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.

Humoral immunostimulation. V. Selection of variant cell lines.

A permanent L-cell variant cell line (LC1) was isolated by the growth of the parent L-cell line (L) in the presence of a cytostimulatory dose (1:200) of rabbit anti-L-cell antiserum (AL) for 9 mo. LC1 differed from L in many aspects: (a) it was larger (1,533 mm3 vs. 1,284 mm3), (b) it grew faster (1.5- to 2-fold), (c) it grew in aggregated fashion, (d) its growth was no longer stimulated by AL, (e) it was almost completely resistant to high concentrations of AL in the presence of complement (C), (f) its original membrane antigens (immunogenic for AL) were redistributed in sparse and patchy clumps as noted by fluorescence microscopy, (g) it contained about 65% of the total original 125I-AL membrane-binding sites (1.4 X 10(7)/cell vs. 2.2 X 10(7)/cell), (h) its AL-binding sites displayed a lower average affinity constant (K = 0.9 X 10(5) M-1 vs. 2.8 X 10(5) M-1), (i) it contained a smaller proportion of high affinity (K greater than 10(6) M-1) binding sites (13% vs 21%), and (j) LC1 was fully immunogenic in that it was readily killed by homologous antiserum (ALC1) and C, whereas L was not similarly affected by ALC1 indicating that LC1 contained new membrane antigens not present on L. Another variant (LC2) was produced by growth of LC1 in a 10-fold higher dose (1:20) of AL (cytotoxic for L) for 1 mo. LC2 was even more resistant to AL in the presence of C, contained 0.84 X 10(7) AL-binding sites/cell with an average affinity constant of 1 X 10(5) M-1 (unchanged from LC1), and was less susceptible than LC1 to lysis in the presence of ALC1 and C. These findings confirm and extend our previous in vitro and in vivo observations dealing with the direct stimulation effects of antibody on tumor cell metabolism and suggest that immunostimulation may be a mechanism of tumor escape from immune control in vivo possibly by immunoselection and antigenic modulation as proposed by other investigators.

Specific binding of leukotriene B4 to a receptor on human polymorphonuclear leukocytes.

In this paper we have described the binding of nanomoler concentrations of [3H]leukotriene B4 (LTB4) to human polymorphonuclear leukocytes. Because up to 80% of the total [3H]LTB4 binding was blocked by excess (greater than 100 times) [14C]LTB4, the majority of binding is specific. Stereospecificity of the LTB4 binding is demonstrated by the diminished relative abilities of the 6-trans-and 12-epi-6-trans- isomers of LTB4 to block [3H]LTB4 binding. With these two isomers 3-10-fold higher than [14C]LTB4 concentrations were needed for equivalent inhibition of [3H]LTB4 binding. This difference is quantitatively less dramatic than the differences between these isomers in many in vitro functional assays such as chemokinesis, chemotaxis, and degranulation. Binding of [3H]FMLP is not blocked at greater than 100-fold excess of LTB4. The binding of [3H]LTB4 to cells appears to be essentially irreversible at 4 degrees C, but not at 37 degrees C where initially bound LTB4 is rapidly converted to metabolites which then enter the medium. These results suggest the presence of a saturable, stereospecific site for LTB4 on PMN. The association of LTB4 binding and the initiation of pharmacological responses to LTB4 will require further studies.

Humoral immunostimulation. I. Increased uptake of (125I)iododeoxyuridine and (3H)thymidine into TNP-cells treated with anti-TNP antibody.

Interaction of microgram quantities of highly purified rabbit anti-TNP antibodies with TNP-substituted HeLa, HEp-2, and L cells caused an intense stimulation of radioactive nucleoside ([(125)I]UdR and [(3)H]TdR) uptake which was maximal 24-72 h after exposure of cells to antibody. The stimulation of nucleoside uptake and presumaly DNA synthesis was shown to be immuno logically mediated because unsubstituted cells were not stimulated by anti-TNP antibody, normal rabbit gamma globulin did not stimulate TNP-cells, and a hapten inhibitor, epsilon-DNP-lysine, prevented the stimulation of TNP-cells by anti-TNP antibody. These findings demonstrate that interaction of antibody with cell surface antigen can alter cell membrane transport, and possibly can enhance cell growth.

Diacylglycerol metabolism in mast cells: a potential role in membrane fusion and arachidonic acid release.

