Studies on a new lymphocyte mitogen from Bordetella pertussis. I. Induction of proliferation and polyclonal antibody formation.

Pertussis B mitogen (PBM), isolated from culture supernatant fluids of Bordetella pertussis, is a potent mitogen for mouse and human lymphocytes. In mice, > 95% of the blast cells recovered from PBM cultures bear surface immunoglobulins. Therefore, PBM seems to induce proliferation of mouse B lymphocytes, but not T cells. The proliferative response observed is nonspecific because cells from all mouse strains tested, including germfree animals, are responsive. Moreover, the mitogenic activity of PBM is independent of T lymphocytes, macrophages, or serum factors. When human peripheral blood or cord blood lymphocytes are cultured in the presence of PBM, a high level of thymidine incorporation by these cells is detected. Furthermore, PBM can induce polyclonal antibody formation by both mouse and human lymphocytes. Despite similar methods of isolation, PBM is distinct from the lymphocytosis-promoting factor of B. pertussis, a previously described T cell mitogen.

The in vitro effects of Bordetella pertussis lymphocytosis-promoting factor on murine lymphocytes. IV. Generation, characterization, and specificity of cytotoxic lymphocytes.

Cytotoxic effector lymphocytes were induced in cultures of mouse spleen or lymph node cells by lymphocytosis promoting factor (LPF). The LPF-activated cytotoxic cells: (a) were not generated unless proliferation occurred; (b) sedimented in the lighter density fraction of a bovine serum albumin gradient; (c) were large, blast-like cells; and (d) were lysed by Thy-1.2 antiserum plus complement and, therefore, were T cells. Neither LPF alone nor supernates from stimulated cultures were cytotoxic. Unlike the situation with concanavalin A and phytohemagglutinin P, LPF-stimulated cytotoxic effector lymphocytes required no further addition of mitogen for maximal cytotoxicity. The effector cells displayed specificity, destroying only allogeneic but not syngeneic normal cells; in the case of tumor cells, both allogeneic and syngeneic cells werelysed in the absence of added mitogen. The reason for differentiated cytotoxicity toward syngeneic tumor and normal cells is not clear but may have some relevance to in vivo tumor rejection initiated by Bordetella pertussis.

The in vitro effects of Bordetella pertussis lymphocytosis-promoting factor on murine lymphocytes. III. B-cell dependence for T-cell proliferation.

The nature of the helper lymphocytes in lymphocytosis-promoting factor (LPF)-induced proliferation was explored. Removal of macrophages from adherent splenocytes by either carbonyl-iron incubation or passage through Sephadex G-10 columns did not affect their synergistic function. Nor did cytolysis with Thy-1.2 antiserum and complement. The helper cells were found to be surface immunoglobulin-positive (sIg+) because they are retained by anti-Ig columns, susceptible to lysis by rabbit anti-mouse immunoglobulin and complement, and occurred in the sIg+ fractions of splenocytes after separation on the fluorescence-activated cell sorter. Further delineation of the surface markers on helper cells showed that complement receptors are not the determining marker for synergistic function. The requirement for B-helper cells in the stimulation of T lymphocytes by LPF is unique for a mouse of T-cell mitogen.

The mitogenic effect of the lymphocytosis promoting factor from Bordetella pertussis on human lymphocytes.

The purified lymphocytosis promoting factor (LPF) from Bordetella pertussis was found to be a potent mitogen for peripheral blood lymphocytes (PBL) from normal adults as well as for cord blood lymphocytes. Proliferation occurred in autologous plasma or fetal calf serum, regardless of previous exposure to pertussis infection or immunization. Only one adult human serum, from a physician constantly working with B. pertussis, inhibited the mitogenic response to LPF and this serum was shown to contain precipitating antibody against LPF. The proliferative effect of LPF was characteristic of a "nonspecific" mitogen and not of antigen stimulation of sensitized cells.LPF, phytohemagglutinin, and concanavalin A were approximately equal in potency although variation occurred depending upon the cell donor. Experiments with lymphocyte subpopulations obtained by rosetting techniques employing sheep erythrocytes, mouse erythrocytes, and sheep erythrocytes coated with antibody and complement suggested the requirement of a multicellular system for LPF mitogencity.PBL from most patients with chronic lymphatic leukemia and lymphosarcoma cell leukemia were even less responsive to LPF than to phytohemagglutinin, whereas PBL from patients with lymphosarcoma usually responded to both mitogens. It can be inferred from the results of experiments with both normal and leukemic cells that LPF, which is a murine thymus-derived (T)-cell mitogen, is also a T-cell mitogen for human PBL. The exact cell requirement and mode of action, however, are as yet unknown.

