Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis

Non-adherent bone marrow-derived cells (NA-BMCs) are a mixed cell population that can give rise to multiple mesenchymal phenotypes and that facilitates hematopoietic recovery. We characterized NA-BMCs by flow cytometry, fibroblast colony-forming units (CFU-f), real-time PCR, and in in vivo experiments. In comparison to adherent cells, NA-BMCs expressed high levels of CD11b⁺ and CD90⁺ within the CD45⁺ cell fraction. CFU-f were significantly declining over the cultivation period, but NA-BMCs were still able to form CFU-f after 5 days. Gene expression analysis of allogeneic NA-BMCs compared to bone marrow (BM) indicates that NA-BMCs contain stromal, mesenchymal, endothelial cells and monocytes, but less osteoid, lymphoid, and erythroid cells, and hematopoietic stem cells. Histopathological data and analysis of weight showed an excellent recovery and organ repair of lethally irradiated mice after NA-BMC transplantation with a normal composition of the BM.     

Interleukin-33 stimulates formation of functional osteoclasts from human CD14+ monocytes

Interleukin (IL)-33 is a recently described pro-inflammatory cytokine. Here we demonstrate IL-33 as a regulator of functional osteoclasts (OCs) from human CD14⁺ monocytes. IL-33 stimulates formation of tartrate-resistant acid phosphatase (TRAP)⁺ multinuclear OCs from monocytes. This action was suppressed by anti-ST2 antibody, suggesting that IL-33 acts through its receptor ST2, but not by the receptor activator of NF-κB ligand (RANKL) decoy, osteoprotegerin, or anti-RANKL antibody. IL-33 stimulated activating phosphorylations of signaling molecules in monocytes that are critical for OC development. These included Syk, phospholipase Cγ2, Gab2, MAP kinases, TAK-1, and NF-κB. IL-33 also enhanced expression of OC differentiation factors including TNF-α receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells cytoplasmic 1, c-Fos, c-Src, cathepsin K, and calcitonin receptor. IL-33 eventually induced bone resorption. This study suggests that the osteoclastogenic property of IL-33 is mediated through TRAF6 as well as the immunoreceptor tyrosine-based activation motif-dependent Syk/PLCγ pathway in human CD14⁺ monocytes.     

Macrophages are important mediators of either tumor- or inflammation-induced lymphangiogenesis

The lymphatic system provides important functions for tissue fluid homeostasis and immune response. Lymphangiogenesis, the formation of new lymphatics, comprises a series of complex cellular events in vitro or in vivo, e.g., proliferation, differentiation, and sprouting. Recent evidence has implied that macrophages act as a direct structural contributor to lymphatic endothelial walls or secret VEGF-C/-D and VEGF-A to initiate lymphangiogenesis in inflamed or tumor tissues. Bone marrow-derived macrophages are versatile cells that express different functional programs in response to exposure to microenvironmental signals, and can be identified by specific expression of a number of proteins, F4/80, CD11b, and CD68. Several causative factors, e.g., NF-κB, IL-1β, TNF-α, SDF-1, M-CSF, especially TonEBP/VEGF-C signaling, may be actively involved in macrophage-induced lymphangiogenesis. Alteration of macrophage phenotype and function has a profound effect on the development and progression of inflammation and malignancy, and macrophage depletion for controlling lymphangiogenesis may provide a novel approach for prevention and treatment of lymphatic-associated diseases.     

Immunoregulatory properties of the cytokine IL-34

Interleukin-34 is a cytokine with only partially understood functions, described for the first time in 2008. Although IL-34 shares very little homology with CSF-1 (CSF1, M-CSF), they share a common receptor CSF-1R (CSF-1R) and IL-34 has also two distinct receptors (PTP-ζ) and CD138 (syndecan-1). To make the situation more complex, IL-34 has also been shown as pairing with CSF-1 to form a heterodimer. Until now, studies have demonstrated that this cytokine is released by some tissues that differ to those where CSF-1 is expressed and is involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. The involvement of IL-34 has been shown in areas as diverse as neuronal protection, autoimmune diseases, infection, cancer, and transplantation. Our recent work has demonstrated a new and possible therapeutic role for IL-34 as a Foxp3⁺ Treg-secreted cytokine mediator of transplant tolerance. In this review, we recapitulate most recent findings on IL-34 and its controversial effects on immune responses and address its immunoregulatory properties and the potential of targeting this cytokine in human.     

