α-1 antitrypsin promotes semimature, IL-10-producing and readily migrating tolerogenic dendritic cells

Tolerogenic IL-10-positive CCR7-positive dendritic cells (DC) promote T regulatory (Treg) cell differentiation upon CCR7-dependent migration to draining lymph nodes (DLN). Indeed, in human DC deficiencies, Treg levels are low. α-1 antitrypsin (AAT) has been shown to reduce inflammatory markers, promote a semimature LPS-induced DC phenotype, facilitate Treg expansion, and protect pancreatic islets from alloimmune and autoimmune responses in mice. However, the mechanism behind these activities of AAT is poorly understood. In this study, we examine interactions among DC, CD4(+) T cells, and AAT in vitro and in vivo. IL-1β/IFN-γ-mediated DC maturation and effect on Treg development were examined using OT-II cells and human AAT (0.5 mg/ml). CCL19/21-dependent migration of isolated DC and resident islet DC was assessed, and CCR7 surface levels were examined. Migration toward DLN was evaluated by FITC skin painting, transgenic GFP skin tissue grafting, and footpad DC injection. AAT-treated stimulated DC displayed reduced MHC class II, CD40, CD86, and IL-6, but produced more IL-10 and maintained inducible CCR7. Upon exposure of CD4(+) T cells to OVA-loaded AAT-treated DC, 2.7-fold more Foxp3(+) Treg cells were obtained. AAT-treated cells displayed enhanced chemokine-dependent migration and low surface CD40. Under AAT treatment (60 mg/kg), DLN contained twice more fluorescence after FITC skin painting and twice more donor DC after footpad injection, whereas migrating DC expressed less CD40, MHC class II, and CD86. Intracellular DC IL-10 was 2-fold higher in the AAT group. Taken together, these results suggest that inducible functional CCR7 is maintained during AAT-mediated anti-inflammatory conditions. Further studies are required to elucidate the mechanism behind the favorable tolerogenic activities of AAT.     

Identification of IFN-γ-producing innate B cells

Although B cells play important roles in the humoral immune response and the regulation of adaptive immunity, B cell subpopulations with unique phenotypes, particularly those with non-classical immune functions, should be further investigated. By challenging mice with Listeria monocytogenes, Escherichia coli, vesicular stomatitis virus and Toll-like receptor ligands, we identified an inducible CD11a^hiFcγRIII^hi B cell subpopulation that is significantly expanded and produces high levels of IFN-γ during the early stage of the immune response. This subpopulation of B cells can promote macrophage activation via generating IFN-γ, thereby facilitating the innate immune response against intracellular bacterial infection. As this new subpopulation is of B cell origin and exhibits the phenotypic characteristics of B cells, we designated these cells as IFN-γ-producing innate B cells. Dendritic cells were essential for the inducible generation of these innate B cells from the follicular B cells via CD40L-CD40 ligation. Increased Bruton''s tyrosine kinase activation was found to be responsible for the increased activation of non-canonical NF-κB pathway in these innate B cells after CD40 ligation, with the consequent induction of additional IFN-γ production. The identification of this new population of innate B cells may contribute to a better understanding of B cell functions in anti-infection immune responses and immune regulation.     

Grenzgänger: adult bone marrow cells populate the brain

While the brain has traditionally been considered a rather secluded site, recent studies suggest that adult bone marrow (BM)-derived stem cells can generate glia and neurons in rodents and humans. Macrophages and microglia are the first to appear in the murine brain after transplantation of genetically marked BM cells. Within weeks after transplantation, some authors have found astrocytes and cells expressing neuronal antigens. We detected cerebellar Purkinje neurons and interneurons, such as basket cells, expressing the green fluorescent protein (GFP) 10–15 months after transplantation of GFP-labeled BM cells. The results push the boundaries of our classic view of lineage restriction.Robert Feulgen Prize Lecture 2003 presented at the 45th Symposium of the Society for Histochemistry in Les Diablerets, Switzerland, on 18 September 2003. The German word "Grenzgänger" means cross-border commuters (French: "frontaliers")     

