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Keywords:

  • Complement system;
  • epithelial tubular cells;
  • IL-17;
  • kidney transplantation;
  • renal acute rejection

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

Acute rejection is still a common complication of kidney transplantation. IL-17 is known to be associated with allograft rejection but the cellular source and the role of this cytokine remains unclear. We investigated IL-17 graft expression in renal transplant recipients with acute antibody-mediated rejection (ABMR), acute T-cell-mediated rejection (TCMR), interstitial fibrosis and tubular atrophy (IFTA) and acute tubular damage due to calcineurin-inhibitor toxicity (CNI). In acute ABMR, tubular IL-17 protein expression was significantly increased compared to TCMR, where most of the IL-17+cells were CD4+graft infiltrating lymphocytes, IFTA and CNI control groups. The tubular expression of IL-17 in acute ABMR colocalized with JAK2 phosphorylation and peritubular capillaries C4d deposition. In addition, IL-17 tubular expression was directly and significantly correlated with the extension of C4d deposits. In cultured proximal tubular cells, C3a induced IL-17 gene and protein expression along with an increased in JAK2 phosphorylation. The inhibition of JAK2 abolished C3a-induced IL-17 expression. The use of steroids and monoclonal antibodies reduced IL-17 expression, JAK2 phosphorylation and C4d deposition in acute ABMR patients. Our data suggest that tubular cells represent a significant source of IL-17 in ABMR and this event might be mediated by the complement system activation featuring this condition.


Abbreviations: 
ABMR

antibody-mediated rejection

TCMR

T-cell-mediated rejection

CNI

calcineurin inhibitor

IFTA

interstitial fibrosis and tubular atrophy

BKV

BK viremia

JAK

Janus kinase

PBMC

peripheral blood mononuclear cells

ELISA

enzyme-linked immunosorbent assay

GAPDH

glyceraldehyde-3-phosphate

IRI

ischemia reperfusion injury

r

correlation coefficient

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

In the last few years the renal transplant research community has realized the prominent role of antibody-mediated rejection (ABMR) among the causes of graft failure. Although the overall incidence of acute graft rejection is progressively reducing, the antibody-mediated form is still a threat for kidney grafts, in particular in the growing population of sensitized patients (1). In addition, the significant improvement in the diagnostic criteria has led to a more frequent recognition of acute (ABMR). Among these criteria, introduced by the Banff classification, the presence of C4d deposition represents the hallmark of this condition (2), further supporting the central role of the complement cascade activation in the pathogenesis of graft lesions in this setting (3–4). ABMR is often associated with glomerular and interstitial inflammation (5–6). Activation of resident cells, in particular tubular epithelial cells, represents a key feature of acute graft rejection as well as of any inflammatory reaction in both native and transplanted kidneys (7–8). Although tubular cells were usually considered ‘only’ as a target of external noxae, including allo-immune response in the transplanted kidney, leading to their damage and subsequent necrosis, it is now clear that they may play a pivotal role in the pathogenesis of the interstitial inflammatory response (9). Indeed, tubular cells, once activated by a suitable stimulus, may modulate the influx and activation of inflammatory and immune cells, through the expression and release of an array of proinflammatory cytokines and chemokines (7–8). It is conceivable that such an activation of tubular cells may also be present in acute ABMR, although, to date, very little is known on their involvement in the pathogenesis of interstitial inflammation in this setting.

Among the proinflammatory cytokines, IL-17 has been shown to play a major role in several pathological conditions. IL-17 is produced by a variety of cell types including subsets of CD8+ T cells, γδ-T cells, NK cells and neutrophils but the predominant source of IL-17 remains the CD4+ T-cell population, where its expression define the Th17 subset (10–11). Interestingly, tubular cells represent a specific target for IL-17. van Kooten et al. reported that IL-17 can induce the expression of MCP-1, IL-6, IL-8 and C3 by cultured proximal tubular cells (7). The same authors also observed a significant increase in IL-17 protein expression within the infiltrating cells in acute TCMR rejection. In addition, different studies in animal models have identified elevated IL-17 mRNA levels in renal allografts (12). However, to date no information is available on IL-17 expression in acute ABMR.

