Elizabeth Gillespie

Histamine release from human leukocytes: studies with deuterium oxide, colchicine, and cytochalasin B

Agents known to interact with either microtubules or microfilaments influenced the antigen-induced release of histamine from the leukocytes of allergic individuals. Deuterium oxide (D(2)O) which stabilizes microtubules and thereby favors their formation enhanced histamine release markedly. Concentrations as low as 5% increased antigen-induced release somewhat while concentrations as high as 75% had no effect on release in the absence of antigen. Enhancement occurred over a wide range of antigen concentrations and was also seen when release was initiated by antibody to IgE or IgG. When the release process was divided into two stages a D(2)O activity could be demonstrated only in the second stage. However, when D(2)O was present in the first stage together with agents which raise cyclic AMP levels and thereby inhibit release it partially reversed this inhibition. Colchicine, demecolcine, and vinblastine, compounds known to disaggregate microtubules, i.e., have an effect opposite to that of D(2)O, inhibited the release of histamine and counteracted the effects of D(2)O. The inhibitory action of colchicine was greater if cells were treated with colchicine before rather than after activation with antigen. Cytochalasin B, a compound which causes the disappearance of microfilaments, had variable effects on histamine release. The most frequently seen response was slight enhancement. Neither D(2)O nor cytochalasin B altered cyclic AMP levels in leukocytes. These observations support and strengthen the view that an intact and functioning microtubule system is directly important for the secretion of histamine from leukocytes and suggest that microfilaments might have multiple indirect effects.

Cyclic AMP metabolism in asthma: Studies with leukocytes and lymphocytes

Abstract The effect of isoproterenol on cyclic AMP (cAMP) levels in lymphocytes and leukocytes from asthmatic and normal individuals has been studied. Lymphocyte cAMP levels rose in response to 10 −8 to 10 −2 M isoproterenol; the dose-response curve was biphasic with a maximum response at concentrations of 10 −6 to 10 −4 M. At all drug concentrations the response of cells from asthmatic individuals was less than the response of cells from normal control subjects. The difference between the two groups was not statistically significant, however. Similarly, basal cAMP levels were lower in the cells of asthmatic as compared with normal individuals, but again the difference was not significant. When these two parameters were combined and the results expressed as absolute cAMP levels after isoproterenol treatment, a significant difference was observed. Both the unstimulated. cAMP levels and the response to isoproterenol of leukocytes from normal and asthmatic subjects were influenced by the incubation medium. Use of a medium buffered with tris as compared with bicarbonate-phosphate resulted in lower basal cAMP levels and increased responsiveness to isoproterenol. Treatment of normal leukocytes with serum from asthmatic individuals did not alter their response to isoproterenol.

Effects of S-lactoylglutathione and inhibitors of glyoxalase I on histamine release from human leukocytes

THE enzyme responsible for the conversion of methylglyoxal to lactic acid by animal tissues was first named glyoxalase1,2. It was later shown that there are two glyoxalase enzymes3. The first (glyoxalase I). converts methylglyoxal and reduced glutathione to S-lactoylglutathione and the second (glyoxalase II). converts this compound to D-lactic acid, regenerating glutathione in the process. These enzymes are very widely distributed4 and as yet have no clearly defined function(s). I describe here the enhancement of anti-IgE-induced histamine release by S-lactoylglutathione and its inhibition by an inhibitor of and an alternative substrate for glyoxalase I. These two compounds should, of course, decrease the production of endogenous S-lactoylglutathione. These experiments were undertaken for two reasons. First, a study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro5 while the release of histamine from leukocytes is a secretory process thought to require an intact microtubule system6,7. Second, concanavalin A has been shown to cause histamine release from human leukocytes8,9 and has also been found to activate the two glyoxalase enzymes in lymphocytes and polymorphonuclear leukocytes10.

