Splenic Stromal Cells Research Articles

Expression of Delta-like 1 in the splenic non-hematopoietic cells is essential for marginal zone B cell development

The Notch ligand Delta-like 1 (Dll1) is critical for the generation of marginal zone (MZ) B cells in the spleen. However, the precise mechanism underlying the differentiation of MZB cells is unclear. To determine whether hematopoietic cells or non-hematopoietic cells provides the Dll1-mediated signals to primitive hematopoietic cells, we transplanted lineage −c-kit +Sca-1 + (KSL) bone marrow cells derived from wild-type (Dll1 +/+) GFP-transgenic mice into lethally irradiated Dll1 conditional knockout (cKO) mice. After transplantation, we examined the kinetics of hematopoietic reconstitution and found that although the frequency of stem/progenitor subsets and of more mature lymphoid, myeloid, and erythroid lineages were normal, the donor-derived hematopoietic cells failed to differentiate into MZB cells. We further demonstrated that while the splenic stromal cells of wild-type mice expressed Dll1 molecule, the splenic stromal cells of recipient Dll1 cKO mice deleted the expression of Dll1. These results suggesting that the expression of Dll1 in splenic non-hematopoietic stromal cells, but not hematopoietic cells, is essential for the development of MZB cells.

Lymphotoxin-Mediated Crosstalk between B Cells and Splenic Stroma Promotes the Initial Type I Interferon Response to Cytomegalovirus

Toll-like receptor (TLR)-dependent pathways control the production of IFNalphabeta, a key cytokine in innate immune control of viruses including mouse cytomegalovirus (MCMV). The lymphotoxin (LT) alphabeta-LTbeta receptor signaling pathway is also critical for defense against MCMV and thought to aid in the IFNbeta response. We find that upon MCMV infection, mice deficient for lymphotoxin (LT)alphabeta signaling cannot mount the initial part of a biphasic IFNalphabeta response, but show normal levels of IFNalphabeta during the sustained phase of infection. Significantly, the LTalphabeta-dependent, IFNalphabeta response is independent of TLR signaling. B, but not T, cells expressing LTbeta are essential for promoting the initial IFNalphabeta response. LTbetaR expression is required strictly in splenic stromal cells for initial IFNalphabeta production to MCMV and is dependent upon the NF-kappaB-inducing kinase (NIK). These results reveal a TLR-independent innate host defense strategy directed by B cells in communication with stromal cells via the LTalphabeta cytokine system.

Infusion of UVB-treated splenic stromal cells induces suppression of β cell antigen-specific T cell responses in NOD mice

Our previous study has demonstrated that transfusion of UVB-irradiation-induced apoptotic β cells effectively prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the limitation of β cell source would preclude the clinical application of this approach. Therefore, in the present study, we have attempted to establish a more practical approach by utilizing apoptotic non-β cells to prevent T1D. We find that apoptotic splenic stromal cells significantly suppress β cell antigen-reactive T cell proliferation in vitro and in vivo. Moreover, β cell antigen-specific T cells primed by β cell antigens in the presence of apoptotic stromal cells have markedly reduced responsiveness to the re-stimulation of the same β cell antigen. We also find that β cell antigen-specific IL-10-producing CD4+ T cells are induced in the presence of apoptotic splenic stromal cells. As expected, transfusion of apoptotic stromal cells effectively protected NOD mice from developing T1D. Furthermore, the proliferation of adoptively transferred β cell antigen-specific TCR-transgenic T cells in pancreatic draining lymph nodes is markedly suppressed in UVB-stroma-treated mice, indicating that UVB-stroma treatment induces immune tolerance to multiple β cell antigens. This study provides an effective and convenient approach for managing T1D by utilizing apoptotic non-β cells.

