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Basic and Experimental Research

Pirfenidone Inhibits T-Cell Activation, Proliferation, Cytokine and Chemokine Production, and Host Alloresponses

Visner, Gary A.1,3; Liu, Fengzhi1; Bizargity, Peyman1; Liu, Hanzhong1; Liu, Kaifeng1; Yang, Jun1; Wang, Liqing2; Hancock, Wayne W.2

Author Information
doi: 10.1097/TP.0b013e3181ae3392
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Abstract

The small molecule, pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone), has protective effects in the experimental models of fibrosis and inflammation (1–4). Pirfenidone (185 Da) is formulated as a water-soluble crystalline powder that readily penetrates into the cells and is well tolerated with relatively minor adverse effects (5, 6). The underlying mechanism of the beneficial effect of pirfenidone in fibrotic disease is not fully understood, although pirfenidone inhibits the expression of fibrogenic cytokines, such as transforming growth factor (TGF)-β, interleukin (IL)-4, and IL-13 (7–9), and the expression of tumor necrosis factor (TNF)-α (10, 11). Pirfenidone also inhibits the expression of intracellular adhesion molecule-1 (12) and enhances the expression of the anti-inflammatory cytokine, IL-10 (13, 14).

Our laboratory and others have shown that pirfenidone has graft protective effects (7, 15–18). As with its anti-fibrotic and anti-inflammatory effects, the beneficial actions of pirfenidone in improving graft survival may result from the inhibition of profibrotic or inflammatory cytokines (19). Although inhibiting profibrotic cytokines, such as TGF-β, may have benefits toward the fibroproliferative aspect of chronic rejection (20, 21), the inhibitory effects of pirfenidone on TGF-β expression could be potentially detrimental to early graft survival with increased acute rejection (22, 23) and potentially long-term graft survival due to reducing the development of inducible regulatory T cells (Tregs) (24). We previously found that pirfenidone prolonged survival and inhibited fibrosis in mouse tracheal and rat lung allograft models (7, 15, 17). The protection afforded by pirfenidone required treatment before the onset of fibrosis to be effective in attenuating the obstructive airway disease and associated fibrosis. These data suggest that pirfenidone has important effects beyond control of fibrosis, including the possibility of having direct effects on lymphocyte activation and function. In this study, we examined whether pirfenidone has immune modulating properties and whether it can directly inhibit T-cell activation or function. Because CD4+ and CD8+ T cells subsets play a role in transplant rejection (25–27), we evaluated the effects of pirfenidone on these subsets of lymphocytes and also its effects on Treg function, and its potential to promote long-term graft survival using a mouse cardiac allograft model.

MATERIALS AND METHODS

Reagents

We purchased carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Carlsbad CA), pirfenidone (CAS 53179-13-8, Department of Chemistry, University of Florida, Gainesville, FL), rapamycin (LC Laboratories, Woburn, MA), TGF-β ELISA assay kit (MB100B R&D systems Inc, Minneapolis, MN), and Luminex Muliplex Panel (Invitrogen Inc, Carlsbad, CA).

Mice

Six- to eight-weeks-old BALB/c (H-2d), C57BL/6 (H-2b) (B6), and C57BL/6/DBA F1 (H-2b/d) mice (The Jackson Laboratory, Bar Harbor, ME) were housed in specific pathogen-free conditions. All protocols were approved by the Institutional Animal Care and Use Committees of the Children’s Hospital of Philadelphia and Children’s Hospital Boston.

