Mammaglobin-A (MGBA), a 10-kD protein, is over expressed in 80% of primary and metastatic human breast cancers. Breast cancer patients demonstrate high frequencies of CD8+ cytotoxic T lymphocytes (CTL) specific to MGBA. Defining CD8+ CTL responses to HLA class I-restricted MGBA-derived epitopes assumes significance in the context of our ongoing efforts to clinically translate vaccine strategies targeting MGBA for prevention and/or treatment of human breast cancers. In this study, we define the CD8+ CTL response to MGBA-derived candidate epitopes presented in the context of HLA-B7, which has a frequency of 17.7% in Caucasian and 15.5% in African American populations. We identified seven MGBA-derived candidate epitopes with high predicted binding scores for HLA-B7 using a computer algorithm. Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-B7 indicated that MGBA B7.3 (VSKTEYKEL), B7.6 (KLLMVLMLA), B7.7 (NPQVSKTEY), and B7.1 (YAGSGCPLL) have the highest HLA-B7 binding affinities. Further, two CD8+ CTL cell lines generated in vitro against T2.B7 cells individually loaded with MGBA-derived candidate epitopes showed significant cytotoxic activity against MGBA B7.1, B7.3, B7.6, and B7.7. In addition, the same CD8+ CTL lines lysed the HLA-B7+/MGBA+ human breast cancer cell line DU-4475 but had no significant cytotoxicity against HLA-B7− or MGBA− breast cancer cell lines. Cold-target inhibition studies strongly suggest that MGBA B7.3 is an immunodominant epitope. In summary, our results define HLA-B7-restriced, MGBA-derived, CD8+ CTL epitopes with all of the necessary features for developing novel vaccine strategies against HLA-B7 expressing breast cancer patients.
Vaccine; CD8 T cell; Mammaglobin-A
The IL-17 axis is implicated in pathogenesis of chronic rejection following human lung transplantation. Using a murine model of obliterative airway disease (OAD), we recently demonstrated that Abs to MHC class I antigens can induce immune responses to self-antigens that contributes to immunopathogenesis of chronic rejection. Using a murine model of OAD, we determined the role of IL-17 family members in induction of autoimmunity leading to OAD following ligation of MHC class I.
Anti-MHC class I or control antibodies (Abs) were administered intrabronchially to wild-type (WT) and IL-17a knock out (IL-17A−/−) C57BL/6.
By day 30, anti-MHC I administered endobronchially in IL-17A−/− mice demonstrated significant reduction in cellular infiltration, a 36.8% reduction in CD4+ T cells, 62.7% in CD11b+ macrophages, 37.5% in degree of fibrosis, 1.94 fold and 2.17 fold decrease in anti-KAT and anti-Col-V respectively when compared to wild type mice. Analysis of lung infiltrating cells in anti-MHC I WT revealed increase in IL-17A (KAT:92+21,Col-V:103+19spm) and IL-17F (KAT:5.03%,Col-V:2.75%) secreting CD4+ T cells. However, administration of anti-MHC I in IL-17A−/− demonstrated increase only in IL-17F for KAT (13.70%) and Col-V (7.08%). Anti-IL-17(A-F) mAb administration following anti-MHC I abrogated OAD in both WT and IL-17A−/−.
Our findings indicate that IL-17A and IL-17F secreted by CD4+Th17 cells specific to lung self-antigens are critical mediators of autoimmunity leading to the pathogenesis of OAD.
