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Glycoprotein 96 (gp96) is an endoplasmic reticulum (ER)-resident heat shock protein. It controls the folding of nascent membrane-spanning and secretory proteins, participates in stress-induced unfolded protein response (UPR) and in pathways leading to proteolysis of damaged proteins through ER-associated degradation pathways and chaperone-mediated autophagy. In addition, gp96 controls the steroid biosynthesis and Ca2+ homeostasis and participates in insulin-IGF/signaling pathways. Besides, owing to its peptide chaperone capacity and ability to interact with antigen-presenting cells, gp96 has been implicated in priming of innate and adaptive immunity. In an attempt to visualize the intensity of ER-stress in thymus and possible participation of gp96 in generation of auto-reactive T cell clones that were detected in regenerating liver, in this study we investigated the dynamics of gp96 expression in partially hepatectomized (pHx) and sham Hx mice. Simultaneously, we detected the thymic expression of receptors responsible for endocytosis of gp96- chaperoned peptides (CD91) and intracellular activation of ER-stress pathways (TLR2), as well as the expression of TGF-β and the distribution of CD4+CD25+FoxP3+ cells. The data have shown that both pHx and sham Hx induced an accelerated apoptosis and hypoplasia in thymus. Partial Hx induced, however, a higher expression of gp96, the translocation of the CD91, TLR2 and TGF-β immunostaining from medulla to cortex and an appearance of Treg cells. The data show that pHx triggers in thymus the ERstress and UPR response and suggest that gp96 participates in the generation of natural Treg cells, which might be involved in the control of liver regeneration in the periphery.

Background: Heat shock protein gp96, has been reported to play important role in the endoplasmic reticulum stress response, acting as a molecular chaperone in the unfolded protein response. Additionally, owing to it’s ability to bind protein and nonprotein molecules with exposed hydrophobic residua, it’s function in antigen presentation and cross-presentation, activation of macrophages and lymphocytes, and activation and maturation of dendritic cells has been also proposed Material and methods: In this study we analyzed the expression of gp96 in the regenerating liver, thymus and spleen after 1/3 partial hepatectomy (pHx) in C57/Bl6 mice. The data, obtained by immunohistochemistry and quantitative real-time PCR, were correlated with phenotype and cytotoxicity of hepatic and splenic mononuclear lymphatic cells against NKT and NK-sensitive targets, as well as with the intensity of apoptosis in the liver and thymus. Results: High time-dependent changes were found in the expression of gp96 protein and mRNA in regenerating liver, thymus, and spleen. Simultaneously, in the liver and thymus augmented the proportion of autoreactive NKT and regulatory T cells, increased the cytotoxicity against the syngeneic thymocytes and augmented the apoptosis in the liver and thymus. Additionally, in thymus and spleen a marked upregulation of TLR2 was noticed. Conclusions: The data suggest that after tissue damages and disturbance of morphostasis gp96 may serve as a natural adjuvant for chaperoning antigenic self peptides and nonprotein molecules into the immune surveillance pathways, resulting in activation of autoreactive NKT and Treg cells that might participate in reestablishment of self tolerance.