T Cells Phenotypic Analysis

Spleen and lymph nodes (para-aortic, mesenteric, and ilioinguinal) were isolated, and single-cell suspensions were prepared in phosphate-buffered saline with 0.1% of bovine serum albumin (Sigma-Aldrich, St Louis Mo). Cells were first incubated with anti-CD16/32 (FcγIII/II receptor antibody) to block nonspecific binding and then stained with anti-CD4, anti–T-cell receptor (TCR) and anti-CD8 (BD Biosciences, San Diego, CA) fluorochrome-conjugated monoclonal antibodies (mAbs). For the identification and phenotypic analysis of Treg cells (TCR+CD4+FOXP3+), we first performed extracellular staining of the cells with anti-TCRβ and anti-CD4 (Invitrogen Inc, Carlsbad CA) mAb. This was followed by intracellular staining with anti-mouse/rat FOXP3 (FJK-16a) antibody using the mouse/rat FOXP3 Staining kit (eBioscience Inc, San Diego, CA) per the manufacturer’s instructions. Viable cells were selected for flow cytometric analysis (LSR II BD, San Jose, CA) based on forward and side scatter light properties. The analysis was performed with FlowJo software (Tree Star software Inc, Portland, OR).

T-Cell Proliferation

Lymphocytes were isolated from spleen and lymph node samples by magnetic cell separation. Briefly, single-cell suspensions were prepared, and red blood cells were removed by hypotonic lysis. Magnetic beads-conjugated mAbs (Miltenyi Biotec Inc, Auburn, CA) were used to separate CD3, CD8, and CD4 subsets. Lymphocytes (1×105) labeled with the tracking fluorochrome CFSE and stimulated with mouse anti-CD3 mAb (1 μg/mL) and anti-CD28 (1 μg/mL), and proliferation assessed 72 hr later by flow cytometry of CFSE dilution.

In Vivo Mixed Lymphocyte Response

Alloreactive T-cell proliferation was assessed using an in vivo allogeneic mixed lymphocyte response (MLR), involving parent to F1 hybrid activation of CFSE-labeled T cells, as previously described (28). Donor cells were isolated from the lymph nodes and the spleens of wild-type C57BL/6 mice (H-2b). Pooled cells were labeled with CFSE, and 4×107 cells were injected intravenously into C57BL/6/DBA F1 (H-2b/d) recipients. Recipients were divided into groups receiving no additional therapy (control), pirfenidone by oral gavages or subcutaneous injection (400 mg/kg/day), rapamycin intraperitoneally (0.1 mg/kg/day), or both pirfenidone and rapamycin treatment. Spleens were harvested from F1 mice after 3 days and splenocytes incubated with fluorochrome-conjugated mAb anti-CD4 and anti-CD8 and biotin-conjugated anti-H-2Kd and anti-H-2Dd− mAb, followed by fluorochrome-conjugated secondary antibody. Donor alloreactive T cells were distinguished from recipient T cells by gating on H-Kd− and H-2Dd− cells. T-cell proliferation was assessed using CFSE division profiles.

Cytokines and Chemokines Assay

CD3+ and CD4+ T cells were stimulated by TCR activation, and cell culture supernatant was collected. The concentrations of interferon (IFN)-γ, IL-1β, TNF-α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p, IL-13, IL-17, intraperitoneally (IP)-10, macrophage inflammatory protein (MIP)-1, Mig, fibroblast growth factor (FGF)-β, granulocyte macrophage-colony stimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF) were measured by Luminex-200 System using the Cytokine Mouse 20-Plex Panel (Invitrogen Inc, Carlsbad, CA) per the manufacturer’s instructions. TGF-β levels were determined from stimulated CD3+ and CD4+ T cells or isolated protein from cardiac allografts using ELISA kit (R&D system Minneapolis, MN) per manufacturer’s directions, and values were within the linear range based on the generation of a standard curve for each individual experiment. There were fourindividual experiments for each group, and each sample was run in duplicate (TGF-β ELISA) or triplicate (mouse cytokine panel). Pirfenidone- and vehicle-treated heart allografts were homogenized in phosphate-buffered saline containing protease inhibitor cocktail (Sigma- Aldrich, St Louis Mo) and centrifuged at 20,000g for 20 min at 4°C, and 200 μg of the protein concentration from supernatant was used for analysis.

