The three proteins that constitute anthrax toxin self-assemble into toxic complexes after one of these proteins, protective antigen (PA), binds to tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2) cellular receptors. The toxin receptor complexes are internalized, and acidic endosomal pH triggers pore formation by PA and translocation of the catalytic subunits into the cytosol. In this study we show that the pH threshold for conversion of the PA prepore to the pore and for translocation differs by approximately a pH unit, depending on whether the TEM8 or CMG2 receptor is used. For TEM8-associated toxin, these events can occur at close to neutral pH values, and they show relatively low sensitivity to ammonium chloride treatment in cells. In contrast, with CMG2-associated toxin, these events require more acidic conditions and are highly sensitive to ammonium chloride. We show, furthermore, that PA dissociates from TEM8 and CMG2 upon pore formation. Our results are consistent with a model in which translocation depends on pore formation and pore formation, in turn, depends on release of PA from its receptor. We propose that because PA binds to CMG2 with much higher affinity than it does to TEM8, a lower pH is needed to attenuate CMG2 binding to allow pore formation. Our results suggest that toxin can form pores at different points in the endocytic pathway, depending on which receptor is used for entry.capillary morphogenesis protein 2 ͉ tumor endothelial marker 8 ͉ toxin entry B acillus anthracis, the causative agent of anthrax, secretes a toxin that is believed to be instrumental in causing anthrax disease symptoms leading to death. Anthrax toxin consists of three proteins, protective antigen (PA), which is a receptorbinding and pore-forming subunit; lethal factor (LF), which is a protease that cleaves mitogen-activated protein kinase kinase family members; and edema factor (EF), which is an adenylate cyclase that raises cAMP levels in cells (1). PA is synthesized as an 83-kDa protein (PA 83 ) for which two cell surface receptors have been identified: tumor endothelial marker 8 (TEM8) (2, 3) and capillary morphogenesis protein 2 (CMG2) (4). Two splice variant mRNAs derived from the TEM8 gene (sv1 and sv2) encode functional anthrax toxin receptors (2, 5). TEM8 expression has been documented in epithelium of the lung, intestine, and skin, the three routes of entry in anthrax infection (6). The CMG2 gene, which has been shown to be broadly expressed in different tissues (4), encodes three protein isoforms, two of which, CMG2 488 and CMG2 489 , are anthrax toxin receptors (4) (H.M.S., unpublished data).Receptor-bound PA 83 is cleaved by a cellular protease to generate a 20-kDa PA 20 subunit and a 63-kDa subunit (PA 63 ). The larger subunit assembles into the heptameric (PA 63 ) 7 prepore in lipid rafts (7-9). EF and LF bind to the prepore, and the toxin-receptor complexes are internalized by clathrindependent endocytosis and by other endocytic mechanisms (9, 10). These complexes are then exp...

The role of thymic stromal cell-derived lymphopoietin (TSLP) in regulating hematopoiesis is poorly characterized, so we investigated its regulatory effects in vivo using TSLP transgenic mice. Overexpression of TSLP disrupted hematopoietic homeostasis by causing imbalances in lymphopoiesis and myelopoiesis. Mice harboring a TSLP transgene had 5-to 700-fold fewer B and T precursors and no detectable pre-B lymphocyte colonyforming activity in the marrow or spleen. Conversely, TSLP transgenic mice possessed 15 to 20 times more splenic myeloid precursors than their littermates, and progenitor activity of the granulocyteerythrocyte-macrophage-megakaryocyte colony-forming units was significantly el- IntroductionGrowth and differentiation of leukocyte progenitors are critical for the establishment and normal function of the mammalian immune system. Appropriate development within the myeloid lineage contributes significantly to protective innate immunity, and the maturation of cells from the lymphoid lineage is essential for adaptive immune responses to foreign antigens. Several studies have shown that failure to maintain homeostasis between myeloid and lymphoid development can cause profound pathophysiologic consequences. [1][2][3][4] Multiple cytokines regulate the homeostatic mechanisms responsible for maintaining a physiologic balance between leukocytes in the lymphoid and myeloid lineages. 5 The cytokine thymic stromal cell-derived lymphopoietin (TSLP) was originally identified as a biologic activity present in conditioned medium from a thymic medullary stromal cell line. 6 This cytokine promoted both proliferation and differentiation of B220 ϩ pro-B cells from committed B220 Ϫ fetal liver progenitors. In long-term bone marrow cultures, TSLP acted at a later stage of B-lineage development leading to an increase in the number of immature B lymphocytes. 7 These studies suggested that TSLP had overlapping functions with a related cytokine, interleukin 7 (IL-7). In fact, TSLP and IL-7 are all members of a hematopoietic cytokine family that includes IL-2, IL-4, IL-9, IL-13, In addition, the receptors for these cytokines share common receptor components.TSLP exerts its biologic effects through its receptor, which is an IL-7R␣ chain and TSLPR heterodimer. Engagement of the TSLPR by its ligand initiates biochemical signals triggering the activation of STAT5 and src-family tyrosine kinases. 7,[11][12][13] Recently, Isaksen et al 12 demonstrated that a single tyrosine residue in the cytoplasmic domain of TSLPR is critical for TSLP-mediated proliferation. Src-family kinases, as well, are important for cell proliferation induced by TSLP. 12 Several src kinases are known oncogenes [14][15][16][17] ; and given that they play an indispensable role in proliferation mediated by TSLP, aberrant expression of this cytokine could result in uncontrolled growth of TSLP-responsive cells and loss of hematopoietic homeostasis. Consequently, it is important to have a clear understanding of the role of TSLP in lymphohematopoiesis. Yet in thi...

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