Gordon D. Wu

Migration of mesenchymal stem cells to heart allografts during chronic rejection

Background. Mesenchymal stem cells (MSC) are pluripotent progenitors for a variety of cell types, including fibroblasts and muscle cells. Their involvement in the tissue repair of allografts during the development of chronic rejection has been hypothesized, but not yet substantiated, by experimental evidence. Methods. Rat MSC were isolated from circulation using an aortic pouch allograft as a trapping device. The plasticity of these cells was examined in differentiation cultures. One of the resulting MSC lines was immortalized and transduced to express a marker lacz gene. The lacz-labeled cells were then transferred to F344 rats bearing Lewis (LEW) cardiac allografts to measure their localization and contribution to graft tissue repair. Results. The MSC isolated from circulation exhibited multipotential for differentiation in culture, developing into various lineages including osteoblasts, lipocytes, chondrocytes, myotubes, and fibroblasts. Intravenous engraftment of the lacz-labeled cells into recipients of heart transplant resulted in migration of the &bgr;-gal+ cells into the lesions of chronic rejection in the cardiac grafts and homing of the cells to the bone marrow. The majority of &bgr;-gal+ cells present in the allografts exhibited fibroblast phenotypes, and a small number of the cells expressed desmin, indicative of myocyte differentiation. Conclusion. MSC vigorously migrated into the site of allograft rejection. This data suggests that they may be attracted to this site to actively participate in tissue repair during chronic rejection. In addition, given the robust migration, the inhibition of MSC differentiation toward fibroblast progeny and induction toward the myocyte lineage may serve as a new strategy for treatment of chronic rejection and allograft tissue repair.

Vascular endothelial cell apoptosis induced by anti-donor non-MHC antibodies: a possible injury pathway contributing to chronic allograft rejection

BACKGROUND Non-major histocompatibility complex (non-MHC) alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized. METHODS The kinetics of alloantibody production and the mechanism by which non-MHC alloantibodies cause graft injury were investigated in a Lewis-to-Fischer 344 (LEW-to-F344) rat model of cardiac transplantation. RESULTS Flow cytometry detected that all the F344 recipients of LEW allografts produced anti-donor immunoglobulin G (IgG) antibodies reactive with LEW lymphocytes and endothelial cells. A sub-group of recipients that rejected their grafts in 30 to 60 days exhibited markedly increased levels of anti-donor IgG antibodies (n = 6, mean fluorescence intensity [MFI]:23.85 +/- 2.7) than recipients with long-surviving allografts (n = 4, MFI:11.23 +/- 0.81; p = 0.00058). Passive transfer of anti-donor sera induced chronic rejection of LEW heart allografts in an immune non-responsiveness model of F344 rats induced by intrathymic inoculation of donor-specific lymphocytes. Immunoglobulin G antibodies purified from the anti-LEW sera exhibited complement-dependent cytotoxicity against LEW vascular endothelial cells in flow-cytometric cytotoxicity assay. The targeted endothelial cells displayed early (annexin V+) and late (TUNEL+) evidence for programmed cell death. Western blot analysis of poly (ADP-ribose) polymerase (PARP) demonstrated that the 25-kD PARP-cleavage fragment was present at the lysates of the vascular endothelial cells treated with anti-donor IgG antibodies, indicating apoptosis-associated caspase activity in these cells. In situ teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining demonstrated that vascular endothelial cell apoptosis was consistently present in all LEW heart allografts with chronic rejection. CONCLUSIONS Non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury through an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.

Endothelial Cells in Co-culture Enhance Embryonic Stem Cell Differentiation to Pancreatic Progenitors and Insulin-Producing Cells through BMP Signaling

