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De Novo Production of K-1 Tubulin-Specific Antibodies: Role in Chronic Lung Allograft Rejection¹

Trudie A. Goers^2,*, Sabarinathan Ramachandran^2,*, Aviva Aloush, Elbert Trulock, G. Alexander Patterson^* and Thalachallour Mohanakumar^3,*,

^* Department of Surgery, Department of Pulmonary Medicine, and Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, MO 63110

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Lung transplantation is the treatment option for a variety of end-stage pulmonary diseases. Posttransplant development of Abs against donor HLA and non-HLA Ags have been associated with acute and chronic rejection of transplanted organs. Development of bronchiolitis obliterans syndrome (BOS) following lung transplantation has been correlated with de novo production of anti-donor-HLA Abs. However, only a portion of the patients with BOS demonstrate detectable anti-donor-HLA Abs. Airway epithelium is considered as a major target for lung allograft rejection. In this study we demonstrate that many BOS⁺ patients (12 of 36) develop Abs reactive to epithelial cell Ag that are distinct from HLA. Furthermore, de novo production of antiepithelial cell Ab precedes clinical onset of BOS. N-terminal sequencing and blastx analysis as well as blocking with K-1 tubulin-specific Ab identified the epithelial Ag as K-1 tubulin. Binding of the de novo-produced anti-K-1 tubulin Abs to the airway epithelial cells resulted in the increased expression of transcription factors (TCF5 and c-Myc), leading to increased expression of fibrogenic growth factors, activation of cell cycle signaling, and fibroproliferation, the central events in immunopathogenesis of BOS following human lung transplantation.

	Introduction

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The development of bronchiolitis obliterans syndrome (BOS)⁴ remains the Achilles heel of human lung transplantation. BOS occurs in nearly all allografts by 10 years after transplant and is the main cause of morbidity and mortality following lung transplantation. BOS is a fibroproliferative process that involves inflammation and fibrosis of the lamina propria and lumen resulting in progressive decline in pulmonary function and eventual allograft failure. Progression of BOS can be slowed but is unresponsive to the current immunosuppressive therapies instituted after transplantation (1).

Many studies in the literature have proposed that the development of BOS involves a final common pathway of alloimmune injury, with subsequent release of immunologic mediators and production of growth factors, leading to luminal occlusion and fibrous scarring of small airways (2). Several studies have shown that the airway epithelial cells (AECs) are the main target for the immunologic insult during the pathogenesis of allograft rejection (3, 4, 5, 6). In vitro studies from our laboratory (7) and others (8) have demonstrated that activation of epithelial cells results in the production of growth factors including TGF-β, epidermal growth factor, basic fibroblast growth factor, and endothelin-1. Exposure to these growth factors results in the activation and proliferation of fibroblasts and smooth muscle cells. More significantly, in vivo studies have revealed a temporal relationship between elevated levels of growth factors and significant fibroblast migration and proliferation within the small airways (9).

Previous studies from our laboratory (10, 11) and others (12) have implicated that the development of anti-donor HLA Abs after lung transplantation predispose patients to the development of chronic rejection. These studies demonstrated that binding of anti-HLA class I Abs stimulated the proliferation of epithelial, endothelial, and smooth muscle cells (7) However, there are many incidences of BOS in patients where Abs to mismatched donor HLA cannot be readily demonstrated, suggesting a role for Abs to non-HLA Ags in the pathogenesis of BOS. The importance of non-HLA Abs in acute as well as chronic rejection has been previously studied in liver, renal, and cardiac allografts (13, 14, 15). In this report, we demonstrate that Abs that recognize the K-1 tubulin expressed on epithelial surface can be defined in human lung transplant recipients undergoing BOS. These Abs bind to AECs, and specific ligation results in increased expression of fibrogenic growth factors, activation of cell cycle signaling, and fibroproliferation. Therefore, we propose a pathogenic role for Abs to K-1 tubulin in the immunopathogenesis of BOS following human lung transplantation.

	Materials and Methods

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Human subjects

Patients who underwent lung transplantation at the Washington University Medical Center/Barnes-Jewish Hospital were enrolled in this study after obtaining informed consent in accordance with a protocol approved by the Institutional Review Board. The mean age at transplantation was 52.0 ± 8.1 years and the male-to-female ratio was 1:1. The end-stage pulmonary pathologies were chronic obstructive pulmonary disease, angiotensin type 1 receptor deficiency, cystic fibrosis, and idiopathic pulmonary fibrosis. Most of the transplants were bilateral. The standard immunosuppression protocol consisted of cyclosporine, azathioprine, and prednisone. After BOS was diagnosed, the immunotherapy protocol was modified to FK506 (tacrolimus), mycophenolate mofetil, and prednisone.

