CD9 is a tetraspan protein that associates with several β1 integrins, including α6β1. Because α6β1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin β), we first asked whether CD9 is present on murine eggs and whether it functions in sperm–egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm–egg binding and fusion in vitro in a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin β disintegrin domain. (Both ligands bind to the egg via α6β1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin α6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, α6A and CD9 both are expressed on the apical epithelial surface at the uterine–oviduct junction. These findings correlate with the observation that fertilin β “knockout” sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin β −/− male mice. Our results suggest that high-avidity binding between fertilin β (ADAM 2) and α6β1 requires cooperation between α6β1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as sperm–egg interactions.
Summary: Technical advances have made it possible to separate hematopoietic tissues such as the bone marrow into ever smaller populations, complicating our understanding of immune system replenishment. Patterns of surface marker expression and transcription profiles as well as results obtained with reporter mice suggest that lymphopoietic cells are not closely synchronized, and there is considerable cell to cell variation. Loss of differentiation options is gradual, and ultimate fate can be established at different stages of lineage progression. For example, individual hematopoietic stem cells can be biased such that some are very poor sources of lymphocytes as contrasted to ones with balanced outputs. Still other hematopoietic stem cells are effective at generating B and T cells but are defective with respect to expansion and difficult to distinguish from early lymphoid progenitors. That diversity carries forward to later events, and similar appearing cells in the immune system can arise from alternate differentiation pathways. In fact, new categories of lymphoid progenitors are still being discovered. Heterogeneity provides adaptability as hematopoiesis can be dramatically altered during infections, influencing numbers and types of cells that are produced.
Abstract Retinoids are known to have potent effects on hemopoietic stem cell integrity, and our objective was to learn whether they influence cells destined to replenish the immune system. Total CD19+ B lineage cells increased substantially in the marrow and spleens of all-trans retinoic acid (ATRA)-treated C57BL6 mice, while lymphoid progenitors were reduced. All B lymphoid progenitors were targets of ATRA in culture and overall cell yields declined without reductions in proliferation. Remarkably, ATRA shortened the time required for primitive progenitors to generate CD19+ cells. PCR analysis and a panel of retinoid acid receptor (RAR)/retinoid X receptor agonist treatments suggested that RARα mediates these responses. The transcription factors EBF1 and Pax-5 were elevated during treatment and ATRA had similar effects on human B cell differentiation. That is, it inhibited the expansion of human progenitor cells and accelerated their differentiation to B lineage cells. There may be previously unsuspected side effects of ATRA therapy, and the new findings suggest retinoids can normally contribute to the lymphopoietic environment in bone marrow.
Stromal cells which grow as an adherent layer of Whitlock-Witte cultures are thought to be an essential component of the lymphohemopoietic microenvironment. Stromal cell lines from bone marrow (BM) and spleen have been obtained by treatment of cultures with 5-fluorouracil and selected for their lymphocyte support capacity by measuring the clonal growth of stromal cell-dependent lymphocyte lines in methyl cellulose. Established stromal cell lines differed significantly from stromal cells in primary Whitlock-Witte cultures with respect to expression of certain hemopoietic cell surface markers. For example, the Thy-1 and Mac-3 antigens were expressed by stromal cell lines obtained from BM and spleen, but not by stromal cells in primary cultures. Features common to all stromal cells include synthesis of actins, the neural adhesion molecule N-CAM, and a variety of collagens. Two types of common leukocyte antigens were not significantly expressed. The proliferation and total protein synthetic capacity of lymphocyte-supportive stromal cell lines was sensitive to ionizing radiation. After exposure of the cells to 200 rads, the incorporation of either [3H]thymidine or [3H]Leucine was reduced to less than 50% of control values, but the growth of lymphocytes was augmented in the presence of an irradiated stromal cell layer. The proliferation of stromal cell lines was also affected by exposure to a variety of growth factors. Addition of epidermal growth factor or endothelial cell growth factor augmented BM or spleen-derived stromal cell proliferation, while interferon-gamma had the opposite effect. In general, but not exclusively, lymphocyte growth was inhibited by factors which augmented the proliferation of stromal cells. Novel methods are described for isolating stromal cells and determining their capacity to support lymphocyte growth in vitro. Evidence is presented that this ability is not restricted to BM-derived stromal cells. The function of stromal cells was not dependent on their ability to proliferate, and this may be modulated by immunoregulatory and other growth factors.