Endothelial cells (ECs) represent the major component of the embryonic pancreatic niche and play a key role in the differentiation of insulin-producing β cells in vivo. However, it is unknown if ECs promote such differentiation in vitro. We investigated whether interaction of ECs with mouse embryoid bodies (EBs) in culture promotes differentiation of pancreatic progenitors and insulin-producing cells and the mechanisms involved. We developed a co-culture system of mouse EBs and human microvascular ECs (HMECs). An increase in the expression of the pancreatic markers PDX-1, Ngn3, Nkx6.1, proinsulin, GLUT-2, and Ptf1a was observed at the interface between EBs and ECs (EB-EC). No expression of these markers was found at the periphery of EBs cultured without ECs or those co-cultured with mouse embryonic fibroblasts (MEFs). At EB-EC interface, proinsulin and Nkx6.1 positive cells co-expressed phospho-Smad1/5/8 (pSmad1/5/8). Therefore, EBs were treated with HMEC conditioned media (HMEC-CM) suspecting soluble factors involved in bone morphogenetic protein (BMP) pathway activation. Upregulation of PDX-1, Ngn3, Nkx6.1, insulin-1, insulin-2, amylin, SUR1, GKS, and amylase as well as down-regulation of SST were detected in treated EBs. In addition, higher expression of BMP-2/-4 and their receptor (BMPR1A) were also found in these EBs. Recombinant human BMP-2 (rhBMP-2) mimicked the effects of the HMEC-CM on EBs. Noggin (NOG), a BMP antagonist, partially inhibited these effects. These results indicate that the differentiation of EBs to pancreatic progenitors and insulin-producing cells can be enhanced by ECs in vitro and that BMP pathway activation is central to this process.

Characteristics of immunoglobulin gene usage of the xenoantibody binding to gal-alpha(1,3)gal target antigens in the gal knockout mouse.

BACKGROUND Natural antibodies that react with galactose-alpha(1,3)galactose [galalpha(1,3)gal] carbohydrate epitopes exist in humans and Old World primates because of the inactivation of the alpha1,3-galactosyltransferase (alpha1,3GT) gene in these species and the subsequent production of antibodies to environmental microbes that express the galalpha(1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-galalpha(1,3)gal antibodies and can be used to study the genetic control of humoral immune responses to this carbohydrate epitope. METHODS Six hybridomas that produce monoclonal antibodies (mAbs) to galalpha(1,3)gal were generated in Gal o/o mice. The mAbs were tested to characterize the binding activity with flow cytometry using pig aortic endothelial cells and ELISA with galalpha(1,3)gal carbohydrates. The VH and VK genes of these hybridomas were cloned, sequenced, and analyzed. RESULTS The mAbs showed distinct patterns of antibody binding to galalpha(1,3)gal antigens. The VH genes that encode the mAb binding activity were restricted to a small number of genes expressed in their germline configuration. Four of six clones used closely related progeny of the same VH germline gene (VH441). Comparison of the mouse gene VH441 to the human gene IGHV3-11, a gene that encodes antibody activity to galalpha(1,3)gal in humans, demonstrates that these two genes share a nonrandom distribution of amino acids used at canonical binding sites within the variable regions (complimentary determining regions 1 and 2) of their immunoglobulin VH genes. CONCLUSIONS These results demonstrate the similarity of the Gal o/o mice and humans in their immune response to galalpha(1,3)gal epitopes. Gal o/o mouse can serve as a useful model for examining the genetic control of antibody/antigen interactions associated with the humoral response to pig xenografts in humans.

Anti-interleukin 6 receptor antibodies attenuate antibody recall responses in a mouse model of allosensitization.

Background Interleukin (IL)-6 is a regulatory cytokine for T helper type 17 (Th17) and Treg cells and a potent stimulus for B/plasma cells. The current study evaluated the effect of IL-6 receptor (IL-6R) blockade with an anti–IL-6R monoclonal (mMR16-1) in alloantibody recall responses. Methods A mouse model of human leukocyte antigen (HLA).A2 sensitization was used for studies to evaluate the efficacy of anti–IL-6R on alloantibody recall responses and to examine the impact of IL-6R blockade on Th17, Treg, follicular T helper (Tfh) and plasma cells using multiparameter flow cytometry, flow antibody binding, and enzyme-linked immunospot (ELISpot) assay. Results Re-exposure of C57BL/6 mice to HLA.A2+ skin allografts resulted in a surge of donor-specific (anti–HLA.A2) immunoglobulin (Ig)G antibodies. Anti–IL-6R treatment significantly decreased but did not eliminate alloantibody responses (IgG mean fluorescence intensity, 486 ± 153 vs. control 792 ± 193, P = 0.0076). Flow cytometry analysis showed that anti–IL-6R treatment resulted in reduction of IL-21+CD4+ (Th17) cells (P = 0.006 vs. control) and CXCR5+CD4+ Tfh cells (P = 0.04), but increased foxp3+CD4+ (Treg) cells in the CD4+ population (P = 0.04 vs. control). The IgG ELISpot experiments showed a significant reduction of IgG spots in the bone marrow and the spleen cells from the anti–IL-6R–treated mice. In vitro treatment of mouse hybridoma (PA2.1) cultures with anti–IL-6R decreased IgG spot formation but had limited effect on cell proliferation. Conclusion The data indicate that anti–IL-6R therapy attenuates alloantibody recall responses by modulating a number of immune regulatory and effector cells, including Th17, Tfh, Treg, and importantly, the long-lived plasma cells in the bone marrow.

