Yutaku Sakaguchi

Analysis of regulatory T cells and IgG4-positive plasma cells among patients of IgG4-related sclerosing cholangitis and autoimmune liver diseases

ObjectivesPatients with autoimmune pancreatitis (AIP) characteristically show elevated serum levels of immunoglobulin G4 (IgG4) and abundant infiltration of IgG4-positive plasmacytes in the involved organs. The most common involved organ showing extrapancreatic lesions is the bile duct, which exhibits sclerosing cholangitis (SC). However, the role of IgG4 in the development of IgG4-related SC (IgG4-SC) remains unclear. To clarify the role of IgG4 in IgG4-SC, we have performed an immunohistochemical analysis of the bile duct.MethodsLaboratory and immunohistochemical findings of liver biopsy specimens obtained from patients with IgG4-SC, primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), and primary biliary cirrhosis (PBC) were compared. The biopsy specimens were first stained with anti-IgG1, anti-IgG4, and anti-Foxp3 (forkhead box P3) antibodies, and the ratio of IgG4-, IgG1-, and Foxp3-positive cells, respectively, to infiltrated mononuclear cells (IgG4/Mono, IgG1/Mono, Foxp3/Mono) was assessed.ResultsThe ratio of IgG4/IgG1-positive plasma cells was significantly higher in specimens obtained from patients with IgG4-SC than in those from patients with PSC, AIH, and PBC. The Foxp3/Mono ratio in patients with PBC was significantly higher than that in patients with IgG4-SC and PSC. In patients with IgG4-SC, the number of Foxp3-positive cells was significantly correlated with the number of IgG4-positive cells; in the other patient groups, there was no correlation.ConclusionsThe IgG4/IgG1 ratio in the liver may be a useful marker for differential diagnosis of IgG4-SC and PSC. In IgG4-SC, abundant infiltration of regulatory T cells (Tregs) may affect the switching of B cells to IgG4-producing plasmacytes, and there is a possibility that the function of Tregs is different in IgG4-SC and other liver diseases (PSC, AIH, and PBC).

Subcutaneous adipose tissue–derived stem cells facilitate colonic mucosal recovery from 2,4,6‐trinitrobenzene sulfonic acid (TNBS)–induced colitis in rats

Background: Adipose tissue–derived stem cells (ADSCs) can be easily obtained from subcutaneous adipose tissue, and ADSCs can be demonstrated to display multilineage developmental plasticity. In this study, using TNBS‐induced colitis rats, we show the feasibility of repairing injured intestinal mucosa with adipose tissue–derived stem cells. Methods: The subcutaneous adipose tissue of F344 rats was obtained and digested by collagenase. The digested tissue was cultured in DMEM containing 10% FBS for 1 month. ADSCs were confirmed to differentiate under appropriate conditions into various lineages of cells, including bone, neural cells, adipocytes, and epithelial cells. HGF, VEGF, TGF‐&bgr;, and adiponectin in the culture supernatants of ADSCs were determined by ELISA. ADSCs (107 cells) were injected into the submucosa of the colon to examine their capacity to repair intestinal mucosa injured by TNBS. Results: In the experimental colitis model, the injection of ADSCs facilitated colonic mucosal repair and reduced the infiltration of inflammatory cells. High levels of HGF, VEGF, and adiponectin were detected in the culture supernatants of ADSCs. Moreover, injected ADSCs distributed to several layers of the colon, and some of them differentiated into mesodermal lineage cells. Conclusions: ADSCs can accelerate the regeneration of injured regions in experimental colitis. HGF, VEGF, and adiponectin might be responsible for the regeneration of injured regions in the colon.

Prevention of graft-versus-host disease by intra-bone marrow injection of donor T cells.

We have recently found that intra‐bone marrow‐bone marrow transplantation (IBM‐BMT) can be used to prevent graft‐versus‐host disease (GvHD), even when intensive donor lymphocyte infusion (DLI) is carried out. In the present study, in conjunction with IBM‐BMT, allogeneic splenic T cells as DLI were also injected into the bone marrow cavity of lethally irradiated (8.5 Gy) recipients. The extent of GvHD was compared with that of recipients that had received allogeneic IBM‐BMT plus i.v. injection of allogeneic T cells (intravenous DLI [IV‐DLI]). GvHD in recipients treated with allogeneic IBM‐BMT plus IBM‐DLI was far milder than in those treated with allogeneic IBM‐BMT plus IV‐DLI. This was confirmed macroscopically and histopathologically. The frequency of regulatory T cells (Tregs) detected as CD4+CD25+ and CD4+Foxp3+ cells was significantly higher in recipients treated with IBM‐BMT plus IBM‐DLI than in those treated with IBM‐BMT plus IV‐DLI. Donor‐derived helper T (Th) cells polarized to Th2 type in recipients treated with IBM‐BMT plus IBM‐DLI, whereas Th1 cells were dominant in recipients treated with IBM‐BMT plus IV‐DLI. Furthermore, the production of transforming growth factor‐β and hepatocyte growth factor from bone marrow stromal cells was enhanced after IBM‐DLI. Thus, IBM‐BMT plus IBM‐DLI seem to preferentially induce Tregs and Th2, resulting in the prevention of GvHD.