Purified rat peritoneal mast cells stimulated with the polycationic histamine-releasing agent compound 48/80 demonstrated a two- to four-fold increase in cellular levels of 1,2-diacylglycerol (DAG) within 1 min as detected by radioactive labeling and direct quantitation experiments. When 2-[1-14C]arachidonoyl-DAG was incubated in the presence of mast-cell homogenates, a rapid conversion to free arachidonate, and to a lesser extent, to monoacylglycerol, triglyceride, and phospholipid was observed. The release of arachidonate was proportional to the amount of broken-cell preparation added and the time of incubation, was prevented by preheating mast-cell preparations, and did not occur when 1-[1-14C]arachidonoyl-phosphatidylcholine was used as substrate, suggesting that the degradation was mediated by an enzyme with Dag-lipase activity. Although much work remains to be done to clarify the precise role of DAG in mast cells, DAG metabolism may be involved in secretion by generating substances which may faciliate membrane fusion and also in arachidonic acid-derived mediator formation by liberating esterified arachidonic acid from mast-cell lipids. Taken together, these studies indicate that the formation of DAG may play a central role in mast-cell function.

Release of arachidonic acid from human lymphocytes in response to mitogenic lectins.

After exposure to mitogenic lectins in vitro, human mononuclear cells (95% lymphocytes) that had been prelabeled with [14C]arachidonic acid rapidly released a portion of their radioactivity in the medium. Most of the released radioactivity was demonstrated to be free arachidonic acid. Although other sources are not excluded, the most important source of cell-bound radioactivity in the release reaction appeared to be phosphatidylinositol, suggesting that at least part of the response is occurring through an increase in phospholipase A2 activity. By gas liquid chromatography, other fatty acids were also shown to be released, but there was considerable selectivity in the response for arachidonic acid. The response was dependent on the availability of free Ca++ in the medium and was enhanced by serum proteins and unlabeled arachidonic acid. Most of the response appeared to be from the the lymphocytes themselves rather than from contaminating cells. The rapid generation of free arachidonic acid in response to mitogenic lectins suggests a possible role for arachidonic acid metabolites in the activation process.

Humoral immunostimulation. IV. Role of complement.

When L cells were treated with anti-L-cell antibody in medium containing heat-inactivated fetal calf serum, nucleoside uptake and cell growth were stimulated. The response was markedly increased when fresh, unheated sera from calves, guinea pigs, humans, mice, or rabbits were also present. The factors in unheated serum responsible for the enhancement of immunostimulation were studied. Using low concentrations of sera deficient in various complement (C) components and low concentrations of antibody no augmentation of immunostimulation was seen with Clr-deficient human serum, C2-deficient human serum, C2,4-deficient human serum, C4-deficient guinea pig serum, C3-C9-depleted guinea pig serum (by administration of cobra venom factor to animals), but stimulation was observed with C5-deficient human serum, C5-deficient mouse serum, and C6-deficient rabbit serum. When the concentration of anti-serum was raised, however, augmentation was observed with C4-deficient guinea pig serum. Thus, at low concentrations of antiserum enhancement appeared to occur through the classical C pathway, whereas at high concentrations of antibody either the classical or alternate C pathways appeared to be involved. Stimulation was specifically restored by purified C2 in C2-deficient serum and by C3 in C3-C9-deficient serum. Under the usual reaction conditions consumption of guinea pig C component C4 could be demonstrated which provided direct evidence for activation of the classical C pathway under conditions leading to immunostimulation. By immunofluorescence, cells treated with antibody and normal human serum had human C3 deposited at the cell surface. Taken together these observations suggest that C activated through C3 by either the classical or alternate pathways has the potential to enhance nucleoside incorporation into DNA and cell growth of cells exposed to limiting amounts of antibody. Although the mechanism of stimulation is unknown, it is likely to involve a direct effect of C3 at the level of the cell membrane.

Morphine: radioimmunoassay.

The development of a radioimmunoassay for morphine is described. The hapten morphine is made antigenic by coupling it to a protein at the phenolic group of the molecule. Extremely low concentrations of morphine (0.5 nanogram) can be measured by this assay procedure.

Radioimmunoassay for creatine kinase isoenzymes.