Thus, the aim of the present study was to evaluate the graft expression of IL-17 in acute ABMR in kidney transplant recipients, in the attempt to define novel cellular sources for this cytokine and to elucidate the molecular mechanisms underlying its expression.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

Patients

In our study we included 50 kidney transplant recipients of deceased and/or living donors, who consecutively underwent renal graft biopsy with the following diagnosis according to the Banff 2007 criteria (4): acute ABMR (n = 20), acute TCMR (n = 10), IFTA (n = 10) and acute tubular damage due to CNI nephrotoxicity (n = 10). In addition, we included in the study two further control groups of patients with biopsy-proven BKV nephropathy (n = 6) and graft pyelonephritis (n = 6).

All patients enrolled received 500 mg of methylprednisolone intraoperatively at the time of transplantation, 125 mg of prednisone daily, with the dose tapered to 25 mg by day 3; 20 mg of a chimeric monoclonal anti-CD25 antibody (Simulect, Novartis, Basel, Switzerland) intravenously on days 0 and 4; mycophenolate mofetil (Cell-Cept, Roche, Basel Switzerland) 1000 mg b.i.d and either cyclosporine A (Neoral, Novartis, Basel, Switzerland) or tacrolimus (Prograf, Astellas, Tokyo, Japan) starting 24 h after reperfusion. All patients with acute ABMR presented donor-specific antibodies as shown by Luminex (Onelambda, Canoga Park, CA, USA). After acute ABMR diagnosis, all patients were treated with steroid pulse therapy (1 g/day on three consecutive days), plasmapheresis (6–10 sessions) and anti-CD3 monoclonal antibodies (Orthoclone, Janssen-Cilag, Cologno Monzese, Italy) at a dose of 5 mg/day for 10–14 days. In four patients intravenous immunoglobulins (IVIG) were added at the end of each plasmapheresis at a dose of 400 mg/kg. Ten out of 20 patients received a second graft biopsy after a median of 4.6 months (range 2.5–6.8) from ABMR diagnosis. The study was carried out according to the Declaration of Helsinki and was approved by our Ethics Committee. Every patient signed an informed consent form agreeing to participate in the study.

Immunohistochemistry

Paraffin-embedded sections of graft biopsies were deparaffinized and underwent epitope unmasking through three microwave (750 W) cycles of 5 min in citrate buffer (pH = 6). The slides were, then, incubated with H2O2 (3%), Triton (0.05%), protein block solution (Dako, Glostrup, Denmark) and with the primary anti-IL-17 antibody specific for IL-17A (1:50) (sc 7927, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and anti-IL-6 antibody (1:400 Abcam, Cambridge, UK). The sections were then incubated with the biotinylated secondary antibody, ABC complex streptavidin/HRP and DAB Chromogen Solution (Dako). The reaction was visualized by a brown precipitate, counterstained with Mayers hematoxylin (blue) and mounted with glycerol (DakoCytomation, Carpintera, CA). Negative controls were obtained incubating serial sections with the blocking solution and then control irrelevant antibody.

The infiltrating cell number was measured in at least 15 high power (×200) fields (HPF) of cortical areas/section (mean 18.3 ± 3.1HPF/section) by two independent observers blinded to the origin of the slides. The final counts were the mean of the two measures. In no case the interobserver variability was higher than 15%. Specific tubular IL-17 immunostaining was quantified using Adobe Photoshop software and expressed as pixel/tubular section.

Confocal laser scanning microscopy

Paraffin-embedded human kidney sections and immortalized human proximal tubular epithelial cells (HK2) were stained or double stained for CD4, IL-17, p-JAK2 (Santa Cruz Biotechnologies), Fluorescein Lotus tetragonolobus lectin (Vector Laboratories Inc., Burlingame, CA, USA) and C4d (Biomedica Gruppe, Wien, Austria). For each experiment 1×105 cells were plated on a cover slip and fixed in 3.7% paraformaldehyde. The slides were incubated with the blocking solution (BSA 2%), primary antibodies (anti-CD4 1:50; anti-IL-17 1:50; anti-Lotus 1:50, anti-p-JAK2 1:100; anti C4d 1:10) and the appropriate secondary antibodies (Alexa Flour 488 and 555 goat antirabbit; AlexaFlour 555 and 488 antimouse, Molecular Probes, Eugene, OR, USA). All sections were counterstained with TO-PRO-3 (Molecular Probes). Specific fluorescence was acquired using the confocal microscope Leica TCS SP2 (Leica, Wetzlar, Germany). The number of IL-17+, CD4+, p-JAK2+ cells was measured in at least 10 high power (×630) fields/sections by two independent observers blinded to the origin of the slides.

