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Keywords:

  • Acute cellular rejection;
  • FK506;
  • infliximab;
  • intestinal transplantation;
  • regeneration;
  • rescue therapy

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

Clinical evidence suggests that recurrent acute cellular rejection (ACR) may trigger chronic rejection and impair outcome after intestinal transplantation. To test this hypothesis and clarify underlying molecular mechanisms, orthotopic/allogenic intestinal transplantation was performed in rats. ACR was allowed to occur in a MHC-disparate combination (BN-LEW) and two rescue strategies (FK506monotherapy vs. FK506+infliximab) were tested against continuous immunosuppression without ACR, with observation for 7/14 and 21 days after transplantation. Both, FK506 and FK506+infliximab rescue therapy reversed ACR and resulted in improved histology and less cellular infiltration. Proinflammatory cytokines and chemotactic mediators in the muscle layer were significantly reduced in FK506 treated groups. Increased levels of CD4, FOXP3 and IL-17 (mRNA) were observed with infliximab. Contractile function improved significantly after FK506 rescue therapy, with a slight benefit from additional infliximab, but did not reach nontransplanted controls. Fibrosis onset was detected in both rescue groups by Sirius-Red staining with concomitant increase of the fibrogenic mediator VEGF. Recovery from ACR could be attained by both rescue therapy regimens, progressing steadily after initiation of immunosuppression. Reversal of ACR, however, resulted in early stage graft fibrosis. Additional infliximab treatment may enhance physiological recovery of the muscle layer and enteric nervous system independent of inflammatory reactions.


Abbreviations: 
ACR

acute cellular rejection

BN

Brown Norway

CB

cuprolinic blue

Cy3

cyanine 3

cDNA

complementary DNA

ED1

antibody recognizing CD68

FOXP3

forkhead box P3 protein

IFN-γ

interferon-gamma

KHB

Krebs–Henseleit bicarbonate solution

LEW

Lewis

MCP-1

monocyte chemoattractant protein-1

MPO

myeloperoxidase

POD

postoperative day

S100b

S100 calcium binding protein B

TNF-α

tumor necrosis factor-alpha

UW

University of Wisconsin

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

After intestinal transplantation, acute and chronic rejection episodes impair the clinical outcome and allogenic rejection is one of the leading causes of graft failure or loss (1–3). Postoperative graft dysmotility occurs in the early phase after intestinal transplantation in response to ischemia/reperfusion injury, surgical manipulation and molecular inflammatory responses triggered by activation of resident macrophages in the intestinal muscle layer (4–6). This temporary dysmotility and the associated molecular changes resolve during the first 1–2 weeks after transplantation and—at first glance—do not seem to induce permanent functional changes in intestinal grafts. Although this is especially true in isogenic animal models of orthotopic SBTX (Lew to Lew), the picture changes in the presence of rejection after allogenic transplantation (BN to Lew). During acute rejection episodes, molecular and cellular inflammation of intestinal grafts with subsequent dysmotility can be observed (6), which also involves activation of resident macrophages in the tunica muscularis of the transplanted intestine (5). In this case, the unspecific inflammation may aggravate the adaptive immune response resulting in severe rejection processes. It is known that multiple acute rejection episodes often result in gradually progressing graft deterioration and loss of function limiting the long-term outcome of intestinal transplant patients. Although ACR may be controlled by a wide array of agents (including steroids and biologicals) or increasing baseline immunosuppression (mostly CNI Inhibitors), the fatal effects of chronic rejection—once initiated—can in most cases not be reversed.

To elucidate the mechanisms of intestinal recovery from ACR in orthotopic intestinal transplantation and to search for the missing link between acute rejection and chronic graft failure, we designed a small animal study with different rescue regimens after ACR in a MHC-disparate high responder model (BN donors–Lew recipients):