Organ transplantation, an accepted treatment for end stage organ failure, is often complicated by allograft rejection and disease recurrence. In this review we will discuss the potential role of microRNAs in allograft immunity especially leading to rejection of the transplanted organ. microRNAs (miRNAs), originally identified in C. elegans, are short non-coding 21–24 nucleotide sequences that bind to its complementary sequences in functional messenger RNAs and inhibits post-translational processes through RNA duplex formation resulting in gene silencing (Lau et al., 2001). Gene specific translational silencing by miRNAs regulates pathways for immune responses such as development of innate immunity, inflammation, T-cell and B-cell differentiation and signaling that are implicated in various stages of allograft rejection. miRNAs also play a role in development of post-transplant complicacies like fibrosis, cirrhosis, carcinogenesis often leading to graft loss and poor patient outcome. Recent advancements in the methods for detecting and quantifying miRNA in tissue biopsies, as well as in serum and urine samples, has led to identification of specific miRNA signatures in patients with allograft rejection and have been utilized to predict allograft status and survival. Therefore, miRNAs play a significant role in post-transplant events including allograft rejection, disease recurrence and tumor development impacting patient outcome.
Significant progress has been made in preventing acute allograft rejection following solid organ transplantation resulting in improved allograft survival. However, long term function still remains disappointing primarily due to chronic allograft rejection. Alloimmune responses primarily defined by the development of antibodies (Abs) to donor mismatched major histocompatibility antigens during the post-transplantation period have been strongly correlated to the development of chronic rejection. In addition, recent studies have demonstrated an important role for autoimmunity including the development of Abs to organ specific self-antigens in the pathogenesis of chronic allograft rejection. Based on this, a new paradigm has evolved indicating a possible cross-talk between the alloimmune responses and autoimmunity leading to chronic rejection. In this review, we will discuss the emerging concept for the role of cellular and humoral immune responses to self-antigens in the immunopathogenesis of chronic allograft rejection which has the potential to develop new strategies for the prevention and/or treatment of chronic rejection.
Alloimmunity; Autoimmunity; Chronic Rejection; Th17; regulatory T-cells and Organ transplantation
Role of non-complement activating antibodies (Abs) to mismatched donor HLA in pathogenesis of chronic lung rejection is not known. We utilized a murine model of obliterative airway disease (OAD) induced by Abs to MHC class I and serum from donor specific Abs (DSA) developed human lung transplantation (LTx) recipients (LTxR) to test the role of non-complement activating Abs in development of OAD and bronchiolitis obliterans syndrome (BOS).
Noncomplement activating anti-MHC (ncAbs) were administered intrabronchially in B.10 mice or in C3 knockout (C3KO) mice. Lungs were analyzed by histopathology. Lymphocytes secreting IL-17, IFN-γ or IL-10 to Collagen V (ColV) and K-alpha 1 tubulin (Kα1T) were enumerated by ELISpot. Serum Abs to ColV and Kα1T determined by ELISA. Cytokine and growth factor expression in lungs was determined by RTPCR. DSA from patients with BOS and control BOS-negative LTxR were analyzed by C1q assay.
Administration of ncAbs in B.10 mice or C3KO resulted in OAD lesions. There were significant increases in IL-17 and IFNγ secreting cells to ColV and Kα1T along with serum Abs to these antigens. There was also augmented expression of MCP-1, IL-6, IL-1β, VEGF, TGFβ, and FGF in ncAbs administered mice by day 3. Among LTxR with BOS only 1/5 had C1q binding DSA.
Complement activation by Abs to MHC class I is not required for development of OAD and human BOS. Therefore, anti-MHC binding to epithelial and endothelial cells can directly activate pro-fibrotic and pro-inflammatory cascades leading to immune response to self-antigens and chronic rejection.
Organ transplantation is the treatment of choice for patients with end-stage organ dysfunction. In spite of advances in understanding of donor and recipient physiology, organ preservation, operative techniques, and immunosuppression long term graft survival still remains a major problem primarily due to chronic rejection. Alloimmune responses to mismatched major histocompatibility antigens have been implicated as an important factor leading to rejection. However, there is increasing evidence pointing towards cross talk between the alloimmune and autoimmune responses creating a local inflammatory milieu which eventually leads to fibrosis and occlusion of the lumen in the transplanted organ i.e., chronic rejection. In this review, we will discuss recent studies and emerging concepts for the interdependence of alloimmune and autoimmune responses in the immunopathogenesis of chronic allograft rejection. The role of autoimmunity in the development of chronic rejection is an intriguing and exciting area of research in the field of solid-organ transplantation with significant potential to develop novel therapeutic strategies towards preventing chronic allograft rejection.
chronic rejection; TH17; regulatory T-cells; autoimmunity
We determined role of donor specific antibodies (DSA) and antibodies (Abs) to self-antigens, collagen-V (Col-V) and K-α1-Tubulin (KAT) in pathogenesis of acute antibody mediated rejection (AMR) and cardiac allograft vasculopathy (CAV) following human heart transplantation (HTx).