Cell culture

Immortalized human proximal tubular epithelial cells (HK2) obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine in a humidified atmosphere with 5% CO2 at 37°C. Cells were plated at approximately 70% confluence and then underwent serum starvation for 24 h. C3a (Merck KG, Darmstadt, Germany) and JAK2 inhibitor (E)-3(6-bromopyridin-2-yl)-2-cyano-N-((S)-1-phenylethyl) acrylamide WP1066 (Sigma Aldrich) were added to serum-free medium for the indicated time period. HK2 cells were processed for IL-17 and p-JAK2 protein expression by immunofluorescence and confocal analysis. In different sets of experiments HK2 were lysed for RNA or protein extraction.

RNA extraction and real time PCR analysis

Total RNA was isolated with RNA easy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions and quantified by NanoDrop® ND-1000 Spectrophotometer and its quality was assessed by electrophoresis on the agarose gel (1%). Half microgram of total RNA was used in a reverse transcription (RT) reaction using the high capacity cDNA RT-Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.

Human IL-17 (sense 5'-GAATCTCCACCGCAATGAGG-3' and antisense 5'CCCACGGACACCAGTATCTT-3') and glyceraldehyde-3 phosphate dehydrogenase (GAPDH), (sense 5'-GAAGGTGAAGGTCGGAGTCA-3' and antisense 5'-GGGTGGAATCATATTGGAA-3') specific primers were used for mRNA amplification and quantification.

Quantitative real-time PCR reactions were performed in triplicate. For each reaction, 2 μL of the cDNA was added to 23 μL of iQTM SYBR Green Supermix buffer (6 mM MgCl2, dNTPs, iTaq DNA polymerase, SYBR Green I, fluorescein and stabilizers) (BIO-RAD Laboratories, CA, USA) and primer pair, in a total volume of 25 μL. Quantification of the mRNA levels was performed on a MiniOpticon Real-Time PCR detection system (BIO-RAD Laboratories). In the PCR reactions the following protocol was used: activation of the polymerase 95°C for 3 min, followed by 45 cycles of 95°C for 10 s, 60°C for 30 s. Melting curves were generated through 60 additional cycles (65°C for 5 s with an increment of 0.5°C/cycle). Gene expression results were obtained as average CT (threshold cycle) values of triplicate samples. IL-17 expression levels in each samples were normalized with GAPDH expression by CFX Manager Software version 1.5 (BIO-RAD) using the 2ΔΔCt method.

Western blotting

The cell monolayer was rinsed twice rapidly with ice-cold PBS and lysed in 100 μL of RIPA buffer (1 mM PMSF, 5 mM EDTA, 1 mM sodium orthovanadate, 150 mM sodium chloride, 8 μg/mL leupeptin, 1.5% Nonidet P-40 and 20 mM tris-HCl [pH 7.4]). The lysates were kept on ice for 30 min and centrifuged at 12 000 RPM at 4°C for 5 min. The supernatants were collected and stored at −20°C until used. Cell extracts containing 40 μg of proteins were subjected to Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) on a 7.5% gel under reducing conditions. Proteins were transferred onto PVDF membrane (Millipore, Badford, MA, USA). The filter was blocked overnight at room temperature (RT) with 2% bovine serum albumin in PBS containing 0.1% tween-20 (T-PBS) and then incubated with polyclonal antiphospho-JAK2 (Tyr1007/1008) antibody (Santa Cruz 1:500 dilution in T-PBS at RT for 2 h); The membranes were washed twice in T-PBS and incubated for 1 h at RT with HRP-conjugated goat antirabbit (Santa Cruz Biotechnology; 1:10 000 dilution in T-PBS). Immune complexes were detected by the ECL enhanced chemiluminescence system (Amersham), as recommended by the manufacturer. The same membranes were then stripped and immunoblotted again with polyclonal anti-JAK2 antibody (Santa Cruz Biotechnology; 1:500 dilution in T-PBS at RT for 2 h) and HRP-conjugated goat antirabbit (1:10 000 dilution in TBS). Intensity of the bands was quantified using Image J 1.34 Software. Protein expression of phosphorylated fraction was normalized to total protein.

