Original Basic Science
Attenuation of Acute Rat Renal Allograft Rejection by Apolipoprotein E-Mimetic Peptide Anna Zakrzewicz,1 Khusin Kutlimuratov,1 Srebrena Atanasova,1 Dariusz Zakrzewicz,2 Martin Reichert,1 Jessica Schmitz,1 Jochen Wilhelm,3 Günter Lochnit,2 Winfried Padberg,1 and Veronika Grau1 Background. In addition to its well-described role in lipid metabolism, apolipoprotein E (ApoE) exerts immunomodulatory functions. A protective role of ApoE and ApoE-mimetic peptide (ApoE(133–149)) application was documented in several inflammatory disorders. In this study, we test the hypothesis that ApoE(133–149) promotes renal allograft survival. Methods. Dark Agouti, Brown Norway, and Fischer 344 kidneys were transplanted to Lewis rats to investigate fatal and reversible acute rejection. Apolipoprotein E expression was assessed in intravascular leukocytes of renal grafts, in graft tissue and in recipient blood plasma. Recipients of Brown Norway kidneys were treated with ApoE(133–149), and graft survival was monitored until day 100. Graft infiltration, cytokine, and chemokine production were analyzed. Results. Intravascular graft leukocytes and renal tissue obtained from animals undergoing reversible acute rejection expressed increased levels of ApoE mRNA, whereas during fatal rejection, ApoE expression was reduced or remained unchanged. Animals treated with ApoE(133–149) showed prolonged allograft survival, which was associated with a reduced infiltration of CD8 and α/β T-cell receptor–expressing cells, diminished Granzyme B mRNA expression, and decreased caspase-3 activation. Conclusions. Endogenous ApoE overexpression and exogenous application of ApoE(133–149) seem to protect renal allografts from fatal acute rejection. This effect was associated with a reduced influx of cytotoxic T cells.
(Transplantation 2015;99: 925–934)
A
polipoprotein E (ApoE) is a multifunctional glycoprotein with a molecular mass of 34 kDa.1 It is primarily synthesized and secreted by the liver, but monocytes/ macrophages have also been shown to produce significant amounts of ApoE.2 In addition to its well-described role in lipid metabolism and cardiovascular disorders, ApoE possesses immunomodulatory properties, which could be of
Received 3 February 2014. Revision requested 27 February 2014. Accepted 22 October 2014. 1
Department of General and Thoracic Surgery, Laboratory of Experimental Surgery, Justus-Liebig-University Giessen, Giessen, Germany.
2
Department of Biochemistry, Justus-Liebig-University Giessen, Giessen, Germany.
3
Department of Internal Medicine II, Justus-Liebig-University Giessen, Giessen, Germany.
This work was supported by the Excellence Cluster Cardio-Pulmonary System (ECCPS) Start-up Fund (to A.Z.). The authors declare no conflict of interest. A.Z. participated in research design, performance of experiments, data analysis and writing of the article. K.K., S.A., M.R., and J.S. participated in performance of experiments, data analysis and editing of the article. D.Z., G.L., and W.P. participated in data analysis and editing of the article. V.G. participated in research design, performance of experiments, data analysis and writing of the article. Correspondence: Anna Zakrzewicz, Dr. hum. biol., Laboratory of Experimental Surgery, Feulgenstrasse 10–12, D-35385 Giessen, Germany. (Anna.Zakrzewicz@chiru. med.uni-giessen.de). Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/15/9905-925 DOI: 10.1097/TP.0000000000000569
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importance in the context of transplantation.1,3 Apolipoprotein E plays a role in regulating innate immunity, including control of monocyte/macrophage activation and function.1,4,5 Interestingly, in vivo stimulation of macrophages from ApoE-deficient mice with interferon (IFN)-γ induces a stronger expression of proinflammatory cytokines, major histocompatibility complex class II molecules and co-stimulatory molecules in comparison to ApoE wild type mice.4 Furthermore, stimulation of macrophages with ApoE promotes their conversion from the proinflammatory M1 to the anti-inflammatory M2 phenotype6 and suppresses the inflammatory response upon LPS stimulation.7 Apolipoprotein E also inhibits platelet activation, aggregation, and regulates function of adaptive immunity including generation of cytotoxic T cells and production of immunoglobulin G.8-10 Apolipoprotein E is composed of 2 distinct functional domains. The N-terminal domain that contains a receptorbinding region located between residues 133 and 149 and the C-terminal domain that includes a lipid-binding region.11-13 Apolipoprotein E binds with high affinity to the low-density lipoprotein superfamily of receptors.14 Apolipoprotein E-mimetic peptide (ApoE(133–149)) corresponding to the low-density lipoprotein receptor binding domain of ApoE is biologically active. It retains the ability of the intact protein to inhibit LPS-stimulated tumor necrosis factor (TNF)-α and nitric oxide (NO) production in microglial cells in vitro5 and suppresses brain and systemic inflammatory responses in LPS-injected mice.15 Furthermore, ApoE(133–149) can directly compete with the binding of ApoE to cell surface receptors.15 Protective effects of exogenous ApoE and ApoE(133–149) were found in the context of several inflammatory disorders,15-24 but their effect on the pathogenesis of acute organ rejection is still unknown. www.transplantjournal.com
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Several animal models for acute rejection of renal allografts have been established. Kidney transplantation in the fully allogenic, Dark Agouti (DA) or Brown Norway (BN) to Lewis (LEW) rat strain combination leads to acute graft destruction 4 to 7 days after transplantation.25-27 Kidneys transplanted in the Fischer 344 (F344) to LEW combination remain functional for at least 6 months but undergo reversible acute rejection, which peaks around day 9 and develop chronic allograft damage in the long run.27-30 During acute rejection, numerous leukocytes, predominantly activated monocytes and T lymphocytes, accumulate in the vasculature of rat renal allografts.31-33 They have been proposed to play a decisive role during organ rejection. To better characterize them and define potential factors involved in the resolution of acute graft rejection, the transcriptome of allograft leukocytes was compared to isograft leukocytes. Microarray analyses revealed several differentially expressed genes. Apolipoprotein E was found among the genes, which were upregulated during reversible acute rejection.33 Given the anti-inflammatory and immunomodulatory activities of ApoE, we hypothesized that ApoE is specifically induced during reversible but not during fatal acute rejection and that ApoE can promote renal allograft survival. To test this hypothesis, we investigated the expression of ApoE in different experimental models of acute rejection and assessed the effect of ApoE(133–149) application on allograft survival.