Effect of bone marrow–derived mesenchymal stromal cells on hepatoma

Background aimsThe aim of the study was to evaluate the effect of mesenchymal stromal cells (MSCs) on tumor cell growth in vitro and in vivo and to elucidate the apoptotic and anti-proliferative mechanisms of MSCs on a hepatocellular carcinoma (HCC) murine model.MethodsThe growth-inhibitory effect of MSCs on the Hepa 1–6 cell line was tested by means of methyl thiazolyl diphenyl-tetrazolium assay. Eighty female mice were randomized into four groups: group 1 consisted of 20 mice that received MSCs only by intrahepatic injection; group 2 consisted of 20 HCC mice induced by inoculation of Hepa 1–6 cells into livers without MSC treatment; group 3 consisted of 20 mice that received MSCs after induction of liver cancer; group 4 consisted of 20 mice that received MSCs after induction of liver cancer on top of induced biliary cirrhosis.ResultsMSCs exhibited a growth-inhibitory effect on Hepa 1–6 murine cell line in vitro. Concerning in vivo study, decreases of serum alanine transaminase, aspartate transaminase and albumin levels after MSC transplantation in groups 2 and 3 were found. Gene expression of α-fetoprotein was significantly downregulated after MSC injection in the HCC groups. We found that gene expression of caspase 3, P21 and P53 was significantly upregulated, whereas gene expression of Bcl-2 and survivin was downregulated in the HCC groups after MSC injection. Liver specimens of the HCC groups confirmed the presence of dysplasia. The histopathological picture was improved after administration of MSCs to groups 2 and 3.ConclusionsMSCs upregulated genes that help apoptosis and downregulated genes that reduce apoptosis. Therefore, MSCs could inhibit cell division of HCC and potentiate their death.     

TRAIL(+) human plasmacytoid dendritic cells kill tumor cells in vitro: mechanisms of imiquimod- and IFN-α-mediated antitumor reactivity

Dendritic cells (DCs) not only exhibit the unique capacity to evoke primary immune responses, but may also acquire TLR-triggered cytotoxic activity. We and others have previously shown that TLR7/8- and TLR9-stimulated plasmacytoid DCs (pDCs) isolated from human peripheral blood express the effector molecule TRAIL. The exact mechanisms through which pDCs acquire and elicit their cytotoxic activity are still not clear. We now show that in the absence of costimulators, TRAIL induction on pDCs occurs with agonists to intracellular TLRs only and is accompanied by a phenotypic as well as functional maturation, as evidenced by a comparatively superior MLR stimulatory capacity. pDCs acquired TRAIL in an IFN-α/β-dependent fashion and, notably, TRAIL expression on pDCs could be induced by IFN-α stimulation alone. At a functional level, both TLR7/8- (imiquimod [IMQ]) and TLR9-stimulated (CpG2216) pDCs lysed Jurkat T cells in a TRAIL- and cell contact-dependent fashion. More importantly, IFN-α-activated pDCs acquired similar cytotoxic properties, independent of TLR stimulation and maturation. Both IMQ- and IFN-α-activated pDCs could also lyse certain melanoma cell lines in a TRAIL-dependent fashion. Interestingly, suboptimal doses of IMQ and IFN-α exhibited synergistic action, leading to optimal TRAIL expression and melanoma cell lysis by pDCs. Our data imply that tumor immunity in patients receiving adjuvant IMQ and/or IFN-α may involve the active participation of cytotoxic pDCs.     

IL-6 and IFN-α from dsRNA-stimulated dendritic cells control expansion of regulatory T cells

Foxp3⁺CD4⁺ regulatory T cells (Treg) control not only autoimmunity but also the effective immune response against RNA virus infections, which produces virus-derived double-stranded RNA (dsRNA). To induce effective anti-viral immunity, it is a key issue to learn how Treg respond to dsRNA in vitro and in vivo. We here showed that synthetic dsRNA, polyI:C, caused peripheral expansion of functional Treg in a TICAM-1- and IL-6-dependent manner in vivo. PolyI:C did not expand Treg directly, but promoted the expansion of naturally occurring Treg indirectly through IL-6 produced from dendritic cells (DCs). In addition, the expansion of Treg by IL-6 was inhibited by IFN-α from polyI:C-stimulated DCs. These data suggest that the balance of IL-6 and IFN-α in the region of RNA virus infection may determine the number of peripheral Treg, which affects the effective immune responses against viruses.     

IFN-γ-primed human bone marrow mesenchymal stem cells induce tumor cell apoptosis in vitro via tumor necrosis factor-related apoptosis-inducing ligand

Human mesenchymal stem cells hold promise as gene therapy vectors for delivery of various genes to solid tumors for either therapeutic or tumor-tracing purposes. However, whether Mesenchymal stem cells support or inhibit tumor growth remains unknown. Herein, we first observed that mesenchymal stem cells primed with IFN-γ selectively induced the death of tumor cell lines, but not normal cells. We further identified that IFN-γ-primed mesenchymal stem cells expressed tumor necrosis factor-related apoptosis-inducing ligand. Tumor-suppressive effect of IFN-γ-primed mesenchymal stem cells could be blocked by activity neutralization or expression reduction of tumor necrosis factor-related apoptosis-inducing ligand. Moreover, mesenchymal stem cells mediated apoptosis of tumor cells by activating caspase-3 in such cells, via a mechanism involving tumor necrosis factor-related apoptosis-inducing ligand. However, when IFN-γ-primed or non-primed mesenchymal stem cells were co-injected into nude mice along with H460 cells, tumor growth was much faster than that of the group receiving only tumor cells (p<0.01) because of the promoting vascularization effect of mesenchymal stem cells, although IFN-γ-primed mesenchymal stem cells also exerted a certain degree of tumor-suppressive effect compared with non-primed cells (2.79±0.9 g versus 2.03±0.6 g). Collectively, our findings show that IFN-γ-primed human mesenchymal stem cells could induce cancer cell apoptosis via TRAIL-mediated pathway. In addition, our data afford a novel explanation of the opposing effects of hMSCs presence on tumor growth in vitro and in vivo. Thus, more attention needs to be paid when seeking to exploit mesenchymal stem cells as a therapeutic option under the condition of malignant tumor.     

