Immunoregulatory function of mesenchymal stem cells

Mesenchymal stem cells (MSC) are a rare subset of stem cells residing in the bone marrow where they closely interact with hematopoietic stem cells and support their growth and differentiation. MSC can differentiate into multiple mesenchymal and non-mesenchymal lineages, providing a promising tool for tissue repair. In addition, MSC suppress many T cell, B cell and NK cell functions and may affect also dendritic cell activities. Due to their limited immunogenicity, MSC are poorly recognized by HLA-incompatible hosts. Based on these unique properties, MSC are currently under investigation for their possible use to treat immuno-mediated diseases. However, both their condition of immunoprivilege and their immunosuppressive function have recently been challenged when analyzed under particular experimental conditions. Thus, it is likely that MSC effects on the immune system may be deeply influenced not only by cell-to-cell interactions, but also by environmental factors shaping their phenotype and functions.     

CENTRAL TOLERANCE MECHANISMS IN CONTROL OF SUSCEPTIBILITY TO AUTOIMMUNE UVEITIC DISEASE

A family of dendritic cells has been identified in situ and in vitro by microscopy and immunolabeling. The members of this family include the dendritic cells isolated from lymphoid organs, Langerhans cells [LC] of the epidermis, veiled cells in afferent lymph, and interdigitating cells [IDC] in the T-cell areas. Some common features to all members of the family are high levels of MHC class II antigens, a lack of most B and T cell markers, and an absence or low levels of macrophage/granulocyte antigens. This review summarizes the markers of mouse dendritic cells as assessed by a panel of monoclonal antibodies, and stresses a few recent findings. 1) In spleen, there are two populations of dendritic cells. More than 75% of isolated cells are 33D1⁺, NLDC145^?, and JI1d^?, while the remainder have the reciprocal phenotype and thus share the NLDC145 antigen of IDC. Thymic dendritic cells, released by collagenase digestion, and epidermal LC also are 33DI^?, NLDC145⁺, J11d⁺. 2) When epidermal LC are placed in culture, there are changes in cell function and phenotype. There is a decrease in Fc-γ receptors and the F4/80 macrophage antigen, an increase in class I and II MHC products and p55 IL-2 receptors, and persistence of the NLDC145 IDC antigen. The cultured LC thereby resembles the IDC. 3) A new antibody N418 shows that dendritic cells express the p150/90 member of the leukocyte β₂ integrin family. Immunolabeling of tissue sections of spleen indicates that N418⁺ dendritic cells not only are present in the periarterial sheaths, the location of IDC, but also in “nests” at the periphery of the T area where 33D1 has been found. The peripheral collections interrupt the marginal zone of macrophages that separates white and red pulp, and places the dendritic cells in the path of T cells as they move through the white pulp. Therefore the members of the dendritic cell family have important markers in common, as well as differences that are associated with state of immunologic function and location.     

Activated NKT cells increase dendritic cell migration and enhance CD8+ T cell responses in the skin

Activated NKT cells produce cytokines such as IL-4 and IFN-gamma that function locally to influence the strength and functional development of antigen-specific T cells. Here we identify an alternative mechanism by which NKT cells influence the strength of T cell responses: through modulation of peripheral dendritic cell (DC) trafficking. NKT cell activation with alpha-galactosylceramide induced high systemic levels of TNF-alpha that mediated increased DC migration from skin to draining lymph nodes. This increased DC trafficking led to a threefold increase in effector T cell priming and in the immune response elicited to antigen challenge when alpha-galactosylceramide was given at the time of immunization of the skin. These studies provide important implications for the use of NKT cell activation strategies to manipulate T cell-mediated responses including responses to cutaneous tumors and graft vs. host disease.     

Influences of the pia mater on the precursors of nerve cells

Summary In the developing cerebellum of 8-day old rats surgical lesions were made. During regeneration of the cerebellum the pia mater was found to penetrate inside the neural tissue. Partially differentiated Purkinje cells and granule cells, that were in close contact with the pial cells, were found atrophied. When the profilerative cells of external granular layer came into contact with the pial cells, they were reduced to a primitive type of epitheloid cells. In this instance epithelio-mesenchymal interaction was found deleterious to the precursors of neurons. However, when the epithelioid cells were freed from the contact with the pial cells by intervening basement membrane, they differentiated into ependymal cells. Such ependymal cells gave rise to small as well as large new ventriculer structures, and structures resembling chorioid plexus.This research was supported by NIH Research Grant NS08817-03. Acknowledgements are due to Sheila Anderson for histology and to Donna Whitehurst for photographic work.     