Mesenchymal stem cells or cardiac progenitors for cardiac repair? A comparative study

In the past, clinical trials transplanting bone marrow–derived mononuclear cells reported a limited improvement in cardiac function. Therefore, the search for stem cells leading to more successful stem cell therapies continues. Good candidates are the so-called cardiac stem cells (CSCs). To date, there is no clear evidence to show if these cells are intrinsic stem cells from the heart or mobilized cells from bone marrow. In this study we performed a comparative study between human mesenchymal stem cells (hMSCs), purified c-kit⁺ CSCs, and cardiosphere-derived cells (CDCs). Our results showed that hMSCs can be discriminated from CSCs by their differentiation capacity towards adipocytes and osteocytes and the expression of CD140b. On the other hand, cardiac progenitors display a greater cardiomyogenic differentiation capacity. Despite a different isolation protocol, no distinction could be made between c-kit⁺ CSCs and CDCs, indicating that they probably derive from the same precursor or even are the same cells.     

Selective effect of burn injury on splenic CD11c+ dendritic cells and CD8α+CD4−CD11c+ dendritic cell subsets

Burn injury causes an immunosuppression associated with suppressed adaptive immune function. Dendritic cells (DCs) are APCs for which signaling via their Toll-like receptors (TLRs) induces their maturation and activation, which is essential for the adaptive immune response. In this study, we examined if burn injury alters the TLR activity of splenic DCs. After injury, we noticed that DC functions were impaired, characterized by a suppressed capacity to prime naive T cells when triggering the TLR4 signaling cascade using specific ligands (LPS or rHSP60). The observed perturbations on LPS-primed DCs isolated from burned mice exhibited significantly diminished IL-12p40 production and enhanced IL-10 secretion-associated impairment in mitogen-activated protein kinase activation. Interestingly, we observed a decrease of TLR4/MD-2 expression on the CD8α⁺ DC subset that persisted following LPS stimulation. The altered TLR4 expression on LPS-stimulated CD8α⁺ DCs was associated with reduced capacity to produce IL-12 after stimulation. Our results suggested that TLR4 reactivity on DCs, especially CD8α⁺ DCs, is disturbed after burn injury.     

Seasonal variation in peripheral blood leukocyte subsets and in serum interleukin-6, and soluble interleukin-2 and-6 receptor concentrations in normal volunteers

This study has been carried out in order to investigate seasonal variation in peripheral blood immune cells, such as leukocytes, monocytes, neutrophils, lymphocytes, CD3⁺ T, CD4⁺ T, CD8⁺ t, CD25⁺ T, CD20⁺ B, and serum interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R) and sIL-2R levels in normal volunteers. Toward this end, 26 normal volunteers (13 men, 13 women) had monthly blood samplings during one calendar year for peripheral blood count, flow cytometric enumeration of peripheral leukocyte subsets and immunoassays of IL-6, sIL-6R and sIL-2R. It was found that most of the immune variables change rhythmically during the seasons as a group phenomenon. Statistically significant yearly variations with seasonal rhythms, i.e. annual rhythms or harmonics, such as semiannual, tetramensual and trimensual rhythms, were found in the number of leukocytes, neutrophils, monocytes, lymphocytes, CD4⁺ T, CD8⁺ T, CD25⁺ T, CD20⁺ B cells, in the CD4⁺/CD8⁺ ratio, and serum IL-6 and sIL-6R levels. It is concluded that the immune system is characterized by a multifrequency time-structure with significant high-amplitude yearly variations in the number of some peripheral blood leukocyte subsets.     

