Background
Alloimmune activation, caused by aberrant T lymphocyte proliferation is one of the key post transplant events in organ transplant recipients. Current immunosuppressive drugs are therefore designed to inhibit T lymphocyte proliferation. Our previous studies have demonstrated that immunosuppressive drugs, cyclosporine (CsA) tacrolimus (TAC), and sirolimus (SRL) besides inhibiting lymphocyte proliferation and IL-2 also induce the expression of TGF-β and other fibrogenic molecules [
1‐
3] leading to nephrotoxicity and chronic rejection. Therefore, there is a need to develop alternate strategies to achieve immunosuppression for increased graft survival with least nephrotoxicity. The most effective immunosuppression can be achieved by the direct inhibition of T lymphocyte proliferation. Since the expression of cyclins and cyclin-dependent kinases and pro-inflammatory cytokines is increased during T lymphocyte proliferation (4), control of T cell proliferation by regulating the expression of cyclins would potentially inhibit allo-immune activation and inflammation. p21WAF1/CIP1 is one of the most potent cyclin kinase inhibitor and therefore has potential to control the expression of cyclins and T cell activation.
We have demonstrated that CsA, TAC and SRL [
4‐
6] induces the expression of cyclin kinase inhibitor p21WAF1/CIP1 and also
in vitro and
in vivo over-expression of p21WAF1/CIP1 in lymphocytes results in decreased response to mitogenic stimuli and greater sensitive to the inhibitory effects of cyclosporine [
7]. The present study was designed to study the expression of cyclins during T cell activation, allograft rejection, and the effect of p21WAF1/CIP1 on mitogenic and allogeneic stimulation, pro-inflammatory cytokines and graft survival in a rat heart transplant model.
Discussion
The experiments performed in this study were designed to understand the role of cyclins on mitogen and allo-stimulation of immune cells and also, if the inhibition of cyclins will correlate with pro-inflammatory cytokines. We also studied if p21WAF1/CIP1 modulation in recipients of cardiac transplantation modulates allo- and mitogenic stimuli and allograft survival. The results demonstrate that during lymphocyte activation, mRNA expression of cyclins and pro-inflammatory cytokines is significantly increased and CsA inhibited lymphocyte activation, mRNA expression of cyclins, pro inflammatory cytokines but induced p21 mRNA and protein expression.
Studies [
9‐
12] have demonstrated that the expression of cyclin D3, cdk6, and cyclin E is activated in IL-2-stimulated T lymphocytes. However, the novel finding of this present study is that mRNA expression of cyclins in activated lymphocytes correlates with that of pro-inflammatory cytokines, and the expression of both the cyclins and pro-inflammatory cytokines is inhibited by immunosuppressive agent CsA. These are novel findings not demonstrated previously. Our results emphasize that the cell cycle progression and inflammation are concerted events thus regulation of cell cycle control could result in decreased inflammation.
Our in vitro findings on the increased expression of cyclins mRNA in activated lymphocytes were reproduced in our in vivo studies. The mRNA expressions of cyclin D3, G and E in lymphocytes (possible predominantly T cells, CsA inhibits proliferation of T lymphocytes) isolated from spleens from untreated recipients of rat cardiac transplant were significantly higher than those treated with cyclosporine. The increased expression of cyclins may represent an uncontrolled allo-immune activation in these rats. Since CsA treatment resulted in the inhibition of allo-immune activation and increased graft survival accompanied by a significant inhibition of mRNA expression of cyclins in CsA treated. This possibly was due to the CsA mediated inhibition of alloimmune activation. These results indicate the presence of an active cell cycle progression during allo-immune activation. Therefore, the control of cell cycle progression should prevent inflammation leading to an improved graft survival. These results are supported by our studies with MLR cultures using lymphocytes from rat heart transplant recipients. An increased proliferation of lymphocytes accompanied increased expression of cyclins and pro-inflammatory cytokine mRNA when responders lymphocytes were used from untreated rats as compared to those from isografts or CsA treated rat heart transplant recipients. Again, these activated lymphocytes were possible predominantly T cells, T lymphocyte proliferation is a key component of allo-immune activation. Therefore, these results lend credence to our thinking that the inhibition of allo-immune activation accompanies decreased expression of cyclins and pro-inflammatory cytokines.
These results confirm that control of cell cycle progression plays a significant role in T cell proliferation/activation. Role of p21 in other aspects of lymphocyte proliferation has been studied. Studies of Balomenos et.al, [
13] Santiago-Raber et al [
14] and Brian et al [
15] demonstrated that T lymphocytes from p21WAF1/CIP1
-/- mice proliferated significantly more than from wild type mice upon stimulation. These results support our studies that p21WAF1/CIP1 modulation alters cell cycle progression and the immune system. Jackson et al [
16] showed that increased levels of p21WAF1/CIP1 at the end of G (1) could prevent cdk-mediated entry into S phase, leading to proliferative unresponsiveness also found in our experiments with p21WAF1/CIP1 over-expressing Jurkat T cells.