Interaction of CD44 and hyaluronic acid enhances biliary epithelial proliferation in cholestatic livers

Biliary epithelia express high levels of CD44 in hepatobiliary diseases. The role of CD44-hyaluronic acid interaction in biliary pathology, however, is unclear. A rat model of hepatic cholestasis induced by bile duct ligation was employed for characterization of hepatic CD44 expression and extracellular hyaluronan distribution. Cell culture experiments were employed to determine whether hyaluronan can regulate cholangiocyte growth through interacting with adhesion molecule CD44. Biliary epithelial cells were found to express the highest level of CD44 mRNA among four major types of nonparenchymal liver cells, including Kupffer, hepatic stellate, and liver sinusoidal endothelial cells isolated from cholestatic livers. CD44-positive biliary epithelia lining the intrahepatic bile ducts were geographically associated with extracellular hyaluronan accumulated in the portal tracts of the livers, suggesting a role for CD44 and hyaluronan in the development of biliary proliferation. Cellular proliferation assays demonstrated that cholangiocyte propagation was accelerated by hyaluronan treatment and antagonized by small interfering RNA CD44 or anti-CD44 antibody. The study provides compelling evidence to suggest that proliferative biliary epithelia lining the intrahepatic bile ducts are a prime source of hepatic CD44. CD44-hyaluronan interaction, by enhancing biliary proliferation, may play a pathogenic role in the development of cholestatic liver diseases.

Evidence for recipient derived fibroblast recruitment and activation during the development of chronic cardiac allograft rejection.

Background. Allograft fibrosis is a prominent feature of chronic rejection. Although intragraft fibroblasts contribute to this process, their origin and exact role remain poorly understood. Methods. Using a rat model of chronic rejection, LEW to F344, cardiac fibroblasts were isolated at the point of rejection and examined in a collagen gel contraction assay to measure fibroblast activation. The allograft microenvironment was examined using immunohistochemistry for fibrogenic markers (transforming growth factor [TGF]-&bgr;, platelet-derived growth factor [PDGF], tissue plasminogen activator [TPA], plasminogen activator inhibitor [PAI]-1, matrix metalloproteinase [MMP]-2, and tissue inhibitor of matrix metalloproteinase [TIMP]-2). The origin of intragraft fibroblasts was studied using female to male allografts followed by polymerase chain reaction [PCR] and in situ hybridization for the male sry gene. Results. The cardiac fibroblasts isolated from allografts with chronic rejection exhibited higher gel contractibility (50.9% ± 6.1% and 68.2% ± 3.8% at 4 and 24 hr) compared with naive cardiac fibroblasts (30.7% ± 3.5% and 55.3% ± 6.6% at 4 and 24 hr; P <0.05 and <0.05, respectively). Immunostaining for TGF-&bgr;, PDGF, TPA, PAI-1, MMP-2 and TIMP-2 was observed in all allografts at the time of rejection. In situ hybridization demonstrated the presence of sry positive cells in female allografts rejected by male recipients. Sixty-five percent of fibroblast colonies (55 of 85) isolated from female heart allografts expressed the male sry gene. Conclusion. Cardiac fibroblasts are activated and exist in a profibrogenic microenvironment in allografts undergoing chronic rejection. A substantial proportion of intragraft fibroblasts are recruited from allograft recipients in this experimental model of chronic cardiac allograft rejection.