We hypothesized that acute rejection could be resolved even after full manifestation by introduction of immunosuppressive therapy (with FK506 or a combination of FK506 and infliximab). Furthermore, we hypothesized that evaluation of molecular and cellular inflammatory responses, neural damage, histopathological integrity and functional assessment of the grafts in the recovery phase could identify the possible underlying mechanisms for chronic graft dysfunction after rescue therapy and subsequent graft recovery from ACR. Finally, we hypothesized that additional infliximab treatment could result in improved recovery and smooth muscle function through supplemental anti-inflammatory effects as well as altered immune responses as suggested by a previous study (7). In summary, the objectives of this study were (1) to establish a standardized recovery model for the testing of different rescue therapy regimens, (2) to evaluate the process of recovery after acute rejection and to discover potential mechanisms for functional graft deterioration as well as immunological priming possibly inducing chronic rejection and (3) to assess the effects of anti-TNF-α therapy with infliximab as an additional component of a rescue strategy in the experimental setting.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

Animals

Inbred male Brown Norway (BN) and Lewis (LEW) rats weighing 180–200 g were obtained from Charles River WIGA GmbH (Sulzfeld, Germany). All experiments were performed in accordance with the federal law regarding the protection of animals. The principles of laboratory animal care (NIH Publication No.8 5–23, revised 1985) were followed. The animals were maintained on a 12-h light/dark cycle and provided with commercially available chow (Altromin, Lage, Germany) and tap water ad libitum.

Surgical procedures and experimental protocol

Orthotopic, allogenic intestinal transplantation from Brown Norway (BN) to Lewis (LEW) rats was performed as previously described (8). Cold and warm ischemia time was approximately 60 and 30 min, respectively. UW solution was used for preservation during cold ischemia. According to results from pilot studies (not shown), immunosuppressive treatment consisted of FK506 at a dose of 2 mg/kg/day (i.m.) and antibiotic treatment with ampicillin (100 mg/kg/day [i.m.]). Physiological saline was used as vehicle control. Recipient rats were divided into eight groups according to the combination of rat strain, protocol of immunosuppression and observation time (seven, 14 and 21 days post-SBTX).

Study groups were set up as follows: nontransplanted/untreated controls (group 1); BN to LEW without immunosuppression, observation for seven days (group 2); BN to LEW, continuous FK506 administration from day 0, observation for 14/21 days (groups 3/4); BN to LEW, FK506 administration after day 7, observation for 14/21 days (groups 5/6); BN to LEW, FK506+infliximab administration after day 7 and observation for 14/21 days (groups 7/8). Antibiotics were given for three days after transplantation in every group, and additionally administered in groups 2, 3 and 4 to achieve reliable survival rates. Operated rats were kept fasting for 24 h before, and at water/glucose solution only for 72 h after transplantation. Rats were sacrificed at the end of the observation periods by isoflurane anesthesia inhalation overdose and intestinal grafts were removed for further analysis. Muscle layers were separated from whole intestinal grafts by manual scratch for mRNA extraction or by under-microscope stripping for histology, histochemistry, immunohistochemistry and in vitro muscle contractility measurement (8).

Histology, histochemistry and immunohistochemistry

For histological analysis, intestinal grafts were fixed overnight with 4% formalin and embedded in paraffin. Fixed tissues were cut into 5 μm sections and stained with hematoxylin and eosin and evaluated with the established grading scheme as reported by Wu et al. (9). Sirius Red (SR) staining with 0.1% Sirius Red solution, prepared from 0.1g Direct Red 80 and 100 mL saturated picric acid, was performed to detect graft fibrosis (10,11). Briefly, slides are deparaffinized in xylene, hydrated in ethanol (100%, 90% and 70%) for 5 min, washed and stained with 0.1% Sirius Red solution for 15 min. Next, slides are washed, and dehydrated in ethanol (70%, 90% and 100%) and xylene for 7 min each. Cuprolinic Blue (CB) staining was performed to detect enteric neurons (12). Briefly, separated intestinal muscle layer was fixed in 4% formalin for 30 min or longer and washed several times with KHB solution. Next, specimen were given a 10 min endogeneous peroxidase block in a 1:4 solution of 3% hydrogen peroxide (H2O2) and methanol followed by five times × 5 min washing in 0.02M potassium phosphate-buffered saline (KPBS), pH 7.4. Incubation in CB solution and subsequent procedure have been described previously (12). Myeloperoxidase (MPO)-positive cells in muscle layer were detected using Hanker-Yates reagent. Separated muscle layer was immersed in a mixture of 10 mg Hanker–Yates reagent, 10 mL KHB and 200 μL 3% H2O2 for 5 min after fixation with 100% ethanol for 10 min. Apoptotic cells in the muscularis propria were quantified using the TdT-mediated dUTP-X nick-end-labeling (TUNEL) method on paraffin-embedded tissue (InSituCellDeathDetectionKit, Roche Diagnostics GmbH, Mannheim, Germany). Immunohistochemical analysis against ED1 was performed as described previously (8). For evaluation, positive stained leukocytes, neurons and area were counted or calculated in five randomly chosen areas in each specimen at a magnification of 100× (MPO) or 200× (Cuprolinic Blue, SR, TUNEL and ED1) with the Nikon TE2000-E microscope (Nicon GmbH, Dusseldorf, Germany). SR positive area was expressed by the percentage of positive area per 200 × 400 μm (width x height, for intralumen upwards) rectangular region of interest (ROI) including whole layer of intestine, which was calculated using the NIS-Elements AR version 3.0 software program (Nikon Instruments Inc., Melville, NY, USA).