The diagnosis of BOS was made according to the International Society for Hearth & Lung Transplantation (ISHLT) standard criteria (16). Patients were diagnosed with BOS (BOS⁺) if either of the following criteria were satisfied: their forced expiratory volume in 1 s (FEV₁) was measured at <80% of the baseline established in their stable postoperative period, or there was histologic evidence of bronchiolitis obliterans. Patients were considered free of BOS (BOS^–) if their FEV₁ remained above the 80% level and their pulmonary histology remained negative for bronchiolitis obliterans. Serum samples were collected from the patients on the day of their transplant before surgery and then in posttransplant months 12, 24, 36, and 48. Further samples were collected at varying follow-ups depending on the patient’s clinical status. All serum samples were processed in our laboratory on their day of collection and then stored at –70°C until further use.

Complement-dependent cytotoxicity assays

The anti-HLA reactivity was determined on an HLA reference panel consisting of T and B lymphocytes from 50 unrelated individuals of known HLA specificity (panel-reactive Abs (PRA)). Briefly, isolated lymphocytes were incubated in the presence of undiluted patient serum (diluted 1/1) for 40 min at 22°C. Then, both three-wash Amos-modified and antiglobulin-augmented complement-dependent cytotoxicity assays were performed according to the National Institutes of Health standard protocols as previously described (10). Patients were considered positive for PRA if there was cytotoxicity against 2% or more of the cells in the panel of lymphocytes in any of the tested sera.

FlowPRA assay

The FlowPRA assay was conducted according to the manufacturer’s instructions (One Lambda). For classes I and II FlowPRA assays, positivity with a single bead produced a clear and distinct peak of fluorescence that contained 2.4 and 2.9% of the total events collected, respectively (i.e., th and th of the bead pool). The cutoff was determined based on the average percentage binding of 20 different normal human sera obtained from healthy volunteers. Therefore, results were recorded as positive when 2.4% of class I and 2.9% of class II beads exhibited a distinct fluorescence peak that was above the fluorescence exhibited by the negative control.

Cell lines

The AEC line was developed from lung biopsies, immortalized by transfection with the pRSV-Tag plasmid, and cultured in small airway growth medium (Cambrex Bio Science). Normal human bronchial epithelial cells were obtained from the American Type Culture Collection and cultured in bronchial epithelial growth medium (Cambrex Bio Science). Endothelial cells were obtained from Cambrex and were cultured in endothelial growth medium (Cambrex Bio Science). Kidney cell lines (KCLs) were developed from the renal cortex of cadaveric kidneys, immortalized by transfection with the pRSV-Tag plasmid, and cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 15% FBS, 2 mM L-glutamine, 25 mM HEPES buffer, 1 mM sodium pyruvate, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. PBLs were isolated by Ficoll gradient centrifugation from healthy donor voluntary blood samples and used for testing on the same day as isolation. All other cell lines were frozen at –70°C until use. Upon thawing, all cell lines were maintained in sterile 5% CO₂ incubation in respective growth media at 37°C.

FACS analysis

Tracheal AECs, endothelial cells, KCLs, and PBLs (1 x 10⁶ cells) were exposed to 100 µl of selected BOS⁺ and BOS^– patient serum samples, panel-reactive human serum (positive control), normal healthy volunteer serum (negative control), or no serum (negative control). After washing, the cells were stained with FITC-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories). Cells were analyzed using a FACScan flow cytometer (BD Biosciences). The data analysis was performed using CellQuest software (BD Biosciences).

Western blot analysis

Crude extracts from the epithelial, endothelial, and PBMC were extracted by resuspending 10 million cells in M-PER protein extraction reagent (Pierce). The whole-cell lysates were then separated under reducing conditions using 4–12% gradient Bis-Tris Gel (Invitrogen) electrophoresis. The proteins were transferred to a nitrocellulose membrane and blocked with TBS with 5% skim milk protein overnight. After washing one time with TBS, the nitrocellulose membrane was either cut into 0.5-cm strips or left intact. Each strip was immunoblotted with a different patient serum (BOS⁺ or BOS^–) or serum from a normal volunteer (negative control) (all at 1/1500) overnight. The serum was washed from the membranes, which were then incubated for 1 h with HRP-conjugated goat anti-human IgG (1/10,000) (Jackson ImmunoResearch Laboratories). The immunoreactivity was detected by using the SuperSignal West Pico Chemiluminescent Substrate Western blot detection system (Pierce).