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Austria), supplemented with 2.7 mM EDTA, 0.1% BSA (Serva, Heidelberg, Germany). To deplete erythrocytes and granulocytes, perfusates were purified by Percoll density centrifugation, and the remaining mononuclear cells were stored under liquid nitrogen until use. RNA Isolation and Complementary DNA Synthesis
Total cellular RNA was extracted from 30 mg kidney tissue or from 5 106 intravascular mononuclear leukocytes harvested from control kidneys, isografts, or allografts on days 4 or 9 after transplantation. RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was reversely transcribed using the M-MLV H−reverse transcriptase and 1 μg of random hexamer primers (Promega, Mannheim, Germany). The reaction was carried out at 40°C for 1 hour. Real-Time Reverse-Transcriptase Polymerase Chain Reaction
Details on reverse transcriptase reaction and primer sequences are available on line data supplement (SDC, http://links.lww.com/TP/B102). Immunohistochemistry
Description of immunohistochemistry is available in the online data supplement, SDC, http://links.lww.com/TP/B102. Protein Isolation and Immunoblotting
MATERIALS AND METHODS Experimental Animals and Kidney Transplantation
Male LEW (RT11), DA (RT1av1) (Janvier, St Berthevin, France), F344 (RT1lv1) (Harlan Winkelmann, Borchen, Germany), and BN rats (Janvier) were kept under conventional conditions. Animals weighing 260 to 300 g were used for transplantation, only BN rats weighed 220 to 240 g. All animals received humane care following the German Law on the Protection of Animals, the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research as well as the National Institute of Health “Guide for the Care and Use of Laboratory Animals.” Experiments were approved by the local authorities (permit number V54-19C20-15(1) GI20/27 No. 51/2010). Kidneys were transplanted orthotopically to totally nephrectomized LEW recipients as described,34 except that the ureter was anastomosed end to end. For allogenic transplantation, DA, BN, or F344 rats and for isogenic transplantation LEW rats were used as donors. Total ischemic times remained below 30 minutes. On days 4, 9, or 100 after transplantation, graft recipients were anesthetized by intraperitoneal application of 60 mg/kg sodium pentobarbital (Narcoren; Merial GmbH, Hallbergmoos, Germany), and blood from the central blood stream was obtained by heart puncture. Before blood collection, animals received intravenous injection of 200 U of heparin (Liguemin N 5000, Roche). Grafts and spleens were removed, cut in small pieces and snap frozen in liquid nitrogen. Isolation of Mononuclear Leukocytes From the Renal Vasculature
Isolation of mononuclear blood cells was described previously in detail.32 Briefly, recipients were anaesthetized, heparinized, and the kidney was extensively perfused with cold Ca2+-free and Mg2+-free phosphate buffered saline (PBS; PAA, Pasching,
Protein extracts from graft tissue were prepared as described previously.35 Protein concentrations were determined using Micro BCA protein Assay kit (Pierce Biotechnology, Rockford, IL). Equal amounts of protein (20 μg) were resolved on 15% reducing sodium dodecyl sulfate (SDS)polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 1 Roti-Block solution (Roth, Karlsruhe, Germany) diluted in 50 mM Tris–HCl, pH 7.6, 0.9% NaCl. Rabbit polyclonal antibodies (Abs) to active caspase-3 (Abcam, Cambridge, UK) were diluted 1:1 000 in 1x Roti-Block (Roth). To ensure equal protein loading, membranes were incubated with mouse monoclonal Abs to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Novus Biologicals, Littleton, CO), 1:20,000. Bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark), 1:5 000, using the chemiluminescent reagent LumiLight Western blotting substrate (Roche, Mannheim, Germany). Densitometric analyses were performed using a digital gel documentation system (Biozym, Hessisch Oldendorf, Germany). Data were divided by the values obtained for GAPDH on the same blot. Synthesis and Administration of Peptide
The 17-amino acid-long ApoE(133–149) derived from the sequence of the receptor binding region of human ApoE (Ac-LRVRLASHLRKLRKRLL-amide) and an 8-amino acid-long control peptide corresponding to the 133 to 140 amino acid region of human ApoE (Ac-LRVRLASHamide) were synthesized by ProImmune (Oxford, UK) to a purity greater than 95%. The amino termini of both peptides were acetylated, and carboxy termini were blocked with an amide moiety. Peptides were reconstituted in sterile isotonic saline at a concentration of 6 μg/μL, aliquoted, and stored at −20°C. Renal allograft recipients
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were treated daily with ApoE(133–149) (6 mg/kg body weight) as published before15 or control peptide by intravenous injections for 10 days starting from the day of renal transplantation. Survival of the animals was monitored until day 100 after transplantation. Quantification of Nitrite/Nitrate
To estimate NO production in the graft, nitrite/nitrate concentration was determined by Griess reaction as previously described.27 Measurement of ApoE Plasma Levels
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in mononuclear leukocytes isolated from renal blood vessels of control animals, isogenic, or allogenic renal transplants. Fatal acute rejection of DA kidneys was accompanied by reduced mRNA expression of ApoE in cells collected from allografts in comparison to the respective isografts or remained at the level of isografts after BN to LEW transplantation. In line with microarray data published before,33 ApoE mRNA was elevated in intravascular leukocytes during reversible acute rejection of F344 kidneys (Figure 1A). Although mRNA expression analyses were performed on the entire intravascular population of mononuclear leukocytes, ApoE was predominantly produced
Apolipoprotein E plasma levels were analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (USCN Life Science, Wuhan, China) according to the manufacturer's instructions. Before measurement, samples were diluted 1:250 with PBS. The detection range was 1.56 to 100 ng/mL. Flow Cytometry
Flow cytometry was performed as described previously. 31,32 A FACS Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany) and the Cell Quest software 3.2.1 (Becton Dickinson) were used and 30000 events were measured for every sample. Microarrays
Microarray and statistical analyses of the data were performed as previously described.33 The data have been deposited in NCBI's Gene Expression Omnibus36 and are accessible through GEO Series accession number GSE58156 at: http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58156. Statistics
Data were analyzed, where applicable, by nonparametric Kruskal-Wallis test followed by the Mann–Whitney rank sum test using SPSS software (SPSS software, Munich, Germany). Kaplan-Meier survival analysis with long rank (Mantel-Cox) test was used for comparison of graft survival between experimental groups (GraphPad software, La Jolla, CA). A P value of 0.05 or less was indicated. RESULTS Accumulation of Mononuclear Leukocytes in the Blood Vessels of the Graft During Acute Rejection
Leukocytes, which accumulated in the vasculature of rat renal allografts during acute rejection, were harvested by intensive perfusion of graft blood vessels on day 4 after DA to LEW and BN to LEW kidney transplantation or on day 9 after F344 to LEW renal transplantation. Perfusates were predominantly composed of monocytes and T cells as published before for LEW, DA, and F344 grafts transplanted to LEW recipients.28,32 Data on the cellular composition of intravascular graft leukocytes from day 4 BN to LEW allografts are summarized in (Table S1, SDC, http://links.lww.com/TP/B102 and expression of the cell surface markers of monocytes are presented in Table S2 (SDC, http://links.lww.com/TP/B102). Differential mRNA Expression of ApoE by Intravascular Leukocytes and Graft Tissue
Apolipoprotein E mRNA expression was analyzed by realtime reverse-transcriptase polymerase chain reaction (RT-PCR)
FIGURE 1. Expression of ApoE during fatal (DA, BN) and reversible acute rejection (F344). ApoE mRNA expression in intravascular mononuclear leukocytes isolated from control kidneys (LEW), renal Iso and Allo (A) and in the tissue of control kidneys, renal isografts and allografts (B) was assessed by real-time RT-PCR. Data are expressed as AU, which are normalized to a mean of one unit in untreated controls. ApoE protein level in plasma obtained from control animals and recipients of isografts and allografts (C) was determined by ELISA. Data were analyzed by nonparametric Kruskal-Wallis test followed by the Mann–Whitney rank sum test. Bars indicate median, whiskers percentiles 25 and 75. F344, Fischer 344; d, day; Iso, isografts; Allo, allografts; AU, arbitrary units.
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by monocytes. Monocytes isolated from control kidneys by perfusion and sorted by magnetic assisted cell sorting (MACS) show similar levels of ApoE mRNA expression like the entire population of mononuclear leukocytes (data not shown). Apolipoprotein E mRNA expression was also detected in graft tissues and did not differ between healthy control kidneys, renal isografts, and allografts on day 4 after DA to LEW transplantation. In contrast, during reversible acute rejection, ApoE was elevated in F344 kidney allografts (Figure 1B). Stable ApoE Plasma Levels During Fatal and Reversible Acute Rejection
The secretory protein ApoE is abundantly present in blood plasma.37 To evaluate a potential effect of acute rejection on the plasma level, ApoE was quantified by ELISA. Analysis of plasma from animals during fatal or at the peak of reversible acute rejection revealed no significant differences in ApoE levels between the groups analyzed (Figure 1C). Prolonged Survival of Renal Allografts Upon Treatment With ApoE(133–149)
To test the protective potential of ApoE on graft survival, totally nephrectomized recipients of BN kidneys were treated with ApoE(133–149) or control peptide. The survival of the animals was monitored until day 100 after transplantation. All control animals died due to acute rejection between days 8 and 16 after transplantation, whereas treatment with ApoE (133–149) prolonged allograft recipient survival (Figure 2). Stable mRNA Expression of Cytokines and inducible NO synthase Upon Treatment With ApoE(133–149)
To study the effect of the ApoE(133–149) treatment on cytokine expression in allografts and spleens, the mRNA of selected cytokines was quantified by real-time RT-PCR on day 4 after transplantation of BN kidneys to LEW rats. No significant difference between the ApoE(133–149)-treated group and controls was found regarding IFN-γ, prointerleukin (IL)-1β, IL-2, IL-12p35, IL-12p40, IL-17 F, IL-23p19, IL-10, TGF-β, and TNF-α mRNA expression in graft tissue (Figure 3A) and spleens (Figure S1, SDC, http://links.lww.com/TP/B102).
FIGURE 2. Survival of Brown Norway to Lewis renal allograft recipients treated with ApoE(133–149) or control peptide (n = 8, per group). ApoE(133–149) or control peptide were administered by daily intravenous injections for ten days starting from the day of renal transplantation. Survival of the animals was monitored daily until day 100 and presented as Kaplan-Meier curve. Long rank Mantel-Cox test was used for comparison of graft survival between experimental groups.