Creatine kinase has three isoenzymes designated MM, MB, and BB, with BB being the grain form and MM the muscle form. Antibodies to BB creatine kinase were obtained by immunization of rabbits with human BB creatine kinase. The antibodies demonstrated specificity for BB and MB creatine kinase (myocardial isoenzyme) but no cross-reactivity with MM creatine kinase. With the use of this antibody, a highly sensitive radioimmunoassay capable of measuring picomolar amounts of MB creatine kinase has been developed. Clinical application of this method should provide a sensitive and specific test for the diagnosis of myocardial infarction.

Stimulation of cells by antibody.

Tumor cell lines exposed to immunoglobulins specific for cell surface antigens developed increased cellular incorporation of [(125)I]iododeoxyuridine and [(3)H]thymidine (up to 200-fold increases over cells treated with normal rabbit immunoglobulins). Antibody-stimulated cells multiplied more rapidly and lived longer than control cells in tissue culture. These observations were made both with cells substituted with 2,4,6-trinitrophenol and purified antibody against 2,4,6-trinitrophenol, and with several cell lines and their respective whole-cell antibodies. Antibodies that were stimulatory at low concentrations were cytotoxic at high concentrations. These observations may have significance in regard to enhancing effects of antibodies on tumor cell growth in vivo.

Radioimmunoassay for prostaglandins.

Antibodies to prostaglandin were obtained by immunization of rabbits with PGA(1), PGA(2), and PGE(1), protein conjugates of prostaglandins. The antibodies demonstrated specificity toward both the cyclopentane ring and the aliphatic side chains. With the use of these antibodies a highly sensitive radio-immunoassay capable of measuring less than picomolar amounts of PGA1, PGA2, and PGE1 has been developed.

Adenosine 3',5'-monophosphate is localized in cerebellar neurons: immunofluorescence evidence.

Adenosine 3',5'-monophosphate is localized in specific cerebellar neurons, as shown by fluorescence immunocytochemistry with a specific rabbit immunoglobulin. Positive staining is exhibited by Purkinje neurons and granule cells. The increase in concentration of cyclic adenosine monophosphate in the cerebellum, which is known to follow decapitation, is represented by greatly increased fluorescence of Purkinje neurons only. These immunofluorescence data provide the first evidence for localization of cyclic adenosine monophosphate in specific neurons and may permit further exploration into the role of this cyclic nucleotide in neuronal function.

Alterations in cyclic adenosine monophosphate metabolism in human bronchial asthma. I. Leukocyte responsiveness to -adrenergic agents.

In an effort to better define the role of betaadrenergic blockade in human bronchial asthma, peripheral blood leukocytes and lymphocytes from individuals with this condition were studied for possible alterations in cyclic AMP metabolism. Using a previously described radioimmunoassay to measure cyclic AMP, cells from asthmatic subjects were shown to have a highly significant decrease in their cyclic AMP response to beta-adrenergic agents (isoproterenol, norepinephrine, and epinephrine) by comparison with normal control cells. The alteration in responsiveness was most marked at the time of severe active asthma and returned toward normal during periods of clinical remission. Evidence was presented to indicate that the reduced response in cells from asthmatic individuals was not due to marked alterations in the proportion of T and B lymphocytes. Five normal volunteers were treated with an oral bronchodilator preparation containing theophylline and ephedrine over a 2 wk period without a significant change in the lymphocyte cyclic AMP response. These results provide unambiguous evidence for altered adrenergic responsiveness in bronchial asthma and indicate that purified peripheral blood lymphocytes should be a suitable in vitro system for further elucidation of the abnormality. Despite the reduction in catecholamine responsiveness in the asthmatic population as a whole, major alterations were largely restricted to individuals with severe, chronic asthma. Conclusive evidence for beta-adrenergic blockade in individuals who have not had recent asthmatic symptoms was not obtained, casting some doubt on the theory that bronchial asthma is due to a congenital derangement of cyclic AMP metabolism. Moreover, transient episodes of bronchospasm were often accompanied by a normal cyclic AMP response indicating that episodes of asthma frequently occur in the absence of easily demonstrable adrenergic blockade.

Aspirin effects on lymphocyte cyclic AMP levels in normal human subjects.