K-1 tubulin was purchased from Abnova and was reconstituted according to the manufacturer’s instructions and then frozen at –70°C until use. Fifty micrograms of the protein was run under reducing conditions through a 4–12% gradient Bis-Tris Gel. The protein was transferred, blocked, and washed as above. The nitrocellulose membrane was then cut into eight equal slices. These membrane slices were incubated overnight with mouse anti-K-1 tubulin mAb (1/100), normal human serum (1/1500), 3 BOS⁺ sera, and 3 BOS^– sera (1/1500). The membranes were washed after incubation. HRP-conjugated goat anti-mouse IgG secondary Ab (1/10,000) (Jackson ImmunoResearch Laboratories) was applied to the K-1 tubulin Ab, while HRP-conjugated goat anti-human IgG (1/1000) (Jackson ImmunoResearch Laboratories) was added to the other samples for 1 h. Detection was achieved as above. To determine the specificity of the Ab reactivity, crude epithelial extracts were electrophoresed and transferred onto nitrocellulose paper. The membranes were blocked overnight with mouse anti- K-1 tubulin mAb or control Ab (1/100). BOS⁺ sera with AEC reactivity were used to probe the membranes and were developed as described above.

Luminex

AECs were cultured in 6-well plates in small airway growth medium to 80% confluency. The growth media was then removed and the cells were maintained in a starvation media (small airway basic medium (Cambrex Bio Science) for 24 h. After this period, the starvation medium was removed and 3 BOS⁺ and 3 BOS^– patients’ sera were individually added to each well (1000 µl). After 1 h of incubation at 37°C, 1000 µl of starvation media was added to the sera in each of the wells and cultured for 24 h. The entire well volume was then collected and frozen at –70°C until use.

Growth factor 4-plex combined with cytokine 20-plex (vascular endothelial growth factor (VEGF), G-CSF, basic fibroblast growth factor, platelet-derived growth factor-BB + fibroblast growth factor basic, GM-CSF, IFN-, IL-1, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40/p70, IL-13, IL-17, IP-10, KC, MCP-1, monokine induced gamma-interferon, MIP-1, TNF-, and VEGF basic) analysis kits were obtained from Biosource International. Millipore multiscreen 96-well filter plates were used for all multiplex growth factor kits. Assays were run in triplicate using 50 µl of sample according to the manufacturer’s protocol. Data were collected using the Luminex-100 system version 1.7, a dual-laser flow analyzer (Luminex). Data analysis was performed using the MasterPlex QT 1.0 system (MiraiBio). A five-parameter regression formula was used to calculate the sample concentrations from the standard curves. All 96 samples were analyzed with the LINCOplex kit (Linco Research).

Gene array

Expression profiles of intracellular signal genes in the isolated AECs were analyzed using the Signal Transduction PathwayFinder gene array (SuperArray Bioscience) as per the manufacturer’s recommendation. Briefly, total RNA was extracted from 10 x 10⁶ cells using TRIzol reagent. The RNA was reverse-transcribed and radiolabeled using an AmpoLabeling-LPR kit (SuperArray Biosciences). The radiolabeled probes were hybridized with GEArray Q series Signal Transduction PathwayFinder gene array overnight at 60°C. Membranes were washed twice with 2x SSC/1% SDS and twice with 0.2x SSC/0.5% SDS and then exposed to x-ray film. After overnight exposure at –70°C, the film was developed. The data obtained from the membranes were analyzed using the GEArray Analyzer software (SuperArray Biosciences). The data were normalized using GAPDH and data were represented as relative expression (gene of interest/GAPDH).

Protein isolation and sequencing

With the identical protocol used in the Western blotting above, the AECs were prepared and the proteins separated under reducing conditions. The proteins were then transferred to a polyvinylidene difluoride membrane. When the transfer was complete, the membrane was stained with Coomassie blue stain for 1 min. The membrane was destained overnight. The band at 48 kDa was sharply excised and dried. The sample was then sent to ProSeq for protein microsequencing determination. In brief, phenylisothiocyanate reacts with the amino acid residue at the amino terminus under basic conditions to form a phenylthiocarbamoyl derivative. Trifluoroacetic acid then cleaves off the first amino acid as its anilinothialinone derivative and leaves the new amino terminus for the next degradation cycle. The ATZ-amino acid is then removed by extraction with n-butyl chloride and converted to a phenylthiohydantoin derivative with 25% trifluoroacetic acid/water. The PTH-amino acid is transferred to a reverse-phase C-18 column for detection at 270 nm. This chromatogram provides standard retention times of the amino acids for comparison with a standard chromatogram. The HPLC chromatograms are collected using a computer data analysis system. This process is repeated sequentially to provide the N-terminal sequence of the protein/peptide.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Sera from BOS⁺ lung transplant recipients develop Abs specifically reactive to AECs