in vitro generation of IFN-γ-producing Listeria-specific T cells is dependent on IFN-γ production by non-NK cells

In vitro 5-day cultures of naive spleen cells with viable Listeria monocytogenes (VLM), but not heat-killed L. monocytogenes, induced CD4⁺ T cells that produced IFN-γ upon secondary antigen stimulation. The VLM-induced Listeria-specific T cells produced IFN-γ but lacked expression of IL-2 and IL-4. To study the role of IFN-γ in the induction of the IFN-γ-producing T cells, we added anti-IFN-γ mAb to the primary culture and analyzed IFN-γ production upon secondary antigen stimulation. Addition of anti-IFN-γ mAb to the culture suppressed generation of IFN-γ-producing CD4⁺ T cells, suggesting that IFN-γ is important in the induction of IFN-γ-producing CD4⁺ T cells. Furthermore, our results showed that depletion of NK cells from spleen cells by anti-asialo GM1 antibody plus complement before culture enhanced induction of IFN-γ-producing CD4⁺ T cells. Although NK cells are known to produce IFN-γ, the results indicate that NK cell-derived IFN-γ may not be important in induction of the Listeria-specific IFN-γ-producing CD4⁺ T cells in the culture system. In addition, we demonstrated that IFN-γ expression was high in CD4⁺ T cells from cultures of spleen cells with VLM at the primary culture level. These results suggest that IFN-γ derived from T cells may enhance production of IFN-γ by CD4⁺ T cells, while NK cells rather suppress the induction of IFN-γ-producing CD4⁺ T cells.     

ERRα regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

The orphan nuclear receptor estrogen-related receptor-α (ERRα) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERRα in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERRα deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERRα deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERRα in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERRα deficient MSCs and enhanced upon ERRα overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERRα. Under adipogenic conditions, ERRα deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERRα in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERRα may play different roles in bone under different physiological conditions.     

DNA synthesis of rat bone marrow mesenchymal stem cells through α1-adrenergic receptors

Multipotential bone marrow mesenchymal stem cells (BMSCs) are important in maintaining the microenvironment of the bone marrow (BM). Sympathetic nerves histologically innervate the BM; however, their role remains unclear. In this study, the effects of norepinephrine on DNA synthesis and the related signaling molecules involved in rBMSCs were examined.mRNA levels of the α1-adrenergic receptor subtypes increased following norepinephrine stimulation (10⁻⁵ M for 30 min). DNA synthesis increased in dose- and time-dependent manners as determined by [³H]thymidine incorporation. Intracellular Ca²⁺ concentration and translocation of protein kinase C from the cytosol to the membrane were also found to be elevated in rBMSCs. Phentolamine was able to suppress translocation of PKC. Norepinephrine also induced phosphorylation of ERK1/2, which was prevented by staurosporine treatment. Pretreatment with PD98059 inhibited ERK1/2 phosphorylation and DNA synthesis in rBMSCs.These findings indicate that norepinephrine stimulates DNA synthesis via α1-adrenergic receptors and downstream Ca²⁺/PKC and ERK1/2 activation in rBMSCs.     

Neutrophils and interferon-α-producing cells: who produces interferon in lupus?

Interferon-α plays a crucial role in the pathogenesis of systemic lupus erythematosus. Nevertheless, the different human cell types producing this cytokine as well as the stimuli inducing its production have not been completely characterized. So far, a subpopulation of dendritic cells activated by immune complexes has been identified as major producers of interferon-α in patients with lupus. However, those cells represent a minor population and some studies have reported the secretion of interferon-α by other cells. On the other hand, more than 50% of blood leukocytes are neutrophils and their functions are still not fully understood. Recent data suggest that neutrophils, though usually not considered interferon-α-producing cells, may represent an unexpected source of this cytokine in response to some lupus stimuli.     

