Identification of Non-HLA Antigens Targeted by Alloreactive Antibodies in Patients Undergoing Chronic Hemodialysis Senada Bilalic,† Michael Veitinger,† Karl-Heinz Ahrer,† Viktoria Gruber,† Maria Zellner,† Christine Brostjan,† Gregor Bartel,‡ Daniel Cejka,‡ Christian Reichel,§ Veronika Jordan,§ Christopher Burghuber,† Ferdinand Mu ¨ hlbacher,† Georg A. Bo ¨ hmig,‡ and Rudolf Oehler*,† Department of Surgery, Medical University of Vienna, A-1090 Vienna, Austria, Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria, and Department Life Sciences, Proteomics, Austrian Research Centers GmbH - ARC, A-2444 Seibersdorf, Austria Received October 16, 2009
The only treatment of end-stage renal disease patients undergoing chronic dialysis is kidney transplantation. However, about half of graft recipients encounter organ loss within ten years after renal transplantation. There is emerging evidence that the presence of alloreactive antibodies against non-HLA antigens in the serum of the recipient prior transplantation is associated with higher incidence of chronic rejection. However, the molecular identity of these antigens is largely unknown. To determine the most common non-HLA antigens, we tested lymphocytic extracts from 20 healthy volunteers with sera of 28 patients on the transplantation waiting list by Western blotting. There was a group of five proteins that was recognized by most sera. Using patient’s own lymphocytes revealed that autoimmunity plays a minor role in this recognition. Two-dimensional Western blotting experiments followed by mass spectrometry identified the antigens as tubulin beta chain, vimentin, lamin-B1, and Rho GDP-dissociation inhibitor 2. A detailed analysis of vimentin expression revealed that the antigenic 60 kDa isoform is underrepresented in patient’s lymphocytes in comparison to those of healthy volunteers. The study revealed that preformed alloreactive antibodies are directed against a small number of specific protein isoforms. Our findings could provide a basis for future improvement of donor-recipient matching. Keywords: Kidney transplantation • humoral organ rejection • non-HLA alloreactive antibodies • antigen identification
Introduction
Thus, patients with a high PRA value have a higher risk that the antigen is also recognized in the graft leading to rejection.
Approximately 25% of end-stage renal disease patients on the kidney transplant waiting list have alloreactive antibodies in their serum.1 Recent large-scale trials show that these patients have a much higher long-term organ failure rate after renal transplantation (RTX) as those without antibodies suggesting that such antibodies contribute considerably to chronic rejection.2 However, which fraction of chronic rejection is antibody-mediated and which cell-mediated remains to be determined. It is assumed that these antibodies are produced in response to immunologic stimuli such as blood transfusion, pregnancy, or previously rejected allografts. Patients with high levels of alloreactive antibodies are therefore qualified as “sensitized”. Transplant candidates on the organ waiting-list are routinely screened for panel reactive antibodies (PRA). Such antibodies detect their antigens in a panel of healthy volunteers.
It is well established that HLA class I and class II are the predominant antigens in transplantation immunology. However, HLA molecules are not the only antigens recognized by PRA. Van der Woude postulated in 1995 a role of alloreactive antibodies in recipient serum which are directed against nonHLA proteins in donor tissue (anti non-HLA antibodies) in late failure of well-HLA-matched renal allografts.3 Terasaki reported that 38% of graft failures are due to anti non-HLA, 18% to anti HLA antibodies, and 43% to nonimmunologic factors.4 Zwirner was first to report on antibodies in kidney recipients reacting with the polymorphic non-HLA MICA antigen system.5 Perrey and co-workers revealed an association of anti non-HLA IgG antibodies directed against endothelial cells with renal transplant failure.6 A recent large scale study showed that anti nonHLA antibodies caused chronic graft loss in HLA-identical siblings.7 Formation of anti non-HLA antibodies is associated with transplant arteriosclerosis and chronic rejection after cardiac or kidney transplantation.8 All this data strongly suggest a significant role of anti non-HLA PRA in chronic rejection of renal allografts. However, the molecular identity of non-HLA antigens of PRA is largely unknown. Identification of non-HLA antigens can be helpful for improved estimation of risk of
* To whom correspondence should be addressed. Rudolf Oehler, Department of Surgery, Medical University of Vienna, AKH (8G9.05), Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail:
[email protected]. Tel.: +43-1-40400-6979. Fax: +43-1-40400-6782. † Department of Surgery, Medical University of Vienna. ‡ Department of Medicine III, Medical University of Vienna. § Austrian Research Centers GmbH - ARC. 10.1021/pr900930d
2010 American Chemical Society
Journal of Proteome Research 2010, 9, 1041–1049 1041 Published on Web 01/14/2010
research articles rejection prior to transplantation and for selection of compatible donors. The present study investigated non-HLA antigens of PRA in a high number of hemodialysis patients on the renal transplantation waiting list. First, we characterized the most commonly targeted non-HLA antigens according to their molecular weight by Western blot experiments and then we identified these antigens by 2DE and Mass spectrometry. Finally, we could show that the antibodies target specific protein isoforms which are underrepresented in patients own tissue.