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Furthermore, the mRNA expression of iNOS revealed no differences upon ApoE(133–149) treatment when compared to control treatment (Figure 3B). In agreement with this finding, nitrite/nitrate concentrations measured by Griess reaction in graft tissues remained stable independent of the treatment (Figure 3C). Differential mRNA Expression of Chemokines Upon Treatment With ApoE(133–149)
To identify changes in chemokine production and to further characterize potential factors differently expressed upon ApoE(133–149) treatment, transcriptional profiling was performed on rat whole genome microarrays using RNA from day 4 allografts treated with ApoE(133–149) or control peptide. The data have been deposited in NCBI's Gene Expression Omnibus36 and are accessible through GEO Series accession number GSE58156. The differential mRNA expression of selected chemokines was further confirmed by real-time RT-PCR. We detected reduced expression of CXCL13 and CCL5 upon ApoE(133–149) treatment, whereas CCL20 was elevated compared to control peptide-treated animals (Figure 3D). No differences were seen in the expressions of CCL4, CCL21, CCL22, and CXCL3 (data not shown). Reduced Number of Graft Infiltrating / T Cell Receptor Expressing Cells Upon Treatment With ApoE(133–149)
To examine the effect of ApoE(133–149) treatment on accumulation of inflammatory cells in grafts during acute rejection, mononuclear leukocytes were identified by immunohistochemical staining (Figure 4A). No significant difference between ApoE(133–149)-treated group and controls was found regarding the number of monocytes/macrophages identified with Abs to a CD68-like antigen, M2 macrophages stained with Abs to CD163, B cells visualized with Abs to CD45RA, and natural killer (NK) cells defined by strong staining with Abs to CD161. However, ApoE(133–149) treatment diminished the number of cells identified with Abs to α/β T-cell receptor (TCR) (Figure 4B). Reduced Interaction Between Monocytes and Platelets Upon Treatment With ApoE(133–149)
The effect of ApoE(133–149) treatment on the interaction of platelets with monocytes and endothelial cells in the vasculature of the graft was investigated by immunohistochemical staining (Figure S2A, SDC, http://links.lww.com/TP/B102). Platelets were identified with Abs to CD61. Antibodies to a CD68-like antigen and rat endothelial cell antigen (RECA)1 were used to detect monocytes and endothelial cells, respectively. Interestingly, immunohistochemical analyses revealed a reduced percentage of platelets interacting with monocytes (Figure S2B, SDC, http://links.lww.com/TP/B102), whereas the percentage of free platelets (Figure S2C, SDC, http://links.lww.com/TP/B102) as well as of platelets interacting with endothelium (Figure S2D, SDC, http://links.lww.com/TP/B102) was not changed. Stable mRNA Expression of T Cell Transcription Factors Upon Treatment With ApoE(133–149)
To further characterize the effect of the treatment with ApoE(133–149) on the T cell compartment, mRNA expression levels of several transcription factors involved in T cell polarization were determined in grafts (Figure 5A) and spleens (Figure S3, SDC, http://links.lww.com/TP/B102) of transplanted
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FIGURE 3. Expression of proinflammatory and anti-inflammatory cytokines, iNOS and chemokines in kidney allografts after treatment with ApoE(133–149). The mRNA expression of IFN-γ, TNF-α, pro-IL-1β, IL-2, IL-12p35, IL-12p40, IL-17 F, IL-23-p19, IL-10, TGF-β (A), iNOS (B), and CCL5, CXCL13, CCL20 (D) was assessed on day 4 post-transplantation by real-time RT-PCR in kidney allografts treated with ApoE (133–149) or control peptide (n = 4, per group). Data are expressed as arbitrary units (AU), which are normalized to a mean of 1 unit in untreated controls. The NOx concentration in renal tissue (C) was determined with Griess reaction as an index of NO production. Data were analyzed by non-parametric Mann–Whitney rank sum test. Bars indicate median, whiskers percentiles 25 and 75. n = 4, per group. NOx, nitrite/nitrate.
animals. No significant difference between the ApoE(133–149)treated group and controls was found regarding Tbet, Gata3, Foxo1, Tcf1, and Foxp3 expressions. However, ApoE(133–149) treatment lowers the mRNA expression of Granzyme B (GrB) in grafts.
CD4pos and CD8pos monocytes/macrophages, sections were double-stained with Abs to a CD68-like antigen. Only single positive cells were quantified. The number of CD4pos CD68-likeneg cells was unchanged, whereas infiltrating CD8pos CD68-likeneg cells were diminished (Figure 5C).
Reduced Graft Infiltration by CD8pos T Cells Upon Treatment With ApoE(133–149)
Diminished Level of Active Caspase-3 Upon Treatment With ApoE(133–149)
Immunohistochemical staining with Abs to detect CD4 and CD8 α-chain was performed (Figure 5B). To exclude
To examine the effect of ApoE(133–149) treatment on caspase-3 activation, detection of active caspase-3 in renal
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FIGURE 4. Leukocytic infiltration of kidney allografts after treatment with ApoE(133–149). Cryostat sections of renal allografts treated with ApoE (133–149) or control peptide were stained on day 4 after transplantation with Abs to a CD68-like antigen to visualize monocytes and macrophages, to CD163 for detection of M2 macrophages. B cells were identified with Abs to CD45RA, NK cells with Abs to CD161 and T cells with Abs to α/β TCR. Bound Abs were detected in brown and sections were lightly counterstained with hemalum (A). Immunopositive leukocytes were counted in renal cortex (n = 4, each) (B). Data were analyzed by nonparametric Mann–Whitney rank sum test. Bars indicate median, whiskers percentiles 25 and 75.
allografts on day 4 after transplantation was performed by immunoblotting (Figure 5D) and quantified by densitometric analysis (Figure 5E). Consistent with the reduced number of CD8pos cells, reduced levels of active caspase-3 were detected upon ApoE(133–149) treatment. Unchanged T Cell Proliferation Upon Treatment With ApoE(133–149)
Recently, our group reported that CD8pos T lymphocytes proliferate in the lumina of the blood vessel of rat renal allografts during the onset of acute rejection.31 Knowing that ApoE suppresses mitogen-activated T-cell proliferation, we tested whether ApoE(133–149) affects intravascular T cell
proliferation. Triple staining was performed with Abs to proliferating cell nuclear antigen (PCNA), to the endothelial marker RECA-1, and to α/β TCR. The PCNApos T cells were quantified inside blood vessels of the grafts (Figure S4A, SDC, http://links.lww.com/TP/B102). Although a high percentage of T cells was PCNApos in the vasculature of grafts, no significant difference between the ApoE(133–149)-treated group and controls was detected (Figure S4B, SDC, http://links.lww.com/TP/B102). Deposition of C4d in Peritubullar Capillaries
Several groups reported that staining of allograft biopsies for C4d could be a reliable marker for the diagnosis of
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FIGURE 5. Expression of transcription factors involved in T cell polarization and T cell function after treatment with ApoE(133–149). The mRNA expression of Tbet, Gata3, Foxo1, Tcf1, Foxp3, and GrB was assessed by real-time RT-PCR in kidney allografts treated with ApoE(133–149) or control peptide on day 4 after transplantation (n = 4, per group) (A). Data are expressed as arbitrary units (AU), which are normalized to a mean of one unit in untreated controls. Renal allografts treated with ApoE(133–149) or control peptide were analyzed on day 4 after transplantation by immunohistochemical staining with Abs to CD4 or CD8 α-chain in blue to detect CD4pos and CD8pos T cells respectively. To exclude CD4pos or CD8pos monocytes/macrophages as a first step staining with Abs to a CD68-like antigen was performed in brown (B). CD4pos CD68-likeneg and CD8pos CD68-likeneg cells were quantified in the renal cortex (n = 4, per group) (C). The level of active caspase-3 upon ApoE(133–149) or control peptide treatment was analyzed in renal tissue by immunoblotting on day 4 after transplantation (D). Detection of GAPDH was performed to ensure equal protein loading. Intensity of the signal was quantified by densitometric analysis (E). Data were analyzed by nonparametric Mann–Whitney rank sum test. Bars indicate median, whiskers percentiles 25 and 75.