ELISA

IL-17 secretion in supernatants of HK2 cells stimulated with C3a was measured by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions. The minimum detectable concentration of IL-17 is less than 15 pg/mL. As positive control, we used supernatants of peripheral blood mononuclear cells (PBMCs) after mitogenic stimulation for 5 days with concanavalin A 5 μg/mL. Cytokine concentration was determined from the regression line for a standard curve generated using highly purified recombinant cytokine at various concentrations performed contemporaneously with each assay. Data were expressed as pg/mL. All samples were assayed in duplicate.

Statistical analysis

Data were expressed as mean ± standard deviation (SD). Differences between groups or conditions were analyzed by paired, unpaired t-test analysis or ANOVA, as appropriate. Pearson's correlation test was used to study the association between two continuous variables. A p value <0.05 was considered statistically significant. The Statview software package (5.0 version) (SAS Inc. Co., Cary, NC, USA) was used for all analyses.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

IL-17 expression in kidney graft with acute rejection

Table 1 summarizes the main clinical features of the patients included in the study.

Table 1.  Main demographic and clinical features of the patients included in the study
 ABMRTCMRCNIIFTA
  1. GB, graft biopsy; MDRD, modification of diet in renal disease; TX, transplant.

  2. ap < 0.05 versus CNI and IFTA.

  3. bp < 0.01 versus CNI.

Patients (no.)20101010
Recipient age (years)41.2 ± 10.9837.1 ± 11.5444.5 ± 11.64 41.8 ± 15.08
Recipient gender (M/F)6/143/75/56/4
Living/ deceased donor3/173/70/100/10
CyclosporinA/tacrolimus8/128/26/46/4
2nd transplant7a100
Serum creatinine (mg/dL) at GB3.71 ± 1.31b3.13 ± 1.27 2.12 ± 0.65 2.48 ± 0.60
aMDRD at GB24.16 ± 11.29b  31 ± 14.0845.6 ± 10.4830.25 ± 10.11
Month GB post TX5.8 (1.03–143.2)7.3 (2.4–81.3)27.4 (6.2–54.4)48.5 (10.1–74)
Graft failure6211

We first investigated the IL-17 protein expression in human renal allograft biopsy samples from patients with acute ABMR and TCMR using graft samples with acute tubular damage due to calcineurin-inhibitor toxicity (CNI) and IFTA as control specimens. Moreover, we investigated the IL-17 expression in biopsies of patients with BKV nephropathy and graft pyelonephritis. IL-17+ graft-infiltrating cells were barely present in IFTA, CNI, BKV nephropathy, pyelonephritis and ABMR (Figures 1A–E). On the contrary, numerous IL-17+ graft-infiltrating cells were present in TCMR (Figure 1F). The number of IL-17+cells was significantly increased in TCMR compared to ABMR (p = 0.001) and control groups (p < 0.0001) (Figure 1G). We then characterized the phenotype of IL-17+ graft-infiltrating cells within the tubulointerstitial area of TCMR grafts and the majority of IL-17-expressing cells were CD4+ (80.1 ± 5.1%, Figures 1H and I).