Suppression Assay

CD4+CD25+ (Treg), CD4+CD25 (Teff), and antigen presenting cells (APCs) were isolated from total splenocytes by using magnetic beads-conjugated mAbs (Miltenyi Biotec Inc). APCs were irradiated (3300 rad) and plated onto 96-well plates. Teff cells (1×105) labeled with CFSE were co-cultured with or without Treg cells at the indicated ratios, and a combination of 1 μg/mL of soluble anti-CD3 provided the polyclonal stimulus. To analyze the effect of pirfenidone on the suppression activity of Treg cells, we used optimum concentration of 1 mM. Proliferation of Teff cells was assessed by CFSE dilution 72 hr later.

Cardiac Transplantation

Heterotopic abdominal cardiac allograft with end-to-side anastomosis of aorta to aorta and pulmonary artery to vena cava (29) were undertaken using BALB/c donors and B6 recipients (5–8 allograft/group). Graft function was monitored daily by palpation, and rejection was confirmed by histology. Allograft recipients were killed, and grafts were harvested for analysis at the time of rejection (lack of heart beat), or in additional recipients at 1 week posttransplant, so as to allow direct comparison of intragraft events. Grafts were removed, fixed in formalin, paraffin embedded, and 5-μ sections were cut and stained with hematoxylin-eosin.

Statistics

Data were expressed as mean±SEM, and statistical analyses were performed using Prism statistical program (Graph Pad, San Diego, CA). We used one-way analysis of variance with Tukey’s Multiple Comparison Test to evaluate the differences between groups, student’s t test to compare two groups, and Kaplan-Meier survival curve for heart allograft survival; a P value less than 0.05 was considered significant.

RESULTS

Pirfenidone Does not Change Percentage of T-Cell Subset at the Steady State Level

Our previous studies using both mouse tracheal and rat lung transplant models showed that pirfenidone treatment had graft protective properties with a reduction in the extent of rejection (7, 15, 17). To determine whether the beneficial effects of pirfenidone on graft survival include modulation of allo responses, we first examined whether pirfenidone has any direct effects on T cells in steady state level. Our initial studies examined whether pirfenidone (400 mg/kg for 7 days) has negative effect on T-cell subsets and Treg percentage in the spleen and lymph nodes in C57BL/6 mice. There are no significant differences in TCR+CD4+, TCR+CD8+, and TCR+ CD4+ Foxp3+ Treg cells in the spleen and the lymph nodes (Fig. 1, Tables 1 and 2) (P>0.05), which indicate that pirfenidone does not have any negative effect on the steady state level of T-cell subsets in mice.

F1-8
FIGURE 1.:
The ratio of TCR+CD4+, TCR+CD8+, and TCR+CD4+foxp3+ T cell. Mice were treated with pirfenidone or vehicle for 7 days, and isolated cells from spleen and lymph nodes were stained for anti-TCRß, CD4, CD8, and Foxp3. The ratio of T-cell subpopulation from the isolated spleen and lymph nodes was assessed by flow cytometry and shown is a representative Dot Plot of CD4+foxp3+ cells ratio among TCR+CD4+ cells isolated from the spleen and lymph nodes.
T1-8
TABLE 1:
Spleen
T2-8
TABLE 2:
Abdominal lymph nodes

Pirfenidone Inhibits T-Cell Proliferation In Vitro and In Vivo

We then tested whether pirfenidone has direct effects on T-cell proliferation. Pirfenidone was found to significantly inhibit TCR-stimulated CD4+ T-cell proliferation in vitro (P<0.01), whereas CD8+ T total cell proliferation was not significantly affected (Fig. 2a). Although we observed no significant inhibition of CD8+ T cells based on responder frequency (percent of original cells that divided), we observed attenuation for both CD4+ and CD8+ T cells proliferation index (number of divisions of proliferating cells) in response to pirfenidone (Fig. 2b) in a dose-dependent manner.