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transplant outcome.38 We detected deposition of C4d in glomelruli as well as on endothelial cells of peritubular capillaries of allografts on day 4 after transplantation (Figure S5A, SDC, http://links.lww.com/TP/B102). Treatment with ApoE(133–149) had no effect on the intensity of the staining (Figure S5B, C, SDC, http://links.lww.com/TP/B102). DISCUSSION Apolipoprotein E as well as ApoE(133–149) have been already shown to exert various immunomodulatory effects.3,5,8,9,15-24,39,40 In the present study, we are the first to demonstrate a protective role of ApoE(133–149) in experimental acute kidney graft rejection. This effect was associated with a reduced influx of α/β TCR-expressing cells and CD8pos cells as well as diminished expression of GrB and decreased levels of active caspase-3. The changes are probably due to a reduction in number of cytotoxic T cells but we cannot formally exclude an additional reduction of the minor population of CD161neg natural killer T (NKT) cells. In our study, intravascular graft leukocytes and renal tissue obtained from animals undergoing reversible acute rejection expressed increased levels of ApoE mRNA compared to isograft recipients, whereas during fatal rejection, ApoE expression was reduced or remained unchanged. The observed changes in ApoE expression cannot be explained by changes in the cellular composition of blood leukocytes and renal blood tissue because during both fatal and reversible acute rejection, the proportion of mononuclear phagocytes increased in a similar way.26-29 These findings together with previously published data describing immunoregulatory properties of ApoE3,5,8,17,39 supported our assumption that elevated levels of ApoE have a protective effect on graft survival. In contrast, no changes in ApoE protein levels were seen in plasma. However, we expect that strong accumulation of monocytes capable to secrete ApoE would result in a local increase of ApoE concentrations inside graft blood vessels.2,28,29 In line with this assumption, transgenic mice expressing ApoE only in monocytes/macrophages were protected from atherosclerosis, even though plasma levels of ApoE remained unchanged and very low. In contrast, transgenic mice with normal ApoE plasma levels but ApoE-null macrophages were more susceptible to this disease.41 The protective potential of ApoE(133–149) treatment on graft survival was tested after renal transplantation in the fully allogenic BN to LEW rat strain combination. This experimental model of fatal acute rejection is frequently used to test new therapeutic approaches.25,32,42 In this model, similar to the DA to LEW or F344 to LEW rat kidney transplantation, the effector phase of acute rejection is accompanied by strong accumulation of mononuclear cells, predominantly monocytes, inside allograft blood vessels. Results of the flow cytometric analyses of the expression of several surface antigens suggested an intermediate state of monocyte activation. Similar to self-limiting acute rejection in F344 to LEW rat strain combination,33 the proportion of major histocompatibility complex IIpos and CD161pos cells increased, whereas only minor changes in the expression of CD4, CD11a, CD18, CD43, and CD71 were detected. Animals treated with ApoE(133–149) showed a prolonged allograft survival, which was associated with a reduced number of infiltrating CD8pos T cells.
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The central role of T lymphocytes in mediating allograft rejection is well established.43-46 CD8pos T cells are abundantly present in acutely rejecting allografts and are thought to be the major effector cells during graft rejection.47,48 Deficiency in CD8pos T cells was described to be protective during acute rejection of heart allografts in a minor histocompatibility antigen-mismatched model.45 In line with this observation, transfer of alloreactive CD8pos T cells primed in vitro caused islet allograft rejection.49 These findings support the importance of CD8pos T cells in acute organ transplant rejection.48,50 Moreover, there is evidence that CD8pos T cells are more resistant to conventional immunosuppression and tolerance induction protocols.51 Consequently, strategies targeting CD8pos T lymphocytes are of interest. In agreement with this finding, ApoE(133–149) treatment also reduced mRNA expression of GrB. Granzymes and perforin are the main effector molecules of cytotoxic T lymphocytes, which include CD8pos, some CD4pos T lymphocytes, NKT, and NK cells.50 It is of importance to note that increased expression of GrB is predictive of the development of acute rejection episodes and has been proposed to serve as a marker for acute rejection.52-55 As the estimated number of CD4pos T cells or NK cells was not affected by peptide treatment, we postulated that reduced GrB expression mirrors the reduced infiltrate of CD8pos T cells. Furthermore, also, caspase-3 activation, an established marker for apoptosis, which reflects activity of cytotoxic cells, was reduced upon ApoE(133–149) treatment. This observation can be correlated with a reduced number of CD8pos T cells. Application of ApoE(133–149), however, did not impair intravascular T cell proliferation, which was described for acute rejection of renal allografts before.31 It is likely that ApoE(133–149) affected the recruitment of CD8pos T cells into the graft. This assumption is in line with the results of chemokine expression. Reduced levels of CCL5 a well-known chemoattractant for T cells, at least partially, explains our observations. In contrast, the observed reduction in CXCL13 mRNA, a B lymphocyte chemoattractant, had no effect on accumulation of B cells in the grafts. Interestingly, although ApoE(133–149) did not affect accumulation of monocytes/macrophages in graft blood vessels and tissue, it reduced the frequency of platelet-monocyte interactions. This might be of importance for graft survival because interactions with platelets induce a proinflammatory phenotype in circulating monocytes.56 It was already reported that ApoE and ApoE(133–149) treatment suppress the release of proinflammatory cytokines in vitro and in vivo.3,6-8 In agreement with these observations, ApoE-deficient mice mount increased systemic inflammatory responses upon LPS injection.16,39 In contrast to these findings, in our experimental setting, ApoE(133–149) did neither affect local nor systemic expression of cytokines involved in Th1-like (IFN-γ, IL-1β, IL-2, IL-12, TNF-α), Th17-like (IL-17F, IL-23) or regulatory (IL-10, TGF-β) immune reactions. In the same line, mRNA expression of iNOS as well as graft NO production and mRNA levels of transcription factors involved in T cell polarization remained stable independent of the treatment. Our experimental study has some limitations. We do not know if our finding would also apply to human transplant recipients. Furthermore, grafts were exclusively investigated during the peak of acute rejection and we ignore mechanisms
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occurring during the onset of the immune reactions. In addition, cytokines and chemokines were analyzed on the mRNA level but protein data are missing. In conclusion, our results support the hypothesis that endogenous ApoE plays a protective role during acute rejection of experimental allografts. Furthermore, we demonstrate that treatment with ApoE(133–149) promotes recipient survival after allogenic kidney transplantation. ApoE(133–149) seems to reduce infiltrating cytotoxic CD8pos T cells but further studies are needed to fully understand its mechanism of action. ACKNOWLEDGMENTS The authors thank Gabriele Fuchs-Moll, Kathrin Petri, and Sigrid Wilker for their expert technical assistance, and Ulrike Berges for help with the art work. REFERENCES
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45. Youssef AR, Otley C, Mathieson PW, Smith RM. Role of CD4+ and CD8+ T cells in murine skin and heart allograft rejection across different antigenic desparities. Transpl Immunol. 2004;13:297. 46. Boisgerault F, Liu Y, Anosova N, et al. Role of CD4+ and CD8+ T cells in allorecognition: lessons from corneal transplantation. J Immunol. 2001; 167:1891–1899. 47. Trentin L, Zambello R, Faggian G, et al. Phenotypic and functional characterization of cytotoxic cells derived from endomyocardial biopsies in human cardiac allografts. Cell Immunol. 1992;141:332–341. 48. Bueno V, Pestana JO. The role of CD8+ T cells during allograft rejection. Braz J Med Biol Res. 2002;35:1247–1258. 49. Diamond AS, Gill RG. An essential contribution by IFN-gamma to CD8+ T cell-mediated rejection of pancreatic islet allografts. J Immunol. 2000; 165:247–255. 50. Choy JC. Granzymes and perforin in solid organ transplant rejection. Cell Death Differ. 2010;17:567–576.
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51. Bierer BE, Hollander G, Fruman D, Burakoff SJ. Cyclosporin A and FK506: molecular mechanisms of immunosuppression and probes for transplantation biology. Curr Opin Immunol. 1993;5:763–773. 52. Kummer JA, Wever PC, Kamp AM, et al. Expression of granzyme A and B proteins by cytotoxic lymphocytes involved in acute renal allograft rejection. Kidney Int. 1995;47:70–77. 53. Lipman ML, Stevens AC, Strom TB. Heightened intragraft CTL gene expression in acutely rejecting renal allografts. J Immunol. 1994;152:5120–5127. 54. Clement MV, Haddad P, Soulie A, et al. Perforin and granzyme B as markers for acute rejection in heart transplantation. Int Immunol. 1991;3:1175–1181. 55. Clement MV, Soulie A, Legros-Maida S, et al. Perforin and granzyme B: predictive markers for acute GVHD or cardiac rejection after bone marrow or heart transplantation. Nouv Rev Fr Hematol. 1991;33:465–470. 56. Passacquale G, Vamadevan P, Pereira L, et al. Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes. PLoS One. 2011;6:e25595.