后肾细胞微环境诱导胚胎干细胞向肾系细胞分化的实验研究

目的: 探讨胚胎后肾细胞微环境诱导胚胎干细胞(ESCs)分化为肾系细胞的作用。方法: 小鼠D3系胚胎干细胞通过悬滴法制备拟胚体(EBs),将EBs细胞与取自孕12.5 d小鼠胚胎的后肾细胞通过间接共培养以诱导其分化,即为共培养诱导组,设EBs细胞自然分化为对照组;采用免疫荧光染色法检测共培养第3、5、7 d后的EBs细胞Pax2、WT-1蛋白表达情况,通过逆转录PCR法检测诱导3 d后的EBs细胞Pax2、WT-1、Lim1、Sall1、Emx2、GDNF、Wnt4、BMP7、Nephl、Nephrin、KSP和CD24 mRNA的表达情况。结果: EBs细胞在诱导的第3 d即出现了全部肾发育相关基因的表达,其中共培养组Pax2、WT-1、Emx2、GDNF、Nephl、Nephrin、KSP和CD24的表达强于对照组;免疫荧光结果显示在诱导3 d后的EBs细胞中可观察到Pax2阳性细胞,阳性细胞数在共培养第5、7 d增多;诱导5 d后可观察到WT-1阳性细胞,对照组未见Pax2或WT-1蛋白阳性细胞出现。结论: 后肾细胞微环境可促进ESCs分化为肾系细胞。     

脂肪来源干细胞诱导分化为神经元样细胞的实验研究

目的： 研究脂肪来源干细胞（ASCs）的分离、纯化、扩增以及在体外定向分化为神经元样细胞的能力。 方法: 获取并培养正常人的ASCs，倒置显微镜下观察其形态；流式细胞仪检测其免疫表型。全反式维甲酸（ATRA）诱导ASCs在体外分化为神经元样细胞，倒置显微镜观察神经元样细胞的形态，逆转录-聚合酶链反应（RT-PCR）检测巢蛋白基因的表达；免疫组织化学染色法和Western blotting法检测神经丝蛋白（NF）和神经元烯醇化酶（NSE）的表达。 结果: ASCs呈纤维样贴壁生长，体外培养易扩增；ASCs表达相关抗原CD29和CD105，不表达CD31、CD34、CD45和HLA-DR；不同扩增代数的ASCs经诱导剂的作用可呈现典型的神经元样细胞形态，巢蛋白基因及NF、NSE均呈阳性表达。 结论: 脂肪组织中分离培养的ASCs具有体外大量扩增并保持低分化状态的特性，以及定向分化为神经元样细胞的能力, 是一种可用于神经系统疾病治疗的种子细胞。     

Accessory phenotype and function of macrophages induced by cyclic adenosine monophosphate

Mature macrophages (Mph) differentiated in culture from normal human peripheral blood monocytes (Mo) exhibit low activity as accessory cells (antigen-presenting cells) in T lymphocyte stimulation. A test system was established based on mitogenicity to quantitate the accessory activity of Mph-derived cells and to follow its changes for several days. The system used accessory cells treated with the oxidative mitogen, sodium periodate. The cells were subsequently co-cultured with pooled human lymphocytes from a cryopreserved stock. DNA synthesis in these cells was used as an indicator of accessory activity. Mph could be converted within 5-6 days into highly active accessory cells if a continuous stimulus of exogenously added dibutyryl cyclic AMP (db-cAMP) was provided. Mph treated by db-cAMP retained a high degree of HLA-DR expression but typical Mph markers such as non-specific esterase, phagocytosis, and expression of Fc-receptors were down-regulated. Acid phosphatase and myeloperoxidase underwent only slight changes, while the monocyte marker 5'-nucleotidase remained undetectable. Morphologically, the cells rounded up and developed veils and dendritiform elongations. In contrast to dendritic cells, Mph-derived accessory cells retained the CD14 antigen characteristic of monocytes and Mph. It is concluded that Mph are able to respond to exogenous stimuli and to convert into a highly active accessory cell. This contrasts to the well-known state of the 'activated Mph' with respect to markers and function. Both states appear to be antagonistically controlled by intracellular second messengers, as the accessory cell phenotype is positively correlated with intracellular cyclic AMP increase, whereas Mph activation correlates with cyclic GMP increase.     