Heat shock proteins (HSPs) are phylogenetically conserved proteins, present in all prokaryotes and eukaryotes that are involved in various physiological and pathological processes. According to their function and size, or cellular localization, they are classified into major classes (small HSPs, HSP40, 60, 70, 90, and 110 families) and several members of a family. Gp96 (known also as glucose-regulated protein 94) is the endoplasmic reticulum (ER)-resident protein belonging to the HSP90 family. It is upregulated in response to glucose starvation and other stressful stimuli that disrupt protein synthesis in the ER, acting as a molecular chaperon involved in the correction of unfolded proteins, in the activation of proteasome-dependent ER-associated degradation of the misfolded proteins and in activation of protein translation that modulate the polypeptide traffic into the ER. In addition, it has been implicated in antigen presentation and MHC class I and II upregulation, in the activation and maturation of dendritic cells and proinflammatory cytokine secretion, as well as in chaperoning of Toll-like receptors, acting as a "danger signal" to the innate and adaptive immunity. Moreover, owing to its specific function in Ca2+ homeostasis and in the insulin-IGF/signaling pathways it was proposed that gp96 participates in mechanisms that are critical for cell growth, differentiation and responses to ER stress. To underline some aspects of these functions, in this survey we will present the data showing the expression of gp96 in the conditions of: 1) normal growth (liver regeneration after partial hepatectomy, syngeneic pregnancy, fetal organogenesis), 2) stress and ageing and 3) autoimmunity (chronic relapsing experimental autoimmune encephalomyelitis ; CR-EAE). Tissue expression of gp96 protein and mRNA was estimated in the liver, thymus and spleen and the data were correlated with phenotype and cytotoxicity of hepatic and splenic mononuclear lymphatic cells against the NKT and NK-sensitive targets. In the model of CR-EAE gp96 expression in the brain was correlated with the intensity of clinical symptoms. The data have shown that gp96 is highly upregulated in fastly proliferating and remodeling tissues (in regenerating liver, at the fetoplacental barrier, during fetal organogenesis), as well as in healing tissues after autoimmune attack (in the brain and spinal cord during the remission phases of EAE). Enhanced level of gp96 expression was also found in the thymus of aged and stress-exposed mice. Moreover, the kinetic studies made in the model of liver regeneration showed that gp96 upregulation was followed by maturation of dendritic cells, accumulation of CD3intermediate/NK1.1+/CD69+cells in the liver and CD4+CD25+Fox3+ cells in the liver and thymus, as well as by augmentation of NKT and NK-mediated cytotoxicity in the liver and in the spleen. The data imply that during the disturbance of morphostasis gp96 may serve as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT, which in cooperation with Treg cells participate in more or less successful reestablishment of self tolerance (Supported by grant 0621341-1337 from Croatian Ministry of Science).

Objectives: Imbalances in homeostasis provoked by the cellular growth at normal conditions, or by various forms of stress, initiate in the mammalian cells a highly conserved heat shock response with increased synthesis of heat shock proteins. They are involved in a variety of chaperoning functions including folding and unfolding of nascent polypeptides, protection against aggregation, specific proteolytic removal of terminally damaged polypeptides, and transport of proteins. Moreover, endoplasmic reticulum (ER)-resident HSP gp96/grp94 acts also as a natural adjuvant for priming innate and adaptive immunity, owing to its ability to stimulate APC cytokine expression and dendritic cell maturation, as well as to chaperone both MHC class I- and class II-restricted epitopes into the cross-presentation pathway of DCs. Methods: Hypothesizing that during the normal growth the discrepancies between the demand and capacities might induce ER stress that changes the gp96 expression, as well as that gp96 as natural carrier of self-peptides might participate in the breakdown of tolerance to self antigens, in this report we investigated the tissue expression of gp96 in fastly proliferating tissues during: 1) embryogenesis, 2) at fetoplacental unit during syngeneic pregnancy and 3) in regenerating liver after partial hepatectomy (pHx). In the latter model, the expression of gp96 protein and mRNA was estimated also in lymphatic organs and the changes were correlated with the appearance of self-reactive NKT and regulatory T cells and the level of cytotoxicity of lymphatic cells against the NKT and NK-sensitive targets. Results: High gp96 overexpression was found at all sites of tissue growth and remodelling (in fetal organs, in syncytiotrophoblast and chorionic vili and in regenerating liver). Moreover, pHx induced marked overexpression of gp96 in the thymus, followed by biphasic changes in the gp96 mRNA and the appearance of cytotoxic NKT and regulatory T cells (in the liver and in the thymus). Conclusion: The data point to the critical contribution of gp96 in mammalian development, embryonic-implantation and reparatory processes, implying that during the disturbance of morphostasis it serves as an endogenous danger-associated signal and a natural adjuvant, which participates in generation of autoreactive NKT and Treg cells, involved in reestablishment of self tolerance.