137 HTx recipients - 60 early period (≤ 12months) and 77 late period (> 12months) patients were enrolled. Circulating DSA was determined using LUMINEX. Abs against Col-I, II, IV, V and KAT were measured using ELISA. Frequency of CD4+T helper cells (CD4+Th) secreting IFN-γ, IL-5, IL-10 or IL-17 specific to self-antigens were determined using ELISPOT.
A significant association between AMR and DSA was demonstrated. Development of DSA in AMR patients correlated well with the development of auto-Abs to Col-V(AMR(+): 383±72μg/mL, AMR(−): 172±49μg/mL, p=0.033) and KAT (AMR(+): 252±49μg/mL, AMR(−): 61±21μg/mL, p=0.014). Patients who developed AMR demonstrated increased frequencies of CD4+Th secreting IFN-γ and IL-5 with reduction in IL-10 specific for Col-V/KAT. Patients diagnosed with CAV also developed DSA and auto-Abs to Col-V (CAV(+): 835±142μg/mL, CAV(−): 242±68μg/mL, p=0.025) and KAT (CAV(+): 768±206μg/mL, CAV(−): 196±72μg/mL, p=0.001) with increased frequencies of CD4+Th secreting IL-17 with reduction in IL-10 specific for Col-V/KAT.
Development of Abs to HLA and self-antigens are associated with increases in CD4+Th secreting IFN-γ and IL-5 in AMR and IL-17 in CAV, with reduction in CD4+Th secreting IL-10 in both AMR and CAV.
Self-antigens; cardiac transplantation; antibody mediated rejection; cardiac allograft vasculopathy
The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2 ± 0.2 kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30 mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98 kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9 Å. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence.
Potassium channel; voltage sensor domain; S4; fluorescence spectroscopy; resonance energy transfer; small unilamellar vesicles
Human breast cancer associated antigen, Mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as breast cancer vaccines. In this study, we define the CD8+ CTL response to Mam-A derived candidate epitopes presented in the context of HLA-A24 (A*2402). HLA-A24 has a frequency of 72% in Japanese, 27% in Asian-Indian and 18% in Caucasian populations. Using HLA-binding prediction algorithm we identified seven HLA-A24 restricted Mam-A-derived candidate epitopes (MAA24.1–7). Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-A2402 (T2.A24) indicated that MAA24.2 (CYAGSGCPL) and MAA24.4 (ETLSNVEVF) have the highest HLA-A24 binding affinity. Further, two CD8+ CTL cell lines generated in vitro against T2.A24 cells individually loaded with Mam-A-derived candidate epitopes showed significant cytotoxic activity against MAA24.2 and MAA24.4. In addition, the same CD8+ CTL lines lysed the HLA-A24+/Mam-A+ stable transfected human breast cancer cell line AU565 and MDA-MB-361. However, these CTLs had no cytotoxicity against HLA-A24−/Mam-A+ and HLA-A24+/Mam-A− breast cancer cell lines. In summary, our results define HLA-A24-restriced, Mam-A-derived, CD8+ CTL epitopes which can potentially be employed for Mam-A-based breast cancer vaccine therapy to breast cancer patients with HLA-A24 phenotype.