In vitro smooth muscle contractility measurement

Mechanical in vitro activity of the mid-jejunum was evaluated using smooth muscle strips of the circular muscle layer as described previously (13). Dose response curves were generated using increasing doses of the muscarinic receptor agonist bethanechol (1–300 mol/L). The contractile response was recorded and analyzed with ADI Chart© software (ADI, Heidelberg, Germany) and calculated as grams per square millimeter per second by conversion of the weight and length of the strip to square millimeters of tissue (g/mm2/s).

Data analysis

Results are expressed as mean ± standard deviation (SD) or standard error (SEM) where indicated. Statistical analysis consisted of one-way ANOVA followed by Bonferroni or Dunnett's multiple comparison tests or, whenever suitable, by the nonparametric Kruskal-Wallis test followed by Mann Whitney's U-test. p ≤ 0.05 was considered significant. SPSS Statistics 17.0 (SPSS GmbH Software, Munich, Germany) was used for statistical analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

Cellular and molecular inflammation

Neutrophil infiltration:  As depicted in Figure 1, the highest levels of neutrophil infiltration were measured in allogenic transplanted animals without immunosuppressive therapy (POD7: 117.50 ± 42.20 cells/field) and in animals with continuous FK506 treatment (POD21: 114.40 ± 26.10 cells/field; Figure 1). The infiltration of MPO-positive cells in animals under rescue therapy and in recovery from acute rejection decreased steadily without significant differences between FK506 monotherapy (POD14: 75.10 ± 38.30 cells/field; POD21: 41.60 ± 41.10 cells/field) and additional infliximab treatment (POD14: 70.20 ± 19.60 cells/field; POD21: 43.70 ± 16.60 cells/field).

image

Figure 1. Infiltration and inflammation. Histograms depicting neutrophil infiltration, infiltration of monocytes/macrophages, chemotactic mediators and pro- and anti-inflammatory cytokines (*p ≤ 0.05 – allogenic controls vs. all other groups; §p ≤ 0.05 vs. nontransplanted controls; #p ≤ 0.05 – rescue therapy groups: POD14 vs. POD21). Data are expressed as mean ± SD.

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Monocytes and macrophages:  The highest levels of ED1-positive monocytes and macrophages in the smooth muscle layer occurred in transplanted animals without immunosuppressive therapy (139.90 ± 9.40 cells/field; p ≤ 0.05 vs. all other groups) and animals under rescue therapy with FK506 only at POD14 (63.50 ± 6.60 cells/field). On POD14, additional infliximab treatment (45.20 ± 5.90 cells/field) led to significantly reduced infiltration compared to FK506 only (p = 0.004) in the rescue therapy groups. On POD21, both rescue strategies (FK506: 36.60 ± 7.50 cells/field/FK506+infliximab: 32.80 ± 3.10 cells/field) resulted in decreased infiltration of ED1-positive cells compared to continuous FK506 therapy (56.30 ± 10.00 cells/field) without significant differences between both rescue regimens.

