Galectin-4 Interaction with CD14 Triggers the Differentiation of Monocytes into Macrophage-like Cells via the MAPK Signaling Pathway

Galectin-4 (Gal-4) is a β-galactoside-binding protein mostly expressed in the gastrointestinal tract of animals. Although intensive functional studies have been done for other galectin isoforms, the immunoregulatory function of Gal-4 still remains ambiguous. Here, we demonstrated that Gal-4 could bind to CD14 on monocytes and induce their differentiation into macrophage-like cells through the MAPK signaling pathway. Gal-4 induced the phenotypic changes on monocytes by altering the expression of various surface molecules, and induced functional changes such as increased cytokine production and matrix metalloproteinase expression and reduced phagocytic capacity. Concomitant with these changes, Gal-4-treated monocytes became adherent and showed elongated morphology with higher expression of macrophage markers. Notably, we found that Gal-4 interacted with CD14 and activated the MAPK signaling cascade. Therefore, these findings suggest that Gal-4 may exert the immunoregulatory functions through the activation and differentiation of monocytes.

Increase in Anti-Gal IgM Level is Associated With Early Graft Failure in Intraportal Porcine Islet Xenotransplantation

BACKGROUND: Anti-Gal is a major antibody induced in non-human primates (NHPs) after xenotransplantation. To understand the mechanism of graft rejection, we investigated the association between anti-Gal responses and graft failure in NHP recipients of porcine islet transplantation (PITx). METHODS: Intraportal PITx was performed in 35 diabetic NHPs, and graft function was monitored. Early graft failure (EGF) was defined as loss of graft function within a month after PITx. Seven, 19, nine NHPs received immunosuppression (IS) without CD40 pathway blockade (Group I), with anti-CD154 (Group II), and with anti-CD40 (Group III), respectively. The anti-Gal levels on day 0 and day 7 of PITx were measured by ELISA. RESULTS: The frequency of EGF was significantly lower in Group II (26.3%) than in Group I (100%, P=0.0012) and Group III (77.8%, P=0.0166). While levels of anti-Gal IgG in Group I and anti-Gal IgM in Group III increased on day 7 compared with day 0 (P=0.0156 and 0.0273), there was no increase in either on day 7 in Group II. The ratio of anti-Gal IgM or IgG level on day 7 to that on day 0 (Ratio7/0) was significantly higher in recipients with EGF than without EGF (P=0.0009 and 0.0027). ROC curve analysis of anti-Gal IgM Ratio7/0 revealed an area under the curve of 0.789 (P=0.0003). CONCLUSIONS: IS with anti-CD154 suppressed anti-Gal responses and prevented EGF in PITx. Anti-Gal IgM Ratio7/0, being associated with EGF, is a predictive marker for EGF.

Erratum: Correction of the Grant Number

We found an error in our published article.

Erratum: Correction of the Grant Number

We found an error in our published article.

Role of Regulatory T Cells in Transferable Immunological Tolerance to Bone Marrow Donor in Murine Mixed Chimerism Model

Constructing a bone marrow chimera prior to graft transplantation can induce donor-specific immune tolerance. Mixed chimerism containing hematopoietic cells of both recipient- and donor-origin has advantages attributed from low dose of total body irradiation. In this study, we explored the mechanism of mixed chimerism supplemented with depletion of Natural Killer cells. Mixed chimerism with C57BL/6 bone marrow cells was induced in recipient BALB/c mice which were given 450 cGy of gamma-ray irradiation (n = 16). As revealed by reduced proliferation and cytokine production in mixed leukocyte reaction and ELISpot assay (24.6 vs 265.5), the allo-immune response to bone marrow donor was reduced. Furthermore, the induction of transferable immunological tolerance was confirmed by adoptive transfer and subsequent acceptance of C57BL/6 skin graft (n = 4). CD4+FoxP3+ regulatory T cells were increased in the recipient compartment of the mixed chimera (19.2% --> 33.8%). This suggests that regulatory T cells may be therapeutically used for the induction of graft-specific tolerance by mixed chimerism.