Vaccine; HLA-A24; CD8 T cell; Mammaglobin-A; Breast Cancer
Mammaglobin-A (Mam-A) is a 10 kD secretory protein that is overexpressed in 80% of primary and metastatic human breast cancers. Previous studies from our laboratory demonstrated that Mam-A cDNA vaccine can induce Mam-A specific CD8 T-cell responses and mediate regression of human breast cancer xenografts in NOD/SCID mice. In this report, we present our results on a phase I clinical trial of a Mam-A cDNA vaccination in breast cancer patients with stage-IV metastatic disease, including the impact of vaccination on the expression of the inducible co-stimulator molecule (ICOS) on CD4 T cells. Specimens from seven patients with stage IV metastatic cancer were available for these analyses. Patients were vaccinated with a Mam-A cDNA vaccine on days 0, 28 and 56, and immune responses were assessed at serial time points following vaccination. At six months following the first vaccination, flow cytometric analysis demonstrated a significant increase in the frequency of CD4+ICOShi T cells from 5 ± 2% pre-vaccination to 23 ± 4% (p<0.001), with a concomitant decrease in the frequency of CD4+Foxp3+ T cells (Treg) from 19 ± 6% to 10 ± 5% (p<0.05). ELISpot analysis of CD4+ICOShi sorted T-cells demonstrated that following vaccination the cytokines produced by Mam-A-specific T cells switched from IL-10 (78 ± 21 spm pre-vaccination to 32 ± 14 spm 5 months post-vaccine p<0.001) to IFN-γ (12 ± 6 spm pre-vaccination to 124 ± 31 spm 5 months post-vaccine p<0.001). The ratio of CD4+ICOShi T cells to CD4+Foxp3+ T cells increased from 0.37 ± 0.12 prior to vaccination to 2.3 ± 0.72 (p=0.021) following vaccination. Further, these activated CD4+ ICOShi T-cells induced preferential lysis of human breast cancer cells expressing Mam-A protein. We conclude that mammaglobin-A cDNA vaccination is associated with specific expansion and activation of CD4+ICOShi T cells, with a concomitant decrease in Treg frequency. These encouraging results strongly suggest that Mam-A cDNA vaccination can induce antitumor immunity in breast cancer patients.
DNA Vaccine; Mammaglobin-A; Breast Cancer; T-cells; ICOS
Alloimmune-induced immune responses to self-antigens are involved in the development of chronic lung allograft rejection. Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) have been shown to modulate autoimmune diseases. This study investigated the effect of modulation of the renin angiotensin aldosterone system (RAAS) a murine model of obliterative airways disease (OAD).
Major histocompatibility complex (MHC) class I antibodies were administered intrabronchially to C57Bl/6 mice on Days 1, 2, 3, and 6, and weekly thereafter. ACEI/ARB (10 mg/kg/day) were administered in water 5 days before antibody administration. Antibodies were analyzed by enzyme-linked immunosorbent assay, cytokines by Luminex, Th-frequency by enzyme-linked immunosorbent spot, and transcription factors by Western blotting and real-time polymerase chain reaction.
Significant decreases (50%–70%) in airway lesions and fibrous deposition were noted in lungs at Day 30 in the animals administered ACEI and ARB vs controls. Antibody concentrations to self-antigens also decreased from 14 ± 21 to 62 ± 18 μg/ml for collagen V and from 263 ± 43 to 84 ± 28 μg/ml for K-α1 tubulin. Th-precursor frequency and cytokine analysis showed increased interleukin (IL)-10 (3-fold increase) and decreased levels of IL-6 (3.4-fold) and IL-17 (4-fold decrease; p < 0.05) in ACEI and ARB groups. There was also messenger RNA level downregulation of tumor necrosis factor-α (8.6-fold) and p38/mitogen-activated protein (MAP)kinase (3.1-fold) in the treatment groups.
