Uptake and intracellular transportation of a bacterial surface protein in lymphoid cells

Some strains of the human pathogen Streptococcus pyogenes express a surface protein called protein H, which is released from the streptococcal surface by a cysteine proteinase produced by the bacteria. Here, we find that soluble protein H binds to the surface of lymphocytes and granulocytes, and that the molecule is taken up by lymphocytes and transported to the perinuclear region. The translocation over the cell membrane is rapid, and the uptake and intracellular transportation is not dependent on actin polymerization. Protein H could be immunoprecipitated from cell extracts and nuclear preparations of lymphocytes, and analysis of molecular interactions between protein H and proteins of different cellular compartments demonstrated a binding to nucleophosmin/ B23, a protein known to shuttle between the cytoplasm and the nucleus, and to the nuclear proteins SET and hnRNP A2/B1. Nucleophosmin/B23 was co-immunoprecipitated with protein H from cell and nuclear extracts, and binding experiments, including kinetic analyses, suggest that protein H dissociating from nucleophosmin/B23 complexes in the perinuclear region or in the nucleus binds to proteins SET and hnRNP A2/B1. Finally, the uptake and intracellular transportation of protein H was found to result in a cytostatic effect on B and T lymphocytes.     

Regulation of JC virus expression in B lymphocytes.

The etiologic agent of progressive multifocal leukoencephalopathy, a subacute demyelinating disease of the central nervous system, is the human polyomavirus JC virus (JCV), which causes a lytic infection of myelin-producing oligodendrocytes. In infected individuals the JCV genome can be detected in brain tissue and B lymphocytes isolated from the blood, bone marrow, or lymph nodes. Using mobility shift assays and a radiolabeled oligonucleotide from the JCV promoter-enhancer region (JCV bp 130 to 160), referred to as domain B, we were able to detect specific bands of the same mobility in nuclear extracts from human fetal glial cells, U-251 glioma cells, different B-cell lines, and in vitro-activated tonsillar B lymphocytes but not from T cells. In addition, a specific shift was detected when using nuclear extracts from freshly isolated tonsillar or lymph node B cells from five AIDS patients, two of whom later developed progressive multifocal leukoencephalopathy. Somewhat surprisingly, the above gel shift was partially inhibited by unlabeled oligonucleotides containing a kappa E2-binding site. UV cross-linking of the protein-DNA complex from either B cells or glial cells and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a 46-kDa band. Transient transfection of a reporter plasmid constructed by fusing a trimer of the domain B sequence to a minimal promoter revealed activity in B lymphocytes and glial cells but not in T cells. Mutational analysis of this region demonstrated that the core TGGC repeat was essential for enhancer activity. Thus, a similar protein in B lymphocytes and glial cells may account for the preferential replication of JCV in these two cell types.     

Lymphocytosis and Histamine Sensitization of Mice by Fractions from Bordetella pertussis

Administration of Bordetella pertussis cell extracts induced in mice hypersensitivity to histamine, as well as pronounced leukocytosis and hypoglycemia. The leukocytosis was mainly caused by an increase in the small lymphocytes in the circulating blood, and it was most pronounced 3 to 4 days after injection of B. pertussis extracts. Rabbit antimouse lymphocyte serum produced a decrease in the lymphocyte count in normal mice, as well as in mice treated with B. pertussis extracts. This depression in lymphocytes was observed whether the antilymphocyte serum was given 1 day or 2 days after the administration of B. pertussis extracts. The increased histamine sensitivity and hypoglycemia of mice treated with B. pertussis extract were not affected by treatment with antilymphocyte serum, although a marked lymphopenia was present. These observations indicate that the three phenomena observed in pertussis-treated mice are independent of each other.     

An Epstein-Barr virus transformation-associated membrane protein interacts with src family tyrosine kinases.

In latently infected growth-transformed human lymphocytes, Epstein-Barr virus (EBV) encodes two integral plasma membrane proteins: LMP1, which constitutively induces B-lymphocyte activation and intercellular adhesion, and LMP2A, which associates with LMP1 and is a tyrosine kinase substrate. We now demonstrate that LMP2A associates with src family protein tyrosine kinases, particularly lyn kinase, in nonionic detergent extracts of transfected B lymphoma cells or in extracts of EBV-transformed B lymphocytes. The LMP2A and tyrosine kinase association is stable in nonionic detergents and includes a 70-kDa cell protein which is also an in vitro or in vivo kinase substrate. This LMP2A association with B-lymphocyte src family tyrosine kinases is likely to be an important pathway in EBV's effects on cell growth.     

