Medullary Stromal Cells Research Articles

Factors influencing the development of Multicystic Dysplastic Kidney (MCDK) following urinary tract obstruction in the fetal lamb.

Creating obstructive uropathy (OU) during glomerulogenesis in the fetal lamb results in multicystic dysplastic kidney (MCDK) at term. We explored this using immunohistochemical techniques. OU was created in fetal lambs at 60-day gestation, ligating the urethra and urachus. The kidneys of MCDK lambs, 60-day gestation fetal lambs, full-term lamb (145days), term sham-operated lambs, and adult ewes were evaluated by HE staining, and immunohistochemistry with paired box genes 2 (PAX2) and CD10. Multiple cysts were found in the MCDK model. CD10 was expressed in proximal tubular epithelial cells, glomerular epithelial cells, and medullary stromal cells in the kidneys of 60-day gestation fetal lambs and full-term lambs and adult ewes. PAX2 expression was found in ureteric buds, C- and S-shaped bodies, epithelial cells of collecting ducts, and Bowman's capsule of fetal kidneys at 60-day gestation, but only in the collecting ducts of full-term fetal lambs and adult ewes. Both CD10 and PAX2 were expressed in the cystic epithelial cells of the MCDK model. PAX2 expression in cystic epithelial cells suggests that cyst formation is associated with disturbed down-regulation of PAX2 in the nephrogenic zone epithelial cells during the renal development in the OU model.

Medullary stromal cells synergize their production and capture of CCL21 for T-cell emigration from neonatal mouse thymus.

The release of newly selected αβT cells from the thymus is key in establishing a functional adaptive immune system. Emigration of the first cohorts of αβT cells produced during the neonatal period is of particular importance, because it initiates formation of the peripheral αβT-cell pool and provides immune protection early in life. Despite this, the cellular and molecular mechanisms of thymus emigration are poorly understood. We examined the involvement of diverse stromal subsets and individual chemokine ligands in this process. First, we demonstrated functional dichotomy in the requirement for CCR7 ligands and identified CCL21, but not CCL19, as an important regulator of neonatal thymus emigration. To explain this ligand-specific requirement, we examined sites of CCL21 production and action and found Ccl21 gene expression and CCL21 protein distribution occurred within anatomically distinct thymic areas. Although Ccl21 transcription was limited to subsets of medullary epithelium, CCL21 protein was captured by mesenchymal stroma consisting of integrin α7+ pericytes and CD34+ adventitial cells at sites of thymic exit. This chemokine compartmentalization involved the heparan sulfate-dependent presentation of CCL21 via its C-terminal extension, explaining the absence of a requirement for CCL19, which lacks this domain and failed to be captured by thymic stroma. Collectively, we identified an important role for CCL21 in neonatal thymus emigration, revealing the importance of this chemokine in initial formation of the peripheral immune system. Moreover, we identified an intrathymic mechanism involving cell-specific production and presentation of CCL21, which demonstrated a functional synergy between thymic epithelial and mesenchymal cells for αβT-cell emigration.

β-Catenin in stromal progenitors controls medullary stromal development

The renal stroma is a population of matrix-producing fibroblast cells that serves as a structural framework for the kidney parenchyma. The stroma also regulates branching morphogenesis and nephrogenesis. In the mature kidney, the stroma forms at least three distinct cell populations: the capsular, cortical, and medullary stroma. These distinct stromal populations have important functions in kidney development, maintenance of kidney function, and disease progression. However, the development, differentiation, and maintenance of the distinct stroma populations are not well defined. Using a mouse model with β-catenin deficiency in the stroma cell population, we demonstrate that β-catenin is not involved in the formation of the stromal progenitors nor in the formation of the cortical stroma population. In contrast, β-catenin does control the differentiation of stromal progenitors to form the medullary stroma. In the absence of stromal β-catenin, there is a marked reduction of medullary stromal markers. As kidney development continues, the maldifferentiated stromal cells locate deeper within the kidney tissue and are eliminated by the activation of an intrinsic apoptotic program. This leads to significant reductions in the medullary stroma population and the lack of medulla formation. Taken together, our results indicate that stromal β-catenin is essential for kidney development by regulating medulla formation through the differentiation of medullary stromal cells.