F2-8
FIGURE 2.:
(a) Suppressive effect of pirfenidone (PFD, 1 mM) on T-cell receptor-stimulated (antigen presenting cell+anti-CD3) CD4+ T-cell proliferation (mean±SEM from six independent experiments, *P<0.01, control to PFD) as assessed by flow cytometry, including all dividing populations (R3). A representative fluorescence-activated cell sorter (FACS) experiment of carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution is shown. (b) CD4+ and CD8+ T-cell proliferation on stimulation with CD3 and CD28 monoclonal antibodies on addition to increasing PFD dose (0, 0.01, 0.1, and 1 mM) (*P<0.05 when compared with untreated group, n=4). A representative FACS experiment of CD4+ and CD8+ T-cell proliferation based on CFSE dilution and illustrating a reduction in cell division proliferation on addition of PFD (note overall shift to the right with PFD use).

To examine the effects of pirfenidone on T-cell allogeneic activation and proliferation in vivo, we used a parent into F1 in vivo one-way MLR assay (28). We found that pirfenidone treatment resulted in attenuation of both CD4+ and CD8+ T cells proliferation. There was a small but consistent reduction in CD4+ and CD8+ T cells proliferation based on responder frequency; however, this was most evident based on proliferation index with a reduction of proliferation at the higher division populations. We also examined whether there was an additive effect of pirfenidone and rapamycin on alloreactive T-cell proliferation. By using low dose rapamycin plus pirfenidone, we observed a greater inhibition of both CD4+ and CD8+ T cells proliferation than when either agent was used alone (Fig. 3).

F3-8
FIGURE 3.:
Results of a parent-F1 hybrid in vivo mixed lymphocyte response illustrating both CD4+ and CD8+ T-cell proliferation (all divisions and last three divisions) in untreated, pirfenidone (PFD) treated (400 mg/kg/day), rapamycin treated (RAP, 0.1 mg/kg/day), or a combination of PFD plus RAP using carboxyfluorescein diacetate succinimidyl ester dilution (*P<0.05 when compared with untreated group, n=4), and a representative fluorescence-activated cell sorter experiment for both CD4+ and CD8+ T-cell proliferation is shown for the experimental groups.

Pirfenidone Significantly Reduced Some Cytokine and Chemokine Expression

Many of pirfenidone actions are believed to occur through its inhibitory actions on cytokine expression. In addition, cytokine production is a component of T-cell activation (30, 31). Therefore, we examined whether pirfenidone inhibited cytokine or chemokine release in response to TCR activation in vitro. We first observed a significant (P<0.05) decrease in multiple cytokines in CD3+ T cells, including those linked with Th1 (IFN-γ, IL-1β, and TNF-α), Th2 (IL-4), and Th17 (IL-17) differentiation, plus at least one profibrotic cytokine FGF-β, and pro- inflammatory chemokines (IP-10, MIP-1, Mig). No significant change in response to pirfenidone was observed in production of IL-2, IL-6, IL-12p, IL-5, IL-10, IL-13, GM-CSF, or VEGF. Because we observed a greater effect of pirfenidone on CD4+ T-cell proliferation in vitro than CD8+ T cells, we also evaluated the effects of pirfenidone on TCR-stimulated cytokine expression by purified CD4+ T cells. We found a significant inhibition of CD4+ T cells production of IFN-γ, IL-1β, IL-4, IL-5, IL-17, IP-10, MIP-1, and Mig in response to pirfenidone (Fig. 4a). Also of note was a lack of inhibition of TGF-β in either purified CD3+ or CD4+ T cells with pirfenidone (Fig. 4b), in contrast to our previous data from both rat lung and mouse tracheal transplant models in which pirfenidone resulted in decreased TGF-β in the peripheral blood and grafts (7, 17). However, similar to our previous studies, there was an inhibition of TGF-β expression of isolated protein from cardiac allografts treated with pirfenidone (Fig. 4c) indicating cell-specific responses of TGF-β to pirfenidone.

F4-8
FIGURE 4.:
(a) Graphs of cytokines and chemokines released from T-cell receptor-stimulated CD3 and CD4+ T cells under control (untreated) and pirfenidone (PFD)-treated conditions. The agents listed are those that showed significant inhibition in response to PFD (P<0.05 with n=4). (b) In addition, the results of transforming growth factor (TGF)-β are shown in which there were no significant difference observed between untreated and treated CD3+ and CD4+ T cells. (c) Graph illustrating TGF-β expression from PFD (n=6) and vehicle (n=6) in isolated protein from PFD and untreated heart allografts 7 days after transplantation. PFD-treated heart allografts significantly (P<0.05) express lower TGF-β compared with vehicle-treated grafts.