Tissue disintegration after injury leads, in the endoplasmic reticulum (ER), to activation of adaptive pathways known as the ER stress response. It is directed to the correction of unfolded proteins and to the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, but induces also a rapid activation of natural and adaptive immunity, since a ER resident heat shock protein-gp96 acts not only as a molecular chaperone, but also as a strong adjuvant, able to cross-present the antigenic peptides onto MHC class I or MHC class II pathways. Analyzing its potential role in processes of normal growth, in mice subjected to 1/3 partial hepatectomy (pHx) we determined the tissue expression of gp96 protein and mRNA in regenerating liver, thymus and spleen, determining simultaneously the phenotypic profile and spontaneous cytotoxic activity of intrahepatic and splenic mononuclear lymphatic cells (MNLC) against NKT- and NK-cells sensitive targets (syngeneic thymocytes and YAC-1) in wild, perforin and FasL deficient mice. The data have shown that pHx induces fast overexpression of gp96 protein and mRNA in hepatocytes, spleen and thymus, with accumulation of CD3intermediate/NK1.1+/CD69+ cells (liver) and Foxp3+CD4+CD25+ cells (liver and thymus). Simultaneously, intrahepatic MNLC acquired the FasLdependent cytotoxic potential against NKT-sensitive targets and both, intrahepatic and splenic MNLC, acquired the perforin-dependent cytotoxic potential against NK-sensitive targets, implying that during the disturbance of morphostasis gp96 serves as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT and regulatory cells, as well as NK cells. Moreover, cell cycle analysis revealed that G2+M phase of regenerating hepatocytes in PKO mice was translocated from the 1st to the 7th p. o. day, as well as that hepatocytes from FasL deficient mice were arrested in G0/G1 phase.

Various kinds of tissue disintegration lead in the endoplasmic reticulum (ER) to activation of adaptive pathways known as the ER stress response, which is directed to correction of unfolded proteins, to the activation of proteasome-dependent ER-associated degradation of the misfolded proteins and to activation of protein translation that modulate the polypeptide traffic into the ER. A crucial role in these events plays gp96, which acts not only as a molecular chaperone, but also as an adjuvant, able to induce the specific immune response against some self and foreign antigens. Material and methods: To illustrate the mulfipotential functions of gp96 in this study we investigated its participation in conditions of: 1) normal growth (liver regeneration after partial hepatectomy, pregnancy and fetal organogenesis), 2) autoimmunity (experimental allergic encephalomyelitis) and 3) pre-malignant growth (cervical intraepithelial neoplastic lesions). Tissue expression of gp96, detected by immunohistochemistry and RT-PCR, was in animal models correlated with phenotype and cytotoxicity of hepatic and splenic mononuclear lymphatic cells against the NKT sensitive (syngeneic thymocytes) and NK-sensitive targets (YAC-1 cells), while in CIN lesions (gradus I-III) it was correlated with the intensity of neoplastic process. Results: Tissue expression of gp96 was highly upregulated in fetal and adult rapidly proliferating tissue (regenerating liver and in several fetal organs), at fetoplacental barrier, in injured tissues after autoimmune attack (on astrocytes, microglia and neurons in the brain and spinal cord during EAE), as well as in dysplastic areas of cervix (on hyperplastic epithelial cells, vascular epithelia, and glandular epithelium). Kinetic studies made in the model of liver regeneration also showed that gp96-overexpression in the liver and thymus was followed by maturation of dendritic cells, accumulation of CD3intermediate/NK1.1+/CD69+cells in the liver and CD4+CD25+Fox3+ cells in the liver and thymus, as well as by augmentation of NKT and NK-mediated cytotoxicity in the liver and in the spleen. Conclusions: The data imply that during the disturbance of morphostasis and structural integrity of the cell gp96 may protect the cells against the proteotoxic damage and serve as a natural adjuvant for chaperoning of antigenic self or oncogenic peptides into the immune surveillance pathways, contributing during the normal growth to survival of cells and reestablishment of self tolerance, and in pathological growth to both development of cancer and initiation of antitumour immune response.