Defining the role of CCR8 in promoting thymocyte development and thymic epithelial homeostasis

Abstract Generation of functional and self-tolerant T cells in the thymus is crucial to preventing autoimmune diseases. Medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present diverse auto-antigens to developing thymocytes; strong TCR reactivity in response to self-antigens causes thymocytes to undergo negative selection, eliminating autoreactive cells from the repertoire. Chemokine receptors expressed on the surface of developing thymocytes have been shown to promote accumulation of post-positive selection thymocytes in the medulla, where they may encounter mTECs and DCs presenting auto-antigens. CCR8 is highly expressed by post-positive selection CD4 thymocytes while its ligands, CCL1 and CCL8 are expressed by mTECs. Thus, CCR8 signaling may promote medullary entry of post-positive selection thymocytes and efficient interactions with mTECs and enforcing central tolerance. Our preliminary data indicate that CCR8−/− thymi have a reduced medullary area, implicating impaired thymocyte:mTEC interactions that impacts normal proliferation and differentiation of mTECs. Furthermore, 2-photon imaging reveals that CCR8 deficiency results in a significant increase in the path straightness of CD4 SP thymocytes, which could impact the efficiency of interactions with medullary stromal cells. Strikingly, CCR8 deficiency results in increased cellularity of CD4SP OTII TCR transgenic thymocytes in a wild-type host, suggesting CCR8 is critical for efficient clonal deletion to endogenous auto-antigens. Thus, our preliminary data indicate that CCR8 contributes to the migratory properties and stromal interactions of post-positive selection thymocytes that promote efficient negative selection.

CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3(+) T Cells.

SummaryCurrent models of Foxp3+ regulatory T cell (Treg) development involve CCR7-mediated migration of thymocytes into the thymus medulla to enable essential interactions with medullary epithelium. However, increased Foxp3+ thymic Treg numbers in Ccr7−/− mice challenge this view, and the role of CCR7 in Treg development, emigration, and/or recirculation is unknown. Here, we have examined CCR7 and Rag2pGFP levels during Treg development and generated Rag2pGFPCcr7−/− mice to study its impact on the intrathymic Treg pool. We reveal surprising developmental heterogeneity in thymocytes described as Treg precursors, showing that they contain recirculating CCR6+CCR7−Rag2pGFP− T cells. Although CCR7 defines bona fide Rag2GFP+ Treg precursors, it is not required for Treg production and emigration. Rather, we show that lack of CCR7 renders the thymus more receptive to Treg thymus homing. Our study reveals a role for CCR7 in limiting Treg recirculation back to the thymus and enables separation of the mechanisms controlling Treg production and thymic recirculation.

The Influence of Ovarian Stromal/Theca Cells During In Vitro Culture on Steroidogenesis, Proliferation and Apoptosis of Granulosa Cells Derived from the Goat Ovary

Early follicular development is closely related to oocyte-granulosa cells-ovarian stromal cells/theca cells. The aim of the present study was to investigate the effects of ovarian cortical, medullary stromal and theca cells on oestradiol and progesterone biosynthesis, proliferation and apoptosis of goat ovary granulosa cells in vitro. Using Transwell coculture system, we evaluated steroidogenesis, cell proliferation and apoptosis, and some molecular expressions regarding steroidogenic enzyme, luteinizing hormone receptor and apoptosis-related genes in granulosa cells. The results indicated that ovarian stromal/theca cells were able to stimulate oestradiol and progesterone production, promote cell proliferation and inhibit apoptosis of granulosa cells. Among all the three kinds of cells, theca cells affected strongly on granulosa cell function, and ovarian medullary stromal cells had the weakest effect on granulosa cells. These findings would provide an important knowledge of cell interaction among follicular cells during follicular development.