Materials and Methods Serum Samples. Serum samples were taken from 28 hemodialysis patients on the renal transplantation waiting list at the General Hospital of Vienna (19 male, 9 female, average age 56 ( 15 years) and from 10 adult healthy volunteers (10 male, average age 35 ( 15 years). Sera were stored at -80 °C in aliquots. The trial was carried out according to the Helsinki Declaration and was approved by the local ethics committee (471/2006). An informed consent was obtained from all patients at the time of enrollment. PRA Testing. The CDC-PRA test (complement-dependent cytotoxicity panel-reactive antibody test) was performed according to the protocol originally described by Terasaki and McClelland.9 The Flow-PRA test (One Lambda Inc., Canoga Park, CA) was performed according to the protocol originally described by Pei et al.10 In brief, sera were incubated for 30 min with a mixture of FlowPRA HLA class I and FlowPRA HLA class II beads. After 30 min incubation, beads were washed and (FITC)-conjugated anti human IgG antibody (One Lambda Inc., Canoga Park, CA) was added at saturating concentrations for another 30 min. Fluorescence intensity was measured using a FACS-Calibur flow cytometer (Becton Dickinson, San Jose, CA). The major bead population was gated on the forward-versus side-scatter dot plot. Then, two gates were set on the FL2 histogram in order to separately analyze HLA class I (FL2 negative) and class II (FL2 high fluorescent) coated populations. Antibody binding to HLA class I or class II beads was analyzed on FL1 (FITC) histograms. Markers were set according to an appropriate nonbinding negative control serum. Results are expressed as percentage of positive events (%PRA). The specificity of HLA antibodies was calculated from staining pattern, using the software CellQuest (Version 3) (One Lambda Inc., Canoga Park, CA). Cell Preparation and Protein Extraction. A panel of lymphocytes isolated from 20 healthy volunteers was used as source of antigens. For lymphocyte preparation peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Paque gradient centrifugation (GE Healthcare, Uppsala, Sweden) from EDTA treated blood. Obtained PBMCs were washed with phosphate-buffered saline without calcium and magnesium (PBS) supplemented with 0.5% BSA (bovine serum albumin). The lymphocytes were enriched by depletion of monocytoid cells through adherence to plastic. Therefore PBMCs were allowed to rest for 1 h in RPMI-1640 cell culture media (1-2 × 106 cells/mL) supplemented with 10% fetal calf serum at standard culture conditions (37 °C, 5% CO2) in a plastic tissue culture flask. Then the supernatant was collected gently avoiding major turbulences or scraping of attached cells. The so harvested nonadherent cells were washed twice in PBS and stored as cell pellets at -80 °C. The purity of lymphocytes was determined by flow cytometry. The contamination with nonlymphocytic cells was always below 20%. Primary endot1042
Journal of Proteome Research • Vol. 9, No. 2, 2010
Bilalic et al. helial cells (ECs) were isolated from human foreskin samples by Dispase digest, purified via anti CD31 Ab-coupled Dynabeads (Invitrogen Life Technologies, Carlsbad, CA) and cultured in fibronectin-containing Microvascular Endothelial Cell Growth Medium EGM2-MV (Cambrex, East Rutherford, NJ) without vascular endothelial growth factor supplementation. Then ECs were washed twice in PBS and stored in aliquots at -80 °C. For protein extraction, cell pellets were lysed in 2 µL/106 cells urea buffer (7 M Urea, 2 M Thiourea, 4% Chaps, 1% Amberlite). After homogenization, the cell lysate was collected and clarified from unsolubilised particles by centrifugation (20 000g for 10 min). The protein content of the samples was quantified using a CBB protein assay kit with BSA (dissolved in urea buffer) as standard protein (Pierce Biotechnology, Rockford, IL). The protein extracts were stored at -80 °C. Gel Electrophoresis. Protein extracts were separated by onedimensional as well as two-dimensional gel electrophoresis (1DE and 2DE, respectively). For 1DE, 20 µg per lane of human lymphocytic protein extract were separated on 9% SDSpolyacrylamide gels according to ref 11. In the 2DE, proteins were separated according to isoelectric point (pI) and molecular weight (MW) of the individual protein. Therefore, 75 µg of proteins were brought up with rehydration solution (7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 1% Servalyt pH 4-7; Serva, Heidelberg, Germany) to a final volume of 250 µL. This mixture was loaded on IPG DryStrips (pH 4-7, 13 cm, GE Healthcare, Uppsala, Sweden) and passively rehydrated for 16 h at room temperature. Isoelectric focusing was carried out on an IPGphor device (GE Healthcare, Uppsala, Sweden) until 20 kVh were reached. Prior to the second dimension IPG strips were equilibrated for 15 min in equilibration solution (50 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS, pH 8.8) containing 1% DTT followed by equilibration for 15 min in equilibration solution containing 2.5% iodoacetamide (Sigma, St. Louis, MO). Thereafter SDS-PAGE was performed on a 10% SDS-polyacrylamide gel. These gels were used for protein transfer onto nitrocellulose membranes or for silver staining. Immunoblotting. After electrophoretic separation proteins were transferred onto a nitrocellulose membrane (Pierce, USA) by wet electro blotting. Transferred proteins were visualized using a ruthenium-(II)-tris (bathophenanthroline disulfonate) (RuBPS) based fluorescence dye using the protocol of,12 modified according to.13 Then free protein binding sites were blocked with dry milk powder (2% in PBS) for at least 1 h. For immunodetection membranes were incubated either with human serum (diluted 1:50) or with mouse anti HLA I (HLA -A, -B,-C) antibodies (MBL, Woburn USA, diluted 1:5000) or goat anti human vimentin polyclonal antibody (Chemicon, Massachusetts USA, diluted 1:2500) in PBS for at least 1 h. Binding of antibodies was detected by fluorescence Western blotting according to.13 For detection of bound antibodies from human serum a Cy5 conjugated goat anti human IgG antibody (Jackson, Immuno-Research laboratories West Baltimore Pike West Grove, PA, diluted 1:5000 or 1:2500) was used. Bound specific anti HLA I antibodies were detected with a horseradish peroxidase conjugated anti mouse IgG antibody (Pierce, Rockford, IL) in presence of ECL Plus Western Blot Detection System (GE Healthcare, Uppsala, Sweden). Bound anti vimentin antibodies were detected with a Cy 5 conjugated anti goat antibody (Jackson, Immuno Research laboratories West Baltimore Pike West Grove, PA, diluted 1:400). Nitrocellulose membranes were scanned using a Typhoon TRIO scanner (GE Healthcare, Uppsala, Sweden) as previously described by.13 SDS
research articles
Non-HLA Antibodies Table 1. Patients List flow PRA pat. no.