Lymphocytosis-promoting factor of Bordetella pertussis: isolation, characterization, and biological activity.

The leukocytosis and lymphocytosis-promoting factor (LPF) of Bordetella pertussis has been isolated in apparently pure form. LPF is a protein essentially free of lipid and carbohydrate with an estimated molecular weight of 67,000-73,600 daltons. Purified LPF induced both histamine sensitization and refractoriness to epinephrine-induced hyperglycemia and was a murine thymus-derived (T-) cell mitogen. Adenyl cyclase activity also appeared to be associated with LPF.

Islet of Langerhans allotransplantation in the rat.

Normoglycemia in rats allotransplanted with islets of Langerhans was studied. It was found that pretreatment with donor liver extract and pertussis followed by a short course of ALS treatment results in much better overall survival of functioning islets of Langerhans allotransplants than with other forms of immunosuppression tested.

The in vitro effects of Bordetella pertussis lymphocytosis-promoting factor on murine lymphocytes. I. Proliferative response.

The lymphocytosis-promoting factor of Bordetella pertussis is a potent mitogen for murine lymphocytes in vitro. The stimulatory response was not the result of specific antigen stimulation. Spleen and lymph node cells were responsive, whereas normal thymocytes were unresponsive. However, DNA replication was induced in cortisone-resistant thymocytes by lymphocytosis-promoting factor (LPF). Bone marrow cells were not stimulated by LPF.

The in vitro effects of Bordetella pertussis lymphocytosis-promoting factor on murine lymphocytes: II. Nature of the responding cells.

The mitogenic response of murine lymphocytes to the lymphocytosis-promoting factor of Bordetella pertussis has been shown to be due to activation of T cells. The selectivity of responsiveness to LPF with respect to the population of T cells which is stimllated, differs from that of PHA as well as Con A, and the surface receptors are different. A population of adherent cells, which does not appear to consist of macrophages or other phagocytic cells, is required for the T-cell response.

Isolation and properties of the leukocytosis- and lymphocytosis-promoting factor of Bordetella pertussis.

The leukocytosis- and lymphocytosis-promoting factor (LPF) of Bordetella pertussis has been isolated to near homogeneity by physical, chemical, and electron microscopical criteria. LPF contains 14.5% nitrogen and is lipid and carbohydrate free. It is apparently composed of four polypeptide subunits. LPF caused leukocytosis and lymphocytosis in "nude" as well as in normal mice. In addition, purified LPF also induced histamine sensitization and hypoglycemia and refractoriness to the hyperglycemic effect of epinephrine. A monospecific LPF antiserum blocked these reactions as well as leukocytosis and lymphocytosis. LPF is clearly distinct from the hemagglutinating pili of B. pertussis.

The effect of Bordetella pertussis on the antibody response in mice to type III pneumococcal polysaccharide.

The effect of an i.p. injection of Bordetella pertussis on the primary humoral immune response in mice to the thymus-independent antigen SIII has been studied. Suppression of the antibody response occurred when pertussis cells were injected at the same time as an optimal immunizing dose of SIII. In contrast, the antibody response to high doses of SIII was enhanced by B. pertussis. When SIII alone was injected, only 19S antibody was detected. However, when B. pertussis was administered with either optimal or high doses of SIII, 7S as well as 19S antibody against SIII was produced.

Schistosoma mansoni: in vitro conversion of cercariae to schistosomula.