Hematopoiesis of immature myeloid dendritic cells in stroma-dependent spleen long-term cultures occurs independently of NF- kB/RelB function

The nuclear factor-kappaB (NF-KB)/RelB transcription factor plays an essential role in development of some dendritic cell (DC) subsets in mice. In this laboratory, immature myeloid DC are produced in vitro in a stroma-dependent murine spleen long-term culture (LTC) system. In LTC, DC differentiate from hematopoietic progenitors maintained within the stromal cell matrix. Expression and function of RelB in development of LTC-DC has been investigated, with a view to assessing the relationship between DC produced in this system and other known subsets of DC. RelB expression by LTC-DC was confirmed by detection of protein by Western blotting, RNA by reverse transcription polymerase chain reaction, and nuclear protein with DNA-binding function in electrophoretic mobility shift assays. The role of RelB in cell development was assessed by addition of antisense RelB oligonucleotides into LTC and colony assays established above STX3 stromal cells. RelB(-/-) mice were also examined for ability to produce LTC, and for presence of DC progenitors in spleen and bone marrow that can generate DC when overlaid on STX3 in cocultures. Functional RelB was detected in both LTC-DC and in STX3 stromal cells. A critical role for RelB in DC differentiation from spleen progenitors was confirmed, because antisense RelB oligonucleotides specifically and completely inhibited production of large differentiated myeloid DC in LTC. Further investigation using RelB(-/-) mice revealed that RelB expression by stromal cells rather than hematopoietic cells was required for production of LTC-DC. This was evidenced by a combination of factors, including 1) inability to generate productive LTC from RelB(-/-) mice; 2) presence of DC precursors in RelB(-/-) bone marrow and spleen, which could produce DC in stromal cocultures; and 3) increased myeloid precursor frequency among RelB(-/-) spleen cells over RelB(+/+) control cell populations. Specific development of fully differentiated, but immature myeloid CD11c(+)CD11b(+)MHC-CII(-)CD8alpha(-)CD40(-) DC in spleen LTC is dependent on expression of activated NF-kappaB/Rel-B. However, this appears to relate to stromal cell function rather than to the function of hematopoietic cells. Altogether these data confirm the importance of splenic stromal cells in myelopoiesis leading to development of immature DC as produced in LTC.

Phenotypic differences between red pulp capillary and sinusoidal endothelia help localizing the open splenic circulation in humans

The distribution of capillaries, sinuses and larger vessels was investigated by immunohistology in paraffin sections of 12 adult human spleens using a panel of antibodies. Double staining for CD34 and CD141 (thrombomodulin) revealed that capillary endothelia in the cords of the splenic red pulp and at the surface of follicles were CD34(+)CD141(-), while red pulp sinus endothelia had the phenotype CD34(-)CD141(+). Only in the direct vicinity of splenic follicles did sinus endothelial cells exhibit both antigens. Thus, splenic sinuses do not replace conventional capillaries, but exist in addition to such vessels. The endothelium in arterioles, venules and larger arteries and veins was uniformly CD34(+)CD141(+). Anti-CD34 and anti-CD141 both additionally reacted with different types of splenic stromal cells. Differential staining of capillaries and sinuses may permit a three-dimensional reconstruction of serial sections to unequivocally delineate the "open" and "closed" splenic circulation in humans.

Concise Review: Dendritic Cell Development in the Context of the Spleen Microenvironment

The dendritic cell (DC) population in spleen comprises a mixture of cells including endogenous DC progenitors, DC precursors migrating in from blood and bone marrow, and DC in different states of differentiation and activation. A role for different microenvironments in supporting the dynamic development of murine DC of different types or lineages is considered here. Recent evidence for production of DC dependent on splenic stromal cells is reviewed in the light of evidence that cell production is dependent on cells comprising an endothelial niche in spleen. The possibility that self-renewing progenitors in spleen give rise to DC with tolerogenic or regulatory rather than immunostimulatory function is considered.

Essential role of stromally induced hedgehog signaling in B-cell malignancies

Interaction of cancer cells with their microenvironment generated by stromal cells is essential for tumor cell survival and influences the localization of tumor growth. Here we demonstrate that hedgehog ligands secreted by bone-marrow, nodal and splenic stromal cells function as survival factors for malignant lymphoma and plasmacytoma cells derived from transgenic Emu-Myc mice or isolated from humans with these malignancies. Hedgehog pathway inhibition in lymphomas induced apoptosis through downregulation of Bcl2, but was independent of p53 or Bmi1 expression. Blockage of hedgehog signaling in vivo inhibited expansion of mouse lymphoma cells in a syngeneic mouse model and reduced tumor mass in mice with fully developed disease. Our data indicate that stromally induced hedgehog signaling may provide an important survival signal for B- and plasma-cell malignancies in vitro and in vivo. Disruption of this interaction by hedgehog pathway inhibition could provide a new strategy in lymphoma and multiple myeloma therapy.