Molecular associations of IA antigens after T-B cell interactions. I. Identification of new molecular associations

In this report we report the identification of novel molecular associations involving the MHC class II (Ia) Ag expressed on the surface of Ag-presenting B lymphocytes. Biosynthetically radiolabeled murine B cells were incubated for 2 h in the presence or absence of T lymphocytes before treatment with the cleavable cross-linking reagent dimethyl-3,3'-dithiobispropionimidate. Anti-Ia immunoprecipitates of solubilized cell extracts revealed novel cross-linked products of Mr 90,000 to 95,000 which, upon cleavage of the cross-linker, could in part be resolved into native Ia and other structures of approximately Mr 67,000. The detection of the cross-linked products was significantly enhanced in B cells that had been co-cultured with T lymphocytes, but not with other cell types, and co-culture with various monoclonal T cell lines resulted in different levels of enhancement. Detection of the 90- to 95-kDa cross-linked products appeared to be independent of the foreign Ag for which the T cells were specific and could be enhanced when either cell type was replaced by a plasma membrane fraction, indicating that it resulted from direct cell-cell contact. These results suggest that some proportion of the Ia glycoproteins expressed on the surface of B cells become associated with other structures of Mr 67,000 upon Ag-non-specific interactions between T and B lymphocytes.     

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.     

Generation of killer lymphocytes in vitro against human autologous leukemia cells with soluble bacterial extraxts

Summary Ten patients with acute lymphoblastic leukemia (ALL) were studied to determine the ability of their remission lymphocytes to kill autologous leukemic blasts (ALB) following in vitro exposure to soluble extracts (SE) of BCG, Staphylococcus aureus (SA) or Listeria monocytogenes (LM). Remission lymphocytes from some patients became markedly cytotoxic to ALB after stimulation with BCG-SE, LM-SE, or SA-SE. These bacterially stimulated lymphocytes, although specifically lytic for ALB, were usually not cytotoxic to autologous remission lymphocytes. Bacterial extracts were able to generate killer lymphocytes at low concentrations. Generally, large amounts either had no stimulatant effect or were less stimulating. Bacteria-stimulated lymphocytes of ALL patients were cytotoxic not only to their leukemia cells, but also to leukemia cells from ALL and AML patients who were allogeneic to stimulated lymphocytes.     

A comparison of the immunosuppressive effects of salivary gland extracts from two laboratory strains of Boophilus microplus

This study addresses three questions related to the immune response of cattle to tick salivary gland extracts. Firstly, is there a difference in the inhibition of proliferation of Concanavalin A (ConA) stimulated bovine lymphocytes induced by salivary gland extracts of the N and Y strains of Boophilus microplus? Second, is there a difference in the development rate of the Y and N tick strains? Third, does the host affect the inhibitory effect of salivary gland extract on the proliferation of ConA stimulated lymphocytes from the two tick strains? Salivary gland extract of the Y strain inhibited in vitro proliferation of lymphocytes stimulated by ConA significantly more than that of the N strain, when each strain was raised on different animals. A difference in the development rate was observed between the tick strains when raised on the same animal, with female ticks of the Y strain developing faster and reaching a greater fully engorged weight than ticks of the N strain. The difference in their rate of development did not appear to contribute to a difference in inhibitory effects of the salivary gland extracts and there was no difference between the inhibitory effects of salivary gland extracts from both strains. However, when Y strain ticks were raised on different animals, there was a significant difference in the inhibition of lymphocyte proliferation between the two salivary gland extracts. Therefore, it was concluded that there is no difference between the inhibitory effects of the two tick strains and that the host has an influence on salivary gland extract composition of B. microplus and its inhibitive properties.     

A specific high-performance liquid chromatography assay for dehydroascorbic acid shows an increased content in CLL lymphocytes

A method for the assay of dehydroascorbic acid using high-performance liquid chromatography with uv detection is described. The dehydroascorbic acid is separated from ascorbic acid and reduced with dithiothreitol, and is then quantitated as ascorbic acid following rechromatography. Since as little as 22 pmol can be detected, sensitivity is at least 40-fold greater than that of other currently available procedures. This method was used to measure the level of dehydroascorbic acid in normal and chronic lymphocytic leukemia lymphocytes. A significantly higher concentration of dehydroascorbic acid was found in leukemic (21.80 +/- 3.55 nmol/10(8) cells, mean +/- SE) than in normal lymphocytes (9.32 +/- 1.15 nmol/10(8) cells) (P less than 0.03). Analysis of extracts from normal B cell lymphocytes revealed comparable dehydroascorbic acid levels to unfractionated lymphocytes, indicating that the elevated level in chronic lymphocytic leukemia was not simply a reflection of the increased percentage of B lymphocytes in this disorder. These studies illustrate that the technique can be used to measure the dehydroascorbic acid content from sources where only scanty material is available or low levels are found.     