干细胞若干定义的相关探讨

背景：近10年来干细胞研究所取得的巨大进展,不仅影响和促进了生物学及其相关基础科学,而且还在医学、药物开发、农业等许多领域得到广泛的应用,目前已成为研究的热点问题。 目的：在干细胞的定义上还存在一些需要探讨的问题,明确定义将有利于干细胞研究的快速发展。 方法：回顾干细胞的发展和概念提出,特别是通过中英文权威定义的比较,提出作者的看法。 结果与结论：干细胞的定义上还存在一些问题值得探讨,特别是一些中英文表述不同影响了理解。例如,“多能干细胞”对应译成两个单词：Pluripotent Stem Cell和Multipotent Stem Cell,然而Pluripotent和Multipotent两个单词的英文意义不同。为了学术交流和沟通,作者提出将Pluripotent Stem Cell称为“万能干细胞”,而保留Multipotent Stem Cell为“多能干细胞”的定义。以上结论供广大同仁探讨,以利于抛砖引玉。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Identification of a CD8α Dendritic Cell Subpopulation in Rat Spleen and Evaluation of Its OX-62 Expression

Rat spleen DC and bone marrow-derived DC were isolated and characterized by morphology and flow cytometry. We found a CD8α⁺ DC subpopulation representing 19–48% (27.4 ± 12.0) of total spleen DC. The OX-62 expression on total spleen DC was 41–59% (51.8 ± 7.5). Myeloid bone marrow-derived DC were negative for CD8α and OX-62. We demonstrated the coexpression of CD8α and OX-62 molecules, at least in a portion CD8α⁺ spleen DC. Both CD8α⁺ and CD8α⁻ spleen DC subpopulations separated by MACS were able to induce an in vivo primary immune response to OVA. The immune response induced by the CD8α⁻ DC subpopulation was higher (P < 0.05). We identified a CD8α⁺ DC subpopulation in rat spleen less effective in inducing an immune response than CD8α⁻ DC. Moreover, our results suggest the presence of DC subpopulations with different lineages in DC preparations based on OX-62 expression.     

Human Osteogenic Sarcoma: Fine Structure of the Osteoblastic Type

The fine structure of representative regions of 13 osteoblastic osteogenic sarcomas was studied. These regions contained four morphologically distinguishable subtypes of osteoblastlike cells. In addition, fibroblastlike and chondroblastlike cells were present, along with multinucleated giant cells, leukocytes, macrophagelike cells, and small populations of histogenetically unclassifiable (but probably neoplastic) cells.

The morphologic evidence was compatible with the view that the variations in appearance among the subgroups of osteobl astlike cells reflected differences in maturation and differentiation of these cells. In at least one subgroup, the morphologic findings suggested that the ceils were capable of manufacturing a secretory product. The multinucleated giant cells occurring in genuine tumor areas appeared to be closely related to neoplastic osteoblasts.

The presence of chondroblastlike cells in the tissues illustrates that cells with a diverging differentiation can occur in an osteoblast-dominated cell population. This agrees with the view that the neoplastic cells originate from a mesenchymal stem cell with potential for multifaceted differentiation.     

Immune cells in term and preterm labor

Labor resembles an inflammatory response that includes secretion of cytokines/chemokines by resident and infiltrating immune cells into reproductive tissues and the maternal/fetal interface. Untimely activation of these inflammatory pathways leads to preterm labor, which can result in preterm birth. Preterm birth is a major determinant of neonatal mortality and morbidity; therefore, the elucidation of the process of labor at a cellular and molecular level is essential for understanding the pathophysiology of preterm labor. Here, we summarize the role of innate and adaptive immune cells in the physiological or pathological activation of labor. We review published literature regarding the role of innate and adaptive immune cells in the cervix, myometrium, fetal membranes, decidua and the fetus in late pregnancy and labor at term and preterm. Accumulating evidence suggests that innate immune cells （neutrophils, macrophages and mast cells） mediate the process of labor by releasing pro-inflammatory factors such as cytokines, chemokines and matrix metalloproteinases. Adaptive immune cells （T-cell subsets and B cells） participate in the maintenance of fetomaternal tolerance during pregnancy, and an alteration in their function or abundance may lead to labor at term or preterm. Also, immune cells that bridge the innate and adaptive immune systems （natural killer T （NKT） cells and dendritic cells （DCs）） seem to participate in the pathophysiology of preterm labor. In conclusion, a balance between innate and adaptive immune cells is required in order to sustain pregnancy; an alteration of this balance will lead to labor at term or preterm.     