Our results demonstrate that modulation of RAAS leads to downregulation of IL-17 through tumor necrosis factor-α– dependant IL-6 through p38/MAPKinase pathway and thus abrogation of anti-MHC–induced OAD.
renin angiotensis aldosterone system; ACE inhibitors; obliterative airway disease; TNF-alpha; TH-17 pathway
Long term survival of the human lung allografts are hindered by chronic rejection, manifested clinically as bronchiolitis obliterans syndrome (BOS). We previously demonstrated significant correlation between the development of antibodies (Abs) to K-α1-tubulin (Kα1T) and BOS. In this study, we investigated the molecular basis for fibrinogenesis mediated by ligation of Kα1T expressed on airway epithelial cells by its specific Abs. Using RT-PCR we demonstrate that normal human bronchial epithelial (NHBE) cells upon ligation of Kα1T with specific Abs caused upregulation of pro-fibrotic growth factors. Western blot analysis of NHBE incubated with Kα1T Abs increased hypoxia inducible factor (HIF-1α). Kα1T Ab-mediated growth factor expression is dependent on HIF-1α as inhibition of HIF-1α returned fibrotic growth factor expression to basal levels. In conclusion, we propose that HIF-1α -mediated upregulation of fibrogenic growth factors induced by ligation of Kα1T Abs is critical for development of fibrosis leading to chronic rejection of lung allograft.
Hepatitis C (HCV) recurrence following orthotopic liver transplantation (OLT) is universal, often with accelerated allograft fibrosis. Donor liver steatosis is frequently encountered and often associated with poor early post-operative outcome. The study’s aim was to test the hypothesis that allograft steatosis alters immune responses to HCV and self-antigens promoting allograft fibrosis.
Forty-eight HCV OLT recipients (OLTr) were enrolled and classified based on amount of allograft macrovesicular steatosis at time of OLT. Group 1-No Steatosis (0–5% steatosis, n=21), Group 2 – Mild (5–35% - n=16), Group 3 – moderate (>35%, n=11). Cells secreting IL-17, IL-10, IFN-γ in response to HCV antigens were enumerated by ELISpot. Serum cytokines were measured by Luminex, antibodies (Abs) to Collagen (Col) I, II, III, IV, V by ELISA.
OLTr of moderate steatotic grafts had the highest incidence of advanced fibrosis in protocol one-year post-OLT biopsy (10.8% vs. 15.8% vs. 36.6%, r = 0.157, p<0.05). OLTr from Groups 2 and 3 had increased HCV specific IL-17 (p<0.05) and IL-10 (p<0.05) with reduced IFN-γ (p<0.05) secreting cells when compared to group 1. This was associated with increase in serum IL-17, IL-10, IL-1β, IL-6, IL-5 and decreased IFN-γ. In addition, there was development of Abs to Col I, II, III and V in OLTr with increased steatosis (p<0.05).
The results demonstrate that allograft steatosis influences post-OLT HCV specific immune responses leading to a IL-17 T-helper response and activation of humoral immune responses to liver associated self antigens which may contribute to allograft fibrosis and poor outcome.
Allograft Steatosis; Hepatitis C; Recurrence; Fibrosis; Liver Transplantation
Liver disease due to hepatitis C virus (HCV) infection is an important health problem worldwide. HCV induced changes in microRNAs (miRNA) are shown to mediate inflammation leading to liver fibrosis. Gene expression analyses identified dysregulation of miRNA-449a in HCV patients but not in alcoholic and non-alcoholic liver diseases. By sequence analysis of the promoter for YKL40, an inflammatory marker upregulated in patients with chronic liver diseases with fibrosis, adjacent binding sites for nuclear factor of Kappa B/P65 and CCAAT/enhancer-binding protein alpha (CEBPα) were identified. P65 interacted with CEBPα to co-operatively activate YKL40 expression through sequence specific DNA binding. In vitro analysis demonstrated that tumor necrosis factor alpha (TNFα) mediated YKL40 expression is regulated by miRNA-449a and its target NOTCH1 in human hepatocytes.NOTCH1 facilitated nuclear localization of P65 in response to TNFα. Further, HCV patients demonstrated upregulation of NOTCH1 along with downregulation of miRNA-449a. Taken together it is demonstrated that miRNA-449a plays an important role in modulating expression of YKL40 through targeting the components of the NOTCH signaling pathway following HCV infection. Therefore, defining transcriptional regulatory mechanisms which control inflammatory responses and fibrosis will be important towards developing strategies to prevent hepatic fibrosis especially following HCV recurrence in liver transplant recipients.