Pirfenidone Does not Interfere With Regulatory T-Cell Function

Becuase pirfenidone had an inhibitory effect on T-cell activation and production of multiple cytokines (both Th1- and Th2-associated), we examined whether pirfenidone had effects on Treg-cell activity. A standard in vitro Treg assay was performed using varying ratios of CD4+CD25+ co-cultured with CFSE-labeled CD4+CD25 T cells plus CD3 mAb and APC. As expected, there was less overall TCR-stimulated proliferation in the pirfenidone-treated group as compared with the control population; however, the suppressive effects of CD4+CD25+ population on effector cells was maintained in the presence of pirfenidone (Fig. 5). Hence, pirfenidone does not seem to affect Treg function.

F5-8
FIGURE 5.:
This is a representative experiment evaluating the effects of pirfenidone (PFD) on regulatory T-cell (Treg) function. The first set illustrates the T-cell receptor-stimulated proliferation of CD4+ CD25, and T effector cells (Teff), with increasing concentration of CD4+CD25+, Treg population. The second set illustrates the results with the addition of PFD. The graph shows the mean of three independent experiments.

Pirfenidone Promotes Long-Term Heart Allograft Survival in Low Dose Rapamycin-Treated Recipient

To examine the effect of pirfenidone on immune responses in a transplant model, we undertook heterotopic vascularized cardiac allografting across a full major histocompatibility complex mismatch, BALB/c (H-2d) donors and B6 (H-2b) recipients. Although low dose of pirfenidone (40 mg/kg) had no effect on allograft survival, a higher dose of pirfenidone (400 mg/kg) significantly (P<0.05) prolonged graft survival (Fig. 6a), and there was a remarkable reduction in the histologic evidence of acute rejection after 7 days. Untreated cardiac allografts showed extensive mononuclear cell infiltration and multifocal myocyte necrosis characteristic of acute cellular rejection, grade 3R, whereas pirfenidone- and rapamycin-treated grafts showed only mild or low grade acute cellular rejection with diffuse interstitial pattern of lymphocytes and little perivascular infiltrate without myocyte damage, grade 1R (Fig. 6b). Similar to the in vivo MLR, the combination of pirfenidone plus a subtherapeutic regimen of rapamycin significantly prolonged allograft survival (P<0.05) and delayed the onset of rejection and the development of key features of chronic rejection, including arteriosclerosis and myocardial fibrosis.

F6-8
FIGURE 6.:
(a) Survival curve of heterotopic heart transplants from BALB/c donors into B6 recipients for untreated (control) and those treated with rapamycin (RAP), pirfenidone (PFD), or the combination of PFD plus RAP (n=5–8 transplants/group). P less than 0.05 for PFD and PFD-/RAP-treated groups when compared with untreated allografts. (b) Representative microphotograph (hematoxylin-eosin stain, ×200) of a normal mouse heart and heart allografts from untreated (vehicle), RAP-, and PFD-treated mice at day 7 posttransplantation.

DISCUSSION

Pirfenidone’s antifibrotic effects have elicited an interest in its use as a potential therapeutic agent to limit the fibroproliferative response of chronic rejection (19, 32). Our laboratory and others have shown previously that pirfenidone has graft protective effects and reduced the development of fibrosis in the lung and tracheal transplant models (7, 15–18). This was associated with an inhibition of the profibrotic cytokine TGF-β and a reduction in collagen formation. However, in addition to antifibrotic effects, pirfenidone also limited the alloimmune or inflammatory response, as reflected by reduction of intragraft neutrophil and lymphocyte recruitment (15). This study was performed to assess whether pirfenidone had a direct effect on T-cell activation and proliferation, thereby having an immunosuppressive role in addition to its known antifibrotic effect. We found that pirfenidone exposure attenuated T-cell proliferation, activation and production of multiple cytokines and chemokines, thereby indicating that the graft protective effects of pirfenidone may be 2-fold, encompassing both the alloimmune and nonalloimmune phases of rejection.