age
sex
HLA-I [%]
HLA-II [%]
pre RTX
blood transfusion
CDC PRA IgG [%]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
65 30 68 43 57 48 70 55 50 57 51 51 49 61 74 76 68 56 60 58 58 46 37 65 54 52 61 52
male male male female female female male male female male male female female male female male male female male male male female male male male male male male
71 33 74 34 8 42 29 69 11 75 80 87 61 18 16 10 1 1 2 7 7 6 1 1 1 0 0 0
75 32 69 31 58 2 40 1 61 86 97 26 4 1 3 1 2 1 1 3 1 2 1 1 1 1 1 0
2 1 1 4 1 3 1 1 0 1 1 0 0 2 0 0 1 0 0 0 0 0 1 1 0 0 1 0
+ + + + + + + + + + + + + + + + + + + + -
84 83 78 70 38 34 34 22 22 12 10 0 0 0 0 0 38 26 4 0 0 0 0 0 0 0 0 0
gels for mass spectrometry (MS) analysis were stained using the MS-compatible silver staining protocol according to.14 The protein of interest were defined in a silver stained gel using Delta 2D-Software (Decodon, Greifswald, Germany). Protein Identification. (I) Tryptic digests: Spots from silverstained 2D-PAGE gels were excised and digested with trypsin according to.15 Briefly, gel plugs were washed in LC-MS grade water for 3 × 15 min, cut into cubes (ca. 1 mm3), and destained (15 min) with a 1:1 (v/v) mixture of 100 mM sodium thiosulfate and 30 mM potassium hexacyanoferrate (III) according to ref 16. Subsequently, the gel pieces were washed and equilibrated in an aqueous solution of 200 mM ammonium hydrogencarbonate (2 × 15 min) as well as reduced (5 mM DTT in 25 mM ammonium hydrogencarbonate; 56 °C, 30 min) and alkylated in the dark (55 mM iodoacetamide in 25 mM ammonium hydrogencarbonate; room temperature, 30 min). After a washing step (25 mM ammonium hydrogencarbonate; 2 × 15 min) the pieces were dehydrated in acetonitrile (3 × 10 min) and then dried in a SpeedVac (40 °C, ca. 15 - 30 min). For in-gel tryptic digestion the cubes were rehydrated with a solution of 10 mM ammonium hydrogencarbonate in 10% (v/v) acetonitrile containing 13 ng/µL trypsin. After overnight incubation at 37 °C the supernatant was collected and the cubes sequentially extracted with 50 mM ammonium hydrogencarbonate, a mixture of acetonitrile and 5% TFA (1:1, v/v), and acetonitrile. The combined extracts were concentrated in a SpeedVac (40 °C) and then redissolved under sonication in 10 µL of 0.1% TFA. Subsequently, the peptide solution was desalted using C18 pipet tips (PerfectPure, Eppendorf; Hamburg, Germany) according to the manufacturer’s instructions and directly eluted with a solution of R-cyano-4-hydroxycinnamic acid in 50% acetonitrile (3 mg/mL) containing 0.1% TFA. (II) Mass spectrometry: Between 0.5 and 1.0 µL of the C18 eluted fraction
were spotted on a stainless steel MALDI-target plate. Peptide mass fingerprints (PMF) and MS/MS-spectra were acquired with a 4800 MALDI TOF/TOF Analyzer (355 nm Nd:YAG laser, 200 Hz). The instrument was controlled by the 4000 Series Explorer (version 3.5) software and operated in positive ion reflector mode with delayed extraction. Typically, 1000 subspectra were recorded for each MS-spectrum and between 1000 and 4000 subspectra for each MS/MS-spectrum. PMF-spectra were internally calibrated using masses of trypsin autolysis peptides (m/z 842.51 and 2211.104). In cases where these masses could not be observed an external default calibration was used instead (generated on a daily basis and with five peptide masses of the 4700 Cal Mix standard, Applied Biosystems). All MS/MS spectra were externally calibrated using five fragment masses of Glu1-Fibrinopeptide B. The minimum S/N for peak detection was set to 10 and only monoisotopic peaks were used (4000 Series Explorer, version 3.5). Usually, a mass resolution between 12 000 and 20 000 (full width half-maximum, fwhm) in the mass range of ca. 0.9 to 2.5 kDa and an average mass accuracy of 10% FlowPRA HLA class I and/or class II reactivity). Two thirds of them showed g10% CDC-PRA reactivity. Lower CDCPRA reactivity was regarded as negligible. Among the 13 FlowPRA-negative patients, two had moderate CDC reactivity. The presence of anti HLA sensitization was tightly associated with a clinical history of presensitization, i.e. one or more prior kidney transplantations, blood transfusions and/or documented pregnancies (12/15 FlowPRA-positive versus 4/13 FlowPRA negative patients). Of notice, one of the two CDCPRA-positive but FlowPRA-negative patients had a history of prior transplantation (Table 1). These data suggest that our patient collective includes subjects with high and subjects with low levels of allo-reactive antibodies. Molecular Weight of Antigens. In a first set of experiments, sera were screened for panel reactivities applying fluorescencebased one-dimensional Western blotting analyses (1D-WB). To establish patterns of non-HLA PRA reactivities lymphocytes isolated from 20 different healthy volunteers (10 women and 1044
Journal of Proteome Research • Vol. 9, No. 2, 2010