The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software.
The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1was significantly upregulated in steatotic liver following I/R.
Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.
Ischemia repurfusion injury; Two dimensional gel electrophoresis; Mass spectrometry; Liver transplantation; Chaperonins; Endoplasmic reticulum (ER) stress
Previous studies have shown that intrabronchial administration of antibodies (Abs) to MHC class I resulted in development of obliterative airway disease (OAD), a correlate of chronic human lung allograft rejection. Since development of Abs specific to mismatched donor HLA class II have also been associated with chronic human lung allograft rejection, we analyzed the role of Abs to MHC class II in inducing OAD. Administration of MHC class II Abs (M5/114) to C57BL/6 mice induced the classical features of OAD even though MHC class II expression is absent de novo on murine lung epithelial and endothelial cells. The induction of OAD was accompanied by enhanced cellular and humoral immune responses to self-antigens (Collagen V and K- α1Tubulin). Further, lung-infiltrating macrophages demonstrated a switch in their phenotype predominance from MΦ1 (F4/80+CD11c+) to MΦ2 (F4/80+CD206+) following administration of Abs and prior to development of OAD. Passive administration of macrophages harvested from animals with OAD but not from naïve animals induced OAD lesions. We conclude that MHC class II Abs induces a phenotype switch of lung infiltrating macrophages from MΦ1 (F4/80+CD11c+) to MΦ2 (F4/80+CD206+) resulting in the breakdown of self-tolerance along with an increase in autoimmune Th17 response leading to OAD.
Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality post lung transplantation (LTx). We sought to determine the relationship between alloimmune responses and autoimmunity, and subsequently how autoimmunity leads to chronic rejection.
We analyzed the development of donor specific antibodies (Abs) in LTx by flow PRA and the development of Abs to K-α1 tubulin (K-α1T) and collagen V (ColV) by ELISA. The frequency of K-α1T and ColV specific T cells that secrete IFN-γ, IL-17 and IL-10 in LTx recipients was measured by ELSIPOT.
In a retrospective analysis of 42 LTx recipients, we demonstrated a strong correlation between development of donor specific anti-HLA Abs, Abs to self-antigens, and BOS (p<0.05). To test the hypothesis that alloimmunity is related to an immune response to self-antigens, we analyzed 103 LTx patients prospectively for the development of donor specific Abs (DSA) and Abs to self-antigens. 42.7% of recipients developed DSA and 30.1% developed Abs to K-α1T and ColV. Development of DSA preceded development of Abs to self-antigens. BOS+ patients had higher frequency of T-cells secreting IL-17 (p<0.01) and IFNγ (p<0.05) with decreased IL-10 (p<0.05) compared to BOS- patients.
Based on these results we propose that alloimmune responses to donor HLA can induce autoimmune responses to airway epithelial self-antigens, characterized by activation of the IL-17 pathway. These immune responses to self-antigens along with alloimmunity contribute to the pathogenesis of BOS. Strategies to prevent development of autoimmunity may be important in preventing the development of chronic rejection.
Hepatic steatosis continues to present a major challenge in liver transplantation. These organs have been shown to have an increased susceptibility to cold ischemia and reperfusion (CIR) injury compared to otherwise comparable lean livers; the mechanisms governing this increased susceptibility to CIR injury are not fully understood. Endoplasmic reticulum (ER) stress is an important link between hepatic steatosis, insulin resistance and the metabolic syndrome. In this study, we investigated ER stress signaling and blockade in the mediation of CIR injury in severely steatotic rodent allografts. Steatotic allografts from genetically leptin-resistant rodents had increased ER stress responses and increased markers of hepatocellular injury following liver transplantation into strain-matched lean recipients. ER stress response components were decreased by the chemical chaperone, TUDCA, resulting in improvement of the allograft injury. TUDCA treatment decreased NF-κB activation, and the pro-inflammatory cytokines IL-6 and IL-1β. However, the predominant response was decreased expression of the ER stress cell death mediator, CHOP. Further, activation of the inflammation-associated caspase 11 was decreased linking ER Stress/CHOP to pro-inflammatory cytokine production following steatotic liver transplantation. These data confirm ER stress in steatotic allografts, and implicate this as a mediating mechanism of inflammation and hepatocyte death in the steatotic liver allograft.