In Vitro Effect of Neopterin on Splenic Mast Cell Development in Senescence Accelerated Mice (SAM)

Abstract Neopterin is produced by monocytes and is a useful biomarker of inflammatory responses. We found that neopterin enhances granulopoiesis, but suppresses B-lymphopoiesis triggered by the positive- and negative regulations of cytokines produced by stromal cells in mice. Furthermore, neopterin suppressed the colony formation of mast cell progenitor (CFU-mast) from bone marrow cells in in vitro culture system. In this study, neopterin was also found to regulate the proliferation and differentiation of splenic CFU-mast in vitro as observed in that from bone marrow, which was confirmed in the mouse model of senescent stromal-cell impairment (SCI). In non- SCI mice (=less senescent stage of SCI mice), neopterin also decreased the number of colonies of interleukin-3 (IL-3)-dependent mast-cell progenitor cells (CFU-mast) from unfractionated spleen cells, but not that from the lineage-negative (fractionated) spleen cell population without stromal cells in a semisolid in vitro culture system. In contrast, in a case of SCI mice, the treatment with neopterin did not decrease the number of colonies of IL-3- dependent mast-cell progenitor cells (CFU-mast) from unfractionated spleen cells. These results suggest that, firstly, neopterin decrease the number of colonies of IL-3-dependent CFU-mast by stimulating splenic stromal cells, and secondly, such neopterin function becomes declined during senescence because of an impaired stromalcell function.

Effect of α-fetoprotein on the count of bone marrow and splenic stromal precursor cells and proliferation of their cultural descendants

The efficiency of cloning of stromal precursor cell increased more than 2-fold in splenic cultures and more than 3-fold in bone marrow cultures 24 h after injection of Profetal preparation to mice in vivo. The number of nucleated cells did not change in the bone marrow and slightly increased in the spleen. Addition of Profetal in vitro 2-fold decreased the efficiency of stromal precursor cells colony formation in mouse splenic cultures and dose-dependently decreased this process in bone marrow cultures derived from these animals, the maximum (5-fold) inhibitory effect was observed in a dose of 50 microg/ml. Addition of Profetal to cultured human bone marrow fibroblasts did not change the content of stromal fibroblasts in cultures. These data indicate the possibility of indirect effect of alpha-fetoprotein on the number of stromal precursor cell in hemopoietic and lymphoid organs.

Balancing immunity and pathology in visceral leishmaniasis

Experimental visceral leishmaniasis (VL) caused by infection with Leishmania donovani results in the development of organ-specific immunity in the two main target tissues of infection, the spleen and the liver. The liver is the site of an acute resolving infection associated with the development of inflammatory granulomas around infected Kupffer cells, and resistance to reinfection. Paradoxically, the spleen is an initial site for the generation of cell-mediated immune responses, but ultimately becomes a site of parasite persistence with associated immunopathological changes. These include splenomegaly and a breakdown in tissue architecture that is postulated to contribute to the immunocompromized status of the host. The progressive development of splenic pathology is largely associated with high levels of TNF and interleukin (IL)-10. Follicular dendritic cell (DC) networks are lost, whereas TNF mediates the destruction of marginal zone macrophages and gp38(+) stromal cells, and IL-10 promotes impaired DC migration into T-cell areas with consequent ineffective T-cell priming. Splenic stromal cell function is also altered, promoting the selective development of IL-10-producing DC with immunoregulatory properties. Ultimately, a fine immunological balance determines responses that effectively promote parasite clearance in the liver and those that promote pathology in the spleen, and future investigation aims to separate these responses to offer further means of parasite control in chronically infected VL patients.