Chemokines and inflammatory and anti-inflammatory cytokines:  The mRNA expression levels of MCP-1 in transplanted animals without treatment (30-fold), measured in RT-PCR, correlated with severe signs of acute rejection which differed significantly from all other groups (p < 0.05). Both groups under continuous FK506 treatment (POD14/21: onefold) presented with significantly decreased levels of MCP-1 gene expression compared to rescue therapy groups and control (7d ACR) group (p < 0.05). Both rescue strategies showed a further significant reduction (p < 0.05) in MCP-1 mRNA expression from POD14 (FK506: sevenfold vs. FK506+infliximab: 11-fold) to POD21 (FK506: twofold vs. FK506+infliximab: threefold). Gene expression of the proinflammatory cytokines IL-6 and TNF-α presented in a similar pattern as for MCP-1, with only slight differences. IL-6 and TNF-α gene expression in the rescue group with FK506 monotherapy at POD21 did not differ significantly from the groups with continuous FK506 treatment. Both cytokines showed a peak expression during recovery from acute rejection on POD14 with subsequent decrease (p < 0.05) on POD21 and no additional benefit of infliximab treatment. In the rescue and the continuous immunosuppression groups, the anti-inflammatory cytokine IL-10 was significantly decreased compared to the 7d ACR group. A further significant reduction between POD14 and POD 21 was observed in the rescue groups with FK506 monotherapy. Additional infliximab treatment did not show significant differences regarding IL-10 expression when comparing POD14 and POD21.

Fibrosis and neural damage

Both rescue groups presented with significantly increased Sirius Red-positive areas, as did the 7d ACR group, when compared to continuous FK506 treatment and nontransplanted controls, which showed no signs of increased fibrosis (Figure 2). Accordingly, expression levels of VEGF mRNA expression were also highest in the rescue groups compared to nontransplanted and allogenic controls. However, in both groups with continuous FK506 treatment, levels of VEGF gene expression were also increased when compared to nontransplanted controls. In the 7d ACR group, the number of neurons detected by Cuprolinic Blue staining was significantly (p < 0.001) reduced (21.88 ± 3.32 neurons/field) compared to nontransplanted controls (54.04 ± 3.10 neurons/field). This neuronal damage was significantly prevented by continuous FK506 treatment (POD14: 40.33 ± 8.73/ POD21: 46.00 ± 7.19 neurons/field) and reconstituted by both rescue therapy regimens (rescue therapy with FK506 monotherapy at POD21: 35.25 ± 3.01 neurons/field and FK506+infliximab on POD21: 42.63 ± 8.04 neurons/field).

image

Figure 2. Fibrosis, neural damage, apoptosis and histological grading. Histograms showing the quantity of Sirius Red-positive areas, neurons and apoptotic signals in the muscularis and histological grading of controls, groups with continuous FK506 therapy and rescue therapy groups and real time RT-PCR results comparing relative mRNA expression of VEGF, and FasL. (A) Sirius Red staining; (B) Cuprolinic Blue staining; (C) H&E staining. Data are expressed as mean ± SD. (*p ≤ 0.05 – vs. nontransplanted controls; $p ≤ 0.05 – vs. continuous FK506 treatment POD14; +p ≤ 0.05 – FK506 monotherapy vs. combination therapy POD14.)

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Acute rejection grading and apoptosis

As illustrated in Figure 2, all animals without immunosuppressive therapy (7d ACR group) developed severe signs of acute rejection and presented with significantly increased mean rejection grading scores (4.50 ± 0.55; p < 0.05) compared to all other groups (Figure 2). Continuous FK506 treatment prevented ACR (mean scores POD14: 2.00 ± 0.00; POD21: 2.25 ± 0.50). Additional infliximab treatment (POD14) did not improve recovery from acute rejection compared to rescue therapy with FK506 monotherapy (mean scores FK506+infliximab POD14: 3.20 ± 0.84 vs. FK506 monotherapy 2.44 ± 0.73; p = 0.147 and FK506+infliximab POD21: 2.86 ± 1.21 vs. FK506 monotherapy 2.91 ± 0.83; p = 0.724).

