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. 2003 Feb;77(3):2182-94.
doi: 10.1128/jvi.77.3.2182-2194.2003.

Cytomegalovirus-mediated upregulation of chemokine expression correlates with the acceleration of chronic rejection in rat heart transplants

Daniel N Streblow  1 , Craig Kreklywich, Qiang Yin, V T De La Melena, Christopher L Corless, Patricia A Smith, Christina Brakebill, Judith W Cook, Cornelis Vink, Cathrien A Bruggeman, Jay A Nelson, Susan L Orloff
Affiliations

Cytomegalovirus-mediated upregulation of chemokine expression correlates with the acceleration of chronic rejection in rat heart transplants

Daniel N Streblow et al. J Virol. 2003 Feb.

Abstract

Cytomegalovirus (CMV) infections have been shown to dramatically affect solid organ transplant graft survival in both human and animal models. Recently, it was demonstrated that rat CMV (RCMV) infection accelerates the development of transplant vascular sclerosis (TVS) in both rat heart and small bowel graft transplants. However, the mechanisms involved in this process are still unclear. In the present study, we determined the kinetics of RCMV-accelerated TVS in a rat heart transplant model. Acute RCMV infection enhances the development of TVS in rat heart allografts, and this process is initiated between 21 and 24 days posttransplantation. The virus is consistently detected in the heart grafts from day 7 until day 35 posttransplantation but is rarely found at the time of graft rejection (day 45 posttransplantation). Grafts from RCMV-infected recipients had upregulation of chemokine expression compared to uninfected controls, and the timing of this increased expression paralleled that of RCMV-accelerated neointimal formation. In addition, graft vessels from RCMV-infected grafts demonstrate the increased infiltration of T cells and macrophages during periods of highest chemokine expression. These results suggest that CMV-induced acceleration of TVS involves the increased graft vascular infiltration of inflammatory cells through enhanced chemokine expression.

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Figures

FIG. 1.
FIG. 1.
Kinetics of RCMV acceleration of rat cardiac allograft TVS. (A) Heterotopic heart allografts were transplanted into Lewis recipients, with or without RCMV infection, and graft tissues were harvested at the predetermined endpoints of PODs 7, 14, 21, 28, 35, and 45. Graft vessels stained with elastin showed endothelialitis in the RCMV-infected, but not in the uninfected, allogeneic recipients at PODs 7 and 14. Average NIs are given below each representative photo. TVS was detected at POD 21 with little difference between infected and uninfected allogeneic recipients. However, at PODs 28, 35, and 45, RCMV-infected-recipient heart graft vessels showed a dramatic increase in the severity of TVS compared to uninfected controls. These data suggest that RCMV accelerates the time to graft rejection between PODs 21 and 35; however, this might be due to events occurring within the first 7 to 14 PODs. Original magnification, ×200. END, endothelium; NH, neointimal hyperplasia; IEL, internal elastic lamina. (B) Graphical comparison of average NIs of graft heart vessels of uninfected and RCMV-infected animals. The x axis shows the days posttransplantation; the y axis shows the mean NI (n is at least four animals).
FIG. 2.
FIG. 2.
RCMV DNA is detectable in native and graft heart tissues, blood, and SMG. Total DNA was prepared from graft and native hearts, SMG, and peripheral blood leukocytes at PODs 7, 14, 21, 28, 35, and 45. Viral DNA was quantitated by using TaqMan PCR techniques specific for RCMV DNA polymerase. These data suggest that RCMV is present within the graft tissues and that SMG is present during all of the stages of RCMV-accelerated TVS. The presence of virus in the blood was detected only through 14 days posttransplantation.
FIG. 3.
FIG. 3.
Host cellular infiltrates are higher in RCMV-infected graft tissues. Heterotopic heart allografts, with or without RCMV infection, were harvested at the times of RCMV-accelerated TVS formation (PODs 24, 28, and 32), and subsequently, tissue sections were immunohistochemically stained to determine the levels and types of infiltrating cells. Average staining indices are given below each picture. NH, neointimal hyperplasia. (A) Immunohistochemical staining of graft heart tissues for the macrophage marker ED1 at day 28 posttransplantation. (B) Immunohistochemical staining of graft heart tissues for T-helper-cell marker CD4 at day 32 posttransplantation. (C) Immunohistochemical staining of graft heart tissues for cytotoxic-T-cell marker CD8 at day 28 posttransplantation. RCMV infection increases the proportion of cellular infiltrates in both the intima and media of graft vessels, which corresponds to the timing of virus-enhanced chemokine expression. Original magnification, ×200.
FIG. 4.
FIG. 4.
Host CC chemokine expression is enhanced in rat heart graft tissues infected with RCMV. (A) RT-PCR TaqMan was used to detect expression of the CC chemokines (MCP-1, MIP-1α, and RANTES) in heart allografts and native hearts. Graft and native hearts from RCMV-infected and uninfected animals were analyzed at PODs 7, 14, 21, 24, 28, 32, 35, and 45 (n = 4). cDNA was produced from total RNA isolated from the graft tissues. RT-PCR TaqMan detection of the ribosomal protein L32 was used as a normalizing control. (B) Virus-specific induction of host CC chemokine expression in native and graft hearts is shown as a fold increase compared to that for uninfected controls. These data suggest that host CC chemokine expression is specifically enhanced during the time that RCMV accelerates graft TVS (days 21 to 32) but resolves as the graft fails (days 35 to 45).
FIG. 5.
FIG. 5.
RCMV infection dramatically increases IFN-γ and IP-10 expression in heart allografts. (A) RT-PCR TaqMan was used to detect expression of the cytokine IFN-γ and the CXC chemokine IP-10 in native hearts and heart allografts. Graft and native hearts from RCMV-infected and uninfected animals were analyzed at PODs 7, 14, 21, 24, 28, 32, 35, and 45 (n = 4). cDNA was produced from total RNA isolated from the graft tissues. RT-PCR TaqMan detection of the ribosomal protein L32 was used as a normalizing control. (B) Virus-specific induction of host IFN-γ and IP-10 expression in native and graft hearts from RCMV-infected recipients compared to that for uninfected controls is shown. These data suggest that IFN-γ induces IP-10, and this occurs at days 28 and 32, corresponding to the acceleration of TVS in the grafts from RCMV-infected recipients.
FIG. 6.
FIG. 6.
RCMV infection increases Fractalkine and Lymphotactin expression in heart allografts. (A) RT-PCR TaqMan was used to detect expression of the chemokines Lymphotactin and Fractalkine in the heart allografts. Native and graft hearts from RCMV-infected and uninfected animals were analyzed at PODs 7, 14, 21, 24, 28, 32, 35, and 45 (n = 4). cDNA was produced from total RNA isolated from the graft tissues. RT-PCR TaqMan detection of the ribosomal protein L32 was used as a normalizing control. (B) Virus-specific induction of the Lymphotactin (C chemokine) and Fractalkine (CX3C chemokine) expression in native and graft hearts is shown (fold increase is the expression of chemokines in grafts from infected recipients compared to that for uninfected controls). These data suggest that expression of both Fractalkine and Lymphotactin is highest during the RCMV acceleration phase of TVS.
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