image

Figure 1. IL-17 expression in kidney graft with acute rejection. IL-17 protein expression was investigated on paraffin-embedded human kidney sections by immunohistochemistry in CNI toxicity (n = 10) (A), IFTA (n = 10) (B), BKV nephropathy (n = 6) (C), graft pyelonephritis (n = 6) (D), ABMR (n = 20) (E) and TCMR (n = 10) (F). The number of infiltrating IL-17+ cells was measured as described in the section ‘Methods’. Results are expressed as mean ± SD of the number of cells/high power field (hpf) (*p = 0.001 compared to acute ABMR; ^p < 0.0001 compared to IFTA, CNI, BKV and Pyelonephritis control groups) (G). The characterization of the phenotype of IL-17+ graft-infiltrating cells within the tubulointerstitial area of grafts with TCMR was performed by confocal microscopy as described in the Methods section. The majority (80.1 ± 5.1%) of the cells expressing IL-17 (green) were also CD4+ (red) (H and I). Tubular epithelial cell staining for IL-17 was prominent in acute ABMR compared with acute TCMR (J and K, respectively). The extent of IL-17 staining was quantified as described in the Methods section (*p < 0.0001 compared to acute TCMR and to control groups ^p < 0.0001) (L). For each group, all images (original magnification ×200 or ×400) are from a single patient and are representative of the whole group of patients. Nuclei were counterstained by hematoxylin.

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As illustrated in Figures 1J–L, a significant increase of IL-17 protein expression was detected in the tubular epithelial cells from patients with ABMR compared to TCMR (p < 0.0001) and control groups (p < 0.0001). The staining with Lotus tetragonolobus lectin (Lotus), a specific marker of renal proximal tubular cells, demonstrated that IL-17 was prevalently, but not exclusively, expressed by these cells (Figures 2A–F).

image

Figure 2. Tubular characterization of IL-17 and analysis of IL-6 expression. Double staining with Lotus tetragonolobus lectin (Lotus) (green) and IL-17 (red), by indirect immunofluorescence and confocal microscopy, showed the localization of the protein within both proximal (Lotus+) (A–C) and distal epithelial cells (Lotus) (D–F). The IL-6 expression was negative in tubular epithelial cells of all groups examined (G–J). On the other hand, IL-6 specific staining was focally present within the interstitial infiltrating cells in the CNI and within the peritubular capillaries of ABMR biopsies (original magnification ×200).

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To define whether the upregulation of IL-17 expression was merely a part of an unspecific proinflammatory response of tubular cells, we investigated the expression of IL-6, a well-known proinflammatory cytokine closely linked to IL-17. Interestingly, IL-6 specific staining was mainly localized within peritubular capillaries. No tubular cells expressed IL-6 in ABMR biopsies. Finally, IL-6 was barely detected in CNI while was absent in IFTA and TCMR groups (Figures 2G–J).

C3a induced IL-17 expression in cultured proximal tubular cells (HK2)

In ABMR biopsies, we observed a close spatial association between IL-17+ tubular sections and C4d deposition within the peritubular capillaries (Figure 3A). In addition, IL-17 protein expression was directly and significantly correlated with the number of C4d+ peritubular capillaries (r = 0.38; p = 0.01).

image

Figure 3. C3a induces IL-17 expression and production by HK2 cells. Double-label immunofluorescence performed in acute ABMR renal biopsy showed a specific colocalization of IL-17 tubular expression (green) and C4d deposits (red) within the peritubular capillaries (A). To-pro-3 was used to counter-stain nuclei (blue). Magnification 63×. IL-17 gene expression in HK2 cells treated for 24 h or 48 h with C3a was evaluated by real-time PCR as described in the method section (B) (*0.002 vs. Basal 24 h, °0.002 vs. Basal 48 h). The effect of C3a on IL-17 protein expression was evaluated by confocal microscopy in cultured HK2 cells (C–G). Quiescent subconfluent HK2 cells were incubated for 24 h (C–D) or 48 h (E–F) with (D and F) or without (C and E) C3a (5×10−7M) (magnification 63×). The quantitative analysis showed a statistically significant increase of IL-17 expression at 48 h (°p = 0.005 vs. basal 24 h) (*p = 0.001 vs. basal 48 h) (G). The effect of C3a on IL-17 secretion was evaluated by ELISA as previously described (*p = 0.02 vs. basal) (H). Data represent mean ± SD of three experiments.