Liver Transplantation; Endoplasmic Reticulum Stress; Hepatic Steatosis; Ischemia-Reperfusion Injury
This study aims to determine the role of antibodies (Abs) to donor mismatched HLA developed during the post-transplant period in inducing defensins and their synergistic role in the pathogenesis of chronic rejection, Bronchiolitis Obliterans Syndrome (BOS), following human lung transplantation (LTx).
Bronchoalveolar Lavage (BAL) and serum from twenty-one BOS+ LTx patients were assayed for β-defensins HNP1-3 (ELISA) and Anti-HLA Abs (Luminex). Human Airway Epithelial Cells (AEC) were treated with anti-HLA Abs, HNP-1/2 or both and the levels of β-defensin was measured by ELISA. Using a mouse model of obliterative airway disease induced by anti-MHC class-I Abs, we quantitatively and qualitatively determined neutrophil infiltration (Myeloperoxidase (MPO) staining) and activity (MPO assay) and defensin levels in the BAL.
In human LTx patients, higher defensin levels correlated with presence of circulating anti-HLA Abs (p<0.05). AEC treated with anti-HLA Abs or HNP-1/2, produced β-defensin with synergistic effects in combination (612±06 vs. 520±23 anti-HLA Ab or 590±10 pg/ml for HNP treatment, p<0.05).Neutrophil numbers (6 fold) and activity (5.5 folds) was higher in the lungs of mice treated with anti-MHC Abs compared to control. Two-fold increase in α-defensin and β-defensin levels was also present in BAL on day 5 following anti-MHC administrations.
Anti-HLA Abs developed during the post-transplant period and α-defensins stimulate β-defensin production by epithelial cells leading to increased cellular infiltration and inflammation. Chronic stimulation of epithelium by Abs to MHC and resulting increased levels of defensins induce growth factor production and epithelial proliferation contributing towards development of chronic rejection following LTx.
Human Neutrophil Peptide; Bronchiolitis Obliterans Syndrome; Bronchoalveolar Lavage; Human Beta Defensin 2; Airway Epithelial Cells
The role of donor specific antibodies (DSA) to mismatched human leukocyte antigen (HLA) and antibodies (Abs) to cardiac self-antigens myosin (MYO) and vimentin (VIM) in the pathogenesis of acute antibody mediated rejection (AMR) in the early posttranplant period (EP,<12months) and cardiac allograft vasculopathy (CAV) in the late posttransplant period (LP,>12months) following heart transplantation (HTx) was studied.
148 HTx recipients (65 EP, 83 LP) were enrolled in the study. Development of DSA was determined by LUMINEX. Circulating Abs against MYO and VIM in sera were measured using ELISA. Frequency of CD4+ T helper cells (CD4+TH) secreting IFN-γ, IL-17, IL-10 or IL-5 specific to either MYO or VIM were analyzed in vitro using ELISPOT assays.
AMR patients were more likely DSA positive (AMR−:15%, AMR+:70%, p=0.03) and demonstrated increased Abs to MYO (AMR−:144±115mcg/ml, AMR+:285±70 mcg/ml, p=0.033) and VIM (AMR−:37±19mcg/ml, AMR+:103±43mcg/ml, p=0.014). AMR patients demonstrated increased IL-5 CD4+TH specific to MYO (5.2±0.9fold, p=0.003) and VIM (7.3±2.9fold, p=0.004) and decreased IL-10 CD4+TH specific to MYO (2.2±0.4 fold, p=0.009) and VIM (1.7±0.2 fold, p=0.03). CAV patients were more likely DSA positive (CAV−: 25%, CAV+:79%, p=0.03) and demonstrated increased Abs to MYO (CAV−:191±120ug/ml, CAV+:550±98ug/ml, p=0.025) and VIM (CAV−: 55±25ug/ml, CAV+: 255±49ug/ml, p=0.001). CAV patients demonstrated increased IL-17 CD4+TH specific to MYO (10.5±7.3fold, p=0.002) and VIM (7.0±3.9 fold, p=0.003).