In lung allografts epithelium has been shown to be the primary target of immune responses. Abs have been postulated to play a crucial role in chronic rejection of allografts. Thirty-six BOS⁺ lung transplant recipients with no detectable anti-HLA Abs by FlowPRA, cytotoxicity, or ELISA were tested by flow cytometry against a panel of 30 PBMCs covering most of the common HLA allelic Ags in the population studied (see Table I). None of the 36 sera recognized any of the PBMCs tested, indicating the absence of any anti-HLA Abs in those sera. To test for the presence of Abs that recognize epithelial cells, we analyzed immunoreactivity of sera from these 36 BOS⁺ and from 36 BOS^– lung transplant recipients and from 10 normal volunteers by flow cytometry against a panel of epithelial cells. None of these sera had detectable anti-HLA Abs by cytotoxicity, ELISA, or FlowPRA methods. However, 12 of the 36 BOS⁺ lung transplant recipients had Abs that specifically bound to epithelial cells (Table I). In contrast, none of the sera from BOS^– patients (36 patients) or normal donors (10 volunteers) reacted to the panel of epithelial cells tested. To test the specificity of the Abs, we analyzed the immunoreactivity of the positive sera against endothelial cells, kidney tubular epithelial cells (KCLs), and PBMCs by flow cytometry. The list of the cell lines tested, their origin, and the HLA phenotypes are summarized in Table II. FACS analysis of the sera from BOS⁺ patients demonstrated a significant binding of the Abs to the AECs (Fig. 1A). However, no binding was observed with the endothelial cells (Fig. 1B), KCLs (Fig. 1C), or the PBMCs (Fig. 1D) tested, suggesting tissue-restricted specificity of the Abs. The demographics of the BOS⁺ patients with and without AEC reactivity is presented in Table III, and no significant difference in age, HLA matches, cause of transplant, or gender was observed in the two groups. These results demonstrate that a portion of lung transplant recipients develop Abs specific for AECs that are distinct from Abs to MHC alloantigens. Gradation of these BOS⁺ patients based on the International Society for Heart & Lung Transplantation guidelines showed that 10 of the 12 BOS⁺ patients were of BOS grade 2 or higher, 1 patient was BOS grade 1, and 1 was BOS grade 0. The higher prevalence of the AEC Ags in the patients with grade 2 or higher suggests that these AEC Ags could play a vital role in the progression of BOS.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. BOS+ patient serum with positive FACS on AEC line. Sera from BOS+ and BOS– patients with no anti-HLA reactivity were tested against epithelial cells, endothelial cells, PBMCs, and macrophages by flow cytometry. A, Epithelial cells; B, endothelial cells; C, PBMCs; and D, KCLs. A representative BOS+ sera with epithelial reactivity is overlaid on a BOS– sera. The dotted line represents the binding of the BOS– sera, and the solid line represents the BOS+ sera.

To determine whether the ligation of cell-surface molecules on the AECs by the Abs developed in BOS⁺ lung transplant recipients, we incubated AECs with Abs, following which growth factor production by the AEC was determined by Luminex assay. It has been demonstrated that increased expression of growth factors can accelerate the fibroproliferation cascade, which is considered to be one of the major pathways toward the induction of chronic rejection in transplanted allografts. To study the expression of growth factors subsequent to binding of antiepithelial Abs, we exposed epithelial cells to BOS⁺ sera with and without antiepithelial reactivity for 1 h at 37°C. Expression of the growth factors in the culture supernatant were quantitated after 24 h of culture using a 4-plex growth factor Luminex assay. Binding of the antiepithelial Ab to the epithelial cells, as shown in Fig. 2, resulted in a significant increase in the expression of VEGF (6-fold, 13 vs 79 ng/ml, p < 0.05), heparin-binding epidermal growth factor-like growth factor (HB-EGF; 2-fold, 27 vs 56 ng/ml, p < 0.05), and TGF-β (2.5-fold, 12 vs 32 ng/ml, p < 0.05) compared with the expression levels observed on exposure to sera with no epithelial-specific reactivity. These results indicate that binding of the antiepithelial Ab to the epithelial cells results in increased production of growth factors that activate the fibroproliferation cascade, a critical event in BOS development in lung transplant recipients. Therefore, these Abs may play a role in the pathogenesis of BOS following lung transplantation.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Binding of antiepithelial Abs to AECs results in increased expression of fibrogenic growth factors. Airway epithelial cells were incubated with sera from BOS+ patients with and without epithelial reactivity for 1 h and cultured 24 h at 37°C. Growth factors secreted in the culture supernatant were analyzed by growth factor Luminex assay. A significant increase was found in the expression of VEGF (6-fold), HB-EGF (2-fold), and TGF-β (2.5-fold) in culture supernatants of AECs exposed to BOS+ sera compared with sera with no epithelial reactivity. The bars represent fold increase observed in means from three different experiments.

