The effect of hydrocortisone and x-irradiation on the lymphocytosis induced by Bordetella pertussis.

1. In mice rendered lymphocytopenic by X-irradiation or hydrocortisone acetate, pertussis vaccine evoked both lymphocytosis and polymorphonuclear leukocytosis. 2. When mice with lymphocytosis induced by pertussis vaccine were X-irradiated, prompt and extensive destruction of circulating as well as tissue small lymphocytes occurred. The devitalized circulating cells were cleared from the blood primarily by the Kupffer's cells of the hepatic sinusoids. 3. Hydrocortisone acetate administered to mice with lymphocytosis did not cause acute lymphopenia nor was there any evidence of destruction of circulating small lymphocytes. However, destruction of these cells within lymphoid tissues was apparent. These observations suggested that adrenal cortical hormones are not "lymphocytolytic" with respect to circulating lymphocytes.

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.

In vivo transmission of drug resistance factors between strains of Staphylococcus aureus.

1. Multiply resistant strains of Staphylococcus aureus often harbor one or more extrachromosomal drug resistant factors as well as temperate prophages capable of mediating generalized transduction. 2. Spontaneous transduction occurs in mixed cultures of such staphylococcal strains, and the extrachromosomal resistance factors are involved more frequently than are chromosomal genes. 3. Spontaneous transduction of extrachromosomal determinants of erythromycin resistance and of linked penicillin-erythromycin resistance occurs in the kidneys of mice in which mixed infection has been induced.

Studies on the leukocytosis and lymphocytosis induced by Bordetella pertussis. I. Radioautographic analysis of the circulating cells in mice undergoing pertussis-induced hyperleukocytosis.

By the use of radioautographic techniques it was shown that the lymphocytosis induced by Bordetella pertussis in mice was not caused by an increased production of lymphocytes but was primarily due to the entry into the circulation of mature cells from tissue pools. The accompanying polymorphonuclear leukocytosis was due to both proliferation of myeloid elements and entry of mature cells from tissue reserves, with the former the predominant mechanism.

Studies on the leukocytosis and lymphocytosis induced by Bordetella pertussis. II. The effect of pertussis vaccine on the thoracic duct lymph and lymphocytes of mice.

The 24 hr volume flow, cell concentration, and total cell output of thoracic duct fluid from mice with pertussis-induced hyperlymphocytosis were markedly reduced when compared with values obtained in normal animals. An increase in the number of circulating lymphocytes occurred in several of the pertussis-treated mice despite the presence of an indwelling thoracic duct cannula. The drainage from such animals also showed a reduced cell concentration and total cell output. It is suggested that lymphocyte recirculation may be minimal in pertussis-induced lymphocytosis, and the evidence obtained also suggests that lymphocytes may enter the blood stream by direct routes during the course of the reaction.

The occurrence and properties of leukocytosis and lymphocytosis-stimulating material in the supernatant fluids of Bordetella pertussis cultures.

1. Leukocytosis- and lymphocytosis-stimulating activity was present in fluid cultures of B. pertussis. The activity was found primarily in the culture supernatant fluid. 2. The sequential changes in the leukocyte response were similar to those previously observed following injection of intact bacteria into mice. 3. Activity was destroyed by heat and was diminished, but not abolished, by prolonged treatment with proteolytic enzymes. 4. A water-insoluble fraction of the culture supernatant fluid was isolated which contained virtually all of the activity. The specific activity was more than 100-fold greater than that of the intact bacteria, and injection of microgram quantities produced a response. 5. The distribution of histamine-sensitizing factor followed that of leukocytosis-stimulating activity. In contrast, mouse protective antigen was localized to the bacterial pellet.

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.

Studies on the ultrastructure of Bordetella pertussis. I. Morphology, origin, and biological activity of structures present in the extracellular fluid of liquid cultures of Bordetella pertussis.

Two distinct particles have been recognized in the extracellular fluid of B. pertussis cultures. Both appeared to arise from the surface (cell wall) of the organism. One of these, a membranous particle, seemed to derive from long projections on the organism composed of the outer membrane of the cell wall. The second particle, a fine filament, was not readily comparable with any previously described bacterial structure. The two particles could be separated from each other by gradient centrifugation in CsCl. Lymphocytosis-promoting factor and histamine-sensitizing activity were only associated with fractions containing the fine filaments.

Studies on the leukocytosis and lymphocytosis induced by Bordetella pertussis. 3. The distribution of transfused lymphocytes in pertussis-treated and normal mice.

Peripheral blood lymphocytes were isolated from normal mice and mice undergoing pertussis-induced lymphocytosis. After labeling in vitro with tritiated uridine the cells were transfused into normal or pertussis-treated mice. It was found that the lymphocytes from pertussis-treated mice entered the lymph nodes of both normal mice and pertussis-treated mice to a significantly lesser extent than did normal lymphocytes which had been transfused into either class of recipient. In addition, an interdependence of changes in the various body compartments examined was found when normal lymphocytes were injected into either type of recipient. However, when pertussis lymphocytes were injected into normal mice there was no interrelationship between the changes in the node with those in the blood, liver, lung, or spleen. In the case of pertussis lymphocytes transfused into pertussis-treated mice no interrelationship between any two compartments was observed. It was concluded that in pertussis-treated mice there is an inhibition of lymphocyte emigration which is primarily the consequence of an effect on the cell.

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.

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.

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.

Distribution of labeled lymph node cells in mice during the lymphocytosis induced by Bordetella pertussis.

The mechanism by which Bordetella pertussis organisms and their products induce lymphocytosis in mice was analyzed in terms of the localization of syngeneic Cr-51-labeled lymph node cells. Labeled lymphoid cells incubated in vitro with the supernatant of B. pertussis cultures and then injected intravenously into normal recipients, or labeled cells injected into pertussis-treated recipients were unable to "home" to lymphoid organs but persisted for long periods in the blood. In animals "equipped" with a population of Cr-51-labeled lymphoid cells, administration of B. pertussis organisms or culture supernatant effected a shift of radioactivity from lymph nodes and spleen into the peripheral blood, coincident with the lymphocytosis. In in vitro experiments it was found that the active principle could bind to both erythrocytes and lymphocytes and could spontaneously elute from these cells onto labeled lymphocytes which were then unable to home efficiently. The data suggest that Bordetella pertussis-induced lymphocytosis involves a reversible attachment of the pertussis factor onto the surfaces of lymphocytes which prevents their recirculation to lymphoid organs. Recirculating lymphocytes are presumably affected as they emerge from lymphoid organs to enter the blood.

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 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.

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.

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.