Various kinds of tissue disintegration lead in the endoplasmic reticulum (ER) to activation of the ER stress response, which is directed to correction of unfolded proteins, to the activation of proteasome-dependent degradation of the misfolded proteins and to activation of protein translation that modulate the polypeptide traffic into the ER. A crucial role in these events plays gp96, which acts not only as a molecular chaperone, but also as an adjuvant, able to induce the specific immune response against some self and foreign antigens. response, which is directed to correction of unfolded proteins, to the activation of proteasome-dependent degradation of the misfolded proteins and to activation of protein translation that modulate the polypeptide traffic into the ER. A crucial role in these events plays gp96, which acts not only as a molecular chaperone, but also as an adjuvant, able to induce the specific immune response against some self and foreign antigens. Trying to estimate its possible link with the development of autoimmunity in this study we analyzed the interplay between the gp96, NKT and Treg in the conditions of: 1) normal growth, induced by partial hepatectomy, 2) psychosocial stress without the tissue lesions and 3) after the treatment with bacterial peptidoglycan-monomer. Tissue expression of gp96 protein and mRNA were estimated in liver, thymus and spleen and the data were correlated with phenotype and cytotoxicity of hepatic and splenic MNLC against the NK and NKT-sensitive targets. All procedures induced fast cytoplasmic overexpression of gp96 protein and mRNA in hepatocytes and in the thymus. Simultaneously, in the liver accumulated NKT and Treg cells, while hepatic and splenic MNLC became highly cytotoxic against syngeneic thymocytes and YAC-1, implying that during the disturbance of morphostasis gp96 acts as a natural adjuvant for chaperoning of self peptides into the immune surveillance pathway, leading to activation of autoreactive NKT and Treg cells that participate in reestablishment of self tolerance.

Introduction: Metallothioneins (MTs), the intracellular, ubiquitous, low molecular cysteine rich proteins are stress-proteins, which participate in cell reactions on different kinds of injuries, as well as in host defense responses, affecting the functions of several immune cells, TLR signaling, expression of MHC proteins and co-stimulatory molecules on dendritic cells and the production and release of pro-inflammatory cytokines. Owing to this the induction MTs dramatically increases in response to tissue injury, infection, inflammation and neoplastic disease. It is however, of particular interest that both nuclear and cytoplasmic localization of MTs might be transiently expressed also in several growing tissues and during fetal development, implying that they might participate in DNA synthesis-and apoptosis-related processes, in regulation of transition from proliferation to the differentiation stage, as well as in several immune-related processes that initiate and coordinate the normal growth. In an attempt to underline the physiological functions of MTs in this study we analyzed the tissue distribution of MT I+II after partial hepatectomy (pHx), as well as in syngeneic pregnancy, looking for the changes in the liver, at the fetoplacental unit and in several fetal tissues. Material and methods: Experiments were done on C57/BL6 mice, which were subjected to 1/3 pHx or matted with syngeneic partner. Mice were sacrified in early postoperative period (1, 2, 6, 12 and 24 hours) after one-third hepatectomy (pHx), or on 16th day of syngeneic pregnancy. MT I/II expression was determined immunohistochemicaly, using monoclonal anti-MT I+II antibody and DAKO EnVision System. Additionally, in the regenerating liver MT-I mRNA and apoptosis were analyzed by RT-PCR and by TUNEL method, respectively. Results: The data have shown that fast liver growth after pHx is followed by marked increases of MT proteins and MT-I mRNA in the regenerating liver, where areas expressing cytoplasmic and nuclear MT immunoreactivity inversely correlated with those containing apoptotic cells. Similar over expression of MT I/II was found in maternal and fetal liver, as well as on the fetoplacental unit, affecting syncytiotrophoblast, cytotrophoblast, stromal and macrophages-like cells within the core of the chorionic vili and some decidual cells. Additionally, a high cytoplasmic and nuclear MT I/II imunoreactivity was noticed in fetal tissues, such as intestine, pancreas, gallbladder, kidneys and skin. Conclusion: The data underline the growth-related properties of MTs, implying that as metal-donors or metal-acceptors they alter the functional state of metal-dependent proteins, such as zinc finger domain-containing transcription factors, DNA synthesis enzymes and signal transduction molecules, regulating cell proliferation and apoptosis, facilitating also the flow of vital nutrient materials to the embryos and providing a barrier to entry of toxic materials from the mother at the fetoplacental unit. (Supported by grant 0621341-1337 from Croatian Ministry of Science).

