Reciprocal role of cyclins and cyclin kinase inhibitor p21WAF1/CIP1 on lymphocyte proliferation, allo-immune activation and inflammation
© Khanna; licensee BioMed Central Ltd. 2005
Received: 09 July 2005
Accepted: 21 September 2005
Published: 21 September 2005
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.
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.
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.
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.
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 . 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.
Preparation of lymphocytes and experimental protocol
Lymphocytes from the normal individuals (n = 4, obtained from Blood Center of Greater Milwaukee, Milwaukee) were separated as described . For mRNA studies, lymphocytes (1 × 106/ml) were cultured with PHA (2 μg/ml), with and without CsA (100 mg/ml) for 4 h, cells were washed twice with PBS and the cells were stored in Trizol at -80 C for RNA preparation.
Rat cardiac transplantation
Hetrotopic heart transplants were performed as described by us . We used Lewis (LEW, RT11) and Wistar-Furth (WF RT1u) rats, which represent complete genetic disparity at both major and minor histocompatibility loci. Isografts were performed in LEW-LEW while allogeneic transplantations were performed in WF-LEW transplant combinations. Immunosuppression was accomplished using CsA at a dosage of 2.5 mg/kg for the whole duration of the experiment described. Rats were monitored daily for evidence of allograft slowing and failure and at the time of rejection, animal s were sacrificed and spleens were used to prepare lymphocytes and organs were snap frozen in liquid nitrogen and stored at -80 C till isolation of RNA.
Detection of mRNA by reverse transcription and polymerase chain reaction (PCR) in allografts and lymphocytes
Total RNA was isolated from lymphocytes with Trizol (Invitrogen, Carlsbad, CA) and tissues with SV RNA isolation kit (Promega, Madison, WI). Purity of RNA was confirmed by a ratio of 260/280 nm. 1 μg of RNA was reverse transcribed into cDNA using a superscript reverse transcription kit from Invitrogen (Carlsbad CA). The amplification of specific mRNA expression was achieved by polymerase chain reaction (PCR) using specific primer sequences for p21WAF1/CIP1, β-actin, IL-2, TNF-α; Cyclin G, Cyclin E, Cyclin D3, IL-6, and IL-10 are described by us [4–6]. The PCR products were resolved in 1% agarose gel electrophoresis, ethidium bromide stained specific bands were visualized under UV light and photographed. The densitometric analysis of specific bands was made using Alpha-Imager (Alpha Innotech Corp, San Leandro, CA) and data are represented as the ratio of the specific gene to β-actin. We performed cycle analysis for each primer pair to select a cycle number for amplification for each gene studied.
Jurkat T cells proliferation assay
Cell proliferation was determined using 3H-thymidine incorporation as previously described (1–2). All assays were performed in triplicate. A total of 3 individual experiments investigating the proliferation of unaltered and p21WAF1/CIP1 Jurkat cells [described in ref. 7] were performed in unstimulated and activated with PHA. Briefly, 200,000 cells were added to each well of a round bottom 96-well plate. PHA (2 μg/ml) was added to the wells, controls were without PHA. The cells were cultured for 64 h at 37°C in 95% air and 5% CO2 enriched environment. The cultures were pulsed with 3H Thymidine (1 μCi/well) for the last 16 h of incubation, cells were harvested and radioactivity counted using a scintillation counter. 3H -Thymidine uptake was expressed as the mean counts per minute of triplicate samples. The magnitude of Jurkat T cells proliferation from unaltered and p21WAF1/CIP1-augmented cells were investigated at rest following mitogen stimulation.
In vivo transfection of p21WAF1/CIP1
p21 sense plasmid DNA
Plasmid DNA was isolated from competent E-coli cells transformed with either the empty pcDNA3.1/Zeo vector (Invitrogen, Carlsbad CA) or the vector containing the full-length p21WAF1/CIP1 gene in the sense direction described by us .
Mixed Lymphocyte Reaction (MLR)
MLR was performed with splenocytes from isografts, untreated and CsA treated transplant recipients (LEW) as responders and irradiated splenocytes from donor strain (WF) as stimulators. The stimulator or responder cells were cultured alone as negative controls. 3H Thymidine uptake was expressed as the median counts per minute of triplicate samples. The extent of proliferation determined the allo-reactivity of among these groups of mice and rats.
Differences between groups were determined using two-tailed unpaired T test with significance considered present at a p value of less than 0.05. Statistical analysis was performed using a software program from GraphPad Software, Inc., San Diego, CA 92121 USA. The results are expressed as M ± SEM.
Effect of inhibition of lymphocyte proliferation on IL-2, cyclins, TNF-α, IL-6 and p21WAF1/CIP1 mRNA
The results obtained from three consecutive experiments as the ratio of each gene with β-actin (Mean ± SEM) are presented in Figure 1B. A statistically significant decrease in pro-inflammatory cytokines IL-2 (p < 0.016), IL-6 (p < 0.02), TNF-α (p < 0.03) and cyclins; Cyclin D3 (p < 0.01) and Cyclin G (p < 0.008) was observed in sharp contrast to a significant increase in p21 (p < 0.03) in lymphocytes activated in the presence of CsA. CsA treatment also resulted in increased expression of p21 protein in activated lymphocytes (Figure 1C). This increase was about 2 fold.
