD8+ and CD4+ infiltrates (P<0.01) compared with Mock grafts. No difference was observed in these T lymphocyte subsets between the CMV-infected allografts with and without GCV treatment (P>0.05, ns). (B) Cells from allografts of Mock, CMV, and GCV animals were stained for CD19+/B220+ markers and evaluated by flow cytometry. CMV-infected grafts showed greater CD19+/B220+ lymphocyte infiltrates (P<0.05) compared with Mock grafts, which was sustained in the GCV grafts compared with CMV grafts (P>0.05, ns) and remained elevated in the GCV grafts compared with the Mock grafts (P<0.05).

FIGURE 4.

Ganciclovir (GCV) attenuates CD11b+ immunostaining in cytomegalovirus (CMV)-infected allo-grafts. Paraffin-embedded allografts from Mock (left column), CMV (middle column) and GCV-treated (right column) animals were stained by hematoxylin-eosin for light microscopy (top row), or immunostained with primary antibodies for CD3+ cells (middle row), or for CD11b+ cells (bottom row) followed by AlexaFluor488-conjugated secondary antibodies (green) and nuclear counterstain (blue) for confocal microscopy. Compared with Mock grafts, CMV-infected allografts showed induction of mononuclear infiltrates that included both CD3+ and CD11b+ cells. GCV treatment reduced the mononuclear and CD11b+ infiltrates but not the CD3+ infiltrates.

FIGURE 5.

Viral loads rise and innate leukocyte infiltrates reaccumulate in allografts after cessation of ganciclovir (GCV). (A) DNA was extracted from allografts of animals that were cytomegalovirus (CMV) infected (CMV, closed squares), GCV treated (GCV, nd), or treated with GCV which was subsequently discontinued for 7 days (post-GCV, closed triangles). Quantitative DNA PCR for murine CMV (MCMV) immediate-early 1 was performed and results depicted as copies/gram tissue. MCMV DNA was detectable in CMV allografts, became undetectable in GCV grafts, and became detectable again after discontinuation of GCV. (B) Leukocytes from CMV, GCV, and post-GCV allografts were analyzed by flow cytometry for CD11c+, CD204+, and CD49b+ cells. CD11b+, CD204+, and CD49b+ leukocytes were lower in GCV grafts compared with CMV grafts (P<0.05), but after cessation of GCV, numbers of these leukocytes increased (post-GCV) to similar numbers as the CMV grafts without GCV treatment (P>0.05, ns).

FIGURE 6.

Ganciclovir (GCV) induces sustained reduction of Gr-1+ myeloid cells in allografts but not in spleen and does not influence the intragraft alloimmune response. (A) Gr-1+ cells were analyzed by flow cytometry in cytomegalovirus (CMV), GCV, and post-GCV allografts. Gr-1 cells decreased in GCV grafts compared with CMV grafts (P<0.05) and remained low after GCV was discontinued (GCV vs. post-GCV, P>0.05, ns). Post-GCV Gr-1+ numbers remained lower than those found in CMV grafts without GCV treatment (P<0.01). (B) Gr-1 cells were analyzed by flow cytometry in spleens from Mock, CMV, GCV, and post-GCV animals to determine whether GCV treatment induces neutropenia. Numbers of Gr-1+ cells were similar in spleens from animals with and without GCV treatment, indicating that GCV did not induce global neutropenia in the host animals. Mock animals were given cyclosporine (white bars), no treatment (checkerboard bars), or GCV without cyclosporine (diagonal hatched bars), and allografts were analyzed for CD45+ (C) and CD11b+ (D) infiltrates. Cyclosporine reduced the alloimmune-associated infiltrates compared with untreated grafts (P<0.05), but GCV did not reduce the alloimmune response in cyclosporine-untreated, CMV-negative animals compared with those not receiving GCV (ns).