Trypanosoma cruzi calreticulin: In vitro modulation of key immunogenic markers of both canine tumors and relevant immune competent cells

Recombinant calreticulin from Trypanosoma cruzi (rTcCalr), the parasite responsible for Chagas’ disease, binds to Canine Transmissible Venereal Tumor (CTVT) cells from primary cultures and to a canine mammary carcinoma cell line. A Complement-binding assay indicated that interaction of the first component C1q with these tumor cells operated independently of the rTcCalr-presence. This apparent independence could be explained by the important structural similarities that exist among rTcCarl, endogenous normal canine and/or mutated calreticulins present in several types of cancer. In phagocytosis assays, tumor cells treated with rTcCalr were readily engulfed by macrophages and, co-cultured with DCs, accelerated their maturation. In addition, DCs maturation, induced by tumor cells co-cultured with rTcCalr, activated T cells more efficiently than DCs, treated or not with LPS. In an apparent paradox, a decrease in MHC Class I expression was observed when these tumor cells were co-cultivated with rTcCalr. This decrease may be related to a down regulation signaling promoting the rescue of MHC I. Possibly, these in vitro assays may be valid correlates of in vivo sceneries. Based on these results, we propose that rTcCalr improves in vitro the immunogenicity of two widely different tumor cell lines, thus suggesting that the interesting properties of rTcCalr to boost immune responses warrant future studies.     

Multiple endocrine cell types in thyroid medullary carcinoma

Summary 10 cases of thyroid medullary carcinoma (TMC) have been studied ultrastructurally and histochemically. Well differentiated calcitonin-producing C cells were present in all tumours, being prevalent in 9 cases. 5-Hydroxytryptamine (5HT) storing cells were found in two cases, somatostatin immunoreactive cells in at least 5 cases and ACTH-immunoreactive cells in 4 cases. Ultrastructurally, at least 3 types of apparently non-C cells were observed. Type 1 cells with large, poorly osmiophilic granules resembling those of gastroenteropancreatic D cells, were present in 6 cases; they appeared to correlate well with somatostatin immunoreactive cells. Type 2 cells with large osmiophilic granules were found in 5 cases; they resembled ACTH-MSH cells of the human pituitary and may correspond to the ACTH-immunoreactive cells of light microscopy. Type 3 cells with small granules and an unknown function were found in 6 cases, always in scarce number. It is concluded that TMC, although mainly made up of C cells, usually contains large proportions of other endocrine cell types.Supported in part by grant N. 75.00630.04 from the Italian National Research Council (C.N.R.). P.F. is a fellow of the Fondazione Anna Villa Rusconi, Varese     

Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor

Mesenchymal stem cells (MSCs) inhibit T‐cell activation and proliferation but their effects on individual T‐cell‐effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4⁺ T cells toward the Th17 phenotype was examined. CD4⁺ T cells exposed to Th17‐skewing conditions exhibited reduced CD25 and IL‐17A expression following MSC co‐culture. Inhibition of IL‐17A production persisted upon re‐stimulation in the absence of MSCs. These effects were attenuated when cell–cell contact was prevented. Th17 cultures from highly purified naïve‐ and memory‐phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX‐2 inhibitor. Media from MSC/Th17 co‐cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC‐mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation‐induced IL‐17A secretion by naturally occurring, effector‐memory Th17 cells from a urinary obstruction model was also inhibited by MSC co‐culture in a COX‐dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T‐cell precursors and inhibit naturally‐occurring Th17 cells derived from a site of inflammation. Suppression entails cell‐contact‐dependent COX‐2 induction resulting in direct Th17 inhibition by PGE2 via EP4.     