Schistosoma mansoni cercariae were incubated for 3-5h at 37 degrees C in various test solutions, and the rate and extent of conversion of the cercariae to schistosomula were determined. Criteria used to identify schistosomula included: (1) loss of cercarial tail, (2) viability of organisms in saline but not in water, (3) pre-acetabular gland evacuation and (4) ability to survive in culture. Incubation of cercariae in rat chamber fluid resulted in organisms which were water sensitive, but retained their tails and pre-acetabular gland contents. Conversion to water sensitivity was not blocked by 0-01 M EDTA.

Lymphocyte depletion induced by cholera toxin; relationship to adrenal cortical function.

Intravenous injection of cholera toxin (choleragen) into mice caused a profound lymphocytopenia associated with marked cellular depletion of the lymph nodes, spleen, and thymus. After administration of 1 mu g of choleragen, lymphocytopenia was mot marked at 24 hr; recovery occurred 6 to 10 days later. Similarly depletion of lymph nodes and spleen was maximal at 24 hr with recovery by 14 days. Choleragen also caused a marked elevation of serum corticosterone and lymphocyte depletion was not observed in adrenalectomized mice. These results suggested that the lymphocytopenic effect of choleragen was mediated by increased production and secretion of adrenal cortical hormones secondary to a rise in intracellular cAMP induced by cholera toxin. The site of action of choleragen may be the adrenal cortex itself and/or the hypothalamic-pituitary system.

The effect of Bordetella pertussis on lymphocyte cyclic AMP metabolism.

Bordetella pertussis culture fractions produce decreased metabolic responses to isoproterenol and epinephrine in mice and rats, suggesting the possibility of systemic beta adrenergic blockade. The present study was undertaken to elucidate the mechanism of the alteration in adrenergic responsiveness and to clarify its relationship to other biological effects of the organism. Lymphocytes were selected as a suitable tissue because of the marked alteration in lymphocyte distribution in pertussis-treated mice and rats, suggesting a change in the surface properties of these cells. Human peripheral blood lymphocytes, purified by nylon fiber chromatography, were studied. In short incubation experiments (20 min or less) B. pertussis did not alter the cyclic AMP response to isoproterenol, prostaglandin E (PGE(1)), or methacholine. However, when cells were preincubated with B. pertussis for 90 min at 37 degrees C, the responses to all three agents were markedly inhibited. Although these observations provide direct confirmation of the ability of B. pertussis to inhibit catecholamine responsiveness, the fact that PGE(1) and methacholine responses were also inhibited suggests that blockade at the level of the beta adrenergic receptor is doubtful. The inhibitory activity was localized in a nondialyzable, protein-rich fraction that is precipitated from B. pertussis culture fluid by ammonium sulfate at 90% of saturation. The bulk of the activity was obtained in the load volume after 50,000 g centrifugation in a cesium chloride gradient, density 1.2-1.5 (fraction 4). Fraction 4 produced a change in lymphocyte hormonal responsiveness at concentrations as low as 5 ng/ml. The relationship between cyclic AMP inhibitory activity in isolated human cells and leukocytosis-producing activity in intact mice was studied. The two activities seemed to parallel one another quite closely until the final Sephadex G-150 fractionation step, in which the two activities were obtained in the same column fraction, but a greater recovery of the leukocytosis-producing activity was obtained. Additional purification will be required to establish conclusively whether the same macromolecule is responsible for both activities. The availability of a bacterial product that markedly inhibits cyclic AMP accumulation in purified lymphocytes may help to clarify the role of cyclic AMP in lymphocyte activation by antigen and nonspecific mitogens.

Interaction of lymphoid and nonlymphoid cells with the lymphocytosis-promoting factor of Bordetella pertussis.

The activity of soluble lymphocytosis-promoting factor (LPF) from Bordetella pertussis decreased after brief in vitro incubation with lymphoid cells or erythrocytes. The LPF activity was found to be associated with the cells used, and injection of the cells produced a leukocytosis and lymphocytosis which completely accounted for the loss of soluble activity. Attachment of LPF to cells was found to be reversible in vitro. It is suggested that reversible binding occurs in vivo.