Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation

Abstract

Background: Immune activation that results due to the aberrant proliferation of lymphocytes leads to inflammation and graft rejection in organ transplant recipients. We hypothesize that the cell cycle control and inflammation are parallel events, inhibition of cellular proliferation by cyclin kinase inhibitor specifically p21 will limit inflammation and prevent allograft rejection.

Methods: We performed in vitro and in vivo studies using lymphocytes, and rat heart transplant model to understand the role of cyclins and p21 on mitogen and allo-induced lymphocyte activation and inflammation. Lymphocyte proliferation was studied by 3H-thymidine uptake assay and mRNA expression was studied RT-PCR.

Results: Activation of allo- and mitogen stimulated lymphocytes resulted in increased expression of cyclins, IL-2 and pro-inflammatory cytokines, which was inhibited by cyclosporine. The over-expression of p21 prolonged graft survival in a completely mismatched rat heart transplant model resulted by inhibiting circulating and intra-graft expression of proinflammatory cytokines.

Conclusion: Cyclins play a significant role in transplant-induced immune activation and p21 over-expression has potential to inhibit T cell activation and inflammation. The results from this study will permit the design of alternate strategies by controlling cell cycle progression to achieve immunosuppression in transplantation.

Figure 1

CsA inhibits cyclins, IL-6 and TNF-α and induces p21WAF1/CIP1 mRNA expression in activated lymphocytes. A). A picture representative of three different experiments demonstrating cyclins (D3 and G), p21WAF1/CIP1, IL-6, TNF-α and induction of mRNA expression in activated lymphocytes (lane 2) with CsA (lanes 3). The lower bands in IL-2 mRNA area are primer dimers. B) Mean ± SEM (n = 3) of the ratio of cyclins, IL-6 and TNF-α and induces p21WAF1/CIP1 with β-actin is shown for lymphocytes either unctivated or activated with and without CsA. The p values signify the statistical significance for mRNA expression in lymphocytes activated with and with out CsA for each gene. C) Western blot analysis for p21 protein in lymphocytes unctivated (lane 1), activated (lane 2) with CsA (lane 3).

Figure 2

In vitro and in vivo mRNA expression of Cyclins relates to alloimmune activation: A: Cyclins; D3, E and G and β-actin mRNA expression in lymphocytes isolated from spleens of rat heart transplant recipients. Untreated (lanes 4,5), CsA treated (lanes 1,2,3). B: Mean ± SEM of the ratio cyclins with β-actin, p values are calculated between densitometric numbers of untreated and CsA treated transplant recipient. C: Cyclin D3 and pro-inflammatory cytokines mRNA expression in lymphocyte from MLR assay using stimulants from donor strain with responders from donor strain (Control) untreated rejecting transplant recipients (A) and CsA treated non rejecting transplant recipients (B). The p values represent the statistical significance between ratio of densitometric numbers for each gene with β-actin from untreated (Group A) vs CsA (Group B) treated transplant recipients.

Figure 3

Over expression of p21WAF1/CIP1 inhibits proliferation and IL-2 mRNA expression in Jurkat T cells: A: A comparison of the proliferation of Jurkat T cells with and without p21 overexpression. B. Il-2 mRNA expression in PHA activated normal and p21 over-expressing Jurkat T cells. An identical expression of house keeping gene β-actin is also shown. C: p21 protein expression in four different clones of p21 overexpressing Jurkat T cells, Untreated (lane 1), p21 overexpressing (lane 2–5) and transfected with empty vector DNA (lane 6).

Figure 4

p21WAF1/CIP1 over-expression prolongs allograft survival: A: p21 Injection of p21 sense plasmid DNA injected mice (set 2not empty vector plasmid DNA induces p21 mRNA expression in heart (h), liver (l), kidney (k) and spleen (s). B: Kaplan-Meyer survival graph for rat cardiac transplant recipients. Significant difference in the survival of allografts in p21WAF1/CIP1 transfected recipients compared to controls (* = p < 0.04) and p21WAF1/CIP1 together with CsA (* * = p < 0.005) can be seen. C: Effect of p21WAF1/CIP1 over-expression and CsA treatment on mRNA expression of IL-2 in lymphocytes and allografts. A significant decreased expression of IL-2 mRNA expression in lymphocytes and allografts compared to controls is shown (* = p < 0.01) and (* * = p < 0.001).