Molecular pathology of myelodysplastic syndromes: Biology of medullary stromal and hematopoietic cells (Review)

Myelodysplastic syndromes (MDS) have been defined as a disease entity based on clinical features and morphological findings. Despite similarities in clinical manifestations, genetic abnormalities occurring in hematopoietic cells are heterogeneous among the syndromes. However, recent investigations have revealed that there are common biological events in the bone marrow of MDS cases. Most notably, excessive apoptosis of hematopoietic cells was observed to be induced by the bone marrow microenvironment. The apoptosis was mediated by paracrine as well as autocrine factors, suggesting that medullary stromal and hematopoietic cells play a role in the pathology of disease. Pro-inflammatory cytokines, such as TNFα, in the bone marrow microenvironment are predominantly paracrine mediators of apoptosis. Regarding autocrine stimulation mechanisms, it has recently been shown that the deregulation of ribosomal protein is capable of initiating a stress response in the hematopoietic cell through a p53-mediated signaling pathway. Thus, both the stromal cells of the bone marrow microenvironment and hematopoietic cells themselves possess a common and characteristic biology in this heterogeneous disease entity.

Vascular Niche and Platelet Production: Stromal Cells Inhibit Proplatelet and Platelet Formation by Human Megakaryocytes (MK), Whereas Endothelial Cells Are Permissive

AbstractAbstract 402The site of platelet production in the human body is still unclear, but several evidence, including the recent demonstration by our team that shear stress in vitro accelerates platelet formation, favour the hypothesis of an intravascular location of the platelet release process. Therefore we have undertaken the following study to compare the effect of two cell types from the human bone marrow microenvironment, stromal medullary cells and endothelial cells, on the final steps of megakaryocyte (MK) maturation and platelet production. Firstly, supporting the goal of our study, we show that entire mature MK can be encountered in the lumen of normal bone marrow sinusoids and therefore enter the circulation of flowing blood; Secondly, we have used a new microcapillary device coated with von Willebrand factor and were able to confirm by videomicroscopy that exposure of human mature MK to shear forces accelerates proplatelet extension and facilitates platelet liberation from the tips of proplatelets. During this process, the nuclear lobes of polyploid MK tend to separate and to form distinct proplatelet units ready to deliver platelets: this observation might explain why massive senescent MK nuclei are only rarely found in human tissues, bone marrow or lung. Then, we used flow cytometry, optical and electron microscopy, to document and visualise the interaction of human medullary stromal cells (HS5), murine medullary stromal cells (MS5), and human endothelial cells (HUVEC) with the final steps of human MK maturation, namely proplatelet formation. MK were grown from umbilical cord blood CD34+ cells in the presence of Stem Cell Factor (SCF) and thrombopoietin and co-cultured with stromal cells between day 10 and day 13 of culture. Stromal cells virtually completely inhibited proplatelet formation from MK, whereas endothelial cells showed no effect and allowed the emission of proplatelets. EM and flow cytometry confirmed that stromal cells blocked proplatelet and platelet formation and showed that this was accompanied with a considerable development of demarcation membranes which coincided with a 60% increase of CD41 expression by MK. In parallel, stromal cells induced a reduction of apoptosis signs with a reduction of annexin V fixation by maturing MK. Since the step of proplatelet formation is accompanied with cytoplasmic apoptotic signs, among which increased phosphatidyl serine exposure, this result is in accordance with the observed blockade of proplatelet extension. Stromal cell secretion include several growth factors, namely SCF and GMCSF. MK were cultured in the presence of stromal cell culture supernatants, and with these growth factors either individually or mixed together, but this had no effects on proplatelet production, suggesting that physical contacts between the two cell types are necessary. In conclusion, our data show that human bone marrow microenvironment (stromal cells) has an inhibitory effect on proplatelet and platelet formation whereas the vascular microenvironment (endothelial cells) is permissive. This may explain the absence of proplatelets in the bone marrow parenchyma and be an additional evidence that platelet formation and release has to mainly occur in the intravascular compartment. Disclosures:No relevant conflicts of interest to declare.

Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development

The Wnt4 gene encodes a secreted signaling molecule controlling the development of several organs, such as the kidney, adrenal gland, ovary, mammary gland and pituitary gland. It is thought to act in the embryonic kidney as an auto-inducer of nephrogenesis controlling mesenchyme-to-epithelium transition, and Wnt4-deficient mice die soon after birth, probably of kidney failure. Given the requirement for Wnt4 signaling in the control of organogenesis, the targeting of Cre recombinase under the control of the Wnt4 promoter would provide a valuable tool for fate mapping and functional genomics. We report here on the generation and characterization of a Wnt4 EGFPCre knock-in allele where the EGFPCre fusion cDNA and Neo selection cassette were targeted into the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates of the E14.5 embryonic kidney that normally express Wnt4 mRNA. Characterization of the pattern of recombination of the floxed Rosa26 LacZ reporter with the Wnt4 EGFPCre allele revealed that in addition to the embryonic kidney, reporter-derived staining was observed in the embryonic gonad, spinal cord, lung and adrenal gland, i.e. the sites of Wnt4 gene expression. Time-lapse fate mapping of the Wnt4 EGFPCre -activated yellow fluorescent protein (YFP) from the Rosa26 locus in organ culture revealed that the cells that had expressed the Wnt4 gene contributed to the nephrons, some of the cells around the stalk of the developing ureter and also certain presumptive medullary stromal cells. Moreover, the time-lapse movies suggested that the first few pretubular cell aggregates may not mature into nephrons but instead appear to disintegrate. In association with this, Rosa26 YFP -positive stromal cells emerge around these disintegrating structures. Such cells may be transient, since their derivatives are neither detected later in the more mature kidney nor is there an overlap of the Wnt4 EGFPCre ; Rosa26 LacZ -marked cells with those of the endothelial cells, the smooth muscle cells or the macrophages. The Wnt4 EGFPCre allele provides a useful new tool for conditional mutagenesis and provides the first time-lapse-based map of the fate of nephron precursor cells.

Wild-type AIRE cooperates with p63 in HLA class II expression of medullary thymic stromal cells

During T cell development in the thymus, autoreactive T cells are deleted through a mechanism that is actively supported by medullary epithelial cells. These epithelial cells possess particular transcription factors including autoimmune regulator (AIRE), which is responsible for regulating expression of self-antigens, as well as p63, a p53-like molecule. Here we present evidence suggesting interaction of AIRE with p63 through a SAND domain and a transactivation domain, respectively. Interestingly an AIRE molecule with a mutated SAND domain of G228W, whose genetic alteration is inherited in an autosomal dominant manner, could not establish a complex with p63 as indicated by immunoprecipitation and molecular modeling analyses. Further in vitro study indicated that the G228W mutation led to downregulation of the transcription levels of CIITA and, accordingly, the cell surface expression of HLA class II molecules in thymic epithelial cells with p63. This indicates novel involvement of AIRE and p63 in the regulation of HLA class II, and suggests that defects in the AIRE–p63 interaction may lead to malfunction of HLA-based selection of self-reactive helper CD4 + T cells in the thymus.

Expression Pattern of Immunoproteasome Subunits in Human Thymus

The expression pattern of immunoproteasomes in human thymus has not been analyzed but may have important consequences during thymic selection. Here we examined the expression patterns of immunoproteasome subunits in fetal and adult thymic tissues by immunohistochemistry and found that all three subunits are expressed in both cortical and medullary stromal cells. These data suggest that thymic selection in human can be affected by peptide repertoires generated by immunoproteasomes.