Monoclonal anti-interleukin-6 receptor antibody attenuates donor-specific antibody responses in a mouse model of allosensitization

Interleukin 6 is an immune regulatory cytokine that impacts the development and maturation of T-cell, B-cell, and antibody producing plasma cells. A monoclonal antibody to the IL-6R (Tocilizumab®) was recently approved by the FDA for treatment of rheumatoid arthritis. Although anti-IL-6R anitbodies can reduce autoantibody levels in human disease, the use of anti-IL-6R for alloantibody suppression has not been examined. Here, we report on our experience with a mousenized rat-anti-mouse IL-6R (mMR16-1) for attenuating donor-specific antibody (DSA) responses. C57BL/6 mice were sensitized with skin allografts from a HLA.A2 transgenic mouse, and treated with intraperitoneal injections of mMR16-1 or control antibody. DSA responses were monitored weekly for 5weeks by measurement of serum anti-HLA.A2 antibodies in a flow cytometric antibody binding assay. Results show that mMR16-1 significantly reduced DSA IgM, IgG2a and IgG1 responses, respectively, while normalizing serum amyloid A (SAA), an acute phase reactant induced by IL-6 (p<0.01 vs. control). mMR16-1 injections increased mononuclear cell apoptosis in the spleens, as detected by annexin V staining and TUNEL. In conclusion, anti-IL6R attenuates de novo DSA responses and suppresses inflammatory markers (SAA). The data indicate that antibody therapy targeting the IL-6/IL-6R pathway may serve as a strategy to suppress DSA generation.

Interleukin-6, A Cytokine Critical to Mediation of Inflammation, Autoimmunity and Allograft Rejection: Therapeutic Implications of IL-6 Receptor Blockade

The success of kidney transplants is limited by the lack of robust improvements in long-term survival. It is now recognized that alloimmune responses are responsible for the majority of allograft failures. Development of novel therapies to decrease allosensitization is critical. The lack of new drug development in kidney transplantation necessitated repurposing drugs initially developed in oncology and autoimmunity. Among these is tocilizumab (anti-IL-6 receptor [IL-6R]) which holds promise for modulating multiple immune pathways responsible for allograft injury and loss. Interleukin-6 is a cytokine critical to proinflammatory and immune regulatory cascades. Emerging data have identified important roles for IL-6 in innate immune responses and adaptive immunity. Excessive IL-6 production is associated with activation of T-helper 17 cell and inhibition of regulatory T cell with attendant inflammation. Plasmablast production of IL-6 is critical for initiation of T follicular helper cells and production of high-affinity IgG. Tocilizumab is the first-in-class drug developed to treat diseases mediated by IL-6. Data are emerging from animal and human studies indicating a critical role for IL-6 in mediation of cell-mediated rejection, antibody-mediated rejection, and chronic allograft vasculopathy. This suggests that anti-IL-6/IL-6R blockade could be effective in modifying T- and B-cell responses to allografts. Initial data from our group suggest anti-IL-6R therapy is of value in desensitization and prevention and treatment of antibody-mediated rejection. In addition, human trials have shown benefits in treatment of graft versus host disease in matched or mismatched stem cell transplants. Here, we explore the biology of IL-6/IL-6R interactions and the evidence for an important role of IL-6 in mediating allograft rejection.

Anti-CD20 antibody suppresses anti-HLA antibody formation in a HLA-A2 transgenic mouse model of sensitization.

BACKGROUND B cell depletion by anti-CD20 antibody is used in desensitization protocols and for treatment of antibody-mediated rejection (AMR). However, little is known about the efficacy and the mechanism(s) of action. METHODS A mouse model of HLA sensitization was used to study the effectiveness of anti-CD20 treatment on B cell depletion and anti-HLA antibody suppression. RESULTS Immunization of C57BL/6 mice with skin grafts from a transgenic C57BL.Tg/HLA-A2.1 mouse resulted in robust production of anti-HLA IgM and IgG antibodies, and accelerated rejection of a secondary skin allograft (within 3 days) featured by intragraft IgG and C4d deposition. Both IgM and IgG alloantibodies are specific to HLA-A2 as well as to a panel of class I HLA, including A1, A3, A25, A26, A29, and A30. These alloantibodies were complement-dependently cytotoxic (CDC) against HLA-A2 expressing target cells. Administration of 2 doses of a mouse-anti-mouse CD20 monoclonal antibody significantly reduced the levels of anti-HLA IgG2a antibodies, suppressed serum CDC, and prolonged survival of the secondary skin allografts. Suppression of anti-HLA IgG antibodies was associated with significant depletion of B220(+)/CD5(-) B cells from the blood, the spleen and the bone marrow of the treated animals. CONCLUSION Anti-CD20 treatment effectively depletes B220(+)/CD5(-) B cells, resulting in potent suppression of anti-HLA IgG and prolongation of skin graft survival. The data are in support for the use of anti-CD20 antibodies in highly-HLA sensitized patients undergoing desensitization and for the treatment of AMR.