10 men) were used as a source of allogeneic antigens. Proteins were extracted, separated according to their molecular weight and transferred to a NC membrane. An example of a RuBPSstained membrane sheet representing the panel of 20 different lymphocyte donors is shown in Figure 1A. Proteins with a molecular weight above 100 kDa or below 25 kDa were not considered in this study because of insufficient separation in this range. For individual testing of all control and patient sera, a total of 38 membrane sheets were made. For each serum, patterns of antibody binding were visualized using fluorescently labeled anti human IgG secondary antibodies. Figure 1B illustrates the results of an experiment evaluating the binding properties of a sample obtained from patient number 13. This serum was established to contain anti HLA class I IgG as shown by Flow-PRA. Accordingly, the 1D-WB showed a distinct band at 42 kDa which corresponded well to that of HLA class I alpha chain protein Figure 1C. In addition, within the lymphocyte protein panel, numerous other bands were detected at different molecular weights on the 1D-WB ranging from 28 to 100 kDa. This indicated that the serum of patient number 13 contained antibodies against HLA class I as well as against a variety of non-HLA class I antigens. Comparing the 1D-WB analyses of the different patient sera revealed that every serum recognized its own specific pattern of non-HLA antigens. Some antigens were only detected by single sera. Others were detected more frequently (a detailed list of the results can be found in the Supporting Information).
research articles
Non-HLA Antibodies Table 2. Frequency of Allo-Reactive Antibodies As Revealed in the 1D Western Blot Panel Test reactivity in patients with reactivity to
flow PRA >10%
flow PRA 23 indicate identity or extensive homology (p < 0.05). Protein scores are derived from ions scores as a nonprobabilistic basis for ranking protein hits.
example is shown in Figure 3B. The 2D-WBs showed a number of different spots which were derived from antibodies binding to antigenic proteins on the membrane. Comparison of the different membranes allowed the identification of the most frequently detected antigens. The antigens at a molecular weight of 28, 49, 53, 60, and 64 kDa could be clearly assigned to single spots on the 2D-WB (ID9444, ID9705, ID9701, ID9604, and ID8913 respectively). In the next step the corresponding protein spots were defined on a silver stained gel by direct comparison of its spot pattern with that of the RuBPS-stained NC-membrane and the Cy5 antibody stained 2D-WB using gelwarping software Delta 2D. The defined spots of interest were cut out from the silver stained gel and the identity of the protein was determined by mass spectrometry. The results are shown in Table 3. The antigens have been identified as tubulin beta chain, vimentin, lamin-B1, and Rho GDP-dissociation inhibitor 2. Two spots were assigned to vimentin (spot IDs 9604 and 9705). This suggested that at least one of them represented an isoform of the primary transcript. Such an isoform may result from post translational modifications. Because the MS experiments could not provide complete sequence coverage for any of the identifications, the presence of such modifications cannot be excluded also for other spots. The main targeted tissue of donor specific antibodies in vivo is the endothelium of the graft rather than donor’s lymphocytes. Therefore we analyzed whether the most commonly detected antigens found in lymphocytes are also recognized in endothelial cells (ECs). Proteins from human microvascular ECs were separated by 2DE and transferred to a nitrocellulose membrane. Incubation of this membrane with the serum of patient no. 9 resulted in a pattern comparable to the one observed for lymphocytes (Figure 3C). All proteins which were most commonly detected in lymphocytes were also detected in ECs. Allospecificity of Anti Vimentin Antibodies. Vimentin is an intermediate filament protein which is normally expressed in all lymphocytes. However, the Western blots shown in Figure 2 revealed that for example the serum of patient no.6 detects the 60 kDa vimentin isoform strongly in the allogeneic setting but not in its own lymphocytes. It cannot be determined whether this is due to the absence of the antigen in the patients’ own lymphocytes or due to modifications of the antigen. Therefore we investigated the vimentin expression pattern in this patient and in the corresponding healthy volunteer in more detail by 2D-WB. Anti vimentin antibodies revealed that this protein is present in different isoforms in the two lymphocytic extracts Figure 4A and B. Interestingly, the 60 kDa isoform of vimentin (ID 9604) was detected only in the lymphocytes of the healthy volunteer (Figure 4A) but not in those of the patient 1046
Journal of Proteome Research • Vol. 9, No. 2, 2010
Figure 4. Analysis of allo- versus autospecificity of anti vimentin antibodies. Lymphocytic proteins from the blood of a HV (A, C) and of patient no.6 (B, D) were separated by 2D gel electrophoresis (pI range 4-7), transferred to a nitrocellulose membrane and immunostained with antibodies against vimentin. A and B show the proteins targeted by vimentin-specific antibodies. For a better comparison between healthy volunteer (A) and patient (B) the corresponding regions were encircled. Only the vimentin relevant region on the membrane is shown. C and D show the corresponding enlargement of the silver stained 2D gels of the same samples. The marked spots are vimentin isoforms recognized by anti vimentin antibody.