Splenic Endothelial Cell Lines Support Development of Dendritic Cells from Bone Marrow

Although growth factors are commonly used to generate dendritic cells (DCs) in vitro, the role of the microenvironment necessary for DC development is still poorly understood. The mixed splenic stromal cell population STX3 defines an in vitro microenvironment supportive of DC development. Dissection of cellular components of the STX3 stroma should provide information about a niche for DC development. STX3 was therefore cloned by single-cell sorting, and a panel of 102 splenic stromal cell lines was established. Four representative splenic stromal cell lines that support hematopoiesis from bone marrow are described here in terms of stromal cell type and DC production. All four stromal lines express the endothelial genes Acvrl1, Cd34, Col18a1, Eng, Flt1, Mcam, and Vcam1 but not Cd31 or Vwf. Three of the four lines form tube-like structures when cultured on Matrigel. Their endothelial maturity correlates with the ability to support myeloid DC development from bone marrow. A fourth cell line, unable to form tube-like structures in Matrigel, produced large granulocytic cells expressing CD11b and CD86 but not CD11c and CD80. Conditioned media from splenic stromal cell lines also support DC production, indicating that soluble growth factors and cytokines produced by stromal lines drive DC development. This article reports characterization of immature endothelial cell lines derived from spleen that are supportive of DC development and predicts the existence of such a cell type in vivo which regulates DC development within spleen.

HETEROGENEITY AMONGST SPLENIC STROMAL CELL LINES WHICH SUPPORT DENDRITIC CELL HEMATOPOIESIS

Long-term cultures (LTC) producing dendritic cells (DC) have been previously established from spleen. LTC support the development of nonadherent cells comprising small DC progenitors and immature DC. Similarly, the splenic stroma STX3, derived from a LTC which ceased DC production, can support DC development from precursors in overlaid bone marrow. The STX3 stroma is an immortalised mixed population of endothelial cells and elongated spindle-shaped cells, thought to be fibroblasts. The stromal cell components of STX3 have been studied here. A panel of 102 cell lines was established by single-cell sorting. A range of clone morphology, including cobblestone cells and elongated spindle-shaped cells, was reflective of heterogeneity in STX3. However, similar expression levels for the endothelial genes ACVRL1/ ALK1, COL18A1, and MCAM in 13 splenic stromal cell lines suggested that both cell types had endothelial origin. The hematopoietic support function of stromal clones was tested in coculture assays with a bone marrow cell overlay. Splenic stromal cell lines with different morphology were both supporters and nonsupporters of hematopoiesis, in terms of foci formation or release of suspension cells. Cloning of STX3 led to the isolation of a panel of splenic endothelial cell lines heterogeneous in terms of morphology and hematopoietic support function.

Stimulating Lymphotoxin β Receptor on the Dendritic Cells Is Critical for Their Homeostasis and Expansion

The increased number of dendritic cells (DCs) inside lymphoid tissue may contribute to the enhanced priming of lymphocytes. The homeostasis of splenic DCs has mostly been attributed to their migration to the spleen via the chemokine microenvironment induced by lymphotoxin beta receptor (LTbetaR) signaling on splenic stromal cells. In this study we show that the lack of direct LTbetaR signaling on DCs is associated with the reduction of the number of DCs in the spleen independently of chemokine gradients. LTbetaR-/- mice have reduced DCs and reduced BrdU incorporation on DCs, and fewer DCs from LTbetaR-/- mice are detected in the spleen. Furthermore, increased expression of LIGHT (homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for herpes virus entry mediator on T cells) on T cells, a member of the TNF family (TNFSF14) and a ligand for LTbetaR, could dramatically increase the number of T cells and DCs, which leads to severe autoimmune diseases in a LTbetaR-dependent fashion. In vitro, LIGHT could directly promote accumulation of bone marrow-derived DCs. Furthermore, intratumor expression of LIGHT can dramatically expand DCs in situ, and inoculation of DCs into tumor tissues enhanced tumor immunity. Therefore, LTbetaR signaling on DCs is required for their homeostasis during physiology and pathological conditions, and increased LIGHT-LTbetaR interaction could stimulate DC expansion for T cell-mediated immunity.