Biomarkers of bone healing induced by a regenerative approach based on expanded bone marrow–derived mesenchymal stromal cells

BackgroundSafety and feasibility of a regenerative strategy based on the use of culture-expanded mesenchymal stromal cells (MSCs) have been investigated in phase 2 trials for the treatment of nonunion and osteonecrosis of the femoral head (ONFH). As part of the clinical study, we aimed to evaluate if bone turnover markers (BTMs) could be useful for predicting the regenerative ability of the cell therapy product.Materials and MethodsThe bone defects of 39 patients (nonunion: n = 26; ONFH: n = 13) were treated with bone marrow–derived MSCs, expanded using a clinical-grade protocol and combined with biphasic calcium phosphate before implantation. Bone formation markers, bone-resorption markers and osteoclast regulatory proteins were measured before treatment (baseline) and after 12 and 24 weeks from surgery. At the same time-points, clinical and radiological controls were performed to evaluate the bone-healing progression.ResultsWe found that C-Propeptide of Type I Procollagen (CICP) and C-terminal telopeptide of type-I collagen (CTX) varied significantly, not only over time, but also according to clinical results. In patients with a good outcome, CICP increased and CTX decreased, and this trend was observed in both nonunion and ONFH. Moreover, collagen biomarkers were able to discriminate healed patients from non-responsive patients with a good diagnostic accuracy.DiscussionCICP and CTX could be valuable biomarkers for monitoring and predicting the regenerative ability of cell products used to stimulate the repair of refractory bone diseases. To be translated in a clinical setting, these results are under validation in a currently ongoing phase 3 clinical trial.     

Regenerative medicine and liver injury: what role for bone marrow derived stem cells?

In recent years, great interest has been aroused by the discovery of the ability of adult stem cells to contribute to regeneration processes and repair of damaged tissues. In particular, bone marrow derived stem cells (BMSCs), the most well known population of multipotent stem cells in adults, have been shown to be able to generate many different committed cellular types. In this review, we systematically organize the numerous hypotheses emerging from the most recent studies, in animal and humans, which evaluated the potentiality of BMSCs to contribute to tissue repair in different types of liver damage. Our aim is to give scientists and clinicians who are interested in regenerative medicine the rational basis for planning future studies on stem cell therapy for liver diseases.     

Interferon γ induced compositional changes in human bone marrow derived mesenchymal stem/stromal cells

Background

Mesenchymal stem/stromal cells (MSC) display a range of immunoregulatory properties which can be enhanced by the exposure to cytokines such interferon γ (IFN-γ). However the compositional changes associated with the ‘licensing’ of these cells have not been clearly defined. The present study was undertaken to provide a detailed comparative proteomic analysis of the compositional changes that occur in human bone marrow derived MSC following 20 h treatment with IFN-γ.

Methods

2D LC MSMS analysis of control and IFN-γ treated cells from 5 different healthy donors provided confident identification of more than 8400 proteins.

Results

In total 210 proteins were shown to be significantly altered in their expression levels (≥|2SD|) following IFN-γ treatment. The changes for several of these proteins were confirmed by flow cytometry. STRING analysis determined that approximately 30% of the altered proteins physically interacted in described interferon mediated processes. Comparison of the list of proteins that were identified as changed in the proteomic analysis with data for the same proteins in the Interferome DB indicated that ~35% of these proteins have not been reported to be IFN-γ responsive in a range of cell types.

Conclusions

This data provides an in depth analysis of the proteome of basal and IFN-γ treated human mesenchymal stem cells and it identifies a number of novel proteins that may contribute to the immunoregulatory capacity if IFN-γ licensed cells.

     

Adenoviral transduction of hTGF-β1 enhances the chondrogenesis of bone marrow derived stromal cells

TGF-β1 plays a necessary and important role in the induction of chondrogenic differentiation of bone marrow stromal cells (BMSCs). In this study, porcine BMSCs were infected with a replication-deficient adenovirus expression vector carrying the hTGF-β1 gene. The transduced BMSCs were cultured as pelleted micromasses in vitro for 21 days, seeded onto disk-shaped PGA scaffolds for 3 days and subsequently implanted into the subcutaneous tissue of mice. BMSCs transduced with AdhTGF-β1 expressed and secreted more hTGF-β1 protein in vitro than those of the control group. Histological and immunohistological examination of the pellets revealed robust chondrogenic differentiation. Tissues made from cells transduced with AdhTGF-β1 exhibited neocartilage formation after 3 weeks in vivo. The neocartilage occupied 42 ± 5% of the total tissue volume which was significantly greater than that of the control group. Furthermore, there was extensive staining for sulfated proteoglycans and type II collagen in the AdhTGF-β1 group compared to controls, and quantification of GAG content showed significantly greater amounts of GAG in experimental groups. The results demonstrate that transfer of hTGF-β1 into BMSCs via adenoviral transduction can induce chondrogenic differentiation in vitro and enhance chondrogenesis in vivo.