(Figure 4B). The absence of this antigenic spot in the patient’s lymphocytes was confirmed by the total protein stain of a corresponding 2D gel Figure 4D. In contrast, the vimentin isoform corresponding to spot ID 9705 (49 kDa) was found in both lymphocytic extracts (see Figure 4A-D). These data indicate that the expression pattern of the different vimentin isoforms is not the same between hemodialysis patients and the healthy volunteers. To investigate whether this is a general effect we analyzed the vimentin expression pattern in 7 patients and five healthy volunteers by 1D-WB. Figure 5A shows that vimentin is present in various isoforms in healthy volunteers in a molecular weight range between 50-60 kDa. Only healthy volunteer no.1 showed an additional band below 42 kDa. The intensity of the single WB bands varied between the different healthy volunteers indicating different expression levels. The
Non-HLA Antibodies
Figure 5. Analysis of vimentin pattern in lymphocytic proteins of hemodialysis patients and of healthy volunteers. Human lymphocytic proteins from blood of 7 hemodialysis patients (patient no. 18, 17, 7, 4, 9, 11, 6) and of 5 healthy volunteers (HV) were separated by 1D gelelectrophoresis, transferred to nitrocellulose membrane, RuBPS stained (B) and immunostained with antibodies against vimentin (A). The marked bands with molecular weight are vimentin bands recognized by anti vimentin antibody.
expression pattern in the patients was different. All patients showed an expression of vimentin isoforms with a molecular weight between 37 and 42 kDa. The vimentin isoforms in the molecular weight range of 50-60 kDa were strongly expressed in only two patients (no. 17 and 7). In the lymphocytic extracts from the other patients these vimentin isoforms were underrepresented. These data show that vimentin is expressed in both, hemodialysis patients and healthy volunteers, but in different isoforms. We found in lymphocytes from healthy volunteers several vimentin isoforms which were absent in patient’s lymphocytes. A re-evaluation of the results of the 1DWestern blot membranes (shown in Figure 1 and summarized in the Supporting Information) revealed that the allo-reactive anti vimentin antibodies present in the sera of these patients recognized only two of the differentially expressed vimentin isoforms (see Supporting Information). Twelve out of 28 patients recognized the 49 kDa as well as the 60 kDa vimentin isoform in at least every second allogeneic lymphocytic extract. Only a few sera showed exclusive binding to a single isoform (two sera to the 49 kDa and three sera to the 60 kDa isoform). No antibody binding to other vimentin isoforms were found.