Impairment of systemic DHA synthesis affects macrophage plasticity and polarization: implications for DHA supplementation during inflammation

Docosahexaenoic acid (DHA) is an omega-3 fatty acid obtained from the diet or synthesized from alpha-linolenic acid through the action of fatty acid elongases (ELOVL) and desaturases. DHA plays important roles in the central nervous system as well as in peripheral organs and is the precursor of several molecules that regulate resolution of inflammation. In the present study, we questioned whether impaired synthesis of DHA affected macrophage plasticity and polarization both in vitro and in vivo models. For this we investigated the activation status and inflammatory response of bone marrow-derived M1 and M2 macrophages obtained from mice deficient of Elovl2 (Elovl2^?/?), a key enzyme for DHA synthesis in mammals. Although both wild type and Elovl2^?/? mice were able to generate efficient M1 and M2 macrophages, M1 cells derived from Elovl2^?/? mice showed an increased expression of key markers (iNOS, CD86 and MARCO) and cytokines (IL-6, IL-12 and IL-23). However, M2 macrophages exhibited upregulated M1-like markers like CD80, CD86 and IL-6, concomitantly with a downregulation of their signature marker CD206. These effects were counteracted in cells obtained from DHA-supplemented animals. Finally, white adipose tissue of Elovl2^?/? mice presented an M1-like pro-inflammatory phenotype. Hence, impairment of systemic DHA synthesis delineates an alteration of M1/M2 macrophages both in vitro and in vivo, with M1 being hyperactive and more pro-inflammatory while M2 less protective, supporting the view that DHA has a key role in controlling the balance between pro- and anti-inflammatory processes.     

<Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> supports Th17 differentiation and limits de novo regulatory T cell induction by directly interfering with T cell receptor signaling

Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4⁺ T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4⁺ T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3⁺ regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4⁺ T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4⁺ T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3⁺ Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4⁺ T cell subsets by altering their TCR downstream signaling.     

Identification of cancer stem cell-like cells from human epithelial ovarian carcinoma cell line

Cancer stem cells (CSCs) play an important role in the development, invasion, and drug resistance of carcinoma, but the exact phenotype and characteristics of ovarian CSCs are still disputable. In this study, we identified cancer stem cell-like cells (CSC-LCs) and investigated their characteristics from the ovarian adenocarcinoma cell line 3AO. Our results showed that CSC-LCs were enriched in sphere-forming test and highly expressed CD44⁺CD24⁻. The spheres and CD24⁻ cells possessed strong tumorigenic ability by transplantation into nonobese diabetic/severe combined immunodeficient mice. CD44⁺CD24⁻ cells expressed stem cell markers and differentiated to CD44⁺CD24⁺ cells by immunofluorescence assay and fluorescence-activated cell-sorting analysis. In vitro experiments verified that CD44⁺CD24⁻ cells were markedly resistant to carboplatin and paclitaxol. In conclusion, our study identifies the CD44⁺CD24⁻ phenotype, self-renewal, high tumorigenicity, differentiation potential, and drug resistance of ovarian CSC-LCs. Our findings may provide the evidence needed to explore a new strategy in the treatment of ovarian cancer.     

T helper 17 cells: discovery, function, and physiological trigger

In the few years since their discovery, T helper 17 cells (T_H17) have been shown to play an important role in host defense against infections, and in tissue inflammation during autoimmunity. T_H17 cells produce IL-17, IL-21, IL-10, and IL-22 cytokines, and thus have broad effects on a variety of tissues. Notably, the requirement for the immunosuppressive cytokine TGF-β along with the pro-inflammatory cytokine IL-6 for T_H17 differentiation supports the intimate relationship between the T_H17 subset and FOXP3⁺ regulatory T cells. Here, we discuss current knowledge on effector functions and differentiation of the T_H17 lineage. Furthermore, we now know of a physiological stimulus for T_H17 differentiation: innate immune recognition of cells undergoing apoptosis as a direct result of infection induces unique development of this subset. As our knowledge of T_H17 and T regulatory cells grows, we are building on a new framework for the understanding of effector T cell differentiation and the biology of CD4⁺ T cell adaptive immune responses.     