Our in vitro studies demonstrated that pirfenidone reduced TCR-stimulated proliferation of CD4+ T cells and a more limited effect on CD8+T-cell proliferation. However, using an in vivo allogeneic model of stimulated T-cell proliferation (parent to F1 model), we observed an inhibitory effect on both CD4+ and CD8+ T cells proliferation. Further evidence of pirfenidone’s inhibition of the allogeneic response was observed by its significant delay of allograft loss in a fully major histocompatibility complex-mismatched murine cardiac allograft model. Interestingly, we observed that pirfenidone with low dose rapamycin also potentiated the inhibition of allogeneic-stimulated T-cell proliferation using the parent to F1 in vivo MLR, and more importantly, further prolonged mouse cardiac allograft survival.

Hirano et al. (8) showed that pirfenidone inhibited the antigen-specific proliferation of the lymph node mononuclear cells in ovalbumin-sensitized mice and the production of the profibrotic Th2 cytokines, IL-4, and IL-13. In addition, pirfenidone was shown to inhibit the expression of the Th1 cytokines, TNF-α and IFN-γ (11, 14). We also found that pirfenidone inhibits a number of proinflammatory cytokines or chemokines from activated lymphocytes including IFN-γ, IL-1β, TNF-α, IL-4, IL-17, FGF-β, IP-10, MIP-1, and Mig. Pirfenidone’s protective effects on graft survival may still be related to its inhibition of a number of these cytokines and chemokines, thereby reducing the inflammatory response, T-cell activity, and possibly trafficking with its effects on chemokines. The suppression of these cytokines and chemokines does not seem to be from a general inactivation of lymphocytes and therefore inhibiting the release of all products because we observed no change in IL-2, IL-6, IL-12p, IL-5, IL-10, IL-13, GM-CSF, and VEGF. In addition, we did not see a decrease in CD25 or CD69 expression on TCR activation of T cells in vitro (data not shown).

As noted, pirfenidone’s antifibrotic effects are attributed commonly to its suppression of TGF-β production. TGF-β is a pleiotropic cytokine with complex regulatory functions including the differentiation and proliferation of a wide variety of cell types, and a central mediator in the development of fibrosis (33–35). TGF-β has an early proinflammatory role in tissue, which can recruit monocytes and granulocytes to injured areas by acting as a chemoattractant (34, 36) but is subsequently anti-inflammatory with effects on a variety of inflammatory cells, which is most apparent on T cells (37, 38). The later inhibitory effects are believed to help resolve inflammation (35). TGF-β also has antiproliferative actions on T cells, largely attributed to inhibition of IL-2 production (39), and plays a role in the development of both natural and inducible Tregs (40). Similar to our previous observations for mouse tracheal and rat lung allografts, pirfenidone inhibited TGF-β expression in heart allografts. Pirfenidone’s inhibition of the early TGF-β and other acute inflammatory mediators could reduce the early transplant response, whereas its effects on the later TGF-β response and profibrotic cytokines reduce the fibroproliferative injury. In contrast to what we observed in the lung or heart tissue, we found that pirfenidone had no suppressive effects on TGF-β release from TCR-activated T cells showing a differential cellular response to pirfenidone. More importantly, because TGF-β can mediate the generation of Tregs, and these cells may play a role in long-term tolerance of allografts (40), it is reassuring that pirfenidone did not interfere with the suppressive effects of CD4+ CD25+ cells/Tregs on T-cell proliferation.

This study supports the potential of pirfenidone as an additive therapeutic agent in prolonging allograft survival. As previously shown, it may reduce the fibroproliferative response that is part of the pathophysiology of chronic rejection. However, it also has inhibitory actions against the alloimmune phase with a reduction in lymphocyte activation and proliferation, and prolongs graft survival, especially in combination with low dose rapamycin. It is encouraging that despite having the ability to reduce TGF-β expression, we did not see an inhibition of TGF-β in activated T cells, and, more importantly, it did not reduce the number of Tregs in the transplant model or interfere with Treg suppressive activities.