To study the intracellular signaling events following binding of the antiepithelial Ab to the epithelial cells we exposed AECs to BOS⁺ sera with and without antiepithelial reactivity for 30 min. Expression profiles of the various signaling cascade intermediates were analyzed by using a PathwayFinder gene array. Gene array analysis presented in Fig. 3 demonstrated a 4-fold increase in the expression of heat shock proteins (HSPs) 27 (0.75 vs 3.1) and 90 (0.67 vs 2.95) as well as PKC (0.9 vs 4.0) after binding of the antiepithelial Abs to the epithelial cells. There was also a >2-fold increase in production of transcription factors TCF5 (0.74 vs 1.98) and c-Myc (1.02 vs 2.17), which are involved in inflammatory response, cell cycle progression, apoptosis, and cellular transformation (Fig. 3). These results demonstrate that binding of the antiepithelial Ab results in the activation of PKC and stress pathways that could result in increased expression of growth factors.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Gene array analysis. Airway epithelial cells were incubated with sera from BOS+ patients with and without epithelial reactivity for 30 min on ice. Expression profile of the signaling intermediates in stress signaling, calcium, and cytoskeleton signaling were analyzed on a Signal Transduction PathwayFinder gene array. The bars represent fold increase observed in means from three different experiments. Gene array analysis demonstrated a 4-fold increase in the expression of HSPs 27 and 90 as well as PKC after binding of the antiepithelial Abs to the epithelial cells. There was also significant increase in production of transcription factors TCF5 and c-Myc (2-fold).

K-1 tubulin is the target Ag recognized by BOS⁺ sera

To identify the Ag recognized by BOS⁺ sera we performed Western blot analysis against the crude antigenic extract of epithelial cells. Western blot analysis demonstrated that the BOS⁺ sera recognized a polypeptide of 48 kDa. No immunoreactivity was observed with the BOS^– sera or the normal human sera (Fig. 4). To identify the epithelial Ag we electroeluted the 48-kDa polypeptide from the crude airway epithelial cell extract and sequenced the polypeptide. The sequence of the epithelial Ag was MRECISIHVGQAGVQIXNA. BLAST analysis of the sequence demonstrated a 100% homology to the K-1 tubulin protein. These results indicate that the epithelial cell target Ag recognized by the BOS⁺ sera is K-1 tubulin.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Molecular characterization of epithelial Ag by Western blot analysis. Total protein extracts from AECs were electrophoresed on a 4–12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with BOS+ sera and BOS– sera (1/1500 dilution). Membrane-bound Abs were detected with HRP-conjugated goat anti-human Ab and developed with chemiluminescent substrate. BOS+ sera with epithelial reactivity recognized a 48-kDa polypeptide. Membrane strips represent one of the BOS+ sera with epithelial reactivity and one from BOS– patient.

To investigate the temporal development of the non-HLA anti-AEC Abs relative to lung allograft transplantation, serial serum samples collected on the day of transplant to time of BOS diagnosis were analyzed by Western blot using purified K-1 tubulin. As seen in Table IV, serum from samples 14.8 ± 11.8 mo (range 6–35 mo) before diagnosis of BOS following transplantation recognize the 48-kDa Ag of AEC by Western blot. Moreover, once detectable Abs to the 48-kDa polypeptide were observed, they persisted in all of the serum samples from patients who developed BOS. These results indicate that the development of antiepithelial Ab precedes the development of BOS in a subset of lung transplant recipients.

