Table 1.
The kinds of reagents.
Table 2.
Primer sequences used for the gene expression analysis.
Figure 1.
Leukocytes in the peripheral blood after bone marrow transplantation (BMT).
The total white blood cell (WBC) count in peripheral blood (A) decreased markedly on day 4, but recovered between day 7 and day 14 in both allogeneic and syngeneic BMT rats. The number of WBCs in the peripheral blood was higher on day 21 in allogeneic BMT rats than in syngeneic BMT rats. WBCs in the peripheral blood decreased again in allogeneic BMT rats on day 28, which may be because of recruitment of WBCs to GVHD organs. Almost all circulating leukocytes in allogeneic BMT rats on day 28 after BMT (B) expressed donor-type RT1Aa, indicating that circulating leukocytes in peripheral blood originated from the donor (Gray; no staining, Black; anti-RT1Aa,b). In peripheral blood, CD6+ T-cells (C), CD8+ T-cells (D), CD4+ T-cells (E), and ED1+ macrophages levels (F) recovered between day 7 and day 21 after BMT in both syngeneic and allogeneic BMT rats. The number of CD6+ T-cells and CD8+ T-cells was significantly higher on day 21 in allogeneic BMT rats than in syngeneic BMT rats. The number of CD4+ T-cells and CD68+ macrophages was similar in both syngeneic and allogeneic BMT rats. *P<0.05, **P<0.01.
Figure 2.
Body weight and semiquantitative score of systemic acute GVHD after bone marrow transplantation (BMT).
Comparison of body weight in percentage (A) on day 0, after radiotherapy and after BMT showed that this parameter decreased in syngeneic and allogeneic BMT rats on day 7 and continued gradually to decrease in allogeneic BMT rats by>20% on day 28. In addition, body weight was significantly lower in allogeneic BMT rats than in syngeneic BMT rats between day 14 and day 28 after BMT. The semiquantitative score of systemic acute GVHD (B) showed that symptoms associated with acute GVHD occurred from day 7 in allogeneic BMT rats, and developed by day 28 with score of 8.7±0.5 (mean ± SD; score 0–10). In contrast, acute GVHD did not develop in syngeneic BMT rats by day 28. *P<0.05.
Figure 3.
Acute GVHD in the skin, liver, and intestine after allogeneic bone marrow transplantation (BMT).
On day 28 after allogeneic BMT, macroscopic findings of the skin indicated erythematous rush and alopecia (A, B). Light microscopic findings showed inflammatory cells, mainly CD3+ T-cells, infiltrating the epidermis and the hair follicle in the dermis, indicating acute GVHD in the skin (C, D: HE stain, E: CD3 stain, C: ×400, D, E: ×800). Inflammatory cells, mainly CD3+ T-cells, infiltrated the portal areas and spread to the hepatic lobules with cholangiolitis and phlebitis in the portal and central veins, indicating acute GVHD in the liver (F, G: HE stain, H: CD3 stain, F: ×400, G, H: ×800). In the colon, erosion and inflammatory cell infiltration was noted with cryptitis and infiltration of CD3+ T-cells, indicating acute GVHD in the colon (I, J: HE stain, K: CD3 stain, I: ×600, J, K: ×800).
Figure 4.
Liver and renal dysfunction after allogeneic bone marrow transplantation (BMT).
Liver function was determined using serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (T-Bil) levels. Serum AST and ALT levels on day 28 were significantly increased in allogeneic BMT rats compared with those in syngeneic BMT rats, although serum T-Bil levels were similar in both groups. Renal function was assessed using serum blood urea nitrogen (BUN), serum creatinine (Cr), and urinary N-acetyl-β-D-glucosaminidase (NAG) levels. Serum BUN and urinary NAG levels on day 28 significantly increased in allogeneic BMT rats compared with those in syngeneic BMT and non-BMT control rats, although serum Cr levels were similar in these groups. Importantly, urinary NAG levels in allogeneic BMT rats were increased while the serum Cr levels were stable. *P<0.05.
Figure 5.
Renal inflammation in acute GVHD after allogeneic bone marrow transplantation (BMT).
In mild renal inflammation (A, PAS stain, ×200), mononuclear cells infiltrated the renal interstitium around the small vessels (arrow). In moderate renal inflammation (B, PAS stain, ×200), infiltration of mononuclear cells expanded into the peritubular interstitium. High magnification of renal inflammation (C, PAS stain, ×400), interstitial cell inflammation was often accompanied by tubulitis (arrow) and peritubular capillaritis (arrowhead). In severe renal inflammation (D, PAS stain, ×200), diffuse interstitial inflammation was noted in the renal cortex.
Figure 6.
Histopathological features in the kidney on day 28 after allogeneic bone marrow transplantation (BMT).
In the kidney, 28 days after allogeneic BMT, tubulitis (A–C), peritubular capillaritis (D–F), acute glomerulitis (G–I), and endarteritis (J–L) were caused by the infiltration of mononuclear cells (A, D, G, J), CD3+ T-cells (B, E, H, K), and ED-1+ macrophages (C, F, I, L), indicating cell-mediated renal injury in acute GVHD. (A, D, G: PAS stain, ×600; J: HE stain, ×1000; B, E, H, K: CD3 stain, ×600; C, F, I, L: ED1 stain, ×600).
Figure 7.
The infiltrating cells in the kidney and the MHC class II expressions in renal tubules.
In the kidney on day 28 in allogeneic (allo-BMT) bone marrow transplantation rats, CD3+ T-cells including CD8+ T-cells, and ED1+ macrophages infiltrated the interstitium. The number of CD3+ T-cells, CD8+ T-cells, and macrophages per ×200 magnification field on day 28 showed that infiltration of these cells in the kidney significantly increased in allogeneic BMT rats compared with that in the non-transplanted (non-BMT) control rats and syngeneic (syn-BMT) bone marrow transplantation control rats. In addition, the expression of MHC class II in renal tubules increased in the kidney on day 28 in allogeneic BMT rats. The expression of MHC class II in renal tubules was significantly increased in allogeneic BMT rats than those in non-BMT control and syngeneic BMT control rats. *P<0.05.
Figure 8.
Infiltrating cells in the kidney in acute GVHD after allogeneic bone marrow transplantation (BMT).
Double immunofluorescence stain by fluorescence antibody technique against CD3+ (A) and CD8+ (B), and their merged image (C) indicated that, in the kidney with acute GVHD on day 28, CD8+ T-cells (arrow in A–C) infiltrated the kidney. In addition, CD4+ T-cells (arrow in D–F) were also noted in inflammation, indicating that not only class I-restricted T cell-mediated reactions but also class II-restricted T cell-mediated reactions developed in renal acute GVHD. Double immunofluorescence stain against RT1Aa,b (G) and CD45 (H), and their merged image (I) indicated that, in the kidney with acute GVHD on day 28, almost all CD45+ leukocytes (arrow in G–I) were expressed rat RT1Aa, b, suggesting the infiltration of donor-type leukocytes in acute renal GVHD.
Figure 9.
Real-time reverse transcription-PCR analysis of cytokines in the kidney after bone marrow transplantation (BMT).
The expression of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) was significantly up-regulated in the kidney on day 28 in allogeneic (Allo) BMT rats compared with that in the syngeneic (Syn) BMT rats. The expressions of interleukin 4 (IL-4) and IL-17 were not significantly different between these 2 groups. *P<0.05.