REFERENCES

1. Shimizu T, Kuroda T, Hata S, et al. Pirfenidone improves renal function and fibrosis in the post-obstructed kidney. Kidney Int 1998; 54: 99.
2. Van Erp C, Irwin NG, Hoey AJ. Long-term administration of pirfenidone improves cardiac function in mdx mice. Muscle Nerve 2006; 34: 327.
3. Lee KW, Everett TH, Rahmutula D, et al. Pirfenidone prevents the development of a vulnerable substrate for atrial fibrillation in a canine model of heart failure. Circulation 2006; 114: 1703.
4. Gurujeyalakshmi G, Hollinger MA, Giri SN. Pirfenidone inhibits PDGF isoforms in bleomycin hamster model of lung fibrosis at the translational level. Am J Physiol 1999; 276(2 Pt 1): L311.
5. Azuma A, Nukiwa T, Tsuboi E, et al. Double-blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2005; 171: 1040.
6. Giri SN, Wang Q, Xie Y, et al. Pharmacokinetics and metabolism of a novel antifibrotic drug pirfenidone, in mice following intravenous administration. Biopharm Drug Dispos 2002; 23: 203.
7. Liu H, Drew P, Gaugler AC, et al. Pirfenidone inhibits lung allograft fibrosis through L-arginine-arginase pathway. Am J Transplant 2005; 5: 1256.
8. Hirano A, Kanehiro A, Ono K, et al. Pirfenidone modulates airway responsiveness, inflammation, and remodeling after repeated challenge. Am J Respir Cell Mol Biol 2006; 35: 366.
9. Shihab FS, Bennett WM, Yi H, et al. Pirfenidone treatment decreases transforming growth factor-beta1 and matrix proteins and ameliorates fibrosis in chronic cyclosporine nephrotoxicity. Am J Transplant 2002; 2: 111.
10. Cain WC, Stuart RW, Lefkowitz DL, et al. Inhibition of tumor necrosis factor and subsequent endotoxin shock by pirfenidone. Int J Immunopharmacol 1998; 20: 685.
11. Hale ML, Margolin SB, Krakauer T, et al. Pirfenidone blocks the in vitro and in vivo effects of staphylococcal enterotoxin B. Infect Immun 2002; 70: 2989.
12. Kaneko M, Inoue H, Nakazawa R, et al. Pirfenidone induces intercellular adhesion molecule-1 (ICAM-1) down-regulation on cultured human synovial fibroblasts. Clin Exp Immunol 1998; 113: 72.
13. Nakazato H, Oku H, Yamane S, et al. A novel anti-fibrotic agent pirfenidone suppresses tumor necrosis factor-alpha at the translational level. Eur J Pharmacol 2002; 446: 177.
14. Oku H, Nakazato H, Horikawa T, et al. Pirfenidone suppresses tumor necrosis factor-alpha, enhances interleukin-10 and protects mice from endotoxic shock. Eur J Pharmacol 2002; 446: 167.
15. Liu H, Drew P, Cheng Y, et al. Pirfenidone inhibits inflammatory responses and ameliorates allograft injury in a rat lung transplant model. J Thorac Cardiovasc Surg 2005; 130: 852.
16. Dosanjh A, Ikonen T, Wan B, et al. Pirfenidone: A novel anti-fibrotic agent and progressive chronic allograft rejection. Pulm Pharmacol Ther 2002; 15: 433.
17. Zhou H, Latham CW, Zander DS, et al. Pirfenidone inhibits obliterative airway disease in mouse tracheal allografts. J Heart Lung Transplant 2005; 24: 1577.
18. McKane BW, Fernandez F, Narayanan K, et al. Pirfenidone inhibits obliterative airway disease in a murine heterotopic tracheal transplant model. Transplantation 2004; 77: 664.
19. Dosanjh A. Pirfenidone: Anti-fibrotic agent with a potential therapeutic role in the management of transplantation patients. Eur J Pharmacol 2006; 536: 219.
20. Di Filippo S, Zeevi A, McDade KK, et al. Impact of TGFbeta1 gene polymorphisms on acute and chronic rejection in pediatric heart transplant allografts. Transplantation 2006; 81: 934.
