Effects of Cholera Toxin on In Vitro Modelsof Immediate and Delayed Hypersensitivity

FURTHEREVIDENCE FORTHE ROLEOF CYCLIC

ADENOSINE3',5'-MONOPHOSPHATE

L. M. LICHTENSTEIN, C. S. HENNEY, H. R. BOURNE,andW. B. GREENOUGH,III

From the Department of Medicine, The Johns Hopkins University School ofMedicine at The Good Samaritan Hospital, Baltimore, Maryland 21212, and theDepartment of Medicine, University of California Medical Center,San Francisco, California 94122

A B S T R A C T Cholera enterotoxin inhibits the antigen-induced, IgE-mediated release of histamine from humanleukocytes and the lysis of allogeneic mastocytoma cellsby splenic lymphocytes from specifically immunized mice.This effect requires a prolonged preincubation time ofthe toxin with the lymphocyte/leukocyte preparations:a demonstrable inhibition requires about 30 min of pre-incubation and the toxin activity is still increasing at90-180 min. Cholera enterotoxin also stimulates adenylcyclase and leads to increased levels of cyclic AMP inthe lymphocyte/leukocyte preparations. The concentra-tion of toxin required for both cyclic AMPaccumulationand inhibition of the biologic responses is about the same(ca. 1 ng/ml), and the time course of cyclic AMPac-cumulation parallels the development of inhibitory ac-tivity. Both activities, inhibition of the in vitro hyper-sensitivity reactions and cyclic AMPaccumulation, areblocked by cholera antitoxin and by a toxoid preparedfrom the toxin (choleragenoid). These are specific an-tagonists in that they do not block the inhibiting activityor rise in cyclic AMPlevels caused by other adenyl cy-clase stimulators. Because cholera enterotoxin has noknown activity other than the stimulation of adenyl cy-clase and because of its unusual time course and theavailability of specific antagonists, this data considerably

This is publication 40 from the O'Neill Research Labora-tories of The Good Samaritan Hospital, Baltimore, Md.

Dr. Lichtenstein is the recipient of Research Career De-velopment Award AI42373 from the National Institute ofAllergy and Infectious Diseases, National Institutes ofHealth.

Received for publication 4 May 1972 and in revised form29 August 1972.

strengthens the hypothesis that the cyclic AMPsysteminfluences the expression of these two forms of hypersen-sitivity phenomena.

INTRODUCTION

Cyclic AMPacts as an intracellular "second messenger"mediating the hormonal control of enzymatic or secre-tory processes in many tissues (1). Several years agowe implicated cyclic AMPas a regulator of the IgE-mediated allergic response, in a model involving the anti-gen-induced release of histamine from basophils of al-lergic patients (2-4). This work has since been con-firmed in several other models of immediate hypersen-sitivity (5-7). Thet release of histamine and other medi-ators is prevented by agents which stimulate productionof cyclic AMP(including prostaglandins, catecholamines,and histamine), block its intracellular degradation(methylxanthines), or mimic the effect of the nucleotide(dibutyryl cyclic AMP) (2-4, 8). More recently, wehave shown that the same agents inhibit the activity oflymphocytes in an in vitro model of cellular immunitywhich involves the lysis of allogeneic mastocytoma cellsby splenic lymphocytes obtained from specifically im-munized mice (9, 10).

In both model systems the measured changes in cyclicAMPcorrelated well with the relative inhibitory effectsof the amines and prostaglandins studied. The evidencewas circumstantial rather than conclusive, however, be-cause in both cases the nucleotide was measured in aheterogeneous cell population, whereas histamine releaseor cytolytic activity involved a subpopulation of cells. In

The Journal of Clinical Investigation Volume 52 March 1973 691

the case of human leukocytes, the histamine-containingbasophils comprised less than 1% of the cells in whichcyclic AMPwas measured. The percentage of lympho-cytes in mouse splenic-cell preparations was larger(> 80% of the leukocytes present), but the actual num-ber of these lymphocytes involved in specific cytolyticactivity was only of the order of 2-4% (11).