CD8<Superscript>+</Superscript> Tregs in autoimmunity: learning “self”-control from experience

Autoreactive CD8⁺ regulatory T cells (Tregs) play important roles as modulators of immune responses against self, and numerical and functional defects in CD8⁺ Tregs have been linked to autoimmunity. Several subsets of CD8⁺ Tregs have been described. However, the origin of these T cells and how they participate in the natural progression of autoimmunity remain poorly defined. We discuss several lines of evidence suggesting that the autoimmune process itself promotes the development of autoregulatory CD8⁺ T cells. We posit that chronic autoantigenic exposure fosters the differentiation of non-pathogenic autoreactive CD8⁺ T cells into antigen-experienced, memory-like autoregulatory T cells, to generate a “negative feedback” regulatory loop capable of countering pathogenic autoreactive effectors. This hypothesis predicts that approaches capable of boosting autoregulatory T cell memory will be able to blunt autoimmunity without compromising systemic immunity.     

Factor XIII subunit A as an intracellular transglutaminase

Over the last 2 decades there has been increasing evidence that the role of factor XIII (FXIII) is not restricted to the area of hemostasis and that its subunit A functions as an intracellular enzyme in platelets and monocytes/macrophages. FXIII is already expressed during compartmentalisation of the precursors of megakaryocyte/platelet and monocyte/macrophage cell lines in the bone marrow. FXIII-A, produced by megakaryocytes, is packaged into budding platelets and is present in huge quantity in circulating ones. It seems very likely that it plays an important role in the cytoskeletal remodelling associated with the activation stages of platelets. FXIII-A can also be detected in blood monocytes and in all subsets of monocyte-derived macrophages throughout the body. FXIII-A is mainly localised in the cytoplasm, in association with cytoskeletal filaments, but at a relatively early stage of macrophage differentiation it also appears transiently in the nucleus. Cytoplasmic expression has a very close relationship with phagocytic activities. Further research is needed to understand the biological significance of its nuclear presentation.     

Endothelial cells as antigen-presenting cells: role in human transplant rejection

The immunological properties of human endothelial cells suggest they perform a pivotal role in acute and chronic rejection following solid organ transplantation. In this review the basic features of acute and chronic rejection are described as are the cellular and molecular requirements for antigen presentation. Traditionally, antigen-presenting cells are considered to be bone marrow-derived cells. However, these conclusions have been derived from rodent models of allograft rejection where bone marrow-derived passenger leukocytes are the only source of donor major histocompatibility complex (MHC) class II in the grafted organ. In contrast, in humans, virtually all the microvascular and small vessel endothelial cells are ‘constitutively’ positive for MHC class II antigens. The phenotypic properties of human endothelial cells, their response to cytokines and their ability to stimulate resting T cells are described. Unlike bone marrow-derived antigen presenting cells (APCs), which utilise B7/CD28 interactions, human endothelial cells utilise lymphocyte function antigen 3 (LFA3)/CD2 pathways to stimulate T cells. They activate a CD45RO + B7-independent subpopulation of T cells. Their effect on allogeneic T cells is compared with other non-bone marrow-derived cells such as fibroblasts, epithelial cells and smooth muscle cells, which are unable to stimulate resting T cells. Evidence is presented suggesting that release of MHC and non-human leukocyte antigens (HLA) from endothelial cells stimulates an alloantibody and autoimmune response leading to chronic rejection. Received 30 March 1998; received after revision 4 May 1998; accepted 4 May 1998     

Blockage of the NLRP3 inflammasome by MCC950 improves anti-tumor immune responses in head and neck squamous cell carcinoma