Apoptosis in the smooth muscle layer was evaluated using TUNEL staining. All transplanted animals presented with significantly increased counts of apoptotic cells in the smooth muscle layer compared to nontransplanted controls (0.47 ± 0.43 signals/10-crypts; p < 0.05). A trend to even further increased apoptotic cell counts was observed in the rescue groups with additional infliximab treatment (FK506+infliximab POD14: 47.97 ± 11.34 signals/10-crypts vs. FK506 monotherapy PDO 14: 32.00 ± 10.20 signals/10-crypts, p > 0.05). Accordingly, in the 7d ACR group mRNA expression of FasL (25-fold increase) was significantly increased compared to almost all other groups with the exception of the rescue group undergoing combination therapy (FK506+infliximab) at POD14, where expression levels were equally high (19-fold increase). Here, the FasL mRNA expression was also found to be significantly increased compared to the other rescue group treated with FK506 monotherapy at POD14 (10-fold). Continuous FK506 treatment (POD14: twofold; POD21: onefold) resulted in significantly lower FasL mRNA expression compared to all rescue groups and similar to nontransplanted controls.

T cell activation/differentiation markers and T cell subtypes

As illustrated in Figure 3, assessment of TGF-β1, CD4, FOXP3 and IL-17 mRNA showed comparable expression patterns (Figure 3). In allogenic transplanted animals without immunosuppression (7d ACR), gene expression for all four parameters was increased when compared to nontransplanted controls and animals under continuous FK506 therapy. In both rescue groups (FK506 monotherapy and FK506+infliximab), mRNA expression of TGF-β1, CD4, FOXP3 and IL-17 was elevated compared to continuous FK506 therapy. Over time, this gene expression dropped in both rescue groups from POD14 to POD21. On POD14, levels of CD4, FOXP3 and IL-17 in the rescue groups were significantly increased when comparing rescue therapy with FK506 monotherapy and additional infliximab treatment (FK506+infliximab: CD4 12-fold; FOXP3: 16-fold; IL-17: 87-fold vs. FK506 only CD4: sixfold; FOXP3: eightfold; IL-17: 14-fold, all p < 0.05, respectively). In allogenic transplanted animals without treatment (7d ACR group), peak levels of gene expression of CD8, IFN-γ, IL-2 and IL-4 m-RNA were observed. The mRNA expression of CD8 and IFN-γ dropped significantly under both rescue strategies from POD14 to POD21. Overall, CD8 mRNA expression levels were strongly increased in animals with acute rejection and decreased in groups after rescue therapy or continuous immunosuppression. In contrast to the previous finding, CD4/FOXP3 and IL-17 m-RNA expression levels reached their respective peaks especially after rescue therapy.

image

Figure 3. T cell activation, differentiation markers of T cell subtypes. Real-time reverse-transcriptase polymerase chain reaction analysis comparing relative mRNA expression of TGF-β1, IFN-γ, IL-2, FOXP3, IL-17, CD4 and CD8 in the muscle layer of controls, groups with continuous FK506 therapy and rescue therapy groups. Data are expressed as mean ± SD. Combined rescue therapy of FK506+infliximab resulted in significantly increased levels of mRNA expression of CD4, FOXP3, IFN-γ and IL-17 in comparison to rescue therapy with FK506 alone. (*p ≤ 0.05 – allogenic controls vs. all other groups; #p ≤ 0.05 –comparison between POD14 and POD21 in rescue groups; +p ≤ 0.05 – combination therapy (POD14) vs. FK506 monotherapy (POD14); §p ≤ 0.05 vs. nontransplanted controls.)

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Muscle function

As illustrated in Figure 4, the 7d ACR group showed a 92% reduction of in vitro contractility under stimulation with 100 μM/L bethanechol (0.62 ± 0.32 g/mm2/s; p = 0.001) compared to nontransplanted controls (7.32 ± 0.84 g/mm2/s; Figure 4). Continuous FK506 treatment effectively prevented graft dysfunction (POD14: 14% reduction −6.27 ± 0.1.36 g/mm2/s; POD21: 23% reduction −5.65 ± 1.05 g/mm2/s vs. nontransplanted controls, p < 0.05). Comparison of both rescue strategies revealed slightly improved graft motor function with additional infliximab treatment (POD14: 72% reduction −2.37 ± 0.91 g/mm2/s; POD21: 42% reduction −4.24 ± 0.52 g/mm2/s) compared to FK506 monotherapy (POD14: 76% reduction −1.77 ± 0.69 g/mm2/s; POD21: 58% reduction −3.04 ± 0.62 g/mm2/s), which was significant on POD 21 (100 μM/L: p < 0.001; 300 μM/L: p < 0.001).

image

Figure 4. Contractile force measurements. Bethanechol dose–response curves of smooth muscle contractile activity (n = 5–9 per group) and mRNA expression levels of iNOS. The contractile response to bethanechol is significantly reduced in transplanted rats after vehicle treatment (7d ACR). Under stimulation with 100/300 μM/L bethanechol, additional infliximab treatment improved smooth muscle function compared to FK506 only to a reduction of 58% to 42% compared to contractility in nontransplanted controls on POD21. Data are expressed as mean ± SD. (+p ≤ 0.05; *p ≤ 0.05 – allogenic controls vs. other groups; #p ≤ 0.05 comparison between POD14 and POD21 in rescue groups.)