Recognition of purified K-1 tubulin protein by BOS⁺ sera

To further confirm the identity of epithelial Ag recognized as the BOS⁺ sera as K-1 tubulin, we performed Western blot analysis using a purified recombinant K-1 tubulin protein. Western blot analysis demonstrated that the purified K-1 tubulin protein was recognized by the BOS⁺ sera with epithelial reactivity (Fig. 5A). However, no reactivity was observed against the purified K-1 tubulin using sera from BOS⁺ patients with no reactivity to AECs, sera from BOS^– patients, and normal human sera (Fig. 5A). To further confirm this observation, two strips of K-1 tubulin were blocked with mouse anti-K-1 tubulin Ab or control Ab and probed with BOS⁺ sera with epithelial reactivity. As seen in Fig. 5B, blocking with K-1 tubulin-specific Ab abolished the immunoreactivity of the BOS⁺ sera. To further confirm that the Ag recognized was K-1 tubulin, we performed Western blot analysis with AEC extracts. The strips were blocked with anti-K-1 tubulin-specific Ab or control Ab overnight. The membranes were then probed with BOS⁺ sera with AEC reactivity. As seen in Fig. 5B, blocking of the AECs with K-1 tubulin-specific Abs completely abolished the binding of the BOS⁺ sera with AEC reactivity to the epithelial cells. To further confirm that K-1 tubulin is expressed on the epithelial cells, we performed a flow cytometry with a K-1 tubulin-specific mouse mAb or control Ab. As seen in Fig. 5B, incubation with anti-K-1 tubulin Ab showed a significant reactivity to the epithelial cells when compared with the control Ab. These observations further confirmed that the posttransplant sera from lung transplant recipients with epithelial-specific reactivity are indeed recognizing K-1 tubulin.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. The epithelial Ag recognized by BOS+ serum reactivity is K-1 tubulin protein. A, Purified recombinant K-1 tubulin protein or crude ACE extracts were electrophoresed and transferred onto nitrocellulose paper and probed with BOS+ sera and BOS– sera. The figure shows a representative BOS+ serum with epithelial reactivity binding to the purified K-1 tubulin protein (lane 1), whereas no reactivity was observed with BOS– sera (lane 2). To further confirm this result, two strips with the purified recombinant K-1 tubulin protein were blocked with anti-K-1 tubulin Ab (lane 3) or control Ab (lane 4) and probed with BOS sera with AEC reactivity. Blocking with specific Ab resulted in abolition of BOS sera reactivity with epithelial cells. Two strips with epithelial cell extracts were blocked with K-1 tubulin-specific Ab (lane 5) or control Ab (lane 6) overnight. The strips were then probed with BOS+ sera with AEC reactivity and developed. Blocking of the epithelial extracts with K-1 tubulin-specific Ab completely abolished the AEC reactivity with BOS+ sera (lane 5). B, To further confirm that K-1 tubulin is expressed on the epithelial cells, we performed a FACS analysis of the epithelial cells following staining with K-1 tubulin-specific Ab or control Ab. Incubation with anti-K-1 tubulin Ab showed a significant reactivity to the epithelial cells when compared with the control Ab. The dotted line represents the control Ab, and the solid line represents the K-1 tubulin-specific Ab.

Sera from BOS⁺ lung transplant recipients with detectable anti-HLA Abs also recognize K-1 tubulin

Development of anti-HLA Abs to the mismatched donor HLA Ags has been shown to correlate with the development of BOS in human lung transplant recipients. However, reactivity of the sera containing anti-HLA Abs to other Ags encoded by non-MHC have been difficult to analyze due to several reasons, including low titer and lack of purified proteins in question. To test the presence anti-K-1 tubulin Abs in the sera from BOS⁺ sera from lung transplant recipients with ant-HLA Ab, we performed Western blot analysis with purified K-1 tubulin protein. Western blot analysis with purified K-1 tubulin protein demonstrated that 5 of 10 BOS⁺ sera with anti-HLA Abs also recognized the K-1 tubulin protein (Table V). The anti-AEC Abs in these patients developed 3 to 14 mo before the clinical diagnosis of BOS. Therefore, 50% of lung transplant recipients with anti-HLA Abs also produce Abs to the autoantigen K-1 tubulin.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In this study we have identified posttransplant development of Abs against the K-1 tubulin in lung transplant recipients with clinical evidence of BOS. This agrees with the emerging data that tissue remodeling following transplantation can expose cryptic self-Ags that can become the target of immune recognition leading to rejection (32, 33, 34). Recent studies by Wilkes have demonstrated that immune responses against collagen V, an auto-Ag, can induce obliterative airway disease in rats following lung transplantation (32) and may also play a pathogenic role in BOS following human lung transplantation. Our studies have shown that collagen V-reactive cells develop during post-lung transplantation in humans and they are regulated by IL-10-producing T regulatory cells (35). The inflammatory status observed during the development of BOS in these patients could also result in the development of polyreactive Abs that recognize the self-Ags.