In purified lymphocytes from the peripheral blood of healthy human subjects who had ingested therapeutic doses of aspirin, there was a significant decrease in resting cyclic AMP levels as well as a partial inhibition of the rise in cyclic AMP with isoproterenol or prostaglandin E1. These changes were seen as early as 30 min after aspirin ingestion and did not appear to result from aspirin effects on lymphocyte recovery, purity, viability, or relative number of thymus- or bone marrow-derived lymphocytes. In contrast, the direct addition of aspirin to suspensions of purified peripheral lymphocytes did not significantly alter their cyclic AMP levels. However, an effect of aspirin could be obtained in vitro if aspirin was added to unprocessed whole blood during the dextran sedimentation phase of the cell purification. Thus the effect of aspirin on lymphocyte cyclic AMP metabolism, may be indirect, through other cells present in the peripheral blood.

Metabolism of arachidonic acid in ionophore-stimulated neutrophils. Esterification of a hydroxylated metabolite into phospholipids.

[14C]Arachidonic acid incubated with human neutrophils was esterified into phospholipids and triglycerides. Stimulation of these labeled neutrophils with ionophore A23187 (2 microM) results in release of [14C]arachidonate from phospholipid and its metabolism to prostaglandin E2 and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), a lipoxygenase product. The released arachidonate is also metabolized to a polar lipid of unknown composition here disignated compound A. 5-HETE was found to be released into the medium and then taken up again by the cells. To determine its metabolic fate, [14C]5-HETE was prepared biosynthetically, purified, and incubated with stimulated, unlabeled neutrophils. Most of the radioactivity entered the cells and was esterified into phospholipids and triglycerides. The radiolabeled complex lipids were saponified, and the released fatty acids cochromatographed with authentic 5-HETE. The esterification of 5-HETE, a hydroxylated fatty acid, into membrane phospholipids may be an example of a more generalized mechanism for altering membrane characteristics.

Radioimmunoassay measurement of prostaglandins E, A, and F in human plasma.

The details of a radioimmunoassay capable of measuring as 5 pg of prostaglandin A, E, and F (PGA, PGE, and PGF) in human and rat plasma are described. Plasma samples are extracted (with 4000 cpm [(3)H] PGE(1) added for calculation of recovery) with an organic solvent system at an apparent pH of 5.8 and then chromatographed on silicic acid columns with increasing concentrations of methanol to separate PGA, PGE, and PGF. Each chromatographed sample is measured by radioimmunoassay, using the homologous antibody and tritiated marker. 40 normal individuals had mean plasma concentrations of PGA, PGE, and PGF of 1062+/-107 pg/ml, 385+/-30 pg/ml, and 141+/-15 pg/ml, respectively. Elevated PGE levels were measured in the plasma of patients with medullary carcinoma of the thyroid, carcinoid, and neuroblastoma. Treatment of rats with indomethacin decreased serum PGE levels by 67%. The radioimmunoassay appears to be of considerable experimental as well as clinical interest.

Alterations in cyclic AMP metabolism in human bronchial asthma. II. Leukocyte and lymphocyte responses to prostaglandins.

In an effort to clarify the basis for the reduced cyclic AMP response to catecholamines in leukocytes and lymphocytes from asthmatic donors the response of these cells to prostaglandins has been examined. Cells with an impaired beta adrenergic response had an essentially unaltered response to prostaglandin E(1) (PGE(1)) indicating the presence of selective beta adrenergic blockade. In contrast to what was observed with cells from asthmatic individuals, in normal control leukocytes with reduced catecholamine responsiveness PGE(1) responses were usually reduced as well, suggesting a different mechanism. The excellent cyclic AMP response to PGE(1) in cells from asthmatic donors would suggest that the defect in catecholamine responsiveness is at the level of the beta adrenergic receptor although a contributory role of altered substrate concentrations or increased phosphodiesterase activity is not formally excluded.

Alterations in cyclic AMP metabolism in human bronchial asthma. 3. Leukocyte and lymphocyte responses to steroids.

On the basis of serial studies the responsiveness of leukocytes and lymphocytes from asthmatic donors to catecholamines was increased during high dose corticosteroid therapy. Similar changes were observed in the cells of normal control subjects given 200 mg of hydrocortisone intravenously. The increase in responsiveness did not appear to be due to changes in lymphocyte subpopulations although this may be a contributing factor. In an effort to elucidate the basis for the improved response, in vitro effects of glucocorticoids on lymphocyte cyclic AMP concentrations were investigated. Glucocorticoids (prednisolone succinate, hydrocortisone, hydrocortisone phosphate, and hydrocortisone succinate) stimulated cyclic AMP accumulation in asthma and normal control lymphocytes, increases occurring within the first 2 min of incubation. In the absence of theophylline, responses were regularly obtained at 10 muM hydrocortisone and usually at 1 muM hydrocortisone but not at submicromolar steroid concentrations. Theophylline potentiated the cyclic AMP response to glucocorticoids and also increased the percentage of positive responses in the 0.01-1.0 muM corticosteroid range. Combinations of 1 muM hydrocortisone and 1 muM epinephrine were sometimes additive or synergistic but in many instances higher glucocorticoid concentrations were needed to obtain augmentation of the catecholamine response. The in vitro glucocorticoid effects may not fully explain their potentiating action in vivo.