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

Although adequate control of acute rejection in the early phase after clinical intestinal transplantation has resulted in improved graft and patient survival (1), the impact of recurrent acute rejection episodes on the development of chronic rejection/graft dysfunction and the recovery processes after ACR are not well understood.

In this study, a standardized recovery model to test different rescue therapy regimens after orthotopic, intestinal transplantation and ACR in a MHC-disparate rat strain combination (BN to Lew) was established. The process of intestinal recovery after ACR, potential mechanisms for functional graft deterioration and an immunological priming effect, possibly inducing chronic rejection, were assessed through different parameters. In addition, the effects of an anti-TNF-α therapy with infliximab as a component of a rescue strategy after ACR in this experimental setting were studied.

Overall, both rescue therapies (FK506 monotherapy as well as FK506+infliximab) were efficacious in reversing ACR in our rodent model. Interestingly, not all parameters returned to normal during the observation periods of 14 and 21 days after intestinal transplantation with ACR and subsequent rescue therapy.

In particular, the evaluation of graft fibrosis by Sirius Red staining showed a remarkable increase of fibrosis after ACR and rescue therapy even as early as two and three weeks after intestinal transplantation. Here, additional infliximab treatment was of no benefit compared to ACR treatment with FK506 monotherapy. The histological signs of persisting graft damage outlasted short term inflammatory reactions (e.g. IL-6 mRNA elevation) and were accompanied with an upregulation of the fibrogenic marker VEGF. As chronic rejection of the intestine is mainly characterized by graft fibrosis, intimal hyperplasia of submucosal arteries and chronic ulcers with neutrophil-rich exudate and granulation tissue (14), it could be speculated that this increase in Sirius Red-positive areas along with the mentioned histological changes does indeed represent early stage fibrotic changes.

Looking at cell infiltration, allogenic transplanted animals without immunosuppressive therapy (7d ACR group) presented with massive infiltration of ED1-positive macrophages and neutrophils (MPO+ cells) into the muscle layer. After initiation of rescue therapy with FK506, this infiltration decreased with ongoing treatment, whereas additional infliximab treatment showed no significant benefits.

The pro- and anti-inflammatory cytokines IL-6, TNF-α, IFNγ and IL-10 presented, as expected, with high mRNA-expression levels in allogenic transplanted animals without treatment (7d ACR group) and persisting, low levels under continuous FK506 therapy. During rescue therapy, a typical pattern in the expression of the aforementioned genes—reflecting recovery from ACR—could be observed: temporary upregulation at first and subsequent significant decrease from POD14 to POD21.

As we have shown previously (5), in vitro contractility reflected the functional impact of ACR on the intestinal grafts. As expected, the 7d ACR group performed worst and continuous FK506 therapy was capable of preserving near to normal enteric motor function (see Figure 4). Both rescue therapies had a significant positive impact on recovering enteric motor function. Furthermore, the mRNA expression of the kinetic mediator iNOS correlated with recovered graft motility under both rescue strategies. Comparing the two rescue therapies, a slight but significant benefit from additional infliximab treatment was documented in smooth muscle contractile function. This beneficial effect on contractility seen after combined rescue therapy (FK506+infliximab) in comparison to FK506 monotherapy cannot be explained by different expression levels of iNOS, as these were comparable (see Figure 4). One possible explanation for improved graft motor function could be the observed improved regeneration and preservation of the enteric nerve system, as illustrated by a higher number of neurons (Cuprolinic Blue staining, Figure 2) with additional infliximab treatment. Although neuroprotective effects of infliximab are speculative at this point, the mentioned benefits of FK506+infliximab treatment were associated with an increase of about 30% in anti-inflammatory IL-10 gene expression levels during rescue therapy in accordance with the mentioned findings.