Early after partial hepatectomy in the regeneration liver accumulated Zn++, Ca++, Mg++ and Fe++, while simultaneously decreased the concentration of all these metals in the spleen and Zn++ in the thymus. The changes were followed by marked upregulation of MT I+II in the liver and spleen.

Owing to the findings that partial hepatectomy (pHx) induces a rapid activation of natural and adaptive immunity, in this study we attempted to analyze the possibilities that glycoprotein gp96 participates in these evens. Namely, this highly conserved endoplasmic reticulum (ER) resident heat shock protein, in involved in correction of the unfolded proteins and in activation of proteasome-dependent ER-associated degradation of misfolded proteins. Furthermore, it is also is a strong adjuvant, able to cross-present onto MHC class I or MHC class II pathways the antigenic peptides, derived from tumor, viral or bacterial antigens, eliciting the antigen-specific immunity. Additionally, it is able to deliver the maturation signal to the dendritic cells, regulating the production of pro-inflammatory cytokines and generation of regulatory cell clones. Analyses were done in early and late phase of liver regeneration i.e. on 1st, 2nd, 6th, 24th and 48th hour after 1/3 pHx or sham operation in C57Bl mice. Gp96 and phenotype of lymphoid cells were detected in regenerating liver, thymus and spleen by immunohistological staining, RT-PCR and FACScan analysis. Simultaneously the cytotoxicity of intrahepatic and splenic mononuclear lymphatic cells (MNLC) obtained pHx donors were tested against syngeneic thymocytes and NK-sensitive targets (YAC-1) in wild, perforin and FasL deficient mice. The data have shown that pHx induces upregulation of mRNA for gp96 in the regenerating liver, thymus and spleen, as well as the fast cytoplasmic over expression of gp96 staining in regenerating hepatocytes, followed by surface expression of gp96 on MNLC in the spleen, and in the thymus. FACS analysis showed that in the regenerating liver after 6th p. o. hour accumulated activated NKT cells (CD3intermediate/NK1.1+/CD69+cells). At that time hepatic MNLC became highly cytotoxic against syngeneic thymocytes, while later (at 24th and 48th hour) increased also the perforin and Fas/L-dependent cytotoxicity of hepatic and splenic MNLC against YAC-1 target. The data imply that during liver regeneration gp96 may serve as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT cells, and NK and LAK cells, which might be involved in reestablishment of previous morphostasis. Supported by grant given by Croatian Ministry of Science.