The NLRP3 inflammasome is a critical innate immune pathway responsible for producing active interleukin (IL)-1β, which is associated with tumor development and immunity. However, the mechanisms regulating the inflammatory microenvironment, tumorigenesis and tumor immunity are unclear. Herein, we show that the NLRP3 inflammasome was over-expressed in human HNSCC tissues and that the IL-1β concentration was increased in the peripheral blood of HNSCC patients. Additionally, elevated NLRP3 inflammasome levels were detected in tumor tissues of Tgfbr1/Pten 2cKO HNSCC mice, and elevated IL-1β levels were detected in the peripheral blood serum, spleen, draining lymph nodes and tumor tissues. Blocking NLRP3 inflammasome activation using MCC950 remarkably reduced IL-1β production in an HNSCC mouse model and reduced the numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). Moreover, inhibiting NLRP3 inflammasome activation increased the numbers of CD4⁺ and CD8⁺ T cells in HNSCC mice. Notably, the numbers of exhausted PD-1⁺ and Tim3⁺ T cells were significantly reduced. A human HNSCC tissue microarray showed that NLRP3 inflammasome expression was correlated with the expression of CD8 and CD4, the Treg marker Foxp3, the MDSC markers CD11b and CD33, and the TAM markers CD68 and CD163, PD-1 and Tim3. Overall, our results demonstrate that the NLRP3 inflammasome/IL-1β pathway promotes tumorigenesis in HNSCC and inactivation of this pathway delays tumor growth, accompanied by decreased immunosuppressive cell accumulation and an increased number of effector T cells. Thus, inhibition of the tumor microenvironment through the NLRP3 inflammasome/IL-1β pathway may provide a novel approach for HNSCC therapy.     

2-Carboxyethylgermanium sesquioxide,a synthetic organogermanium compound,as an inducer of contrasuppressor T cells

2-Carboxyethylgermanium sesquioxide (Ge-132), a synthesized organogermanium compound with immunomodulaing activities, was shown to be an inducer of anti-suppressor T cells in normal mice. The suppressor cell activity of T6S cells, a clone of burn-induced CD8⁺ IL-4-producing suppressor T cells, was clearly inhibited when a mixed lymphocyte-tumor cell reaction of the clone was conducted with splenic mononuclear cells from mice treated orally with a 100 mg/kg dose of Ge-132. The activity of anti-suppressor cells was demonstrated in spleens of mice 2 days after treatment with Ge-132 and reached its peak on day 3. The anti-suppressor cells induced by the compound were of a contrasuppressor T cell-linage, because they were characterized as CD4⁺ CD28⁺ TCR/⁺ Vicia villosa lectin-adherent T cells. These cells produced IFN- but did not produce IL-2, IL-4, IL-6 or IL-10 in their culture fluids. CD4⁺ anti-suppressor T cells induced by Ge-132 may be different from other subsets of CD4⁺ T cells because Th1 and Th2 cells generated in our laboratory did not adhere toVicia villosa lectin-coated petri dishes, and each produced specific cytokines. Th1 cells produced IFN- and IL-2 while Th2 cells produce IL-4 and IL-10 in vitro. These results suggest that Ge-132 may be useful as an inducer of contrasuppressor T cells in immunocompromised individuals bearing suppressor T cells. To eliminate suppressor T cells from immunocompromised hosts may result in improved resistance from various opportunistic infections.     

Nestin-expressing progenitor cells: function,identity and therapeutic implications

The neuroepithelial stem cell protein, or Nestin, is a cytoskeletal intermediate filament initially characterized in neural stem cells. However, current extensive evidence obtained in in vivo models and humans shows presence of Nestin⁺ cells with progenitor and/or regulatory functions in a number of additional tissues, remarkably bone marrow. This review presents the current knowledge on the role of Nestin in essential stem cell functions, including self-renewal/proliferation, differentiation and migration, in the context of the cytoskeleton. We further discuss the available in vivo models for the study of Nestin⁺ cells and their progeny, their function and elusive nature in nervous system and bone marrow, and their potential mechanistic role and promising therapeutic value in preclinical models of disease. Future improved in vivo models and detection methods will allow to determine the true essence of Nestin⁺ cells and confirm their potential application as therapeutic target in a range of diseases.     