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To establish a possible role of the activated complement cascade on IL-17 expression, we stimulated HK2 cells, an immortalized proximal tubular epithelial cell line, with C3a in a range of concentrations that reproduces its levels during complement activation (13). A significant increase of IL-17 mRNA abundance after C3a stimulation for 48 h at a concentration of 5×10−7M was observed, as quantified by real-time PCR (Figure 3B). Confocal analysis showed that C3a incubation for 24 and 48 h caused an increased expression of IL-17 with a prominent cytoplasmic localization (Figures 3C–F). The quantitative analysis demonstrated a statistically significant increase of IL-17 at 48 h compared to basal levels (p = 0.005 vs. basal 24 h) (p = 0.001 vs. basal 48 h) (Figure 3G). To confirm this observation and in order to evaluate the ability of tubular epithelial cells to secrete IL-17, we performed an ELISA on the cell supernatant. HK2 incubated with C3a for 48 h presented a significant production of IL-17 compared with basal condition (p = 0.02) (Figure 3H).

JAK2 phosphorylation and complement-dependent IL-17 expression

To investigate the signaling pathway activated in the local production of IL-17, we studied the phosphorylation and subsequent activation of JAK2, a cytoplasmic tyrosine kinase implicated in the expression of IL-17 in several cell lines. Acute ABMR was characterized by a marked increase of JAK2 phosphorylation at the tubular level with an apical and cytoplasmatic localization (p = 0.0009) (Figures 4A–F). Phospho-JAK2 specifically colocalized with peritubular C4d deposits (Figures 4G–I). Interestingly, JAK2 phosphorylation was directly and significantly correlated with both IL-17 expression (r = 0.4; p = 0.008) and C4d deposition (r = 0.35; p = 0.01).

image

Figure 4. Acute ABMR is characterized by tubular JAK2 phosphorylation. The phosphorylation of JAK2 in CNI (A), IFTA (B), ABMR (C and D) and TCMR (E) graft biopsies was investigated by immunohistochemistry. Phospho-JAK2 levels were increased within the tubular epithelial cells in acute ABMR. The phospho-JAK2 specific staining was quantified as described in the section ‘Methods’ and expressed as mean ± SD (*p = 0.0009) (F). The colocalization in acute ABMR biopsies of C4d (G, green) deposits within the peritubular capillaries and phospho-JAK2 (H, red, merge in I) in tubular epithelial cells was investigated by immunofluorescence/confocal microscopy (Original magnification 950×). Nuclei were stained with To-pro-3 (blue).

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Therefore, we analyzed the effect of C3a on JAK2 phosphorylation in HK2 cells. As shown in Figures 5A–F, confocal analysis showed a time-dependent increase of JAK2 phosphorylation with a peak after 30 min of C3a incubation (p = 0.01). These data were confirmed by Western blotting analysis, revealing a statistical significant increase in JAK2 phosphorylation 30 min of C3a incubation compared with baseline (p = 0.02) (Figures 5G–H). Figures 6A–D showed the colocalization of phospho-JAK2/IL-17 proteins at the tubular level in the acute ABMR renal biopsy. To define the role of JAK2 in complement-mediated IL-17 expression we evaluated C3a-induced IL-17 production in the presence and in the absence of (E)-3(6-bromopyridin-2-yl)-2-cyano-N-((S)-1-phenylethyl)-acrylamide (WP1066), a specific JAK2 inhibitor. As shown in Figures 6E–I, JAK2 inhibition caused a significant (p = 0.02) reduction in C3a-induced IL-17 protein expression.

image

Figure 5. C3a induces JAK2 phosphorylation in HK2. JAK2 phosphorylation in cultured HK2 cells was investigated by confocal microscopy (A–F) and western blotting (G–H). In both cases quantification of the specific signal (F and H) demonstrated a significant increase of phospho-JAK2 at 30 min of incubation with C3a (5×10−7M) (*p = 0.01 vs. basal, **p = 0.02 vs. basal). Results are the mean ± SD of at least three experiments.

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image

Figure 6. C3a-induced IL-17 expression in tubular cells is JAK2-dependent. The colocalization in acute ABMR renal biopsy of phospho-JAK2 (red) and IL-17 (green) specific signals at the tubular level was investigated by confocal microscopy (A, magnification 40×). Arrow indicates a pJAK2+/IL-17+ tubular epithelial cell enlarged in B–D. Nuclei are highlighted with To-pro-3 in blue. The effect of JAK2 inhibition on C3a-induced IL-17 expression was investigated by confocal microscopy (E–I). Panels E–H illustrate a representative experiment (E, unstimulated; F, C3a at a concentration of 5×10−7M; G, WP1066 at a concentration of 3 μM plus C3a; H, WP1066 alone). Quantification of specific IL-17 protein expression was obtained as described in the Methods section (*p = 0.004 vs. basal; °p = 0.02 vs. WP1066+C3a). Data represent mean ± SD of three sets of experiments.