Discussion The present study characterizes the antigens of frequent nonHLA allo-reactive antibodies in a group of 28 hemodialysis patients on the kidney transplantation waiting list. We could show that in spite of considerable interindividual variation in the presence of such antibodies there is a group of five nonHLA proteins which is recognized by sera of most patients. These non-HLA antigenic proteins are located intracellularly and most of them are part of the cytoskeleton. Interindividual variations in the expression of these proteins seem to contribute to the antibody reactivity. The 1D Western blot analysis showed that patients’ sera contained antibodies against a variety of non-HLA antigens prior to transplantation. Some non-HLA antigens were only recognized by sera of single patients and some antigens were detected more frequently. We determined antibody binding to lymphocytic extracts of 20 healthy volunteers. To our knowledge, this is the first study which investigated the antibody frequency on a molecular level in such detail. We focused our
research articles attention on common non-HLA antigens and selected therefore the five most frequently targeted antigens (molecular weight of 28, 49, 53, 60, and 64 kDa). In contrast, all sera from healthy volunteers showed no antibody binding to any of the 20 lymphocytic extracts. This suggests that allo-reactive antibodies in hemodialysis patients are disease-associated. It is well established that the probability of developing allo-reactive antibodies increases with the frequency of exposure to foreign antigens from other subjects. A previous study showed a relationship between allo-reactive antibodies and the number of preceding transplantations.17 Allo-reactive anti HLA antibodies were detected in only 17% of patients waiting for a first transplant, but in 64% waiting for a second, in 84% waiting for a third, and in 92% waiting for a fourth graft. Fifteen of our 28 patients had previous transplantations. Correspondingly, about 75% of them were positive in the Flow PRA test. These results suggest that most of our patients on the waiting list produce serum alloreactive antibodies prior to transplantation due to previously rejected grafts. However, some Flow PRA positive patients had no previous transplantations. Allo-reactive antibodies can also be formed in response to blood transfusions. Lymphocytotoxic B-cell reactivity has been detected in a previous study in 20% of waiting list patients who were treated with one blood transfusion, 40% after 10 transfusions, and 70% after 20 transfusions.1 Thus, the PRA detected in our patients could be caused also at least in part by blood transfusions. Two of the 3 Flow PRA positive patients without previous transplantations had blood transfusions. The statistical analysis of the frequency of antibodies against the five most commonly recognized antigens revealed that only two (the 28 and 60 kDa antigen) correlated with the HLA-specific Flow-PRA value. The other antibodies were independent of the anti HLA-antibody status. This indicates that the Flow-PRA test is not suited to evaluate the presence of all classes of frequent non-HLA antibodies. Alloimmunity as well as autoimmunity to self-antigens have been hypothesized to play an important role in immunopathogenesis of chronic rejection of transplanted organs.18 There are numerous descriptions of antigen responses to autoantigens in renal and heart transplantation.19–21 Using cell extracts from patients own lymphocytes we investigated whether the five most common non-HLA antibodies were autoantibodies. Our data showed that only one patient had no antibody binding to autoantigens while the six other investigated sera had weaker binding in the autogeneic than in the allogeneic setting. Alloreactive antibodies bind either not at all or to a weaker extent to the autologous lymphocyte setting. It has to be noted that in 1D as well in 2D electrophoresis, protein denaturing techniques were used, which might have a major influence on presentation of autoimmune or alloimmune antigeneic epitopes. Most of our patients had pretransplantations and blood transfusions. Therefore, we hypothesize that alloimmunity may have been developed de novo after organ transplantation and blood transfusions. The antibody formation of the weak autoimmunity might be related to the primary disease of patients. About 37% of patients had diabetes and 1 patient had rheumatoid disease. Both diseases have been connected to the formation of autoantibodies.22,23 The present study identified the antigens of the five most common allo-reactive antibodies as tubulin beta chain, vimentin isoforms, lamin-B1 and Rho GDP-dissociation inhibitor 2. Vimentin, lamin B1 and tubulin beta chain are cytoskeletal proteins. Rho GDP-dissociation inhibitor 2 is a regulatory Journal of Proteome Research • Vol. 9, No. 2, 2010 1047
research articles protein. Formation of anti vimentin antibodies after heart transplantation is well-known to be associated with the development of cardiac graft vasculopathy.24 It has been reported that anti vimentin antibodies are associated with heart and kidney graft rejection. Vimentin was found to reach circulation during kidney damage in arterioles and glomeruli capillaries.25 This suggests that anti vimentin antibodies in circulation may find their antigen and be effective in case of tissue damage. Correspondingly, anti vimentin antibodies in renal transplanted monkeys are associated with the development of chronic graft vasculopathy.26 In heart transplanted patients anti vimentin antibodies are strongly associated with cardiac allograft rejection and clinical poor outcome.27 The present study is the first that reveals the existence of anti vimentin antibodies already before renal transplantation most likely due to previous transplantations or blood transfusions. Hong et al. showed that patients with pancreatic cancer had autoantibodies against a single isoform of vimentin.28 We have demonstrated that dialysis patients had antibodies against a specific vimentin isoform (60 kDa) only present in allogeneic lymphocytes. This isoform was highly expressed in all investigated healthy volunteers. However, its expression was down regulated in most patient samples. This suggests an influence of the disease on the expression pattern of the different vimentin isoforms. The patient samples showed different vimentin isoforms in the molecular weight range of 37-42 kDa which were absent in most healthy volunteers. This could be due to a disease-related proteolytic cleavage of the 50-60 kDa isoforms. Further experiments are needed to clarify the cause for the differential expression of vimentin isoforms between healthy volunteers and hemodialysis patients. The data suggest that the production of antibodies against the 60 kDa isoform might have been induced by a prior contact of the patients’ immune system with this isoform in the course of RTX or blood transfusion. The induction of antibodies against the allospecific isoform of vimentin (60 kDa) may result in cross reactivity to the autologous vimentin isoform (49 kDa) leading to autoimmunity. There is also alternative explanation for the absence of the 60 kDa isoform in patient’s lymphocytes. The antibodies in patients might be originally autoreactive. Their presence in the circulation might have contributed to a targeted destruction of lymphocytes with the 60 kDa vimentin isoform. The Rho GDP dissociation inhibitor proteins (RhoGDI) are regulatory proteins that act primarily by controlling the cellular distribution and activity of Rho GTPases.29 GTPbinding proteins (G-proteins) have been shown to play significant regulatory roles in cell proliferation, survival, and demise.30 RhoGDI β is expressed in hematopoietic tissue, including bone marrow, thymus, spleen and lymph nodes. Proteins of the Rho family are involved in the transactivation of several steroid receptors.31,32 It has been shown that mineral corticoid receptor, a member of the steroid receptor family, has a major pathophysiological role in the progression of kidney diseases.33,34 Autoantibodies against Rho GDP dissociation inhibitor II were previously described in acute leukemia patients.35 The present study is the first which shows that also dialysis patients have allo-reactive antibodies against Rho GDP dissociation inhibitor β. In conclusion, our current study characterizes the frequency and molecular identity of non-HLA antigens of alloreactive antibodies from hemodialysis patients on the renal transplantation waiting list. It is well accepted that the presence of non-HLA allo-reactive antibodies prior to trans1048
Journal of Proteome Research • Vol. 9, No. 2, 2010
Bilalic et al. plantation is associated with impaired long-term graft survival. The results are still preliminary and need to be approved in a larger prospective trial. But with a total of 28 patients they provide a good guess of the situation in the patient population. Our results could be useful for the analysis of possible associations with the specific antigens. Our findings could provide a basis for future studies clarifying the role of non HLA antibodies in clinical transplantation. In addition, the knowledge on specific antigens could also be useful for targeted plasma apheresis to remove specific antibodies from recipients’ circulation. Abbreviations: PRA, panel reactive antibodies; CDC, complement-dependent cytotoxicity; HLA, human leukocyte antigen; RuBPS, ruthenium(II) tris(bathophenantroline disulfonate); NC, nitro cellulose; ECs, endothelial cells.