MICROTUBULES, CYCLIC AMP, CALCIUM, AND SECRETION*†

It is well established that within cells microtubules are in a dynamic equilibrium with the subunit protein (tubulin) of which they are formed. In 1971 I studied the effects of cyclic A M P (CAMP) and calcium on colchicine binding to soluble material in tissue slices, reasoning that colchicine would bind only to tubulin and that it should be possible to measure changes in the microtubulesubunit equilibrium by this approach. Both calcium and c A M P affected colchicine binding in the three tissues studied: rat liver, spleen and pancreas. As the calcium concentration was increased from 0 to 20 mM, colchicine binding gradually decreased. In the presence of 2 mM calcium c A M P had a biphasic effect on colchicine binding, low concentrations of nucleotide decreased binding whereas higher concentrations increased i t . This biphasic effect was not seen in the absence of calcium or in the presence of 20 mM calcium. These observations led to the hypothesis illustrated in FIGURE 1 . It was suggested that microtubules exist in two forms (designated A and B) and that it is the repeated interconversion of these two forms that leads to sustained activity of the microtubule system. Calcium and c A M P were considered to control the two reactions A + B and B + A respectively and it was assumed that only one microtubule form ( A ) breaks down to subunits. The major conclusions of this study were that both c A M P and calcium influenced (presumably controlled) microtubule function and that the relative concentration of the two was important for activity. In other words, in the presence of high levels of c A M P relative to calcium microtubules would be predominately in the A form and inactive, whereas in the presence of high levels of calcium relative to cyclic AMP, the B form would predominate and again inactivity would result. This hypothesis defines the relationships that should exist between c A M P and calcium on the one hand and agents that affect microtubules on the other. The present paper describes experiments on secretion in several systems that were designed to determine if these relationships d o indeed exist. This approach requires the assumption that the microtubule system is a point of control of secretory processes.

IgE-induced changes in human basophil cyclic AMP levels.

We studied a unique patient with 77% basophils, not different from normal by a number of criteria, in order to measure the changes in cyclic AMP level associated with IgE-mediated histamine release. In accordance with previous hypothesis and circumstantial evidence, anti-IgE challenge led to a significant fall in the cyclic AMP level which preceded histamine release.

In Vivo Suppression of the Immune Response to Alloantigen by Cholera Enterotoxin

The immune response of C57BL/6 mice to allogeneic (DBA/2) mastocytoma cell suspensions was profoundly suppressed by intraperitoneal administration of 1 mug cholera enterotoxin 4 days after antigenic stimulation. The immune response assayed 11 days after antigen showed decreased cytolytically active thymusderived (T) lymphocytes and markedly depressed serumagglutinating titers. A comparable suppression of the immune response to skin allografts (DBA/2-->C57BL/6) was also effected by cholera toxin administration, although there was no prolongation of allograft survival. The mechanism of the immune suppression is apparently related to the known adenylate cyclase stimulatory activities of choleragen.

HEAVY WATER ENHANCES IgE-MEDIATED HISTAMINE RELEASE FROM HUMAN LEUKOCYTES: EVIDENCE FOR MICROTUBULE INVOLVEMENT.

Summary Deuterium oxide, an agent known to stabilize microtubules, greatly enhances antigen-induced histamine release from human leukocytes. Concentrations as low as 7% are effective and enhancement occurs over a wide range of antigen concentrations. The effects of D2O are partially reversed by colchicine, a drug known to disrupt microtubules. These findings strengthen the view that microtubules are involved in the secretory mechanism by which the mediators of the allergic response leave the IgE containing target cells. We thank J. Harrison and A. Sobotka for their excellent technical assistance.

The mechanism of breakdown of tubulin in vitro

It is well established that in vitro tubulin is unstable in that it gradually loses its ability to bind colchicine [ 1,2] . The half time of this change has been estimated to be 11 h at 0°C [3]. During the course of experiments designed to study the assembly of microtubules, it was noted that incubation with dithiothreitol (DTT) decreased the extent of conversion of tubulin to the form that does not bind colchicine. This chance observation led to a systematic study of the effects of both reducing and oxidizing agents on the loss of colchicine binding activity by purified tubulin preparations. This loss of colchicine binding activity will be termed breakdown, although it apparently is unclear whether or not it is actually a result of the dissociation of the tubulin dimer into its two constituent monomer proteins. In contrast to the inhibitory effect of DTT, reduced glutathione (GSH) modestly increased the breakdown of tubulin occurring in 90 min. Oxidized glutathione (GSSG) had no apparent effect alone, while combinations of GSH and GSSG increased the extent of this breakdown more than did GSH alone. These results suggest (1) that the mechanism of breakdown of tubulin in vitro involves both the cleavage and the formation of disulfide bonds, and (2) that the tubulin dimer can exist in vitro in several different biochemical forms.

Pharmacological Control of Mediator Release from Leukocytes

Basophils and mast cells are the only two cell types known that are capable of tightly binding IgE antibody to their cell surfaces. Antigen specific for this IgE antibody can, by interacting with it, cause the release of histamine and other mediators of anaphylaxis from these two cell types. The properties and pharmacological control of mediator release from mast cells are described in Chapter 7. This chapter will deal almost exclusively with release from circulating basophils, and will consider largely the properties of histamine release, since much more is known about the release of this substance than about the release of other mediators. Two themes will be stressed: The first is that the release of histamine from basophils is a process of secretion analogous to the secretion (exocytosis) of hormones and enzymes (i.e., endocrine and exocrine secretion). The second is that this release process is negatively modulated by cyclic AMP (cAMP), and that perhaps histamine, which raises cAMP through interaction with histamine H-2 receptors, plays a special role as a modulator of this response and other immune responses.