Consequently, we have sought to design critical experi-ments which could disprove the hypothesis that cyclicAMP inhibits both immediate and cellular hypersensi-tivity reactions in vitro. For this purpose we used theenterotoxin of Vibrio cholerae, which stimulates adenylcyclase activity and the production of cyclic AMP inmany tissues (12-15), including gut mucosa, where thisaction is probably the pathogenic basis of cholera (16).

We found that cholera enterotoxin stimulated cyclicAMP production and inhibited both histamine release

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FIGURE 1 (a) Inhibition of IgE-mediated histamine releasefrom human leukocytes incubated with cholera enterotoxinfor 5 and 60 min before antigen addition. Each curve is aseparate experiment except those depicted by the closedsquares at 5 and 60 min preincubation, which were carriedout simultaneously. (b) Time course of cholera entertoxins'inhibition of histamine release. Cells were preincubated for30, 60, or 90 min with the stated concentrations of choleraenterotoxin before antigen addition. Two separate experi-ments are shown: (0) and (U) and (El) and (0) werecarried out together.

and lymphocyte-mediated cytolysis. The effects on cyclicAMPand cell function required low molar concentrationsof the toxin, were prevented by specific antagonists, andexhibited a distinctive time course, different from thatof any other compound so far tested. These results addconsiderable weight to the postulated causal relation be-twee intracellular cyclic AMP and the inhibition ofin vitro models of immediate and delayed hypersensi-tivity.

In an accompanying paper we report further observa-tions related to the unique mechanism of action of choleraenterotoxin, and results of experiments in which thetoxin was used to test two other hypotheses that implicatecyclic AMPas a regulator of leukocyte function (17).

METHODSHistamine release. Leukocytes from donors sensitive to

ragweed and grass were separated from the other formedelements of the blood by dextran sedimentation, washed sev-eral times, and resuspended in a serum-free Tris buffer con-taining calcium, magnesium, and crystallized human albumin.The cells were then challenged with either antigen E ofragweed or grass group I antigen and the histamine releasedfrom these cells into the supernatant fluid measured by thefluorometric assay described by Shore et al. (18-20). Thesetechniques have previously been described in detail. Wehavecurrently used May's modification of our earlier method (21,22). All experimental points are carried out in duplicate; thevalues agree within ±5-10%o.

Lymphocytic-mediated cytolysis. The system employedwas that originally described by 'Brunner, Mauel, Cerottini,and Chapuis (23). Briefly, CmT BE mice were injected intra-peritonally with 107 DBA/2 mastocytoma cells. 11 days laterthe immune mice were sacrificed, and splenic lymphoid cellsuspensions prepared. 10,000,000 viable lymphocytes were in-cubated at 370C for varying periods of time with 10' 'Cr-labeled mastocytoma cells. Total cytolysis was evaluated bymeasurement of 'Cr release and specific cytolysis by correc-tion for the 'Cr released in the presence of 107 normal (i.e.,nonimmune) CD7 B1 lymphocytes. These methods have pre-viously been described in detail (11, 23).

Cyclic AMPmeasurements. The method of Gilman wasused (24). Its application to the present systems has beendescribed previously (4, 10), and a modification of the ex-traction procedure is described in detail in the accompanyingpaper (17).

Cholera enterotoxin, toxoid, and antitoxin. Cholera en-terotoxin (choleragen) and the naturally occurring toxoid(choleragenoid) were generously supplied by Dr. RichardA. Finkelstein (25). The antiserum employed was raised byimmunization of a dog with choleragen.2 This serum had 1354antitoxin U by comparison to a standard cholera antitoxinmade by The Swiss Serum and Vaccine Institute and dis-tributed by Dr. John Seale (National Institutes of HealthStandard).

RESULTSImmediate hypersensitivity. Cholera enterotoxin in-

hibits the IgE-mediated release of histamine from thehuman leukocytes of allergic individuals. Two variables

'Pierce, N. F. Personal communication.

692 L. M. Lichtenstein, C. S. Henney, H. R. Bourne, and W. B. Greenough, III

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rectly with time; the cells cannot be studied for longerthan 90 min without a decrease in their viability, so it isnot clear when this effect levels off.