Acknowledgment. The study was supported by the European Society for Organ Transplantation (ESOT Junior Basic Science Grants JBSG 01.001) and by the “Medical Scientific Fund of the Mayor of the City of Vienna” (No.08056). Supporting Information Available: Supplemental Table and spectra. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Susal, C.; Opelz, G. Options for immunologic support of renal transplantation through the HLA and immunology laboratories. Am. J. Transplant. 2007, 7 (6), 1450–6. (2) Terasaki, P. I.; Cai, J. Humoral theory of transplantation: further evidence. Curr. Opin. Immunol. 2005, 17 (5), 541–5. (3) Hourmant, M.; Buzelin, F.; Dantal, J.; van Dixhoorn, M.; Le Forestier, M.; Coste, M.; Cantarovich, D.; Moreau, A.; Bignon, J. D.; van der Woude, F.; et al. Late acute failure of well-HLA-matched renal allografts with capillary congestion and arteriolar thrombi. Transplantation 1995, 60 (11), 1252–60. (4) Terasaki, P. I. Deduction of the fraction of immunologic and nonimmunologic failure in cadaver donor transplants. Clin. Transpl. 2003, 449–52. (5) Zwirner, N. W.; Marcos, C. Y.; Mirbaha, F.; Zou, Y.; Stastny, P. Identification of MICA as a new polymorphic alloantigen recognized by antibodies in sera of organ transplant recipients. Hum. Immunol. 2000, 61 (9), 917–24. (6) Perrey, C.; Brenchley, P. E.; Johnson, R. W.; Martin, S. An association between antibodies specific for endothelial cells and renal transplant failure. Transpl. Immunol. 1998, 6 (2), 101–6. (7) Opelz, G. Non-HLA transplantation immunity revealed by lymphocytotoxic antibodies. Lancet 2005, 365 (9470), 1570–6. (8) Rose, M. L. Long-term effects of damage to the endothelium and chronic rejection. J. Heart Lung Transplant. 2004, 23 (9 Suppl), S240–3. (9) Terasaki, P. I.; McClelland, J. D. Microdroplet Assay of Human Serum Cytotoxins. Nature 1964, 204, 998–1000. (10) Pei, R.; Lee, J.; Chen, T.; Rojo, S.; Terasaki, P. I. Flow cytometric detection of HLA antibodies using a spectrum of microbeads. Hum. Immunol. 1999, 60 (12), 1293–302. (11) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227 (259), 680–5. (12) Rabilloud, T.; Strub, J. M.; Luche, S.; van Dorsselaer, A.; Lunardi, J. A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels. Proteomics 2001, 1 (5), 699–704. (13) Zellner, M.; Babeluk, R.; Diestinger, M.; Pirchegger, P.; Skeledzic, S.; Oehler, R. Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples. Electrophoresis 2008, 29 (17), 3621–7. (14) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68 (5), 850–8. (15) Shevchenko, A.; Tomas, H.; Havlis, J.; Olsen, J. V.; Mann, M. Ingel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 2006, 1 (6), 2856–60.