Intracellular signalling of initiation of DNA synthesis: effect of cell-free extracts from anti-receptor-stimulated B lymphocytes on spleen cell nuclei

To study the relatively late intracellular signals involved in the proliferative response of B lymphocytes to antibodies specific for surface membrane immunoglobulins, extracts from antibody activated cells were mixed with Xenopus laevis splenic nuclei, and the incorporation of thymidine 5'-triphosphate into DNA was assessed. The slight incorporation observed with either nuclei or extract alone was markedly enhanced upon mixing the two entities when the extract was derived from cells cultured with but not without anti-receptor antibody. The appearance of active extract correlated well with the culture requirements necessary for the induction of B lymphocyte proliferation and, as revealed by time course studies, the active component arises relatively late in the activation process. Moreover, the appearance of active extracts is independent of DNA synthesis but is dependent on protein synthesis as judged from studies with metabolic inhibitors. Appropriate homogenization of activated cells yielded nuclei and cytoplasm with 85% of the activity confined to nuclei. In addition, purified active extracts exhibited DNA binding although the active component was readily distinguishable from polymerase alpha by chromatographic techniques. It is tentatively concluded that the active component represents either some replication protein other than polymerase or some earlier signal necessary to induce the formation or utilization of replicating proteins.     

Arginine vasopressin in the cytoplasm and nuclear fraction of lymphocytes from healthy donors and patients with depression or schizophrenia

We investigated whether cytoplasmic or nuclear extracts of human peripheral blood lymphocytes contain AVP in samples from healthy controls and patients diagnosed as depressed or schizophrenic. Both the cytoplasmic and nuclear extracts contained AVP as determined by radioimmunoassay. AVP and other peptides were detected in the purified samples by matrix-assisted laser desorption/ionization time of flight mass spectrometry. It is the first time that AVP has been characterized in human lymphocytes of patients with depression or schizophrenia. This finding demonstrates the presence of another important component within the potential regulatory loop between immune and neuro-endocrine tissues.     

Extracts of regenerating Planarians regulate proliferation of vertebrate cells

The effects of extracts from intact and regenerating planarians on cell behaviour in culture were studied. The extracts were added to the culture of Chinese hamster fibroblasts and to the primary culture of human lymphocytes. Some extracts contained active agents which influenced the proliferation of fibroblasts increasing or decreasing the mitotic index. The extracts exerted no effect on the mitotic index of lymphocytes. When the extracts were added to the lymphocyte culture together with phytohemagglutinin, which induces the proliferation, the mitotic index somewhat increased. The extracts of regenerating planarians contain factors which activate and inhibit cell proliferation in culture. The active factors stimulated, rather than induced proliferation.     

The specific and endogenous mitotic inhibitor of lymphocytes (chalone)

Aqueous extracts of various lymphoid tissues, but not of non-lymphoid tissues, contain a species-non-specific but cell-specific inhibitor of the transformation and DNA synthesis of PHA-stimulated human lymphocytes which is apparently not cytotoxic and is reversible. This activity is found in similar molecular weight fractions from pure lymphocytes obtained in culture and hence appears to be endogenous to the lymphocyte itself. This specific and endogenous mitotic inhibitor does not appear to be a result of competitive lectin-binding, thymidine pool size dilution, phosphorylation, destruction of thymidine, or the direct immunosuppressive effects of thymidine upon the lymphocytes themselves. Rather, it appears to be a result of the effects of a protein contained in the crude ultrafiltrate from lymphoid tissues whose properties correspond to those originally described by Bullough & Laurence for a ‘chalone’. The chalone activity from thymus appears to be specific for T cells rather than B cells.     

In vitro induction of lymphocyte responsiveness by a Strongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected with Strongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults of S. vulgaris. In addition, S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by the S. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary for S. vulgaris mitogen-induced transformation of spleen cells. The S. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells and S. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae of S. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that the S. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the beta-globulinaemia, of which IgG(T) is a major component, in S vulgaris infected horses.     