Cellular Networks of Human Thymic Medullary Stromas Coordinated by p53-Related Transcription Factors

The final elimination step of self-reactive T cells occurs in the medulla of the thymus where a complex framework provided by stromal cells supports an optimal milieu for their selection. Here we present evidence that tight junctions (TJs) widely join medullary stromal cells of the human thymus. Occludin (OCLN) and claudin-1 (CLDN-1) of TJ-associated molecules were dominantly expressed in medullary thymic epithelial cells (mTECs), and CLDN-4 and CLDN-7 were also localized in some mTECs near Hassall's corpuscles. Interestingly, p53-like transcription factors were found to upregulate OCLN and CLDN-1 in human TEC lines, as recently suggested in the regulation of mTEC function. Furthermore, dendritic cells (DCs) of the medulla, with a major role for selection of thymocytes, expressed CLDN-1 and OCLN as well, implying that the interposition of DCs within the mTEC scaffold is also helped by TJs. Analysis of freeze-fracture replicas of the thymus revealed TJ strand structures in the vicinity of gap junction plaques through which small molecules might move, as implied by dye-transfer analysis of a medullary cell line. Thus, it is thought that p53-like molecules regulate TJ-associated interactions of medullary stromal cells and that this mechanism might be associated with an intercellular communication network, probably for preserving the medullary niches.

Immunofluorescence and confocal laser scanning microscopy of chronic myeloproliferative disorders on archival formaldehyde-fixed bone marrow.

Spatial analysis of the histoarchitecture and photographic documentation at high resolution are the principal advantages of confocal laser scanning microscopy (CLSM) over conventional fluorescence microscopy (CFM) if combined with appropriate software. Restrictions for the use of CFM and CLSM, on the other hand, include nonspecific background fluorescence, fading of photolabile fluorochromes, and both tissue-specific and fixation-induced autofluorescence. Most of those shortcomings can now be avoided. Autofluorescence, the most limiting factor of high-resolution CLSM, was recently controlled also for paraffin sections of archival formaldehyde-fixed tissues. This allowed the present study on cytoskeletal fibers and extracellular matrix proteins in both neoplastic cells of myeloproliferative disorders and in medullary stromal cells using CLSM under proper autofluorescence control. By multiple fluorescence labeling, we found that the intracellular smooth muscle alpha-actin (SMA) fibers and the two extracellular adhesive matrix proteins tenascin and fibronectin vary in their presence in stromal and/or myeloid cells according to the degree of bone marrow fibrosis in chronic myeloproliferative disorders (CMPDs). CLSM offers further insight in our attempts to understand a complex interplay between the two cellular compartments.

RNA and protein expression of the murine autoimmune regulator gene (Aire) in normal, RelB-deficient and in NOD mouse.

Mutations in the putative transcription factor autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiosis-ectodermal dystrophy (APECED; OMIM#240300), a monogenic recessively inherited disease characterized by destructive autoimmune diseases of the endocrine organs, chronic candidiosis of mucous membranes and ectodermal dystrophies. In this study the expression of murine homolog for AIRE protein, Aire, was detected in a fraction of thymic medullary epithelial cells. Subcellularly, in thymus the protein appears as concentrated into nuclear dot-like structures, whereas in transfected cells the protein is also bound along a cytosolic fibrillar network. By RT-PCR Aire mRNA was detected in thymus, lymph node, spleen and testis although the second round PCR amplified Aire specific band from most mouse tissues analyzed. Furthermore, the Aire mRNA was detected in dendritic cell (DC) populations isolated from thymus and spleen, representing both myeloid- and lymphoid-related lineages of DC. We also demonstrate that the Aire protein is absent in the thymus of RelB-deficient mouse and in NOD thymus most of the Aire positive cells showed an abnormal morphology. These results suggest that the Aire protein is associated with the normal development and/or action of a subset of thymic medullary stromal cells involved in tolerance induction.

A novel anti-Ep-CAM antibody to analyze the organization of thymic medulla in autoimmunity.