The results from this study are of significance because p21 is one of the most potent regulators of the cell cycle and is known to inhibit cell proliferation in two different ways. p21 binds to Cdk2 and inhibits PCNA (proliferating cell nuclear antigen), which is an auxiliary protein in DNA polymerase needed for DNA synthesis and nucleotide excise-n- repair [
17]. PCNA has 6 binding sites for p21 [
18]. Studies also [
19] demonstrated that the PCNA binding and inhibitory activities reside in the C-terminal domain of p21, compared to the location of the CDK inhibitory activity in the conserved N-terminal domain. The authors also concluded that the CDK and PCNA inhibitory domains prevented DNA replication suggested a dual function of p21 as a cell-cycle inhibitor in vivo. We conducted these studies exclusively with cyclin kinase inhibitor p21WAF1/CIP1, though p53 and cyclin kinase inhibitors (p27, p16) have been shown to inhibit cell cycle yet p16 and p21WAF1/CIP1 inhibit cell cycle progression through distinct mechanisms [
20]. The specific target for p16 is the Cdk/4cyclin D complex and in a tumor model, p21WAF1/CIP1 and p16 did not show additive or synergistic effects [
21]. Furthermore in contrast to p21WAF1/CIP1, the expression of p27 is not under transcriptional control and its mRNA expression remains unchanged during cell cycle [
22]. Also, high levels of p27 but not p21WAF1/CIP1 are observed in most quiescent cells and the inhibition of p27 levels precedes the progression of cell cycle [
23]. Though both p21WAF1/CIP1 and p27 are critical in the response of cells to mitogens, p21WAF1/CIP1 provides a better balance between cyclins and cyclin kinase inhibitors [
24] stressing its significance in inhibition of proliferation/immunosuppression. It is therefore possible that p21WAF1/CIP1 over-expression could interrupt the cell cycle progress and also prevent inflammation. It is well known that during T cell activation, expression of pro-inflammatory cytokines IFN-γ, TNF-α and IL-6 is significantly increased. Since T cells are the key mediators of allo-immune activation, this increased expression of cytokines in organ transplant recipient results in graft rejection [
25‐
27]. Our results demonstrate a parallel increase in the expression of cyclins and pro-inflammatory cytokines. Therefore an inhibition/regulation of cell cycle progression of immune cells by over-expression of cyclin kinase inhibitor p21WAF1/CIP1 would decrease both allo-immune activation and inflammation in transplant recipients.
We also demonstrate that rats transfected with p21WAF1/CIP1 plasmid DNA over expressed p21WAF1/CIP1 mRNA in different tissues. The recipients of cardiac allograft animals who received intra-muscular injection of p21WAF1/CIP1 sense plasmid DNA had significantly increased graft survival compared to the recipients transfected with empty plasmid DNA. These very preliminary studies suggest that p21 overexpression can prolong graft survival to a degree comparable to prolongation by CsA. Further studies will surely be required to confirm and quantify the effect of p21 on graft survival. We also present unique results that the expression of IL-2 mRNA was significantly decreased in both lymphocytes and allografts isolated from p21WAF1/CIP1 over-expressing recipients of rat heart transplants.
Our method of using plasmid DNA to obtain
in vitro and
in vivo transfection of p21WAF1/CIP1 is based on the data supporting the efficacy of intra-muscular injection of plasmid DNA for a number of genes [
28]. A number of studies [
29‐
32] have demonstrated that non-viral plasmid DNA provides a simple, safe, and viable alternative for gene therapy involving muscle tissue resulting in high level of expression. More significantly plasmids do not induce neutralizing immunity, which permits repeated administration. Rauh et al [
33] tested the hypothesis that intramuscular injection of naked DNA could result in the distribution remote from the site of needle placement, facilitating intramuscular gene transfer. Using transcutaneous ultrasound imaging the authors demonstrated that a solution of plasmid DNA administered by direct intramuscular injection into the skeletal muscles of the limb is distributed well beyond the site of needle entry and persisted for 8–10 weeks. Therefore, based on these and our own studies [
7], we believe that the p21WAF1/CIP1 over-expression can be obtained through intramuscular injections of plasmid DNA, which could result in the decreased responsiveness of T lymphocytes to allo-and mitogenic stimuli.
In summary, the results from this study uniquely demonstrate that during lymphocyte activation, expression of cyclins is increased and the inhibition of lymphocyte activation by cyclosporine inhibits the expression of cyclins and increases the expression of cyclin kinase inhibitor p21WAF1/CIP1. The expression of cyclins correlates with that of pro-inflammatory cytokines like TNF-α and IFN-γ
in vitro in activated lymphocytes and
in vivo in lymphocytes from animals with rejecting rat heart transplants. These studies demonstrate that cyclins and pro-inflammatory cytokines are key mediators of allo-immune activation and the alteration of p21WAF1/CIP1 expression can modulate lymphocyte proliferation and allo-immune activation. These studies uniquely provide evidence on the role of cell cycle control molecules on allo-immune activation and will allow the development of alternate strategies to obtain improved graft survival in organ transplantation. Moreover based on our previously published studies, the results presented in this study, and our recently published study [
34] we believe that p21WAF1/CIP1 might provide better immunosuppression with least side effects observed with the currently clinically used immunosuppressive drugs for the organ transplant recipients.