Application of the Multiplex Cytokine Analysis to Monitor Xenogeneic Immune Responses to the Porcine Islet Graft in Non-Human Primate

Non-human primate studies must be conducted prior to the clinical trial of xenotransplantation. In order to develop clinically applicable immune-modulatory regimen through non-human primate studies, close monitoring of xenogeneic immune responses is required. We adopted multiplex cytokine analysis in assessment of the immune status during the course of pig-to-non-human primate islet transplantation. This study aimed to assess the feasibility of this multiplex cytokine assay in the development of immune-modulatory regimen. Using this assay, we were able to detect different cytokines with a minimal usage of blood samples, and this allowed us to detect various immunological situations in the recipients. Detection of TNF-alpha surge (347.8 pg/mL) guided us to block TNF-alpha in the early phase of transplantation. Supportive information for in vivo efficacy of cytokine neutralizing antibody could be speculated by in vitro neutralization assay (1,250 pg/mL --> 0 pg/mL). In addition, periodic monitoring of cytokines in peripheral blood allowed the detection of the infection episode prior to other routine assays. These benefits of multiplex cytokine assay may be generally applied to other pre-clinical research, which is a prerequisite for clinical trials.

Functional Characteristics of C-terminal Lysine to Cysteine Mutant Form of CTLA-4Ig

CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation.

Current Status and Future Perspectives of Xenotransplantation / 대한이식학회지

Xenotransplantation using pigs as the transplant source holds great promise to resolve the severe shortage of human organ donors. Although stem-cell-derived organ and tissue regeneration have a potential to solve this as well for the future, it still remains as very early experimental phase. Likewise, artificial organs and mechanical devices have been simply used for bridge therapy to transplant. Therefore, xenotransplantation might provide the most imminent solution to the scarcity of human organ donors. In the last two decades, major progress has been made in understanding the mechanisms of xenografts rejection, zoonotic infections including porcine endogenous retrovirus (PERV) and production of genetically engineered pigs including alpha1,3-galactosyltransferase-deficient pigs. With these elaborations, it is now on the threshold of first clinical application. Particularly promising first target is porcine pancreatic islet xenotransplantation. Graft survival has been prolonged to almost one year in the non-human primate study and is waiting for the development of relatively non-toxic or clinically applicable immunosuppressive or tolerance-inducing regimens. This review highlights the currently known obstacles to translate xenotransplantation into clinical therapies and the possible strategies to overcome these hurdles, as well as current status and future perspective for clinical xenotransplantaion.

Isolation of ethanol-induced genes in pancreatic beta-cells by representational difference analysis (RDA)

Recent epidemiological studies suggest that alcohol consumption is one of the risk factors leading to type 2 diabetes, but the direct effect of ethanol on beta-cell gene expression is not known. Here, using cDNA RDA method, we isolated 43 ethanol-induced genes in pancreatic beta-cells, and confirmed their differential expression by Northern blot or semi-quantitative RT-PCR. These genes were further categorized by the functional criteria based on the published data; Translation, Transcription, Metabolism, Signal transduction, Transport, Structure, Cytoskeleton, Regulation, or Putative/Unknown genes. The effects of each gene on beta-cell function need to be further investigated, however, the present data strongly suggest that these genes might be related to the metabolic alterations caused by ethanol as indicated in earlier study. In particular, RPS3 gene expression was increased by ethanol, glucosamine, and cytokines, implying that ethanol might decrease the metabolic activity by oxidative stress in beta-cells. Therefore, cloning of these genes in full-length and the detailed studies of each gene on beta-cell functions might provide clues on the pathophysiology of type 2 diabetes caused by alcohol.

Human beta-defensin 2 is induced by interleukin-1b in the cornealepithelial cells

Mammalian epithelia produce the various antimicrobial peptides against the bacterial or viral infection, thereby acting as the active immune modulators in the innate immunity. In this study, we examined the effects of the various proinflammatory cytokines or LPS on cell viability and antimicrobial beta-defensin gene expressions in human corneal epithelial cells. Results showed that the cytokines or LPS did not exert severe cytotoxic effects on the cells, and that beta-defensin 1 was constitutively expressed, while beta-defensin 2 was specifically induced by IL-1beta, supporting the idea that these cytokines or LPS involve the defense mechanism in the cornea. Furthermore, the reporter and gel shift assay to define the induction mechanism of beta-defensin 2 by IL-1beta demonstrated that the most proximal NF-kB site on the promoter region of beta-defensin 2 was not critical for the process. Data obtained from the normal or patients with the varying ocular diseases showed that our in vitro results were relevant in the clinical settings. Our results clearly demonstrated that beta-defensin 1 and 2 are important antimicrobial peptides in the corneal tissues, and that the mechanistic induction process of beta-defensin 2 by IL-1beta is not solely dependent on proximal NF-kB site activation, thus suggesting that the long distal portion of the promoter is needed for the full responsiveness toward IL-1beta.