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Attenuation of Acute Rat Renal Allograft Rejection by Apolipoprotein E-Mimetic Peptide Anna Zakrzewicz,1 Khusin Kutlimuratov,1 Srebrena Atanasova,1 Dariusz Zakrzewicz,2 Martin Reichert,1 Jessica Schmitz,1 Jochen Wilhelm,3 Günter Lochnit,2 Winfried Padberg,1 and Veronika Grau1 Background. In addition to its well-described role in lipid metabolism, apolipoprotein E (ApoE) exerts immunomodulatory functions. A protective role of ApoE and ApoE-mimetic peptide (ApoE(133–149)) application was documented in several inflammatory disorders. In this study, we test the hypothesis that ApoE(133–149) promotes renal allograft survival. Methods. Dark Agouti, Brown Norway, and Fischer 344 kidneys were transplanted to Lewis rats to investigate fatal and reversible acute rejection. Apolipoprotein E expression was assessed in intravascular leukocytes of renal grafts, in graft tissue and in recipient blood plasma. Recipients of Brown Norway kidneys were treated with ApoE(133–149), and graft survival was monitored until day 100. Graft infiltration, cytokine, and chemokine production were analyzed. Results. Intravascular graft leukocytes and renal tissue obtained from animals undergoing reversible acute rejection expressed increased levels of ApoE mRNA, whereas during fatal rejection, ApoE expression was reduced or remained unchanged. Animals treated with ApoE(133–149) showed prolonged allograft survival, which was associated with a reduced infiltration of CD8 and α/β T-cell receptor–expressing cells, diminished Granzyme B mRNA expression, and decreased caspase-3 activation. Conclusions. Endogenous ApoE overexpression and exogenous application of ApoE(133–149) seem to protect renal allografts from fatal acute rejection. This effect was associated with a reduced influx of cytotoxic T cells.
(Transplantation 2015;99: 925–934)
A
polipoprotein E (ApoE) is a multifunctional glycoprotein with a molecular mass of 34 kDa.1 It is primarily synthesized and secreted by the liver, but monocytes/ macrophages have also been shown to produce significant amounts of ApoE.2 In addition to its well-described role in lipid metabolism and cardiovascular disorders, ApoE possesses immunomodulatory properties, which could be of
Received 3 February 2014. Revision requested 27 February 2014. Accepted 22 October 2014. 1
Department of General and Thoracic Surgery, Laboratory of Experimental Surgery, Justus-Liebig-University Giessen, Giessen, Germany.
2
Department of Biochemistry, Justus-Liebig-University Giessen, Giessen, Germany.
3
Department of Internal Medicine II, Justus-Liebig-University Giessen, Giessen, Germany.
This work was supported by the Excellence Cluster Cardio-Pulmonary System (ECCPS) Start-up Fund (to A.Z.). The authors declare no conflict of interest. A.Z. participated in research design, performance of experiments, data analysis and writing of the article. K.K., S.A., M.R., and J.S. participated in performance of experiments, data analysis and editing of the article. D.Z., G.L., and W.P. participated in data analysis and editing of the article. V.G. participated in research design, performance of experiments, data analysis and writing of the article. Correspondence: Anna Zakrzewicz, Dr. hum. biol., Laboratory of Experimental Surgery, Feulgenstrasse 10–12, D-35385 Giessen, Germany. (Anna.Zakrzewicz@chiru. med.uni-giessen.de). Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/15/9905-925 DOI: 10.1097/TP.0000000000000569
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importance in the context of transplantation.1,3 Apolipoprotein E plays a role in regulating innate immunity, including control of monocyte/macrophage activation and function.1,4,5 Interestingly, in vivo stimulation of macrophages from ApoE-deficient mice with interferon (IFN)-γ induces a stronger expression of proinflammatory cytokines, major histocompatibility complex class II molecules and co-stimulatory molecules in comparison to ApoE wild type mice.4 Furthermore, stimulation of macrophages with ApoE promotes their conversion from the proinflammatory M1 to the anti-inflammatory M2 phenotype6 and suppresses the inflammatory response upon LPS stimulation.7 Apolipoprotein E also inhibits platelet activation, aggregation, and regulates function of adaptive immunity including generation of cytotoxic T cells and production of immunoglobulin G.8-10 Apolipoprotein E is composed of 2 distinct functional domains. The N-terminal domain that contains a receptorbinding region located between residues 133 and 149 and the C-terminal domain that includes a lipid-binding region.11-13 Apolipoprotein E binds with high affinity to the low-density lipoprotein superfamily of receptors.14 Apolipoprotein E-mimetic peptide (ApoE(133–149)) corresponding to the low-density lipoprotein receptor binding domain of ApoE is biologically active. It retains the ability of the intact protein to inhibit LPS-stimulated tumor necrosis factor (TNF)-α and nitric oxide (NO) production in microglial cells in vitro5 and suppresses brain and systemic inflammatory responses in LPS-injected mice.15 Furthermore, ApoE(133–149) can directly compete with the binding of ApoE to cell surface receptors.15 Protective effects of exogenous ApoE and ApoE(133–149) were found in the context of several inflammatory disorders,15-24 but their effect on the pathogenesis of acute organ rejection is still unknown. www.transplantjournal.com
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Several animal models for acute rejection of renal allografts have been established. Kidney transplantation in the fully allogenic, Dark Agouti (DA) or Brown Norway (BN) to Lewis (LEW) rat strain combination leads to acute graft destruction 4 to 7 days after transplantation.25-27 Kidneys transplanted in the Fischer 344 (F344) to LEW combination remain functional for at least 6 months but undergo reversible acute rejection, which peaks around day 9 and develop chronic allograft damage in the long run.27-30 During acute rejection, numerous leukocytes, predominantly activated monocytes and T lymphocytes, accumulate in the vasculature of rat renal allografts.31-33 They have been proposed to play a decisive role during organ rejection. To better characterize them and define potential factors involved in the resolution of acute graft rejection, the transcriptome of allograft leukocytes was compared to isograft leukocytes. Microarray analyses revealed several differentially expressed genes. Apolipoprotein E was found among the genes, which were upregulated during reversible acute rejection.33 Given the anti-inflammatory and immunomodulatory activities of ApoE, we hypothesized that ApoE is specifically induced during reversible but not during fatal acute rejection and that ApoE can promote renal allograft survival. To test this hypothesis, we investigated the expression of ApoE in different experimental models of acute rejection and assessed the effect of ApoE(133–149) application on allograft survival.