Contribution of stromal cells to the migration, function and retention of plasma cells in human spleen: potential roles of CXCL12, IL‐6 and CD54

Plasma cells (PC) localize to discrete areas of secondary lymphoid tissue and bone marrow (BM). The positioning of PC in different sites is believed to be regulated by chemokines and adhesion molecules expressed by accessory cells in the lymphoid tissue microenvironment. However, the mechanisms responsible for the positioning of PC within the red pulp (RP) of human spleen have not been elucidated. Therefore, we examined the contribution of human splenic stromal cells to the migration and function of human PC. Splenic PC expressed the chemokine receptor CXCR4 and responded to its ligand CXCL12. In contrast, PC lacked CXCR5 and CCR7, and consequently exhibited minimal migration towards CXCL13 and CCL21. Splenic stromal cells proved to be a rich source of CXCL12, and could induce the migration of human B cells. Furthermore, they supported Ig production by splenic PC mainly by secreting IL-6. Lastly, a striking difference between splenic and BM PC was the constitutive expression of CD11a by only splenic PC. Notably, splenic stromal cells expressed high levels of CD54, the counter-structure of CD11a, and splenic PC were positioned adjacent to stromal cells in the RP. Thus, we propose that stromal cells attract PC to the RP and contribute to their retention and function through the combined expression of CXCL12, CD54 and IL-6.

Splenic stroma drives mature dendritic cells to differentiate into regulatory dendritic cells.

The fates of dendritic cells (DCs) after antigen presentation have been studied extensively, but the influence of lymphoid microenvironments on DCs is mostly unknown. Here, using splenic stromal cells to mimic the immune microenvironment, we show that contact with stromal cells promoted mature DCs to proliferate in a fibronectin-dependent way and that both stromal cell contact and stromal cell-derived transforming growth factor-beta induced their differentiation into a new regulatory DC subset. We have identified an in vivo counterpart in the spleen with similar phenotype and functions. These differentiated DCs secreted nitric oxide, which mediated the suppression of T cell proliferation in response to antigen presentation by mature DCs. Thus, our findings identify an important mechanism by which the microenvironment regulates immune responses.

Immunological Characterization of Tristetraprolin as a Low Abundance, Inducible, Stable Cytosolic Protein

Tristetraprolin (TTP) is a zinc finger protein that can bind to AU-rich elements within certain mRNAs, resulting in deadenylation and destabilization of those mRNAs. Its physiological targets include the mRNAs encoding the cytokines tumor necrosis factor alpha (TNF) and granulocyte-macrophage colony-stimulating factor. TTP was originally identified on the basis of its massive but transient increase in mRNA levels following mitogen stimulation of fibroblasts. It has been difficult to reconcile this transient mRNA profile with the presumed continuing "need" for TTP protein, for example, to reverse the effects of lipopolysaccharide (LPS)-stimulated TNF secretion. To investigate this and other questions concerning endogenous TTP protein in cells and tissues, we raised a high titer rabbit antiserum against full-length mouse TTP. TTP could be detected on immunoblots of mouse cytosolic tissue extracts; it was most highly expressed in spleen, but its concentration in that tissue was only about 1.5 nm. TTP could be detected readily in splenic macrophages and stromal cells from LPS-injected rats. In both LPS-treated RAW 264.7 macrophages and fetal calf serum-treated mouse embryonic fibroblasts, TTP protein was stable after induction, with minimal degradation occurring for several hours after treatment of the cells with cycloheximide. The biosynthesis of TTP was accompanied by large changes in electrophoretic mobility consistent with progressive phosphorylation. Confocal microscopy revealed that TTP accumulated in a vesicular pattern in the cytosol of the LPS-stimulated RAW 264.7 cells, and was occasionally seen in the cytosol of unstimulated dividing cells. Gel filtration of the endogenous protein suggested that its predominant structure was monomeric. TTP appears to be a low abundance, cytosolic protein in unstimulated cells and tissues, but once induced is relatively stable, in contrast to its very labile mRNA.

Effect of platelet growth factors on proliferation of guinea pig bone marrow and splenic stromal precursor cells and proliferation of cultural descendants from bone marrow precursor cells.

Elimination of platelets from guinea pig splenocyte suspension (lacking megakaryocytes) with EDTA considerable reduces the efficiency cloning of splenic stromal precursor cells. It means that platelet-derived growth factors are necessary for stromal precursor cells from different organs (bone marrow, thymus, and spleen). The dependence on the platelet growth factors can vary within a wide range in descendants from cultured bone marrow precursor cells (passaged bone marrow fibroblasts at different stages of differentiation.

Identification of progenitor cells in long-term spleen stromal cultures that produce immature dendritic cells.