In lung allografts, immune responses targeted against the epithelial cells have been thought to play a crucial role in chronic rejection of the transplanted lungs (3, 4, 5, 6). Using primary airway epithelial cell as targets, we analyzed the presence of antiepithelial Abs in a subset of lung transplant recipients with no detectable anti-HLA Abs by both complement-dependent assays as well as solid-phase assays (ELISA and flow cytometry) for the detection of Abs against HLA. Our results indicate that a subset of BOS⁺, anti-HLA Ab-negative patients’ status after lung transplantation (33%) developed Abs against a cell-surface Ag expressed on AECs. One possible explanation for the absence of detectable circulating anti-HLA Abs could be that these Abs are bound to the target organ, thereby reducing the circulating levels of anti-HLA Abs to the undetectable levels. However, our observation that these antiepithelial Abs with no detectable anti-HLA Abs in in vitro studies induce fibrogenic growth factors that lead to fibroproliferation, a central event in the development of BOS, indicate the involvement of the these antiepithelial Abs in BOS pathogenesis. Moreover, we also observed that 5 of the 10 BOS⁺ patients with anti-HLA Abs also recognized the Ag expressed on the AECs. Therefore, a large proportion of lung transplant recipients with BOS (37%) develop Abs reactive to AECs. In summary, based on our screening criteria, we observed that 16 of 40 of the BOS1⁺ patients have anti-AEC reactivity whereas 1 of 6 BOS0⁺ patients demonstrated anti-AEC reactivity. However, none of the BOS^– patients demonstrated anti-AEC reactivity at the dilution tested. Twenty-one percent of the BOS1⁺ patients that lacked anti-AEC Abs had detectable antidonor Abs. However, 38% of the BOS1⁺ patients had no detectable Abs to the donor or the airway epithelial Ag tested. Epithelial specificity of the sera from the lung transplant recipients with antiepithelial reactivity is clearly evident because these sera did not react against PBMC covering most of the well-defined HLA specificities and they were also not reactive to endothelial cells and tubular epithelial cells of kidney origin.

This study complements other earlier reports that have demonstrated that lung transplant recipients with preexisting antiepithelial Abs have an increased rate of lung allograft rejection in 3 mo compared with controls, and 32% of lung transplant recipients with BOS developed Abs specific for AECs (36). Additionally, non-MHC Abs have been suggested to play an important role in the rejection of both human cardiac and renal transplants (37, 38, 39, 40). In this study we have not measured the T cell-dependent responses against the epithelial Ag or the donor Ags, which could play a crucial role in the development of BOS in the 38% of the BOS1⁺ patients who have no detectable B cell-dependent responses or Ab production against the epithelial Ags or donor Ags tested. In this study, we demonstrated that development of the antiepithelial Ab preceded the clinical diagnosis of BOS by 11.9 ± 8.9 mo. The observation that antiepithelial Ab precedes the development of BOS is in concurrence with the observations that anti-HLA Ab development also precedes the clinical onset of BOS (41). These findings provide credence to the view that Ab may play a pathogenic role, and that immune monitoring during the posttransplant period may define human lung transplant recipients who are at higher risk for the development of BOS. Furthermore, it is likely that early identification of this cohort of patients may allow change in immunosuppression, which is directed to humoral immunity against the allograft.

BOS is associated histologically with epithelial injury, bronchocentric mononuclear inflammation, fibrosis, and proliferation of the lamina propria and obliteration of small airways. Gene array and Luminex assay results provided in this paper indicated a 4-fold up-regulation of HSPs 27 and 90 and constituents of the calcium homeostasis pathway and a 2-fold up-regulation in transcription factors TCF5 and c-Myc. There was also a 2-, 3-, and 6-fold increase in HB-EGF, TGF-β, and VEGF production by AECs, respectively, after exposure to the epithelial cell-specific sera from BOS⁺ lung transplant recipients. These results strongly suggest that the binding of the Ab to K-1 tubulin to AEC activates a PKC-driven calcium maintenance pathway that is regulated by HSPs 27 and 90. These pathways can culminate in increased cellular mitosis, proliferation, and growth factor production (Fig. 3). It significant that TGF-β, PDGF, and endothelin-1 have been found to be significantly elevated in human lung transplant recipients with chronic rejection, and VEGF has been shown to be elevated in rat allograft models of rejection (42, 43). TCF5 or CCAAT/enhancer binding protein (C/EBP) β is a bZIP transcriptions factor that can homodimerize and bind to DNA regulatory regions. TCF5 has been shown to play an important role in the regulation of immune and inflammatory response genes. It has been shown to bind to the IL-1 response element in the IL-6 gene, as well as to regulatory regions of several acute-phase and cytokine genes (44). Earlier studies in the literature have also demonstrated a novel role for C/EBP-β in IL-1β-induced connective tissue disease (45). TCF5 has also been shown to bind the promoter and upstream element of the collagen gene and stimulates its production (46). Taken together, it is reasonable to postulate that ligation of AEC K-1 tubulin by specific Abs can result in growth factor production, stimulation of collagen production, as well as cytokine gene up-regulation, which are considered to be central events in the immunopathogenesis of BOS.