Organization of microenvironments through which lymphocytes migrate is crucial for the generation of optimal immune responses and development [1]. In peripheral lymphoid organs, chemokines secreted by stromal cells are required for the constitution of T and B cell areas [2]. In the thymus, immature thymocytes attach to fibronectin via of α5β1 integrins [3]. Then, upon ligation of the preTCR, they upregulate CCR9 which triggers their attraction towards TECK-producing epithelial cells in the cortex and the medulla [4, 5]. Positively selected thymocytes lose CCR9 and acquire CCR4 and 7 which condition their migration towards chemokine gradient secreted by medullary stromal cells [6]. In re1Bdeficient mice, there is a complete disorganization of the cortico-medullary structure associated with a poor negative selection process [7–9]. These mice lack activated epithelial and myeloid-derived dendritic cells and, consequently, the expression of costimulatory or MHC molecules and the secretion of the medullary chemokine TCA4 are very low [10]. This animal model provides thus a useful link between the organization of a functional niche for negative selection and the activation of stromal cells. Using a panel of mAb directed at various types of stromal cells, we have found that the reactivity of some mAb was severely reduced in re1B-deficient thymuses. One of them was found to recognize the 106/JAM molecule, a novel member of the IgSF highly expressed by endothelial cells and most MHC class II-expressing stromal cells in thymus and peripheral lymphoid organs [11]. This molecule plays a stabilizing role in the formation of tight junctions [12] and is acquired upon tissue colonization by macrophages and dendritic cells. In this report, we describe the molecular cloning of the Ep-CAM molecules identified by the mAb 29 [8] used to define relB-dependent thymic medullary stromal cells in normal and autoimmune-prone animals.

Distinct roles of prostaglandin H synthases 1 and 2 in T-cell development.

Prostaglandin G and H synthases, or cyclooxygenases (COXs), catalyze the formation of prostaglandins (PGs). Whereas COX-1 is diffusely expressed in lymphoid cells in embryonic day 15.5 thymus, COX-2 expression is sparse, apparently limited to stromal cells. By contrast, COX-2 is predominant in a subset of medullary stromal cells in three- to five-week-old mice. The isozymes also differ in their contributions to lymphocyte development. Thus, experiments with selective COX-1 inhibitors in thymic lobes from normal and recombinase-activating gene-1 knockout mice support a role for this isoform in the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP). Concordant data were obtained in COX-1 knockouts. Pharmacological inhibition and genetic deletion of COX-2, by contrast, support its role during early thymocyte proliferation and differentiation and, later, during maturation of the CD4 helper T-cell lineage. PGE2, but not other PGs, can rescue the effects of inhibition of either isoform, although it acts through distinct EP receptor subtypes. COX-dependent PG generation may represent a mechanism of thymic stromal support for T-cell development.

Thymocytes and RelB-dependent medullary epithelial cells provide growth-promoting and organization signals, respectively, to thymic medullary stromal cells.

The thymic medulla is composed of distinct epithelial cell subsets, defined in this report by the reactivity of two novel antibodies, 95 and 29, raised against mouse thymic epithelial cell lines. These antibodies were used to probe the development of medulla in wild-type or mutant thymuses. In CD3 epsilon-deficient mice where thymocyte maturation is arrested at the CD4- CD8- stage, few scattered 95+ and 29+ epithelial cells are found. When few mature thymocytes develop as in CD3- zeta/eta mice, expansion and organization of 95+ but not 29+ cells, becomes detectable. In RelB-deficient mice, T cell maturation proceeds normally but negative selection is inefficient due to the lack of thymic medulla and dendritic cells. Strikingly, 29+ epithelial cells are absent and 95+ medullary epithelial cells are scattered throughout the thymus, intermingling with CDR1+ cortical epithelium. In chimeric mice lacking only dendritic cells, the corticomedullary junction persists and both 95+ and 29+ epithelial cells are localized in the medulla. These results suggest that two types of signals are required for development of thymic medulla. A growth signal depends upon the presence of maturing thymocytes, but organization of the thymic medulla requires the presence of activated 29+ medullary epithelial cells.

Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice.