Injury or removal of liver tissue leads to fast growth of liver remnant, involving changes in gene expression, growth factor production and morphological structure. Concomitantly, the breakdown of previously maintained tolerance and the exposure of self antigens lead to the activation of pre-immune and immune repertoire, which participate in surveillance against aberrant cells and re-establishment of previous morphostasis. Since, in these events, important biological function might have tissue minerals which are affecting the structural integrity and enzyme activities, transduction signals, transcription and replication factors during cell proliferation and apoptosis, as well as, the development and maintenance of immune functions and cytokine production, in this study we analyzed tissue dynamics of zinc, iron, magnesium and calcium in the liver, thymus, spleen and submandibular gland in intact and partially hepatectomized (pHx) mice on the 1st, 2nd, 7th and 15th day after 1/3 pHx, using the microwave digestion and inductivity coupled plasma spectrometry. The data have shown that tissue content of Zn++, Fe++, Mg++ and Ca++ is significantly affected by pHx. In early phase of regeneration in the liver significantly increased the concentration of Zn++ and Ca++, while simultaneously, the concentration of Zn++, Ca++ and Mg++ decreased in the thymus and in the spleen. In the latter phase of regeneration (on the 7th and 15th p. o. day), however, a marked accumulation of Zn++, Mg++ and Ca++ was noticed particularly in the thymus. Furthermore, when the mineral content found in estimated organs was, in each mouse, correlated with the intensity of liver regeneration, in early phase of liver growth (24h after pHx) a very high positive correlation was found between the concentration of Zn++ and the concentration of Ca++ in thymus and the index of liver growth (r=0.99 ; p

Introduction: Owing to the findings that partial hepatectomy (pHx) induces a rapid activation of natural and adaptive immunity during which in the regenerating liver accumulate potentially autoreactive NKT cells, in this study we attempted to analyze the possibilities that heat shock protein gp96 participates in these evens. Namely, this highly conserved monomorphic glycoprotein, as molecular chaperone has the unique ability to target the peptides into the endogenous presentation pathway of APCs, representing them as MHC I-peptide complexes for recognition by antigen-specific T lymphocytes via T cell receptor. Furthermore, gp96 delivers also the maturation signal to the dendritic cells, inducing the expression of MHC antigens and co-stimulatory molecules, leading to induction of pro-inflammatory cytokines, simultaneously with the generation of immune response to gp96-chaperoned peptides. Material & Method: Analysis were done in early phase of liver regeneration i.e. on 1st, 2nd and 6th hour after 1/3 pHx in C57Bl mice. Gp96 and phenotype of lymphoid cells were detected in regenerating liver, thymus and spleen by immunohistological staining and FACScan analysis. Simultaneously the cytotoxicity of intrahepatic and splenic mononuclear lymphatic cells (MNLC) obtained pHx donors was tested against syngeneic thymocytes and NK-sensitive targets (YAC-1). Results: The data have shown that in intact mice gp96 is moderately expressed in the liver, as well as in the thymus and spleen. However, as early as 1h after pHx, upregulation of gp96 staining occurred, resulting at 6th p. o. in an impressive cytoplasmic overexpression in regenerating hepatocytes. On the 2nd p. o. hour increased also the surface expression of gp96 on MNLC in the spleen, as well as in the thymus, particularly at the cortico-medullar conjunction. FACS analysis showed that in the regenerating liver after 6th p. o. hour accumulated activated NKT cells (CD3intermediate/NK1.1+/CD69+cells). At that time hepatic MNLC became highly cytotoxic against syngeneic thymocytes, while later (at 24th and 48th hour) increased also the cytotoxicity of hepatic and splenic MNLC against YAC-1 target. Conclusion: The data imply that during liver regeneration gp96 may serve as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT cells, NK and LAK cells, which might be involved in the control of hepatic proliferation and reestablishment of previous morphostasis. (Supported by grants from Croatian Ministry of Science, project No 0062018).

The data showed that early after pHx is activated a highly interconnected network of growth-factors and cytokine-regulated pathways, in which metallothioneins in the liver, thymus and spleen probably regulate a DNA synthesis-related processes, and transduction signals through abstraction or donation of metals such as Zn++, Fe++, Mg++ and Ca++.((Supported by grants from Croatian Ministry of Science, projects No 0062018 and 0062002).