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Austria), supplemented with 2.7 mM EDTA, 0.1% BSA (Serva, Heidelberg, Germany). To deplete erythrocytes and granulocytes, perfusates were purified by Percoll density centrifugation, and the remaining mononuclear cells were stored under liquid nitrogen until use. RNA Isolation and Complementary DNA Synthesis
Total cellular RNA was extracted from 30 mg kidney tissue or from 5 106 intravascular mononuclear leukocytes harvested from control kidneys, isografts, or allografts on days 4 or 9 after transplantation. RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was reversely transcribed using the M-MLV H−reverse transcriptase and 1 μg of random hexamer primers (Promega, Mannheim, Germany). The reaction was carried out at 40°C for 1 hour. Real-Time Reverse-Transcriptase Polymerase Chain Reaction
Details on reverse transcriptase reaction and primer sequences are available on line data supplement (SDC, http://links.lww.com/TP/B102). Immunohistochemistry
Description of immunohistochemistry is available in the online data supplement, SDC, http://links.lww.com/TP/B102. Protein Isolation and Immunoblotting
MATERIALS AND METHODS Experimental Animals and Kidney Transplantation
Male LEW (RT11), DA (RT1av1) (Janvier, St Berthevin, France), F344 (RT1lv1) (Harlan Winkelmann, Borchen, Germany), and BN rats (Janvier) were kept under conventional conditions. Animals weighing 260 to 300 g were used for transplantation, only BN rats weighed 220 to 240 g. All animals received humane care following the German Law on the Protection of Animals, the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research as well as the National Institute of Health “Guide for the Care and Use of Laboratory Animals.” Experiments were approved by the local authorities (permit number V54-19C20-15(1) GI20/27 No. 51/2010). Kidneys were transplanted orthotopically to totally nephrectomized LEW recipients as described,34 except that the ureter was anastomosed end to end. For allogenic transplantation, DA, BN, or F344 rats and for isogenic transplantation LEW rats were used as donors. Total ischemic times remained below 30 minutes. On days 4, 9, or 100 after transplantation, graft recipients were anesthetized by intraperitoneal application of 60 mg/kg sodium pentobarbital (Narcoren; Merial GmbH, Hallbergmoos, Germany), and blood from the central blood stream was obtained by heart puncture. Before blood collection, animals received intravenous injection of 200 U of heparin (Liguemin N 5000, Roche). Grafts and spleens were removed, cut in small pieces and snap frozen in liquid nitrogen. Isolation of Mononuclear Leukocytes From the Renal Vasculature
Isolation of mononuclear blood cells was described previously in detail.32 Briefly, recipients were anaesthetized, heparinized, and the kidney was extensively perfused with cold Ca2+-free and Mg2+-free phosphate buffered saline (PBS; PAA, Pasching,
Protein extracts from graft tissue were prepared as described previously.35 Protein concentrations were determined using Micro BCA protein Assay kit (Pierce Biotechnology, Rockford, IL). Equal amounts of protein (20 μg) were resolved on 15% reducing sodium dodecyl sulfate (SDS)polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 1 Roti-Block solution (Roth, Karlsruhe, Germany) diluted in 50 mM Tris–HCl, pH 7.6, 0.9% NaCl. Rabbit polyclonal antibodies (Abs) to active caspase-3 (Abcam, Cambridge, UK) were diluted 1:1 000 in 1x Roti-Block (Roth). To ensure equal protein loading, membranes were incubated with mouse monoclonal Abs to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Novus Biologicals, Littleton, CO), 1:20,000. Bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark), 1:5 000, using the chemiluminescent reagent LumiLight Western blotting substrate (Roche, Mannheim, Germany). Densitometric analyses were performed using a digital gel documentation system (Biozym, Hessisch Oldendorf, Germany). Data were divided by the values obtained for GAPDH on the same blot. Synthesis and Administration of Peptide
The 17-amino acid-long ApoE(133–149) derived from the sequence of the receptor binding region of human ApoE (Ac-LRVRLASHLRKLRKRLL-amide) and an 8-amino acid-long control peptide corresponding to the 133 to 140 amino acid region of human ApoE (Ac-LRVRLASHamide) were synthesized by ProImmune (Oxford, UK) to a purity greater than 95%. The amino termini of both peptides were acetylated, and carboxy termini were blocked with an amide moiety. Peptides were reconstituted in sterile isotonic saline at a concentration of 6 μg/μL, aliquoted, and stored at −20°C. Renal allograft recipients
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were treated daily with ApoE(133–149) (6 mg/kg body weight) as published before15 or control peptide by intravenous injections for 10 days starting from the day of renal transplantation. Survival of the animals was monitored until day 100 after transplantation. Quantification of Nitrite/Nitrate
To estimate NO production in the graft, nitrite/nitrate concentration was determined by Griess reaction as previously described.27 Measurement of ApoE Plasma Levels
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in mononuclear leukocytes isolated from renal blood vessels of control animals, isogenic, or allogenic renal transplants. Fatal acute rejection of DA kidneys was accompanied by reduced mRNA expression of ApoE in cells collected from allografts in comparison to the respective isografts or remained at the level of isografts after BN to LEW transplantation. In line with microarray data published before,33 ApoE mRNA was elevated in intravascular leukocytes during reversible acute rejection of F344 kidneys (Figure 1A). Although mRNA expression analyses were performed on the entire intravascular population of mononuclear leukocytes, ApoE was predominantly produced
Apolipoprotein E plasma levels were analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (USCN Life Science, Wuhan, China) according to the manufacturer's instructions. Before measurement, samples were diluted 1:250 with PBS. The detection range was 1.56 to 100 ng/mL. Flow Cytometry
Flow cytometry was performed as described previously. 31,32 A FACS Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany) and the Cell Quest software 3.2.1 (Becton Dickinson) were used and 30000 events were measured for every sample. Microarrays
Microarray and statistical analyses of the data were performed as previously described.33 The data have been deposited in NCBI's Gene Expression Omnibus36 and are accessible through GEO Series accession number GSE58156 at: http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE58156. Statistics
Data were analyzed, where applicable, by nonparametric Kruskal-Wallis test followed by the Mann–Whitney rank sum test using SPSS software (SPSS software, Munich, Germany). Kaplan-Meier survival analysis with long rank (Mantel-Cox) test was used for comparison of graft survival between experimental groups (GraphPad software, La Jolla, CA). A P value of 0.05 or less was indicated. RESULTS Accumulation of Mononuclear Leukocytes in the Blood Vessels of the Graft During Acute Rejection
Leukocytes, which accumulated in the vasculature of rat renal allografts during acute rejection, were harvested by intensive perfusion of graft blood vessels on day 4 after DA to LEW and BN to LEW kidney transplantation or on day 9 after F344 to LEW renal transplantation. Perfusates were predominantly composed of monocytes and T cells as published before for LEW, DA, and F344 grafts transplanted to LEW recipients.28,32 Data on the cellular composition of intravascular graft leukocytes from day 4 BN to LEW allografts are summarized in (Table S1, SDC, http://links.lww.com/TP/B102 and expression of the cell surface markers of monocytes are presented in Table S2 (SDC, http://links.lww.com/TP/B102). Differential mRNA Expression of ApoE by Intravascular Leukocytes and Graft Tissue
Apolipoprotein E mRNA expression was analyzed by realtime reverse-transcriptase polymerase chain reaction (RT-PCR)
FIGURE 1. Expression of ApoE during fatal (DA, BN) and reversible acute rejection (F344). ApoE mRNA expression in intravascular mononuclear leukocytes isolated from control kidneys (LEW), renal Iso and Allo (A) and in the tissue of control kidneys, renal isografts and allografts (B) was assessed by real-time RT-PCR. Data are expressed as AU, which are normalized to a mean of one unit in untreated controls. ApoE protein level in plasma obtained from control animals and recipients of isografts and allografts (C) was determined by ELISA. Data were analyzed by nonparametric Kruskal-Wallis test followed by the Mann–Whitney rank sum test. Bars indicate median, whiskers percentiles 25 and 75. F344, Fischer 344; d, day; Iso, isografts; Allo, allografts; AU, arbitrary units.
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by monocytes. Monocytes isolated from control kidneys by perfusion and sorted by magnetic assisted cell sorting (MACS) show similar levels of ApoE mRNA expression like the entire population of mononuclear leukocytes (data not shown). Apolipoprotein E mRNA expression was also detected in graft tissues and did not differ between healthy control kidneys, renal isografts, and allografts on day 4 after DA to LEW transplantation. In contrast, during reversible acute rejection, ApoE was elevated in F344 kidney allografts (Figure 1B). Stable ApoE Plasma Levels During Fatal and Reversible Acute Rejection
The secretory protein ApoE is abundantly present in blood plasma.37 To evaluate a potential effect of acute rejection on the plasma level, ApoE was quantified by ELISA. Analysis of plasma from animals during fatal or at the peak of reversible acute rejection revealed no significant differences in ApoE levels between the groups analyzed (Figure 1C). Prolonged Survival of Renal Allografts Upon Treatment With ApoE(133–149)
To test the protective potential of ApoE on graft survival, totally nephrectomized recipients of BN kidneys were treated with ApoE(133–149) or control peptide. The survival of the animals was monitored until day 100 after transplantation. All control animals died due to acute rejection between days 8 and 16 after transplantation, whereas treatment with ApoE (133–149) prolonged allograft recipient survival (Figure 2). Stable mRNA Expression of Cytokines and inducible NO synthase Upon Treatment With ApoE(133–149)
To study the effect of the ApoE(133–149) treatment on cytokine expression in allografts and spleens, the mRNA of selected cytokines was quantified by real-time RT-PCR on day 4 after transplantation of BN kidneys to LEW rats. No significant difference between the ApoE(133–149)-treated group and controls was found regarding IFN-γ, prointerleukin (IL)-1β, IL-2, IL-12p35, IL-12p40, IL-17 F, IL-23p19, IL-10, TGF-β, and TNF-α mRNA expression in graft tissue (Figure 3A) and spleens (Figure S1, SDC, http://links.lww.com/TP/B102).
FIGURE 2. Survival of Brown Norway to Lewis renal allograft recipients treated with ApoE(133–149) or control peptide (n = 8, per group). ApoE(133–149) or control peptide were administered by daily intravenous injections for ten days starting from the day of renal transplantation. Survival of the animals was monitored daily until day 100 and presented as Kaplan-Meier curve. Long rank Mantel-Cox test was used for comparison of graft survival between experimental groups.