Yet, this study revealed a tendency to increased apoptosis (high signaling in TUNEL staining and upregulated FasL mRNA expression as well as reduced MPO+ infiltrate) under combined rescue treatment (FK506+infliximab), which reflects known (side-) effects of anti-TNF-α agents (15). Whether these findings are due to changes in regulatory mechanisms maintaining intestinal cell homeostasis rather than increased graft damage or an unwanted side effect of infliximab during recovery from ACR is unclear. Further studies will have to assess whether this apoptosis affects mainly infiltrating, pro-inflammatory cell populations, T-lymphocytes (16) or also smooth muscle cells, or other compartments. Although the method of action of infliximab and other TNF-α inhibitors is not completely understood, they are postulated to act via TNF receptors on target cells and not on TNF-α transcription. Hence, mRNA expression of TNF-α was not reduced in the infliximab treatment groups per se, as was to be expected (see Figure 1; 17). T cell activation markers, when assessed with RT-PCR on mRNA level, presented in a distinct pattern when comparing both rescue strategies: TGF-β1, IFN-γ, CD4 and CD8 showed the same decrease over time (POD14-POD21) during rescue therapy as cytokines (IL-6), chemokines (MCP-1) and kinetic mediators (iNOS) during recovery of the intestinal grafts. CD4, FOXP3, TGF-β1 and IL-17 gene expression levels showed a significant increase under additional infliximab treatment, potentially demonstrating an induction of regulatory processes at POD14 after rescue therapy. CD8 gene expression, possibly indicating increased induction of a cytotoxic T cell response, seemed to be correlated with acute rejection in accordance with high levels of IL-2 and decreased motor function on POD7 in allogenic transplanted animals (7d ACR group), along with histological signs of acute rejection. These findings possibly indicate the induction of regulatory T cells as key players in the resolution of ACR episodes. A postulated T-reg induction as previously described after infliximab treatment (18) may also be one of the underlying mechanisms of the slightly improved graft outcome (regarding contractile function and neuroprotection) after two weeks of combined rescue therapy. In a murine model of TNF-α driven arthritis, T-reg frequency increased with inflammation but failed to control the inflammatory process, whereas TNF-α-inhibiting strategies restored the suppressor activity of T-reg (19). Although the presented mRNA expression levels hint at a possible action in this respect, verification of our results by flow cytometry is required.

In clinical transplantation, occurrence of an ACR episode within 30 days posttransplant and a higher number of ACR episodes have been associated with progression to chronic rejection (14). Our findings clearly demonstrate increased graft fibrosis after the treatment and recovery from acute rejection, even after only one episode of rejection in this experimental setting. Although ACR frequency has decreased from historic levels of 80% to 20–40% in the early phase after intestinal transplantation (20), rejection episodes remain one of the most frequent complications after intestinal transplantation in the clinical setting.

Therefore, this study highlights the importance of avoiding these acute rejection episodes regarding development of chronic rejection with increased graft fibrosis. Nevertheless, our findings of improved graft motor function after rescue therapy seem to be independent of increased graft fibrosis at this early stage, indicating and confirming acute rejection with its inflammatory and immunological sequelae as the main cause of acute graft motor dysfunction after intestinal transplantation.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

This study establishes a model for the observation of regenerative processes after successful rescue therapy from ACR following orthotopic, allogenic intestinal transplantation. Our results demonstrate that initial molecular and functional recovery after acute rejection could be achieved through introduction of the CNI inhibitor FK506. Additional anti-TNF-α treatment with infliximab had discrete effects, mainly on smooth muscle contractile function and enteric neuroprotection associated with possible involvement of regulatory T cells. However, rescue from ACR resulted in increased graft fibrosis and was unable to restore smooth muscle function to levels observed under continuous immunosuppression without signs of ACR.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

This research was supported by a grant from the Deutsche Forschungsgemeinschaft (KFO 115/1-2, Teilprojekt 3) and by the Bonfor-Program of the University of Bonn (O-112.0045).

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure
  10. References
  11. Supporting Information

1. Drugs, reagents and solutions.

2. Real-time reverse transcription polymerase chain reaction.

FilenameFormatSizeDescription
AJT_4262_sm_suppmat.doc37KSupporting info item

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