research articles
Non-HLA Antibodies (16) Gharahdaghi, F.; Weinberg, C. R.; Meagher, D. A.; Imai, B. S.; Mische, S. M. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 1999, 20 (3), 601–5. (17) Opelz, G.; Graver, B.; Mickey, M. R.; Terasaki, P. I. Lymphocytotoxic antibody responses to transfusions in potential kidney transplant recipients. Transplantation 1981, 32 (3), 177–83. (18) Barber, L. D.; Whitelegg, A.; Madrigal, J. A.; Banner, N. R.; Rose, M. L. Detection of vimentin-specific autoreactive CD8+ T cells in cardiac transplant patients. Transplantation 2004, 77 (10), 1604–9. (19) Caldas, C.; Luna, E.; Spadafora-Ferreira, M.; Porto, G.; Iwai, L. K.; Oshiro, S. E.; Monteiro, S. M.; Fonseca, J. A.; Lemos, F.; Hammer, J.; Ho, P. L.; Kalil, J.; Coelho, V. Cellular autoreactivity against heat shock protein 60 in renal transplant patients: peripheral and graftinfiltrating responses. Clin. Exp. Immunol. 2006, 146 (1), 66–75. (20) Dunn, M. J.; Rose, M. L.; Latif, N.; Bradd, S.; Lovegrove, C.; Seymour, C.; Pomerance, A.; Yacoub, M. H. Demonstration by western blotting of antiheart antibodies before and after cardiac transplantation. Transplantation 1991, 51 (4), 806–12. (21) Warraich, R. S.; Pomerance, A.; Stanley, A.; Banner, N. R.; Dunn, M. J.; Yacoub, M. H. Cardiac myosin autoantibodies and acute rejection after heart transplantation in patients with dilated cardiomyopathy. Transplantation 2000, 69 (8), 1609–17. (22) Atassi, M. Z.; Casali, P. Molecular mechanisms of autoimmunity. Autoimmunity 2008, 41 (2), 123–32. (23) van Boekel, M. A.; Vossenaar, E. R.; van den Hoogen, F. H.; van Venrooij, W. J. Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value. Arthritis Res. 2002, 4 (2), 87–93. (24) Jurcevic, S.; Ainsworth, M. E.; Pomerance, A.; Smith, J. D.; Robinson, D. R.; Dunn, M. J.; Yacoub, M. H.; Rose, M. L. Antivimentin antibodies are an independent predictor of transplantassociated coronary artery disease after cardiac transplantation. Transplantation 2001, 71 (7), 886–92. (25) Bravo, J.; Quiroz, Y.; Pons, H.; Parra, G.; Herrera-Acosta, J.; Johnson, R. J.; Rodriguez-Iturbe, B. Vimentin and heat shock protein expression are induced in the kidney by angiotensin and by nitric oxide inhibition. Kidney Int. Suppl. 2003, (86), S46–51.
(26) Jonker, M.; Danskine, A.; Haanstra, K.; Wubben, J.; Kondova, I.; Kuhn, E. M.; Rose, M. The autoimmune response to vimentin after renal transplantation in nonhuman primates is immunosuppression dependent. Transplantation 2005, 80 (3), 385–93. (27) Alvarez-Marquez, A.; Aguilera, I.; Blanco, R. M.; Pascual, D.; Encarnacion-Carrizosa, M.; Alvarez-Lopez, M. R.; Wichmann, I.; Nunez-Roldan, A. Positive association of anticytoskeletal endothelial cell antibodies and cardiac allograft rejection. Hum. Immunol. 2008, 69 (3), 143–8. (28) Hong, S. H.; Misek, D. E.; Wang, H.; Puravs, E.; Hinderer, R.; Giordano, T. J.; Greenson, J. K.; Brenner, D. E.; Simeone, D. M.; Logsdon, C. D.; Hanash, S. M. Identification of a Specific Vimentin Isoform That Induces an Antibody Response in Pancreatic Cancer. Biomark Insights 2006, 1, 175–83. (29) Olofsson, B. Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling. Cell Signal. 1999, 11 (8), 545–54. (30) Sasaki, T.; Takai, Y. The Rho small G protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control. Biochem. Biophys. Res. Commun. 1998, 245 (3), 641–5. (31) Rubino, D.; Driggers, P.; Arbit, D.; Kemp, L.; Miller, B.; Coso, O.; Pagliai, K.; Gray, K.; Gutkind, S.; Segars, J. Characterization of Brx, a novel Dbl family member that modulates estrogen receptor action. Oncogene 1998, 16 (19), 2513–26. (32) Su, L. F.; Knoblauch, R.; Garabedian, M. J. Rho GTPases as modulators of the estrogen receptor transcriptional response. J. Biol. Chem. 2001, 276 (5), 3231–7. (33) Chrysostomou, A.; Becker, G. Spironolactone in addition to ACE inhibition to reduce proteinuria in patients with chronic renal disease. N. Engl. J. Med. 2001, 345 (12), 925–6. (34) Williams, G. H.; Burgess, E.; Kolloch, R. E.; Ruilope, L. M.; Niegowska, J.; Kipnes, M. S.; Roniker, B.; Patrick, J. L.; Krause, S. L. Efficacy of eplerenone versus enalapril as monotherapy in systemic hypertension. Am. J. Cardiol. 2004, 93 (8), 990–6. (35) Cui, J. W.; Li, W. H.; Wang, J.; Li, A. L.; Li, H. Y.; Wang, H. X.; He, K.; Li, W.; Kang, L. H.; Yu, M.; Shen, B. F.; Wang, G. J.; Zhang, X. M. Proteomics-based identification of human acute leukemia antigens that induce humoral immune response. Mol. Cell. Proteomics 2005, 4 (11), 1718–24.
PR900930D
Journal of Proteome Research • Vol. 9, No. 2, 2010 1049