山羊羔淋巴集结的研究

对不同发育时期山羊羔淋巴集结、简称ＰＰ的显微和亚显微结构的观察显示羊ＰＰ的发生和组织学特征与鸟类法氏囊极为相似。同时,羊羔ＰＰ提取液可提高仔兔和仔鸡血清抗体的滴度,促进淋巴组织的发育。实验结果表明山羊ＰＰ是Ｂ淋巴细胞发生的重要部位,山羊ＰＰ中含有类似法氏囊素的物质。     

Increased phosphorylation of a specific protein in mitogen-stimulated lymphocytes

The phosphorylation of proteins from murine splenic lymphocytes was studied. When the phosphorylation of proteins in extracts from Concanavalin A (ConA)-treated lymphocytes was compared with that of resting lymphocytes, there was only one detectable difference between them. A protein of 135,000 mol. wt was highly phosphorylatable in extracts from ConA-treated cells while phosphate incorporation into this protein was slight in extracts from untreated cells. This effect could be observed 12 h after ConA treatment and was maximal during S phase. This soluble protein was also phosphorylated in intact lymphocytes.     

Increased assembly of clathrin occurs in response to mitogenic activation of murine lymphocytes

The unassembled (soluble) and assembled (particulate) pools of clathrin in murine lymphocytes have been separated by centrifugation, and specifically quantified by immunoblotting of cellular extracts with an anticlathrin heavy chain monoclonal antibody. In resting spleen lymphocytes only 25-30% of the total cellular clathrin was found to be present in an assembled form. Upon activation of lymphocytes with B or T cell mitogens (lipopolysaccharide or concanavalin A), the levels of assembled clathrin increased to 60% of the total. These changes in the levels of assembled clathrin were not due to an increase in total cellular clathrin concentration following lymphocyte activation, but rather to changes in the steady state ratio of assembled to unassembled clathrin. The increase in assembled clathrin preceded the expression of transferrin receptors, as measured by the cell surface binding of an antitransferrin receptor monoclonal antibody, and maximal DNA synthesis, indicating that clathrin assembly occurs early after lymphocyte activation and precedes cell division. Immunofluorescence analysis of activated lymphocytes with an anti-clathrin heavy chain monoclonal antibody revealed a punctuate staining pattern characteristic of coated pits and vesicles. Activated B lymphocytes displayed particularly prominent staining in the perinuclear region compared to T cells, suggesting that clathrin assembly may be important for B cell functions such as immunoglobulin synthesis or secretion. These results suggest that in lymphocytes, clathrin assembly is a dynamic process that is triggered by mitogenic stimuli.     

Regulation of lymphocyte production in the bone marrow. I. Turnover of small lymphocytes in mice depleted of B lymphocytes by treatment with anti-IgM antibodies

To examine the concept that the genesis of lymphocytes in the bone marrow may be regulated by homeostatic feedback signals from peripheral B lymphocytes or their products, lymphocyte production was measured in mice selectively depleted of B lymphocytes by repeated administration of anti-IgM antibodies from birth. The turnover of small lymphocytes was quantitated radioautographically after DNA labeling by continuous infusion of 3H-thymidine. In the femoral marrow of anti-IgM-treated mice, the number of small lymphocytes was reduced and their turnover time was shorter than in control mice, presumably reflecting the premature elimination from the marrow of maturing cells about to express surface IgM. The absolute number of small lymphocytes being produced per femur in unit time, however, was identical in anti-IgM-treated and control mice. Lymphocyte production in the thymus was also unaffected by anti-IgM suppression whereas in the spleen the turnover of small lymphocytes was reduced due to the lack of young immigrant B lymphocytes from the bone marrow. The results demonstrate that the normal large-scale production of lymphocytes in mouse bone marrow is independent of the magnitude of the peripheral pool of B lymphocytes or the level of circulating immunoglobulins, suggesting the process is not subject to feedback control. Some implications for the genesis and diversity of primary B lymphocytes are discussed.     

Characterization of the murine T cell receptor for IgE (Fc epsilon RII). Demonstration of shared and unshared epitopes with the B cell Fc epsilon RII

Antigenic relationships between the low affinity Fc epsilon R present on murine B and T lymphocytes were studied. A rat mAb (B3B4) and two polyclonal antisera produced by immunizing with the murine B lymphocyte Fc epsilon RII were examined for their ability to inhibit binding of IgE to murine B or T lymphocytes, using an IgE-specific rosette assay. One polyclonal antiserum (goat-anti-mouse Fc epsilon R) inhibited binding of IgE to both B and T lymphocytes, whereas another polyclonal antiserum (rabbit-anti-mouse Fc epsilon R) and the rat mAb inhibited the binding of IgE to B lymphocytes but did not influence the binding of IgE to T lymphocytes. When lymphocytes were surface labeled with 125I, 49-kDa and 38-kDa IgE-binding proteins were immunoprecipitated from B lymphocyte lysates by B3B4 and from B and T lymphocyte lysates by the goat antiserum. Taken together, these results suggest that the Fc epsilon R present on murine B and T lymphocytes are structurally related receptors that share some, but not all, epitopes.