Roles for interleukin-2 (IL-2) and IL-4 in the generation of allocytotoxic T cells in the primary and secondary responses in vitro

Roles for interleukin-2(IL-2) and IL-4 in the generation of murine allocytotoxine T lymphocytes (allo-CTL) in the primary and secondary responses were studied in vitro. The generation of allo-CTL in the primary response was inhibited by anti-IL-2 monoclonal antibody (mAb), but was not inhibited by anti-IL-4 mAb. On the other hand, the generation of allo-CTL in the secondary response was partially inhibited by either anti-IL-2 or anti-IL-4 mAb, and it was almost completely inhibited by the combination of two mAbs. CD8⁺ cell-depleted splenocytes produced IL-2, but not IL-4, in response to alloantigens in the primary response, and these cells produced both IL-2 and IL-4 in the secondary response. Both exogenous IL-2 and IL-4 induced functionally active allo-CTL in the primary response from CD4⁺ cell-depleted splenocytes when these cells were stimulated with T cell-depleted allogeneic cells. These results suggest that the allo-CTL induction in the primary response is IL:-2-dependent and secondary allo-CTL induction is both IL-2 and IL-4-dependent, because unprimed CD4⁺ T cells produce IL-2, but not IL-4, whereas primed cells produce both IL-2 and IL-4 in response to alloantigens.     

Differential chemotactic potential of mouse platelet basic protein for thymocyte subsets

Mouse platelet basic protein (CXCL7/mPBP) was cloned from thymic stromal cells and further identification indicated that it was expressed in thymic monocytes/macrophages (Mo/Ms). Recombinant mPBP was chemoattractive for target cells of polymorphonuclear leucocytes, peritoneal Mo/Ms and splenic lymphocytes with distinct potencies. CXCR2 was identified to be a cognate receptor for mPBP. Mouse thymocyte subsets of CD4^-CD8^- double-negative (DN), CD4⁺CD8⁺ double-positive (DP), CD4⁺CD8^- single-positive (CD4SP) and CD4^-CD8⁺ single-positive (CD8SP) expressed cell surface CXCR2 with different positive percentages and expression levels. mPBP was chemoattractive for thymocyte subsets with the potency order DN>DP> CD8SP>CD4SP, consistent with the levels of CXCR2 expressed on the respective cells. Thus, mPBP in thymus is functionally redundant with chemokine CXCL12/ SDF-1. Moreover, our finding that thymic Mo/Ms can produce mPBP implies that they may have other functions apart from acting as scavengers in thymus.Received 25 March 2004; received after revision 10 May 2004; accepted 8 June 2004     

Memory CD4<Superscript>+</Superscript> T Cells: fate determination,positive feedback and plasticity

Naïve CD4⁺ T cells undergo massive cell proliferation upon encountering their cognate ligand. This proliferation depends upon appropriate cues from the antigen-presenting cells that have processed the antigen and present the peptide to the T cells, and requires the establishment of a cytokine environment that can support such proliferation. Expansion of antigen-specific CD4⁺ T cells needs to be coupled with differentiation into one of several effector/regulatory phenotypes if the priming event is to result in cells that can initially act to control the particular pathogen that elicited the response, and later to serve as memory cells to insure an appropriate response upon reintroduction of the pathogen. Here, we discuss the initiation of T helper lineage commitment, the positive feedback regulation by the cytokine environment to enhance and stabilize the differentiation into distinct T helper subsets, and the biological significance of CD4⁺ T cell plasticity and long-term CD4⁺ T cell memory.