The effect of cholera enterotoxin on cyclic AMPlevelsin human leukocytes after 90 min incubation is shownin Fig. 2a. The results are entirely concordant with theinhibition data: a definite effect is seen with concentra-tions as low as 0.1-0.3 ng/ml; 1 ng/ml provides a strongstimulus and the effect levels off at about 10 ng/ml as inthe inhibition dose-response curves. Fig. 2b shows thatthe time course of cyclic AMPaccumulation in the leu-kocytes after treatment with cholera enterotoxin issimilar to the inhibition time course: little activity at30 min and an increase which is still continuing at 90min. The activity of cholera enterotoxin on cyclic AMPis potentiated by 10' M theophylline, an agent which atthis concentration inhibits cyclic AMP destruction byphosphodiestrase (data not shown).

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FIGURE 2 Effect of cholera enterotoxin on accumulation ofcyclic AMPin leukocytes. (a) Effect of varying concentra-tions of cholera enterotoxin on cyclic AMPcontent of leuko-cytes from five subjects, incubated for 90 min. The originof each dotted line represents cyclic AMPcontent of leuko-cytes incubated for 90 min in the absence of toxin. (b) Timecourse of accumulation of cyclic AMP in leukocytes of onesubject exposed to 10 ng/ml cholera enterotoxin (0), 1.0ng/ml (A), or in the absence of the toxin (U). All valuesare the mean of duplicate determinations, differing by notmore than 10%.

are important: time and concentration. This is illustratedin the dose-response curves shown in Fig. la, in whichvarying concentrations of cholera enterotoxin were in-cubated with the leukocytes for either 5 or 60 min at370C before antigen was added to initiate histamine re-lease. At 60 min there was a profound effect, with 50%inhibition occurring usually at about 0.5-3.0 ng/ml, whilelittle inhibition was noted after the shorter preincuba-tion, even at 1000 ng/ml. Similar dose-response curvesof inhibition have been noted in more than 10 differentexperiments with the cells of different donors. The timedependency of cholera enterotoxin activity is demon-strated more clearly in Fig. lb which shows two ex-periments in which cells were preincubated with 2 and0.2 ng/ml of cholera enterotoxin for 30, 60, and 90 minbefore the start of the reaction. Inhibition increased di-

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Cholera Toxin: Immediate and Delayed Hypersensitivity

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FIGURE 4 Inhibition of cholera enterotoxin-induced accumu-lation of cyclic AMP in leukocytes by specific antagonists.Leukocytes were incubated for 90 min in the presence ofcholera enterotoxin, 10 ng/ml, and increasing concentrationsof canine antiserum (*) or choleragenoid (U) before assayof cyclic AMP. The cells were exposed to the antagonistsimmediately before the addition of toxin. In this experimentleukocytes incubated in the absence of cholera enterotoxincontained 4.5 pmol cyclic AMP per 107 cells. Each valueshown is the mean of duplicate determinations, which dif-fered by no more than 10%.

In order to demonstrate the specificity of cholera en-terotoxin its effects were blocked by first treating the cellswith choleragenoid and with an antitoxin raised in dogs.In the experiments shown in Fig. 3a, cells were treatedwith 2.5 ng/ml of cholera enterotoxin together with vary-ing levels of the toxoid. After 60 min preincubation,antigen was added. The toxin without toxoid caused, inthese two cases shown, 80 and 84% inhibition; we haveplotted the percent inhibition of toxin activity by thetoxoid against the concentration of choleragenoid: 50%neutralization of toxin activity was noted at about 7 and15 ng/ml of toxoid, a three- and sixfold multiple (byweight) of the toxin level used. A similar experimentcarried out with the antitoxin is shown in Fig. 3b: 50%oinhibition was noted at a 1.5 and 3.0 X 10" dilution ofthe antiserum. The effects of choleragenoid and antitoxinon cyclic AMPaccumulation in human leukocytes stimu-lated by cholera enterotoxin are shown in Fig. 4: 50%inhibition of the cyclic AMPincrease is noted at about10 ng/ml of toxoid and at a 2-3 X 10" dilution of theantitoxin. Further studies with the choleragenoid (notshown) indicate that it is specific for cholera toxin inthe sense that it does not block the action of prostaglandinEl or of isoproterenol. Similarly, the toxin is not af-fected by the beta blocker propranolol which inhibits theactivity of isoproterenol.