Human lymphocytic metabolism. Effects of cyclic and noncyclic nucleotides on stimulation by phytohemagglutinin.

The effects of extracellular nucleotides and agents which elevate intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP) concentrations on human lymphocyte metabolism have been studied. Aminophylline, isoproterenol, and prostaglandins, all of which elevate lymphocyte cyclic AMP levels, inhibited incorporation of (3)H-labeled thymidine, uridine, and leucine into the DNA, RNA, and protein of phytohemagglutinin (PHA)-stimulated lymphocytes. Aminophylline inhibition was maximal only when the inhibitor was added within 1 hr after exposure of cells to PHA, suggesting that a relatively early step in the lymphocyte transformation process may be affected. The addition of various nucleotides to the culture medium also inhibited incorporation of labeled precursors. The best inhibitor, dibutyryl cyclic AMP (DU cyclic AMP), produced maximal inhibition only if present during the 1st hr after initial exposure to PHA. Among the various cyclic nucleotides derivatives of guanosine and adenine were the most effective inhibitors (substantial inhibition at 0.1 mM concentrations). However, the inhibition was not specific for nucleotides containing the cyclic phosphodiester moiety since the tri-, di-, and monophosphates of adenosine and guanosine were equally effective in diminishing thymidine uptake. The above inhibitions were not due to secondary effects of the inhibitors on the interaction of PHA with lymphocytes as judged by (125)I-labeled PHA binding studies.Low concentrations (1-10 mumoles/liter) of cyclic AMP produced slight stimulation of thymidine-(3)H uptake in resting lymphocytes (lymphocytes not stimulated with PHA). However, the effects were quite small as compared with those produced by PHA itself. Attempts to demonstrate increased thymidine uptake 48 hr after pulsing lymphocytes with aminophylline or isoproterenol were unsuccessful. The relationship of these observations to a possible regulatory role for cyclic AMP in PHA-stimulated lymphocytes is discussed.

In vitro antibody-enzyme conjugates with specific bactericidal activity.

IgG with antibacterial antibody opsonic activity was isolated from rabbit antisera produced by intravenous hyperimmunization with several test strains of pneumococci, Group A beta-hemolytic streptococci, Staphylococcus aureus, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli. Antibody-enzyme conjugates were prepared, using diethylmalonimidate to couple glucose oxidase to IgG antibacterial antibody preparations. Opsonic human IgG obtained from serum of patients with subacute bacterial endocarditis was also conjugated to glucose oxidase. Antibody-enzyme conjugates retained combining specificity for test bacteria as demonstrated by indirect immunofluorescence. In vitro test for bactericidal activity of antibody-enzyme conjugates utilized potassium iodide, lactoperoxidase, and glucose as cofactors. Under these conditions glucose oxidase conjugated to antibody generates hydrogen peroxide, and lactoperoxidase enzyme catalyzes the reduction of hydrogen peroxide with simultaneous oxidation of I(-) and halogenation and killing of test bacteria. Potent in vitro bactericidal activity of this system was repeatedly demonstrated for antibody-enzyme conjugates against pneumococci, streptococci, S. aureus, P. mirabilis, and E. coli. However, no bactericidal effect was demonstrable with antibody-enzyme conjugates and two test strains of P. aeruginosa. Bactericidal activity of antibody-enzyme conjugates appeared to parallel original opsonic potency of unconjugated IgG preparations. Antibody-enzyme conjugates at concentrations as low as 0.01 mg/ml were capable of intense bactericidal activity producing substantial drops in surviving bacterial counts within 30-60 min after initiation of assay. These in vitro bactericidal systems indicate that the concept of antibacterial antibody-enzyme conjugates may possibly be adaptable as a mechanism for treatment of patients with leukocyte dysfunction or fulminant bacteremia.