Induction of Senile Osteoporosis in Normal Mice by Intra‐Bone Marrow‐Bone Marrow Transplantation from Osteoporosis‐Prone Mice

A P6 substrain of the senescence accelerated mouse (SAMP6) spontaneously develops osteoporosis early in life. These mice show the clinical signs of osteoporosis, such as elevated levels of urinary deoxypyridinoline (Dpd), decreased bone mineral density (BMD), and a significant loss of trabecular and cortical bone thickness at 12 months of age. Here, we describe the transfer of osteoporosis to a normal strain by the injection of bone marrow cells from SAMP6 donors directly into the bone marrow cavity (intra‐bone marrow‐bone marrow transplantation [IBM‐BMT]). More than 1 month after IBM‐BMT, hematolymphoid cells were completely reconstituted by donor‐derived cells, and bone marrow stromal cells that could differentiate into osteocytes were also found to be of donor origin. In addition, the recipient C57BL/6 mouse showed the features of osteoporosis in the trabecular bone. Decreases in BMD and increases in urinary Dpd were also observed. When the message levels of cytokines (interleukin [IL]‐11, IL‐6, receptor activator of NF‐κB ligand [RANKL], osteoprotegerin, macrophage–colony‐stimulating factor, and insulin‐like growth factor‐1) were examined by reverse transcription‐polymerase chain reaction (RT‐PCR) and real‐time RT‐PCR analysis, IL‐6 and IL‐11 were reduced to a level similar to that in SAMP6 mice, whereas that of RANKL was increased. These findings indicate that not only the hemopoietic system but also the bone marrow microenvironment are reconstituted as a result of IBM‐BMT, and suggest that the development of senile osteoporosis might be attributable to “stem cell disorders.”

The role of CD19+CD24highCD38high and CD19+CD24highCD27+ regulatory B cells in patients with type 1 autoimmune pancreatitis

BACKGROUND Patients with type 1 autoimmune pancreatitis (AIP) have several immunologic and histologic abnormalities. It is known that depletion of B cells by rituximab is effective for treatment of IgG4-related disease (IgG4-RD) such as type 1 AIP, suggesting that B cells may be a key player in IgG4-RD. However, the role of regulatory B cells (Bregs) in type 1 AIP is unclear, and the objective of this paper is to clarify the role of Bregs in the pathophysiology of type 1 AIP by analyzing circulating Bregs. METHOD We recruited 21 patients with type 1 AIP as determined by the International Consensus Diagnostic Criteria for AIP (ICDC). No patients received corticosteroid treatments. For comparison, we recruited 14 patients with chronic pancreatitis (CP), 20 patients with pancreatic cancer, and 25 healthy subjects as controls. We analyzed Bregs as CD19+ CD24high CD38high and CD19+ CD24high CD27+ from peripheral blood by flow cytometry. RESULTS In peripheral blood, CD19+ CD24high CD38high Bregs were significantly increased in type 1 AIP patients compared with CP, pancreatic cancer, and healthy controls. Although not significant different, CD19+ CD24high CD27+ Bregs of type 1 AIP were decreased compared to those of other groups. IL-10(+) B cells were not significantly different from type 1 AIP patients and healthy controls. In untreated type 1 AIP patients, the number of CD19+ CD24high CD38high Bregs and IgG4 were not correlated. CONCLUSIONS Our data suggested that CD19+ CD24high CD38high Bregs seemed to increase reactively to suppress the disease activity, and are consistent with the hypothesis that CD19+ CD24high CD27+ Bregs might be involved in the development of type 1 AIP, although it still remains unclear whether the decrease of CD19+ CD24high CD27+ cells is cause or effect of AIP.

The Wistar Bonn Kobori rat, a unique animal model for autoimmune pancreatitis with extrapancreatic exocrinopathy

The male Wistar Bonn/Kobori (WBN/Kob) rat is known to be a unique animal model for chronic pancreatitis with widely distributed fibrosis and degeneration of parenchyma because of the infiltration of lymphocytes. In this report, we show that female (but not male) rats develop dacryoadenitis at 3 months of age, and that both male and female WBN/Kob rats develop sialoadenitis, thyroiditis, sclerotic cholangitis and tubulointerstitial nephritis over 18 months of age. The infiltration of CD8+ cells and the deposits of tissue‐specific IgG2b were observed in the injured pancreas and lachrymal glands. Furthermore, the number of regulatory T cells (defined as CD4+ Forkhead box P3+ cells) decreased in the periphery of both male and female WBN/Kob rats, suggesting that the onset of these diseases is attributable, at least, to the failure in the maintenance of peripheral immune tolerance. These features show clearly that WBN/Kob rats are a useful animal model for autoimmune pancreatitis and Sjøgren‐like syndrome or multi‐focal fibrosclerosis in humans. We also show that these autoimmune diseases can be prevented by a newly devised strategy of bone marrow transplantation (BMT) in which bone marrow cells are injected directly into the bone marrow cavity: intrabone marrow–BMT.

Long-term donor-specific tolerance in rat cardiac allografts by intrabone marrow injection of donor bone marrow cells.