Expression of cyclins in lymphocytes from rejecting and non-rejecting rats
Correlation of the expression of cyclins and pro-inflammatory cytokines in allo-immune
To confirm that the increased expression of cyclins in lymphocytes from rejecting rats was due to alloimmune activation, we performed mixed lymphocyte reaction (MLR) using spleen cells from donor animals (WF) as stimulators and splenocytes from recipients (LEW) as responders. Three groups were studied; isografts (control), untreated allografts (A), and CsA-treated allografts (B). After five-day MLR lymphocytes were harvested and washed. RNA was prepared; reverse transcribed to cDNA, and was amplified by RT-PCR for cyclin D3, IFN-γ, TNF-α, IL-6 and IL-10 mRNA. As shown in the Figure 2C the mRNA expression of cyclin D3 was higher in lymphocytes from untreated allografts group A as compared to lymphocytes from isografts group B. This increased expression of cyclin D3 correlated with the pro-inflammatory cytokines IFN-γ and TNF-α mRNA expression. The expression of IL-6 and IL-10 mRNA was not statistically significant between groups A and B. CsA treatment decreased cyclin D3 expression by 50% and inhibited statistically significant (p < 0.03) IFN-γ mRNA expression. Lymphocyte activation in MLR assay was quantified by 3H-thymidine uptake assay. Proliferation of lymphocytes from untreated allografts was significantly higher (two tailed p value = 0.02) compared to CsA- treated allografts (Mean ± SEM of counts per minute, n = 3, 11796 ± 728 vs 7575 ± 360). These results support our conclusions from rat transplant studies that the alloimmune results in increased expression of cyclins mRNA that correlates with production of pro-inflammatory cytokines. Allo-immune activation is demonstrated by increased lymphocyte proliferation from MLR assay, which decreased in CsA treated animals.
p21WAF1/CIP1 over-expression, lymphocyte proliferation and IL-2 expression
Effect of p21WAF1/CIP1 over-expression on graft survival in a rat heart transplant model
p21WAF1/CIP1 over-expression in Rats
We then studied the effect of modulation of p21WAF1/CIP1 on alloimmunity in a rat heart transplant recipients. We have extensive experience using the completely MHC mismatched WF (RTlu) into LEW (RTll) strain combination and have well defined thresholds of cyclosporine-based immunosuppression. In this model, animals reject within 7 to 10 days in the absence of immunosuppression and as late as 180 days with immunosuppression (CsA 2.5 mg/kg). A total of 12 rat transplants divided into four groups (A-D) were performed. Recipients in Group A were given one intramuscular injection of empty vector plasmid DNA (1 mg); Group B rats were given a daily dose of CsA (2.5 mg/kg). Rats in Group C rats received three weekly injections of p21WAF1/CIP1 sense plasmid DNA (0.5 mg); and Group D rats were given one intra-muscular injection of p21WAF1/CIP1 sense plasmid DNA and a daily injection of CsA (2.5 mg/Kg). The allografts were followed by palpitation, and an arbitrary scale of 1–4 was used to rate the heartbeat to determine the time of graft rejection. The rats were sacrificed when a heartbeat of 1–2 was recorded, which was considered as a cutoff for rejection. Though the number of transplants is low, yet as shown in Figure 4B, p21WAF1/CIP1 alone (*p < 0.04) or in combination with CsA (**p < 0.005) significantly prolonged the graft survival.
To confirm that this effect was due to the inhibition of alloimmune activation, we studied the expression mRNA of IL-2 in lymphocytes isolated from spleens and heart allografts. We also examined the expression of IL-10 mRNA in lymphocytes and allografts. The results are shown in the Figure 4C. The expression of IL-2 mRNA both in lymphocytes and allografts was higher in animals injected with empty vector plasmid DNA demonstrating increased allo-immune activation. IL-2 mRNA expression decreased significantly in recipients treated with p21WAF1/CIP1 sense plasmid DNA alone or together with CsA. The expression of IL-2, correlated with rejection, which indicated an increased immune activity due to allo-immune response resulting in the rejection as compared to p21WAF1/CIP1 or p21WAF1/CIP1 /CsA treated recipients. We did not observe any significant changes in the expression of IL-10 mRNA in allografts, which decreased in animals treated with p21WAF1/CIP1 sense plasmid DNA alone or with CsA, however it did not reach a level of significance (Figure 4C).
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,  Santiago-Raber et al  and Brian et al  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  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 . PCNA has 6 binding sites for p21 . Studies also  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 . 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 . Furthermore in contrast to p21WAF1/CIP1, the expression of p27 is not under transcriptional control and its mRNA expression remains unchanged during cell cycle . 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 . 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  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 . 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  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 , 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  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.
Author is thankful to Mr. Matthew Plummer for skillful technical assistance. This work was supported in part by research grant from the National Institutes of Health RO1AI41703.
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