Normal B lymphocyte development but impaired T cell maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice

The transmembrane tyrosine phosphatase CD45 is expressed in multiple isoforms on all nucleated hematopoietic cells, resulting from alternative splicing of variable exons. We generated mice with a mutation in the variable CD45 exon 6, using homologous recombination. In mice homozygous for the CD45-exon6 mutation, B cells and most T cells did not express CD45. Development of B cells appeared normal, although Ig mu-induced proliferation was completely abrogated. Thymocyte maturation was blocked at the transitional stage from immature CD4+CD8+ to mature CD4+ or CD8+ cells, and only a few T cells could be detected in peripheral lymphoid organs. Clonal deletion of superantigen-reactive T cells still occurred. Cytotoxic T cell responses to lymphocytic choriomeningitis virus were absent in CD45-exon6-/- mice. These data imply that CD45 is differentially required for the development and function of B and T lymphocytes.     

bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice

We have assessed during B cell development, the regulation and function of bcl-x, a member of the bcl-2 family of apoptosis regulatory genes. Here we show that Bcl-xL, a product of bcl-x, is expressed in pre-B cells but downregulated at the immature and mature stages of B cell development. Bcl-xL but not Bcl-2 is rapidly induced in peripheral B cells upon surface immunoglobulin M (IgM) cross-linking, CD40 signaling, or LPS stimulation. Transgenic mice that overexpressed Bcl-xL within the B cell lineage exhibited marked accumulation of peripheral B cells in lymphoid organs and enhanced survival of developing and mature B cells. B cell survival was further increased by simultaneous expression of bcl-xL and bcl-2 transgenes. These studies demonstrate that Bcl-2 and Bcl-xL are regulated differentially during B cell development and activation of mature B cells. Induction of Bcl-xL after signaling through surface IgM and CD40 appears to provide mature B cells with an additional protective mechanism against apoptotic signals associated with antigen-induced activation and proliferation.     

CD40 expression and function in murine B cell ontogeny

The CD40 antigen, a member of the nerve growth factor/tumor necrosis factor receptor family, is expressed on all mature B lymphocytes and plays a crucial role in B cell activation, T cell-dependent antigen-driven isotype switching and germinal center formation. We have analyzed CD40 expression and function during mouse B cell development by examining B cell precursors in normal mice and in transgenic animals in which B cell development is frozen at discrete stages. These models included RAG-2-/- mice, and transgenic littermates that express a mu heavy chain and/or the bcl-2 proto-oncogene transgene. CD40 was undetectable at the pro-B cell stage, but was expressed, although at low levels, on pre-B cells. However, pre-B cells failed to respond to CD40 triggering either by expression of CD23 or by proliferation in the presence of IL-4. Overexpression of bcl-2 increased the density of CD40 expression on pre-B cells: these cells respond to CD40 ligation by expressing CD23 and by proliferating in the presence of IL-4.     

Impaired B cell maturation in mice lacking Bruton''s tyrosine kinase (Btk) and CD40

Septicemia in crustaceans may occur occasionally due to Gram-negative opportunistic bacteria, especially under conditions of intensive aquaculture. The lipopolysaccharide (LPS) endotoxin induces in mammals septic shock and the activation by LPS of hormone release through the hypothalamo-pituitary axis is well known. In crustaceans an increase in circulating Crustacean hyperglycemic hormone and hyperglycemia are reported to result from exposure to several environmental stressors but the metabolic and hormonal effects of LPS in vivo are undescribed. A sublethal dose of LPS (Sigma, Escherichia coli 0111:B4) was injected into at least five individuals of species representative of crustacean taxa and life habits: Squilla mantis (Stomatopoda); the Decapoda Crangon crangon and Palaemon elegans (Caridea), Nephrops norvegicus (Astacidea), Munida rugosa and Paguristes oculatus (Anomura), Pilumnus hirtellus, Macropipus vernalis, Parthenope massena, and Ilia nucleus (Brachyura). Within 3 hr an increase in blood sugar developed ranging from 26.00 +/- 8.37 sd mg/dl in M. rugosa to 201.50 +/- 95. 91 sd mg/dl in P. oculatus and a significant increase of 79% in M. rugosa up to 1300% in P. hirtellus over control levels was observed. The involvement of eyestalk hormones in this generalized response was tested on S. mantis, M. vernalis, and P. elegans; LPS injected into eyestalkless animals did not elicit a significant hyperglycemic response compared with saline-injected controls. Eyestalkless animals injected with one eyestalk equivalent homogenate in saline from untreated animals did show a change in color from red to normal likely due to red pigment concentrating hormone and a hyperglycemic response within 2 hr. Eyestalkless animals injected with homogenate from LPS-treated shrimps showed the change in color but not the hyperglycemic response. It is concluded that LPS directly, or cytokines circulated upon challenge by the endotoxin, may act on the medulla terminalis X-organ-sinus gland complex and release CHH selectively eliciting an hyperglycemic stress response, after which CHH stores become relatively depleted.     

Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice

Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1(TK-/-)) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1(TK-/-) mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.     

CD4+ T cell responses in mice lacking MHC class II molecules specifically on B cells

The role of B lymphocytes in initiating and maintaining a CD4+ T cell response has been examined using a variety of strategies, but remains controversial because of weaknesses inherent to each of the approaches. Here, we address this issue by measuring CD4+ T cell priming both in mutant mice devoid of B cells and in chimeric animals lacking major histocompatibility complex class II molecules specifically on B cells. We find that peptide and some protein antigens do not require B cells expressing class II molecules, nor B cells themselves, to efficiently prime. This could be demonstrated by the usual lymph node proliferation assay, a rather indirect in vitro measure of priming, and by a direct ex vivo assay of population expansion and activation marker expression. Interestingly, one protein antigen, conalbumin, could not prime in the absence of B cells, but could in the presence of B cells devoid of class II molecules. This finding constrains the possible mechanisms whereby B lymphocytes contribute to the initiation of a CD4+ T cell response, arguing against the importance of surface immunoglobulin-mediated antigen presentation by B cells.     

Synergistic effects of human B and T lymphocyte mitogens induce uniform, high levels of immunoglobulin secretion among normal donors

Synergistic effects of B-cell mitogen Staphylococcus bacteria strain Cowan I (Cowan I) plus T-cell activator pokeweed mitogen (PWM) in generating immunoglobulin-secreting cells (ISC) from human peripheral blood mononuclear cells (MNC) were investigated. ISC were assayed by reverse plaque-forming cells uing protein A-coated red blood cells. Low concentrations of PWM plus Cowan I gave superadditive effects on ISI induction, generating 3-20 times as many ISC as optimal amounts of either mitogen alone. The mitogens together and separately showed similar kinetics of ISC; synergy was observed at every day tested. IgM ISC represented 10% of initial MNC from all cell donors tested even if the donors were low responders to either mitogen alone. The numbers of ISC obtained are higher than previous reports and more uniform among donors, making this a superlative method for studies on normal human immunoglobulin secretion.     

CD38 ligation in human B cell progenitors triggers tyrosine phosphorylation of CD19 and association of CD19 with lyn and phosphatidylinositol 3-kinase

CD38 is a 45-kDa transmembrane glycoprotein highly expressed in lymphoid progenitors. Ligation of CD38 with specific Abs inhibits growth and induces apoptosis in human immature B cells. CD38 ligation also triggers tyrosine phosphorylation of syk, c-cbl, and phospholipase C-gamma and activates phosphatidylinositol 3-kinase (PI3-K). In the present study, we investigated whether the cell surface membrane molecules used in B cell receptor-mediated signaling, such as Ig alpha, Ig beta, and CD19, could be involved in the CD38-mediated signaling cascade. In the B cell receptor-negative immature B cell lines RS4;11, 380, and REH, Ig alpha and Ig beta were expressed exclusively in the cytoplasm and were not tyrosine phosphorylated after CD38 ligation. By contrast, CD19 was markedly tyrosine phosphorylated and was associated with lyn and PI3-K. PI3-K activation appears to be directly linked to the growth-arresting effects of CD38 ligation, which are reduced by PI3-K inhibitors. Ligation of either CD38 or CD19 resulted in a similar pattern of protein tyrosine phosphorylation; both signaling pathways caused tyrosine phosphorylation of c-cbl. Levels of CD38 surface expression were not affected by prolonged incubation with anti-CD19 Ab, while CD19 expression markedly decreased. These results indicate that CD19 is a major component of the CD38 signaling cascade in B cell precursors, serving as a cell surface membrane docking site for cytoplasmic kinases. CD38 and CD19 are not physically linked, but activate an overlapping set of kinases in human immature B cells.     