Dendritic cells (DC) are produced continuously by a unique, long-term culture (LTC) system in which hemopoiesis is supported by a splenic stromal cell layer in the absence of added growth factors. Flow cytometric analysis reveals the production of two distinct cell subsets. The more predominant large-cell subset resembles highly endocytic DC that are large, granular, and possess membrane extensions. They also express high levels of the DC markers CD11c, CD11b, DEC-205, and CD80 on their cell surface. They do not resemble mature DC because they express low levels of MHC type II and CD86 molecules, as well as c-kit and Fc receptor (FcR). These are known characteristics of immature DC. Small cells are smaller and less granular than large cells, with negative to low expression of CD11c, DEC-205, and CD86. A majority of small cells express varying levels of CD11b and CD80. Subpopulations of small cells express low levels of c-kit, FcR, and MHC type II, and only a 20% subpopulation is weakly endocytic. Upon transfer to an irradiated stromal layer, cells within the small subset proliferate and differentiate to resemble the large cells in size, complexity, membrane extensions, and CD11c and CD86 expression. The two cell subsets produced in LTC are developmentally linked, with the heterogeneous small-cell subset containing progenitors of the larger homogeneous, immature DC subset. LTC represent a valuable model system for studying DC development from hemopoietic progenitors.

Selective inhibition of hepatoma cells using diphtheria toxin A under the control of the promoter/enhancer region of the human alpha-fetoprotein gene.

We constructed a plasmid containing human α‐fetoprotein (AFP) promoter/enhancer to direct the cell type‐specific expression of diphtheria toxin fragment A (DTA), designated as pAF‐DTA, to AFP‐producing hepatocellular carcinoma cells. The transfection was carried out with cationic liposomes (DMRIE‐C) and the expression of the DTA gene was confirmed by a northern blot analysis. When pAF‐DTA was transfected, the growth of AFP‐positive HuH‐7 cells was inhibited, whereas growth inhibition was not observed in AFP‐negative MKN45 cells. In this experiment, the secretion of AFP was similarly suppressed, but the secretion of carcinoembryonic antigen from MKN45 was not altered. pAF‐DTA could also exert its growth inhibitory effect on PLC, a cell line with a low level of AFP. However, no inhibitory effect of pAF‐DTA was observed on the proliferation of primary hepatocyte cells. Furthermore, transfection experiments in which HuH‐7 and splenic stromal cells were co‐cultured revealed the growth inhibition by pAF‐DTA to be selective in HuH‐7 cells. Finally, the growth of HuH‐7 transplanted on BALB/c nu/nu mice was inhibited by the direct injection of pAF‐DTA/liposome complex into a tumor mass. These results suggest that use of pAF‐DTA may be potentially useful as a novel approach for the selective treatment of tumor cells producing AFP even at low levels, without affecting other types of cells.

Spleen stromal cells support haemopoiesis and in vitro growth of dendritic cells from bone marrow

Long-term stroma-dependent cultures from murine spleen have been previously shown to support dendritic cell (DC) development in vitro. Secondary cultures have now been established using a splenic stromal cell layer overlaid with cells from different lymphoid sites. Cells resembling DCs can be generated in vitro from unfractionated murine lymphoid cells in the absence of added growth factors. Bone marrow (BM) cultures are the most successful but some cultures have been derived from spleen and thymus. Large numbers of mononuclear cells with dendritic morphology can be generated from BM within 2 weeks and cell production in many cultures has been maintained for at least 6 months. A significant proportion of cells binds antibodies specific for DC markers. No lymphoid cells, mast cells or granulocytes are detectable in culture by antibody and histochemical staining and light and electron microscopy. As with cells generated in primary cultures, cells grown in secondary cultures are equally potent stimulators of both syngeneic and allogeneic mixed lymphocyte reactions, confirming their function as antigen-presenting cells. They are also capable of endocytosing and presenting protein antigen to the D10.G4.1 Th2 clone and to unprimed T cells. This study confirmed the presence of DC precursors in multiple lymphoid sites which can be induced to proliferate in the presence of a spleen stromal cell monolayer. The secondary culture system provides an ideal in vitro model for investigation of the development of DC from different tissue sites. It also provides a stable and continuous resource of cells for further studies on DC development.