The presence of DSA in AMR and CAV is significantly associated with development of Abs to MYO and VIM in post-HTx patients. The induction of high CD4+TH specific to cardiac self-antigens that predominantly secrete IL-5 and IL-17 play a significant role in the development of Abs to self-antigens leading to AMR and CAV respectively.
Long term function of human lung allografts is hindered by development of chronic rejection manifested as Bronchiolitis Obliterans Syndrome (BOS). We have previously identified the development of antibodies (Abs) following lung transplantation to K-α1-tubulin (KAT), an epithelial surface gap junction cytoskeletal protein, in patients who develop BOS. However, the biochemical and molecular basis of the interactions and signaling cascades mediated by KAT Abs are yet to be defined. In this report, we investigated the biophysical basis of the epithelial cell membrane surface interaction between KAT and its specific Abs. Towards this, we analyzed the role of the lipid raft domains in the membrane interactions which lead to cell signaling and ultimately increased growth factor expression. Normal human bronchial epithelial (NHBE) cells, upon specific ligation with Abs to KAT obtained either from the serum of BOS(+) patients or monoclonal KAT Abs, resulted in upregulation of growth factors VEGF, PDGF, and bFGF( 6.4 ± 1.1, 3.2 ± 0.9, and 3.4 ± 1.1 fold increase, respectively) all of which are important in the pathogenesis of BOS. To define the role for lipid raft in augmenting surface interactions, we analyzed the changes in the growth factor expression pattern upon depletion and enrichment with lipid raft following the ligation of the epithelial cell membranes with Abs specific for KAT. NHBE cells cultured in the presence of β-methyl cyclodextran (βMCD) had significantly reduced growth factor expression (1.3 ± 0.3,vs βMCD untreated being 6.4 ± 1.1 fold increase) upon stimulation with KAT Abs. Depletion of cholesterol on NHBE cells upon treatment with βMCD also resulted in decreased partitioning of caveolin in the membrane fraction indicating a decrease in raft-domains. In conclusion, our results demonstrate an important role for lipid raft-mediated ligation of Abs to KAT on the epithelial cell membrane, which results in the upregulation of growth factor cascades involved in the pathogenesis of BOS following human lung transplantation.
Lipid rafts; K-α-tubulin; Chronic rejection; Lung Transplantation
Purpose of review
Recent studies demonstrate an increasing role for alloimmune responses in the disruption of self-tolerance leading to immune responses to self-antigens that play a role in the immunopathogenesis of chronic rejection following solid organ transplantation. This review summarizes recent studies and implications for the alloimmune-response-induced de-novo development of autoimmune responses following solid organ transplantations.
Immediately following organ transplantation, several factors lead to enduring an inflammatory milieu. Studies from our laboratory and others have demonstrated that development of antihuman leukocyte antigen antibodies precedes the development of chronic rejection. Using an in-vivo murine model, we have demonstrated that administration of anti-major histocompatibility complex (MHC) class I directly into the native lungs leads to chronic rejection pathology. Further, the in-vitro ligation of epithelial cell surface MHC class I molecules by specific anti-MHC can lead to cell activation and production of fibrinogenic growth factors.
On the basis of these findings, we hypothesized that alloimmune responses can lead to autoimmunity, thus playing an important role in chronic rejection. Characterization of both the temporal occurrence and functional significance of antibodies to self-antigens may provide insight into the pathogenesis of chronic rejection and these antibodies can serve as clinically useful biomarkers.
alloimmunity; antibody mediated rejection; autoimmunity; chronic rejection; organ transplantation