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Pharmacologic treatment reduced IL-17/pJAK2 expression in patients with acute ABMR

Finally, we investigated IL-17 expression, C4d deposition and JAK2 phosphorylation in graft biopsies of 10 ABMR patients before and after treatment with steroids and monoclonal antibodies. There was a statistically significant reduction in IL-17 production at tubular epithelial cell level in the post-treatment biopsies (p = 0.02) (Figures 7 A–C). Interestingly, we observed a simultaneous and significant decrease of p-JAK2 protein (p = 0.0005) and the total abrogation of C4d deposition within the peritubular capillaries (p < 0.0001) (Figures 7 D–F).

image

Figure 7. Pharmacological treatment blunts the complement-JAK2-IL-17 axis in ABMR graft biopsies. IL-17 protein expression in acute ABMR biopsies before (A) and after (B) pharmacological treatment was investigated by immunohistochemistry. Quantification of the specific signal demonstrated a significant reduction after treatment (C) (°p = 0.02). JAK2 phosphorylation (red) and C4d deposition (green) before (D) and after (E) treatment were investigated by double immunofluorescence staining and confocal microscopy. The quantification of both signals demonstrated a statistically significant reduction after treatment (*p < 0.0001 vs. ABMR-pre; **p = 0.0005 vs. ABMR-pre) (F).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

In the present study we demonstrate, for the first time, that tubular epithelial cells may represent a significant source for IL-17 in ABMR and that this event is directly linked to the activation of the complement cascade. In addition, we identified the activation of JAK2 as the potential signaling pathway involved in tubular IL-17 expression.

In the setting of acute rejection IL-17 has always been associated with the activation of the Th17 response and there are several lines of evidence supporting the hypothesis that such a response may represent a key pathogenic mechanism in the development of acute graft rejection (14). Several authors demonstrated the expression of IL-17 within the infiltrating cells of rejecting graft (15,16). In most of the cases, the IL-17+ cells were identified as CD4+ lymphocyte (11). Our results confirm this observation in the acute TCMR biopsies. Indeed, in our population more than 80% of the IL-17-expressing cells were Th17 lymphocytes. Yuan et al. clearly demonstrated the key pathogenic role of this T-cell subset in the setting of acute rejection in a model of heart transplantation in mice lacking Th1 cells (17). These mice presented an accelerated form of rejection with an infiltration predominantly constituted by IL-17-producing CD4+ T cells. Despite a well-defined role in the pathogenesis of TCMR, no information is available on the role of IL-17 in the development of acute ABMR. This observation led us to investigate whether IL-17 expression could be detected in this scenario. Although a few infiltrating cells were IL-17+, tubular epithelial cells represented the main source of this cytokine in our population of acute ABMR. Originally, IL-17 was thought to be produced exclusively by T cells, but it is now well known that a variety of innate cells, including macrophages, granulocytes, dendritic cells, natural killer cells, lymphoid tissue inducer and γδ-T cells, may produce this proinflammatory cytokine (18). So far, there is only one report of an extraimmune expression of IL-17. Takahashi et al. observed that Paneth cells produce IL-17 in response to systemic infusion of TNF-alpha (19). This unique epithelial cell lineage, with phagocytic activity, located in the crypts of the small intestine has been previously suggested to present several leukocyte-like functions and, through the release of IL-17, they might modulate the innate immune response in the setting of inflammatory bowel disease (20).