Because a large proportion of lung transplant recipients with BOS reacted to AECs, it was evident that the reactivity was not directed to mismatched donor HLA Ags. Therefore, we set out to determine biochemical and molecular nature of the Ag defined using Abs specific for AEC. Western blot analysis of the crude airway epithelial cell extracts with patient sera demonstrating epithelial reactivity identified a 48-kDa protein. Further sequencing and BLAST analysis of the eluted 48-kDa protein showed a 100% homology to the K-1 tubulin protein. Thus, for the first time, we were able to define the biochemical and molecular nature of the AEC Ag defined by the sera from post-lung transplantation. Western blot analysis of purified K-1 tubulin protein with sera from patients with antiepithelial Ab further confirmed that the identified protein is K-1 tubulin. K-1 tubulin is a 50-kDa protein in its usual polyglycosylated, posttranslational form. It is found in cells as one of six different isotypes. Its usual cellular functions include GTP binding, GTPase activity, maintaining cellular structure in the form of microtubules, and microtubule-based intracellular movement. Most of these known functions would indicate that K-1 tubulin is not prone to act at the cell surface. However, earlier studies have identified auto-Abs against K-1 tubulin in small-cell lung cancer and breast cancer (47), as well as in postcardiac transplant fatal cardiomyopathy (48), indicating that this protein can reach the cell surface and that it is immunogenic under selected circumstances. This concept has been verified in recent studies demonstrating that the intermediate filament proteins are expressed on the cell surface following activation or apoptosis. The alternate hypothesis is that the indolent inflammatory environment that is present in the posttransplant period may cause an injury-recovery pattern at the level of the airway epithelium. This form of chronic injury may lead to destruction and turnover of the AECs, thereby exposing AEC proteins to the surveillance immune system with resultant Ab production. One potential limitation of this study is the absence of results using a cell line deficient in the K-1 tubulin expression. This raises the possibility that the Abs to the K-1 tubulin can bind to other Ags expressed on the airway epithelial cells to induce the fibrotic changes observed. However, blocking experiments used in this study with mAbs to K-1 tubulin strongly favor this as the target Ag recognized on the epithelial cells.

In conclusion, we have demonstrated that a large proportion (37%) of lung transplant recipients develops anti-K-1 tubulin Abs during the posttransplant period, and this is strongly associated with the development of BOS or chronic rejection of the lung allograft. We further demonstrate that binding of the anti-K-1 tubulin to the epithelial cells resulted in the increased expression of TCF5, a transcription factor involved in the regulation of inflammatory response genes and fibroproliferation cascade. The increased levels of TCF5 and c-Myc can result in the increased expression of fibrogenic growth factors, HB-EGF, TGF-β, and VEGF, all of which can initiate and sustain the fibroproliferation cascade, a central event in the immunopathogenesis of BOS following human lung transplantation.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflicts of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant HL56643 (to T.M.) and National Institutes of Health Training Grant 5T32AI07163 (to T.A.G.).

² T.A.G. and S.R. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. T. Mohanakumar, Washington University School of Medicine, Department of Surgery, Box 8109-3328 CSRB, 660 South Euclid Avenue, Saint Louis, MO 63110. E-mail address: kumart{at}wustl.edu

⁴ Abbreviations used in this paper: BOS, bronchiolitis obliterans syndrome; AEC, airway epithelial cell; HB-EGF, heparin-binding epidermal growth factor-like growth factor; HSP, heat shock protein; K-1, keratinocyte-alpha 1; KCL, kidney cell line; PKC, phosphokinase C; PRA, panel-reactive Abs; VEGF, vascular endothelial growth factor.

Received for publication June 27, 2007. Accepted for publication January 27, 2008.

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