Tamoxifen down-regulates CD36 messenger RNA levels in normal and neoplastic human breast tissues

Tamoxifen (TAM) exerts a long-term suppressive effect on human breast cancer cell proliferation. To determine whether the effects of TAM are mediated by specific gene activation or repression, normal and tumoral human breast tissues obtained before and during TAM treatment were analyzed by differential display technique. Total RNA for differential display analysis was obtained from breast tissues from two women with the diagnosis of estrogen receptor-positive stage II (T2N1M0) infiltrating ductal carcinoma, made by incisional biopsy, followed by modified radical mastectomy performed after a 30-day treatment with TAM (20 mg/day). One 202-bp cDNA band, AP5-1, was present in normal and tumoral biopsy samples, but was absent in breast tissue obtained during TAM treatment, and was confirmed by Northern hybridization, which showed a 2.7-kb band in both patients. The differentially expressed cDNA fragment showed 99% homology to Homo sapiens CD36 gene, a glycoprotein that acts as a receptor for the extracellular matrix proteins thrombospondin-1, collagen types I and IV, and oxidized low-density lipoprotein. These results indicate that the down-regulation of CD36 induced by TAM might represent alternative or additional mechanisms of action of this drug affecting the functions of thrombospondin-1, which is involved in hematogenous tumor spread, invasion and angiogenesis, and oxidized low-density lipoprotein, playing a role in inhibition of arteriosclerosis. The multiple functions affected by the down-regulation of CD36 by TAM warrant the need for additional studies.     

Characterization of human endotoxin lipopolysaccharide receptor CD14 expression in transgenic mice

CD14 is a major receptor for the bacterial endotoxin LPS. Since CD14 is specifically and highly expressed on the surface of monocytic cells, it has been used as a monocyte/macrophage differentiation marker. To identify elements that are critical for the direction of the tissue-specific expression of CD14, an 80-kb genomic DNA fragment containing the coding region of the CD14 gene, as well as a considerable amount of both upstream and downstream sequence, was used to generate transgenic mice. The analysis of mice from six different founder lines demonstrated that this genomic DNA fragment was sufficient to direct human CD14 gene expression in a monocyte-specific manner among hematopoietic cells. Furthermore, the data lead us to a new finding that CD14 is highly expressed in the human liver, a primary organ involved in the acute phase response. These transgenic mice provide a useful model to analyze the biological function of human CD14.     

The IL-2 receptor complex: expression and function on normal and leukemic B cells

The interleukin 2 receptor (IL-2R) is composed of at least three different chains that may be variably expressed on normal and neoplastic lymphocytes. While considerable knowledge has been gained on the expression of the IL-2R alpha chain on human leukemic cells of different origin, less is known of the beta and gamma chains. In view of the highly pleiotropic functions exerted by IL-2, the IL-2-IL-2R interactions may play an important role in various leukemias. In this review we focus on the expression and function of the IL-2R complex on human normal and leukemic B cells. Possible implications in the expansion of the neoplastic clone and in the clinical course of the disease are discussed.     

CD1 hearing-impaired mice. I: Distortion product otoacoustic emission levels, cochlear function and morphology

The levels of distortion product otoacoustic emissions (DPOAEs) were measured in a strain of hearing-impaired mutant mice (CD1) at various stages of outer hair cell impairment and compared to those of a control inbred strain (CBA/J). Parallel measurements of cochlear potentials and auditory brainstem evoked responses (ABRs) were performed and surface preparations of organs of Corti were observed using phalloidin staining of filamentous actin. Comparison of DPOAEs (elicited by stimulus levels of 60 and 70 dB SPL) with standard functional tests allowed the categorization of CD1 ears into two groups on the basis of the presence or absence of DPOAE, which corresponded to mean ABR thresholds greater or less than 40 dB nHL respectively. When adopting ABR threshold as the gold standard, this procedure yielded rates of false-positives and -negatives ranging from 5 to 16%. However, individual predictions of electrophysiological function from DPOAE levels were not accurate, owing to their large variance, and attempts to optimize stimulus levels did not reduce this variance. In contrast, the profiles of DPOAE level vs. f2 exhibited large correlations with ABR threshold profiles as a function of f2. It was also noteworthy that the mean levels of DPOAEs in CD1 mice recorded in frequency intervals with normal ABR thresholds were significantly smaller than those of CBA/J mice. Although hearing loss was revealed early both by DPOAEs and by other functional tests, surface preparations often remained normal until about 3-4 months of age.