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Furthermore, the mRNA expression of iNOS revealed no differences upon ApoE(133–149) treatment when compared to control treatment (Figure 3B). In agreement with this finding, nitrite/nitrate concentrations measured by Griess reaction in graft tissues remained stable independent of the treatment (Figure 3C). Differential mRNA Expression of Chemokines Upon Treatment With ApoE(133–149)
To identify changes in chemokine production and to further characterize potential factors differently expressed upon ApoE(133–149) treatment, transcriptional profiling was performed on rat whole genome microarrays using RNA from day 4 allografts treated with ApoE(133–149) or control peptide. The data have been deposited in NCBI's Gene Expression Omnibus36 and are accessible through GEO Series accession number GSE58156. The differential mRNA expression of selected chemokines was further confirmed by real-time RT-PCR. We detected reduced expression of CXCL13 and CCL5 upon ApoE(133–149) treatment, whereas CCL20 was elevated compared to control peptide-treated animals (Figure 3D). No differences were seen in the expressions of CCL4, CCL21, CCL22, and CXCL3 (data not shown). Reduced Number of Graft Infiltrating / T Cell Receptor Expressing Cells Upon Treatment With ApoE(133–149)
To examine the effect of ApoE(133–149) treatment on accumulation of inflammatory cells in grafts during acute rejection, mononuclear leukocytes were identified by immunohistochemical staining (Figure 4A). No significant difference between ApoE(133–149)-treated group and controls was found regarding the number of monocytes/macrophages identified with Abs to a CD68-like antigen, M2 macrophages stained with Abs to CD163, B cells visualized with Abs to CD45RA, and natural killer (NK) cells defined by strong staining with Abs to CD161. However, ApoE(133–149) treatment diminished the number of cells identified with Abs to α/β T-cell receptor (TCR) (Figure 4B). Reduced Interaction Between Monocytes and Platelets Upon Treatment With ApoE(133–149)
The effect of ApoE(133–149) treatment on the interaction of platelets with monocytes and endothelial cells in the vasculature of the graft was investigated by immunohistochemical staining (Figure S2A, SDC, http://links.lww.com/TP/B102). Platelets were identified with Abs to CD61. Antibodies to a CD68-like antigen and rat endothelial cell antigen (RECA)1 were used to detect monocytes and endothelial cells, respectively. Interestingly, immunohistochemical analyses revealed a reduced percentage of platelets interacting with monocytes (Figure S2B, SDC, http://links.lww.com/TP/B102), whereas the percentage of free platelets (Figure S2C, SDC, http://links.lww.com/TP/B102) as well as of platelets interacting with endothelium (Figure S2D, SDC, http://links.lww.com/TP/B102) was not changed. Stable mRNA Expression of T Cell Transcription Factors Upon Treatment With ApoE(133–149)
To further characterize the effect of the treatment with ApoE(133–149) on the T cell compartment, mRNA expression levels of several transcription factors involved in T cell polarization were determined in grafts (Figure 5A) and spleens (Figure S3, SDC, http://links.lww.com/TP/B102) of transplanted
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FIGURE 3. Expression of proinflammatory and anti-inflammatory cytokines, iNOS and chemokines in kidney allografts after treatment with ApoE(133–149). The mRNA expression of IFN-γ, TNF-α, pro-IL-1β, IL-2, IL-12p35, IL-12p40, IL-17 F, IL-23-p19, IL-10, TGF-β (A), iNOS (B), and CCL5, CXCL13, CCL20 (D) was assessed on day 4 post-transplantation by real-time RT-PCR in kidney allografts treated with ApoE (133–149) or control peptide (n = 4, per group). Data are expressed as arbitrary units (AU), which are normalized to a mean of 1 unit in untreated controls. The NOx concentration in renal tissue (C) was determined with Griess reaction as an index of NO production. Data were analyzed by non-parametric Mann–Whitney rank sum test. Bars indicate median, whiskers percentiles 25 and 75. n = 4, per group. NOx, nitrite/nitrate.
animals. No significant difference between the ApoE(133–149)treated group and controls was found regarding Tbet, Gata3, Foxo1, Tcf1, and Foxp3 expressions. However, ApoE(133–149) treatment lowers the mRNA expression of Granzyme B (GrB) in grafts.
CD4pos and CD8pos monocytes/macrophages, sections were double-stained with Abs to a CD68-like antigen. Only single positive cells were quantified. The number of CD4pos CD68-likeneg cells was unchanged, whereas infiltrating CD8pos CD68-likeneg cells were diminished (Figure 5C).
Reduced Graft Infiltration by CD8pos T Cells Upon Treatment With ApoE(133–149)
Diminished Level of Active Caspase-3 Upon Treatment With ApoE(133–149)
Immunohistochemical staining with Abs to detect CD4 and CD8 α-chain was performed (Figure 5B). To exclude
To examine the effect of ApoE(133–149) treatment on caspase-3 activation, detection of active caspase-3 in renal
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FIGURE 4. Leukocytic infiltration of kidney allografts after treatment with ApoE(133–149). Cryostat sections of renal allografts treated with ApoE (133–149) or control peptide were stained on day 4 after transplantation with Abs to a CD68-like antigen to visualize monocytes and macrophages, to CD163 for detection of M2 macrophages. B cells were identified with Abs to CD45RA, NK cells with Abs to CD161 and T cells with Abs to α/β TCR. Bound Abs were detected in brown and sections were lightly counterstained with hemalum (A). Immunopositive leukocytes were counted in renal cortex (n = 4, each) (B). Data were analyzed by nonparametric Mann–Whitney rank sum test. Bars indicate median, whiskers percentiles 25 and 75.
allografts on day 4 after transplantation was performed by immunoblotting (Figure 5D) and quantified by densitometric analysis (Figure 5E). Consistent with the reduced number of CD8pos cells, reduced levels of active caspase-3 were detected upon ApoE(133–149) treatment. Unchanged T Cell Proliferation Upon Treatment With ApoE(133–149)
Recently, our group reported that CD8pos T lymphocytes proliferate in the lumina of the blood vessel of rat renal allografts during the onset of acute rejection.31 Knowing that ApoE suppresses mitogen-activated T-cell proliferation, we tested whether ApoE(133–149) affects intravascular T cell
proliferation. Triple staining was performed with Abs to proliferating cell nuclear antigen (PCNA), to the endothelial marker RECA-1, and to α/β TCR. The PCNApos T cells were quantified inside blood vessels of the grafts (Figure S4A, SDC, http://links.lww.com/TP/B102). Although a high percentage of T cells was PCNApos in the vasculature of grafts, no significant difference between the ApoE(133–149)-treated group and controls was detected (Figure S4B, SDC, http://links.lww.com/TP/B102). Deposition of C4d in Peritubullar Capillaries
Several groups reported that staining of allograft biopsies for C4d could be a reliable marker for the diagnosis of
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FIGURE 5. Expression of transcription factors involved in T cell polarization and T cell function after treatment with ApoE(133–149). The mRNA expression of Tbet, Gata3, Foxo1, Tcf1, Foxp3, and GrB was assessed by real-time RT-PCR in kidney allografts treated with ApoE(133–149) or control peptide on day 4 after transplantation (n = 4, per group) (A). Data are expressed as arbitrary units (AU), which are normalized to a mean of one unit in untreated controls. Renal allografts treated with ApoE(133–149) or control peptide were analyzed on day 4 after transplantation by immunohistochemical staining with Abs to CD4 or CD8 α-chain in blue to detect CD4pos and CD8pos T cells respectively. To exclude CD4pos or CD8pos monocytes/macrophages as a first step staining with Abs to a CD68-like antigen was performed in brown (B). CD4pos CD68-likeneg and CD8pos CD68-likeneg cells were quantified in the renal cortex (n = 4, per group) (C). The level of active caspase-3 upon ApoE(133–149) or control peptide treatment was analyzed in renal tissue by immunoblotting on day 4 after transplantation (D). Detection of GAPDH was performed to ensure equal protein loading. Intensity of the signal was quantified by densitometric analysis (E). Data were analyzed by nonparametric Mann–Whitney rank sum test. Bars indicate median, whiskers percentiles 25 and 75.