Background. Donor-specific central tolerance in cardiac allograft can be induced by hematopoietic chimerism via conventional intravenous bone marrow transplantation (IV-BMT). However, there are problems with IV-BMT, such as the risk of graft failure and of the toxicity from conditioning regimens. Methods. A new method for heart transplantation is presented. This method consists of administration of fludarabine phosphate (50 mg/kg) and fractionated low-dose irradiation (3.5 Gy×2 or 4.0 Gy×2), followed by intrabone marrow injection of whole bone marrow cells (IBM-BMT) plus heterotopic heart transplantation. Results. Cardiac allografts with IBM-BMT were accepted and survived long-term (>10 months) showing neither acute rejection nor chronic rejection including cardiac allograft vasculopathy by such conditioning regimens. In contrast, cardiac allografts with conventional IV-BMT were rejected within 1 month after the treatment with irradiation of 3.5 Gy×2 or within 3 months after the treatment with irradiation of 4.0 Gy×2. Macrochimerism (>70%) was favorably established and stably maintained by IBM-BMT but not IV-BMT. Low levels of transient mixed chimerism (<7%) were induced by IV-BMT with fludarabine plus 4.0 Gy×2, but the chimerism was lost within 1 month after the treatment. Conclusions. These findings indicate that IBM-BMT is a feasible strategy for the induction of persistent donor-specific tolerance, enables the use of reduced radiation doses as conditioning regimens, and obviates the need for immunosuppressants.

Extensive Studies on Perfusion Method Plus Intra‐Bone Marrow‐Bone Marrow Transplantation Using Cynomolgus Monkeys

The collection of bone marrow cells (BMCs) using a perfusion method has been advantageous not only because of the low contamination of BMCs with T cells from the peripheral blood but also the enrichment of stromal cells, which support hemopoiesis. Before the application of this new method to humans, its safety needed to be confirmed using cynomolgus monkeys. We therefore performed the perfusion method on more than 100 cynomolgus monkeys using the long bones (such as the humerus and femur) and also the iliac bones (for human application); in the more than 150 trials to date, there have been no accidental deaths. Furthermore, the technical safety of a new method for the intra‐bone marrow (IBM) injection of BMCs (termed IBM‐bone marrow transplantation) has also been confirmed using 30 monkeys.

Establishment of animal models for three types of pancreatitis and analyses of regeneration mechanisms.

Objectives: To investigate the mechanisms underlying the onset and progress of pancreatitis, 3 animal models (chronic, acute, and severe pancreatitis) were established by double ligature of the pancreatic duct, injection with cerulein, or injection with cerulein + double ligature of the pancreatic duct. Methods: We prepared a control and 3 experimental groups: group 1 (untreated control), group 2 (a chronic pancreatitis model; the pancreatic tail was exposed through a midline incision, and the pancreatic duct from this part was double-ligated), group 3 (an acute pancreatitis model; cerulein was intraperitoneally injected 7 times on day 0), and group 4 (a severe pancreatitis model; the double ligature of the pancreatic duct plus injection of cerulein). Results: Kinetic observations of survival rate, relative pancreatic weight, and the macroscopical and microscopical diagnoses and observations of the changes in endocrine function clearly show that these 3 murine models of pancreatitis can serve as human models for chronic, acute, and severe pancreatitis. Furthermore, pancreas duodenum homeobox 1, cytokeratin 19, and Ki67 are expressed at the site of injury in the pancreas, resulting from the injection with cerulein and/or double ligature of the pancreatic ducts and indicating that there remains a tissue-regenerative capacity. Conclusions: These 3 mouse models could serve as human models for chronic, acute, and severe pancreatitis. Furthermore, cells of the epithelial lineage might participate in tissue regeneration in chronic, acute, and severe pancreatitis.

Regulatory T Cells in Type 1 Autoimmune Pancreatitis

Autoimmune pancreatitis (AIP) is a newly recognized pancreatic disorder. Recently, International Consensus Diagnostic Criteria for AIP (ICDC) was published. In this ICDC, AIP was classified into Type 1 and Type 2. Patients with Type 1 AIP have several immunologic and histologic abnormalities specific to the disease, including increased levels of serum IgG4 and storiform fibrosis with infiltration of lymphocytes and IgG4-positive plasmacytes in the involved organs. Among the involved organs showing extrapancreatic lesions, the bile duct is the most common, exhibiting sclerosing cholangitis (IgG4-SC). However, the role of IgG4 is unclear. Recently, it has been reported that regulatory T cells (Tregs) are involved in both the development of various autoimmune diseases and the shift of B cells toward IgG4, producing plasmacytes. Our study showed that Tregs were increased in the pancreas with Type 1 AIP and IgG4-SC compared with control. In the patients with Type 1 AIP and IgG4-SC, the numbers of infiltrated Tregs were significantly positively correlated with IgG4-positive plasma cells. In Type 1 AIP, inducible costimulatory molecule (ICOS)+ and IL-10+ Tregs significantly increased compared with control groups. Our data suggest that increased quantities of ICOS+ Tregs may influence IgG4 production via IL-10 in Type 1 AIP.