21. Shittu EA, Yang CP, Roberts IS, et al. Inhibition of chronic vascular rejection by donor-specific blood transfusion is associated with a reduction in transforming growth factor-beta1 expression. Transplantation 2002; 73: 1573.
22. Qin L, Chavin KD, Ding Y, et al. Gene transfer for transplantation. Prolongation of allograft survival with transforming growth factor-beta 1. Ann Surg 1994; 220: 508.
23. Hodge G, Hodge S, Chambers D, et al. Acute lung transplant rejection is associated with localized increase in T-cell IFN gamma and TNFalpha proinflammatory cytokines in the airways. Transplantation 2007; 84: 1452.
24. Csencsits K, Wood SC, Lu G, et al. Transforming growth factor-beta1 gene transfer is associated with the development of regulatory cells. Am J Transplant 2005; 5: 2378.
25. Hall BM. Cells mediating allograft rejection. Transplantation 1991; 51: 1141.
26. Bishop DK, Shelby J, Eichwald EJ. Mobilization of T lymphocytes following cardiac transplantation. Evidence that CD4-positive cells are required for cytotoxic T lymphocyte activation, inflammatory endothelial development, graft infiltration, and acute allograft rejection. Transplantation 1992; 53: 849.
27. Nozaki T, Rosenblum JM, Ishii D, et al. CD4 T cell-mediated rejection of cardiac allografts in B cell-deficient mice. J Immunol 2008; 181: 5257.
28. Suchin EJ, Langmuir PB, Palmer E, et al. Quantifying the frequency of alloreactive T cells in vivo: New answers to an old question. J Immunol 2001; 166: 973.
29. Corry RJ, Winn HJ, Russell PS. Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection. Transplantation 1973; 16: 343.
30. Frenken LA, Koene RA, Tax WJ. The role of antibody isotype in IFN-gamma and IL-2 production during anti-CD3-induced T cell proliferation. Transplantation 1991; 51: 881.
31. Hobbs MV, Weigle WO, Noonan DJ, et al. Patterns of cytokine gene expression by CD4+ T cells from young and old mice. J Immunol 1993; 150(8 Pt 1): 3602.
32. Dosanjh AK, Wan B, Throndset W, et al. Pirfenidone: A novel antifibrotic agent with implications for the treatment of obliterative bronchiolitis. Transplant Proc 1998; 30: 1910.
33. Antoshina E, Ostrowski LE. TGF beta 1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures. In Vitro Cell Dev Biol Anim 1997; 33: 212.
34. Wahl SM, Hunt DA, Wakefield LM, et al. Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci USA 1987; 84: 5788.
35. Li MO, Wan YY, Sanjabi S, et al. Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol 2006; 24: 99.
36. Wiseman DM, Polverini PJ, Kamp DW, et al. Transforming growth factor-beta (TGF beta) is chemotactic for human monocytes and induces their expression of angiogenic activity. Biochem Biophys Res Commun 1988; 157: 793.
37. Prud’homme GJ. Pathobiology of transforming growth factor beta in cancer, fibrosis and immunologic disease, and therapeutic considerations. Lab Invest 2007; 87: 1077.
38. Rubtsov YP, Rudensky AY. TGF beta signalling in control of T-cell-mediated self-reactivity. Nat Rev Immunol 2007; 7: 443.
39. Suda T, D’Ovidio F, Daddi N, et al. Recipient intramuscular gene transfer of active transforming growth factor-beta1 attenuates acute lung rejection. Ann Thorac Surg 2001; 71: 1651.
40. Jonuleit H, Adema G, Schmitt E. Immune regulation by regulatory T cells: Implications for transplantation. Transpl Immunol 2003; 11: 267.
Keywords:

Rodent; T cells; Transplantation

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