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transplant outcome.38 We detected deposition of C4d in glomelruli as well as on endothelial cells of peritubular capillaries of allografts on day 4 after transplantation (Figure S5A, SDC, http://links.lww.com/TP/B102). Treatment with ApoE(133–149) had no effect on the intensity of the staining (Figure S5B, C, SDC, http://links.lww.com/TP/B102). DISCUSSION Apolipoprotein E as well as ApoE(133–149) have been already shown to exert various immunomodulatory effects.3,5,8,9,15-24,39,40 In the present study, we are the first to demonstrate a protective role of ApoE(133–149) in experimental acute kidney graft rejection. This effect was associated with a reduced influx of α/β TCR-expressing cells and CD8pos cells as well as diminished expression of GrB and decreased levels of active caspase-3. The changes are probably due to a reduction in number of cytotoxic T cells but we cannot formally exclude an additional reduction of the minor population of CD161neg natural killer T (NKT) cells. In our study, intravascular graft leukocytes and renal tissue obtained from animals undergoing reversible acute rejection expressed increased levels of ApoE mRNA compared to isograft recipients, whereas during fatal rejection, ApoE expression was reduced or remained unchanged. The observed changes in ApoE expression cannot be explained by changes in the cellular composition of blood leukocytes and renal blood tissue because during both fatal and reversible acute rejection, the proportion of mononuclear phagocytes increased in a similar way.26-29 These findings together with previously published data describing immunoregulatory properties of ApoE3,5,8,17,39 supported our assumption that elevated levels of ApoE have a protective effect on graft survival. In contrast, no changes in ApoE protein levels were seen in plasma. However, we expect that strong accumulation of monocytes capable to secrete ApoE would result in a local increase of ApoE concentrations inside graft blood vessels.2,28,29 In line with this assumption, transgenic mice expressing ApoE only in monocytes/macrophages were protected from atherosclerosis, even though plasma levels of ApoE remained unchanged and very low. In contrast, transgenic mice with normal ApoE plasma levels but ApoE-null macrophages were more susceptible to this disease.41 The protective potential of ApoE(133–149) treatment on graft survival was tested after renal transplantation in the fully allogenic BN to LEW rat strain combination. This experimental model of fatal acute rejection is frequently used to test new therapeutic approaches.25,32,42 In this model, similar to the DA to LEW or F344 to LEW rat kidney transplantation, the effector phase of acute rejection is accompanied by strong accumulation of mononuclear cells, predominantly monocytes, inside allograft blood vessels. Results of the flow cytometric analyses of the expression of several surface antigens suggested an intermediate state of monocyte activation. Similar to self-limiting acute rejection in F344 to LEW rat strain combination,33 the proportion of major histocompatibility complex IIpos and CD161pos cells increased, whereas only minor changes in the expression of CD4, CD11a, CD18, CD43, and CD71 were detected. Animals treated with ApoE(133–149) showed a prolonged allograft survival, which was associated with a reduced number of infiltrating CD8pos T cells.
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The central role of T lymphocytes in mediating allograft rejection is well established.43-46 CD8pos T cells are abundantly present in acutely rejecting allografts and are thought to be the major effector cells during graft rejection.47,48 Deficiency in CD8pos T cells was described to be protective during acute rejection of heart allografts in a minor histocompatibility antigen-mismatched model.45 In line with this observation, transfer of alloreactive CD8pos T cells primed in vitro caused islet allograft rejection.49 These findings support the importance of CD8pos T cells in acute organ transplant rejection.48,50 Moreover, there is evidence that CD8pos T cells are more resistant to conventional immunosuppression and tolerance induction protocols.51 Consequently, strategies targeting CD8pos T lymphocytes are of interest. In agreement with this finding, ApoE(133–149) treatment also reduced mRNA expression of GrB. Granzymes and perforin are the main effector molecules of cytotoxic T lymphocytes, which include CD8pos, some CD4pos T lymphocytes, NKT, and NK cells.50 It is of importance to note that increased expression of GrB is predictive of the development of acute rejection episodes and has been proposed to serve as a marker for acute rejection.52-55 As the estimated number of CD4pos T cells or NK cells was not affected by peptide treatment, we postulated that reduced GrB expression mirrors the reduced infiltrate of CD8pos T cells. Furthermore, also, caspase-3 activation, an established marker for apoptosis, which reflects activity of cytotoxic cells, was reduced upon ApoE(133–149) treatment. This observation can be correlated with a reduced number of CD8pos T cells. Application of ApoE(133–149), however, did not impair intravascular T cell proliferation, which was described for acute rejection of renal allografts before.31 It is likely that ApoE(133–149) affected the recruitment of CD8pos T cells into the graft. This assumption is in line with the results of chemokine expression. Reduced levels of CCL5 a well-known chemoattractant for T cells, at least partially, explains our observations. In contrast, the observed reduction in CXCL13 mRNA, a B lymphocyte chemoattractant, had no effect on accumulation of B cells in the grafts. Interestingly, although ApoE(133–149) did not affect accumulation of monocytes/macrophages in graft blood vessels and tissue, it reduced the frequency of platelet-monocyte interactions. This might be of importance for graft survival because interactions with platelets induce a proinflammatory phenotype in circulating monocytes.56 It was already reported that ApoE and ApoE(133–149) treatment suppress the release of proinflammatory cytokines in vitro and in vivo.3,6-8 In agreement with these observations, ApoE-deficient mice mount increased systemic inflammatory responses upon LPS injection.16,39 In contrast to these findings, in our experimental setting, ApoE(133–149) did neither affect local nor systemic expression of cytokines involved in Th1-like (IFN-γ, IL-1β, IL-2, IL-12, TNF-α), Th17-like (IL-17F, IL-23) or regulatory (IL-10, TGF-β) immune reactions. In the same line, mRNA expression of iNOS as well as graft NO production and mRNA levels of transcription factors involved in T cell polarization remained stable independent of the treatment. Our experimental study has some limitations. We do not know if our finding would also apply to human transplant recipients. Furthermore, grafts were exclusively investigated during the peak of acute rejection and we ignore mechanisms
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occurring during the onset of the immune reactions. In addition, cytokines and chemokines were analyzed on the mRNA level but protein data are missing. In conclusion, our results support the hypothesis that endogenous ApoE plays a protective role during acute rejection of experimental allografts. Furthermore, we demonstrate that treatment with ApoE(133–149) promotes recipient survival after allogenic kidney transplantation. ApoE(133–149) seems to reduce infiltrating cytotoxic CD8pos T cells but further studies are needed to fully understand its mechanism of action. ACKNOWLEDGMENTS The authors thank Gabriele Fuchs-Moll, Kathrin Petri, and Sigrid Wilker for their expert technical assistance, and Ulrike Berges for help with the art work. REFERENCES
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45. Youssef AR, Otley C, Mathieson PW, Smith RM. Role of CD4+ and CD8+ T cells in murine skin and heart allograft rejection across different antigenic desparities. Transpl Immunol. 2004;13:297. 46. Boisgerault F, Liu Y, Anosova N, et al. Role of CD4+ and CD8+ T cells in allorecognition: lessons from corneal transplantation. J Immunol. 2001; 167:1891–1899. 47. Trentin L, Zambello R, Faggian G, et al. Phenotypic and functional characterization of cytotoxic cells derived from endomyocardial biopsies in human cardiac allografts. Cell Immunol. 1992;141:332–341. 48. Bueno V, Pestana JO. The role of CD8+ T cells during allograft rejection. Braz J Med Biol Res. 2002;35:1247–1258. 49. Diamond AS, Gill RG. An essential contribution by IFN-gamma to CD8+ T cell-mediated rejection of pancreatic islet allografts. J Immunol. 2000; 165:247–255. 50. Choy JC. Granzymes and perforin in solid organ transplant rejection. Cell Death Differ. 2010;17:567–576.
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51. Bierer BE, Hollander G, Fruman D, Burakoff SJ. Cyclosporin A and FK506: molecular mechanisms of immunosuppression and probes for transplantation biology. Curr Opin Immunol. 1993;5:763–773. 52. Kummer JA, Wever PC, Kamp AM, et al. Expression of granzyme A and B proteins by cytotoxic lymphocytes involved in acute renal allograft rejection. Kidney Int. 1995;47:70–77. 53. Lipman ML, Stevens AC, Strom TB. Heightened intragraft CTL gene expression in acutely rejecting renal allografts. J Immunol. 1994;152:5120–5127. 54. Clement MV, Haddad P, Soulie A, et al. Perforin and granzyme B as markers for acute rejection in heart transplantation. Int Immunol. 1991;3:1175–1181. 55. Clement MV, Soulie A, Legros-Maida S, et al. Perforin and granzyme B: predictive markers for acute GVHD or cardiac rejection after bone marrow or heart transplantation. Nouv Rev Fr Hematol. 1991;33:465–470. 56. Passacquale G, Vamadevan P, Pereira L, et al. Monocyte-platelet interaction induces a pro-inflammatory phenotype in circulating monocytes. PLoS One. 2011;6:e25595.
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