Several recent pieces of evidence suggest that IL-17 may modulate the innate and, to some extent, also the adaptative immune response. IL-17 can act on antigen-presenting cells, such as macrophages and subsets of dendritic cells, which express specific IL-17 receptors (21,22). IL-17 has been shown to directly induce the production of IL-23, IL-1, IL-6 and TGFβ by antigen-presenting cells (21), which are crucial factors for development of Th17 cells. In this manner, tubular IL-17 could influence the generation of antigen-driven Th17 cells and exacerbate the graft-specific allo-immune response. On the other hand, IL-17 is a potent activator of neutrophils. Ectopic expression of IL-17 may stimulate a strong neutrophilic response and IL-17 deficiencies in mice are associated with neutrophil defects leading to disease susceptibility (23–25). Thus, it is conceivable that IL-17 released by tubular epithelial cells may contribute along with the activation of the complement cascade in the recruitment of neutrophils within the peritubular capillaries, a feature of acute ABMR. Finally, IL-17 may act in an autocrine and paracrine fashion on proximal tubular epithelial cells promoting the secretion of different cytokines and chemokines as demonstrated by van Kooten et al. (7). Interestingly, our study demonstrates that IL-17 expression by tubular epithelial cells is independent from tubular damage, a common characteristic of the four groups examined, thus suggesting the presence of a specific mechanism exclusively featuring ABMR.

The activation of the complement cascade is one of the main events in the pathogenesis of acute ABMR (1). In particular, several lines of evidence suggested a key role in this setting for C3 (26). The tubular expression of C3 is significantly increased in acute rejection and the locally produced C3 contributes to acute rejection of a kidney graft (27,28). Braun et al. demonstrated that tubular cells express a specific receptor for C3a, the active fragment released by the enzymatic cleavage of C3 by the C3 convertase (29). The same authors also demonstrated that C3a induces the expression of several genes in cultured tubular epithelial cells. However, our observation of an increased tubular expression of IL-17 in response to C3a is the first evidence of C3a-dependent modulation of a cytokine directly involved in the pathogenesis of acute rejection and establishes, at least within the kidney graft, a specific link between two key soluble mediators of the innate immunity. In addition, the newly synthesized IL-17 may induce C3 expression in proximal tubular epithelial cells, priming a positive feed-back loop, which may amplify the local innate response.

The last aim of our study was also to understand the molecular mechanisms underlying the tubular expression of IL-17. Based on the observation that IL-23, the main cytokine known to regulate the Th17 response, exerts its effects on IL-17 expression through the activation of the JAK2-STAT3 axis (30), we decided to focus our attention on this specific cytoplasmic tyrosine kinase. The close spatial relationship between IL-17 expression and JAK2 phosphorylation and subsequent activation, as well as their direct quantitative correlation, confirmed at least in part our hypothesis.

The in vitro results demonstrating the activation of JAK2 in response to C3a and the significant reduction in C3a-induced IL-17 expression upon JAK2 inhibition further corroborated the observation in vivo. JAK2 is activated by several proinflammatory cytokines including IL-6 (31). However, in our study we could not demonstrate any association at the tubular level between IL-6 protein expression and JAK2 phosphorylation.

In this context, a limit of our in vivo study is that we cannot exclude that the phosphorylation of JAK2 observed in acute ABMR biopsies might, at least in part, be due to the paracrine effect of IL-17 on proximal tubular cells. In fact, JAK2 is a key step in the signaling pathway activated by this proinflammatory cytokine in several cell types (32). Interestingly, JAK2 inhibition has been suggested as a new therapy for inflammatory diseases such as arthritis. The use of a specific JAK2 inhibitor in a murine model of arthritis has been shown to reduce the Th17 specific immune response. Interestingly, different specific inhibitors for JAK2 are coming into the clinical arena for the treatment of hematologic neoplasia, including polycytemia and myelofibrosis (33). Thus, JAK2 may represent an easily exploitable target for the treatment of acute ABMR, a clinical condition that represents a therapeutic challenge in the field of transplantation.

In conclusion, we demonstrated, for the first time, that renal tubular epithelial cells represent a significant source of IL-17 in acute ABMR through the activation of the complement system and JAK2 phosphorylation. This event may represent a suitable therapeutic target in this scenario.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

We thank Vincenzo Gesualdo from the Renal, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari ‘A. Moro’ for the excellent technical help in processing kidney graft biopsies. This study was supported by Ministero dell’Istruzione dell’Università e della ricerca Scientifica (PRIN 2008 granted to G. Grandaliano), University of Bari (Fondi di Ateneo 2007 and 2008).

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

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

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References