Thymic development of T lymphocytes progresses as a consequence of both TCR-mediated and non-TCR-mediated interactions between thymocytes and stromal cells. As relB-deficient mice appear to lack thymic medullary epithelium and mature dendritic cells, we studied the effect of this "cortex-only" thymus on T cell development. Two major consequences were observed. First, in both relB mutant and TCR transgenic/relB mutant mice, positive selection of both TCR alpha beta and delta gamma T cells appeared to proceed normally, with export of fully functional T cells to the periphery, suggesting that the thymic medullary stromal cells are not required for full maturation of T cells nor is an organized medullary compartment required for accumulation of mature single positive CD4 and CD8 T cells. Second, thymic negative selection was impaired, as evidenced by significant autoreactive proliferative responses to normal spleen stimulators. Peripheral T cells in relB mutant mice showed an unusually high proportion of CD69+ and CD44high cells. While some of these cells may be autoreactive T cells, most of the cells appeared to be activated by cytokines produced by relB mutant nonlymphoid cells, as the effect is minimized in relB mutant bone marrow chimeras. In sum, while the TCR-mediated steps in T cell maturation require both thymic cortex and medulla (epithelium and dendritic cells) for normal positive and negative selection of the repertoire, non-TCR-mediated interactions in the thymic cortex alone are sufficient to generate mature functional T cells.

Identification and functional analysis of Ly-6A/E as a thymic and bone marrow stromal antigen.

We recently described an mAb (MTS23) reactive with a membrane Ag expressed on a subset of thymic medullary stromal cells. This Ag is also constitutively expressed at high levels on peripheral B cells, macrophages, and thymic and splenic dendritic cells of C57BL/6 mice. A number of stromal cell lines derived from thymus and bone marrow also stain with MTS23, but thymocytes and peripheral T cells only weakly express the Ag detected by MTS23. Here we show that the molecule detected by MTS23 is a member of the Ly-6 family of phosphatidylinositol-anchored membrane proteins. Treatment of stromal cells with phosphatidylinositol-phospholipase C before staining completely abolished expression. Using transient expression of 293T cells and a cDNA library of a stromal cell line cloned into the pEF-BOS vector, a cDNA encoding the MTS23-target Ag was isolated. Partial sequencing and restriction enzyme mapping revealed that it represents the Ly-6A/E protein. While the physiologic significance of the presence of Ly-6 molecules on stromal cells is not clear, it has been known for some time that, at least in lymphocytes, cellular activation events can be induced upon Ly-6 engagement. We now demonstrate that Ly-6 also functions as a signal transduction molecule on stromal cells, in that granulocyte-macrophage CSF can be produced by a variety of stromal cell lines upon mAb-mediated cross-linking of Ly-6. Together with the dramatic up-regulation of Ly-6 expression on stromal cells upon IFN-gamma treatment, this is the first indication of a biologic function of an Ly-6 gene product on nonhemopoietic cells. The results suggest that Ly-6 may play a role in the cross-talk between lymphocyte precursors and stromal cells.

A study of the structure, function and distribution of beta 5 integrins using novel anti-beta 5 monoclonal antibodies.

Here, we have utilized six new anti-human beta 5 monoclonal antibodies to perform a detailed investigation of the structure, function and distribution of beta 5 integrins. Monoclonal anti-beta 5 specificity was confirmed by reactivity with beta 5-transfected CHO cells, by direct binding to the beta 5 subunit (immunoblotting), and by immunodepletion experiments using polyclonal anti-beta 5 serum. The beta 5 subunit was predominantly associated with the alpha v subunit, although on some cell lines, the level of beta 5 exceeded that of alpha v for unknown reasons. Cell adhesion studies showed that the adhesive function of beta 5 could be stimulated, inhibited or unaltered by different anti-beta 5 monoclonal antibodies. The beta 5 subunit was involved in adhesion to both vitronectin and fibronectin and, at least for K562 cells, fibronectin appeared to be the preferred ligand. Flow-cytometry studies showed that the beta 5 subunit was expressed at moderate to high levels on all adherent cell lines examined, was absent from all lymphoid cell lines, and was only weakly expressed on myeloid cell lines. Staining of thymic sections showed the distribution of beta 5 on blood vessels, Hassal's corpuscles, cortical and medullary stromal cells, and basement membranes. Skin sections showed beta 5 on the basal layer of the epidermis and on some dermal blood vessel walls, and kidney sections showed staining of glomerular regions, juxta glomerular apparatus, proximal convoluted tubules and collecting tubules, and at least one anti-beta 5 antibody also stained epithelial cells of proximal tubules.