The role of core TNF/LIGHT family members in lymph node homeostasis and remodeling

Lymph nodes (LNs) maintain active homeostasis at steady state. However, in response to changes in the local environment, such as local infection, cancer, vaccination, and autoimmune disease, dramatic remodeling of LN occurs. This remodeling includes changes in size, lymph and blood flow, immune cell trafficking and cellularity, lymphatic and blood vessel growth and activation, as well as microarchitecture. Therefore, inflammatory conditions often lead to enlarged nodes; after local inflammation resolves, LNs actively regress in size and return to steady state. Remodeling of lymphatic vessels (LVs) and blood vessels (BVs) during both the expansion and regression phases are key steps in controlling LN size as well as function. The cells, membrane-associated molecules, and soluble cytokines that are essential for LV and BV homeostasis as well as dynamic changes in the expansion and regression phases have not been well defined. Understanding the underlying cellular and molecular mechanisms behind LN remodeling would help us to better control undesired immune responses (e.g. inflammation and autoimmune diseases) or promote desired responses (e.g. antitumor immunity and vaccination). In this review, we focus on how the closely related tumor necrosis factor (TNF) members: LIGHT (TNFSF14), lymphotoxin-αβ, and TNF-α contribute to the remodeling of LNs at various stages of inflammation.     

Increased numbers of cytokeratin-positive interstitial reticulum cells (CIRC) in reactive,inflammatory and neoplastic lymphadenopathies: hyperplasia or induced expression?

A total of 291 enlarged lymph nodes showing a range of reactive-inflammatory processes, primary and metastatic neoplasms were studied to determine the distribution and immunoprofile of their cytokeratin-positive interstitial reticulum cells (CIRC) in comparison with normal nodes. In 258/291 nodes (89%), CIRC numbers were distinctly increased in the subcapsular, paracortical and, occasionally, in the medullary zones; often, these increased CIRC formed networks around follicles, sinuses and vessels. CIRC had comparatively small, irregularly shaped bodies and dendritic processes; occasionally, giant forms were noted. CIRC contained cytokeratins (CK) 8 and 18 but not 19, as shown by immunohistochemistry, and by gel electrophoresis with subsequent immunoblotting. They co-expressed vimentin consistently, alpha-smooth-muscle actin frequently, and desmin less frequently. They did not contain desmoplakins, Factor VIII, S-100, LCA, B and T lymphocyte- and macrophage-associated antigens, chromogranin A, synaptophysin or the A-80 glycoprotein. We found no clear correlation between the increased CIRC and given nodal disease processes. However, CIRC were most abundant in nodes free of but draining malignant tumours; bizarre CIRC assemblies were noted in HIV lymphadenopathy. CIRC appear to represent a subset of the so-called fibroblastic reticulum cells of lymph nodes. Their function remains undetermined; their increase in diverse lymphadenopathies suggests that they partake in nodal reactions to injury. It remains unclear whether the increase in CIRC relative number is due to proliferation or to CK gene induction processes but their presence and potential capability to undergo hyperplasia with dysplastic forms should alert pathologists to possible diagnostic pitfalls. In addition, we discuss that CIRC may undergo transformation and represent the cell of origin of certain CK-positive tumours restricted to lymph nodes.     

Effect of hypophysectomy on endocrine cell types in rat gastrointestinal mucosa

The effect of hypophysectomy on the gastrointestinal tract was studied in the rat 8 wk after operation, particularly regarding the frequency and distribution of serotonin, somatostatin and gastrin-immunoreactive cells. Body weight, the length of the intestine and the thickness of the mucosa of the antrum and small intestine were all reduced in the hypophysectomised rats compared with sham-operated and untreated controls. In the hypophysectomised animals the serotonin-immunoreactive cells were fewer in the antrum and caecum, whereas they were more numerous in the proximal large intestine. There were fewer gastrin-immunoreactive cells in the antrum, while the somatostatin-immunoreactive cells were more numerous in the antrum and caecum. The significant influence of hypophysectomy on the gastrointestinal tract could be direct, but could also be associated with the marked effect of pituitary deficiency on endocrine cells, known to exert both trophic and antitrophic actions. However, it could also be an indirect effect on metabolism, resulting in lower food intake, other endocrine cell systems, and growth factors.