Lymphocyte-mediated cytotoxicity. Lymphocyte-medi-ated cytolysis requires the specific interaction of twocell types: an effector lymphoid cell and an antigen-bearing target cell. The destruction of target cells (as

measured by "1Cr release) is a specific lytic event char-acterized kinetically by a linear destruction of target cellsresulting from interactions of a single target cell with asingle "sensitized" lymphocyte (a "one-hit destruction)(11). Compared with the in vitro release of histamine,the destruction of target cells is a relatively slow process,somewhere between 1 and 2 h being required beforespecific cytolysis is demonstrable even at a ratio of 100lymphocytes to each target cell. In spite of these vastkinetic differences, all of the cyclic AMPactive agentsstudied thus far are similar in the two systems (10).Isoproterenol, for example, has an effect which israpidly dissipated in in vitro cytolysis inasmuch as thecyclic AMP levels rise and fall rapidly (17). Cholera

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FIGURE 5 (a) Inhibition of specific lymphocyte-mediatedcytolysis of mastocytoma target cells by cholera entero-toxin. Target cell lysis is reflected by 'Cr release (seemethods). The lymphocytes were exposed to the noted con-centrations of toxin for 1, 2, or 3 h before the addition oftarget cells; inhibition of lysis was measured four hours laterand was calculated relative to uninhibited controls. (b) Ef-fect of cholera enterotoxin on the accumulation of cyclicAMP in spleen-cell suspensions. The cells were exposed tothe noted concentrations of toxin for 1 or 3 h before thecyclic AMPlevel was measured. All values are the mean ofduplicate determinations, differing by not more than 10%.

694 L. M. Lichtenstein, C. S. Henney, H. R. Bourne, and W. B. Greenough, III

I

TABLE I

Effect of Cholera Toxin on Lymphocyte-Mediated Cytolysis

Meanpercent

inhibitionof specific

Target cells Lymphocytes Inhibitor Cytolysis* cytolysist cAMP

% -i-SD pnol/10710 DBA/2 mastocytoma cells Normal C57BINone 8.8+1.5

Immune§ C57B1 None 63.2 +7.1 6.9

Toxinll 16.8+2.2 85.3 80.0Toxoid¶ 64.1+4.2 0 9.2

1oxin + toxoid 49.6+5.9 25.0 16.0Antitoxin** 62.5 +2.0 < 5 5.0

'1'oxin + antitoxin 62.0+3.1

We have previously shown that there is completeagreement between the effects of the catecholamine series(isoproterenol, epinephrine, norepinephrine, and phenyl-ephrine) as to their known beta agonist activity, theirinhibition of histamine release, and their stimulation ofadenyl cyclase. The beta-blocker propranolol blocks boththe inhibition of the allergic response and the cyclasestimulation. Similarly, in both hypersensitivity systems,the prostaglandin series, PGE1 and PGE2 and PGFla,shows complete concordance between the decreased im-munologic response and increased cyclase activity (4,10). Kinetic studies with these agents, presented in theaccompanying paper, are similarly in agreement (17).With cholera enterotoxin acting on human leukocytes noeffect on cyclic AMPis seen until about 30 min and theeffect rises linearly at 60 and 90 min. Inhibition of hista-mine release after incubation of these cells with the toxinfollows entirely similar kinetics. In cell-mediated spe-cific immune cytolysis there is a linear rise with timein the cyclic AMPlevel and the inhibition of target celllysis. These time course correlations add another dimen-sion to the postulated causal association between in-creased cyclic AMPlevels and in vitro manifestations ofboth immediate and delayed hypersensitivity.

The specificity of the response to cholera enterotoxin isamply demonstrated by the ability of the antitoxin andcholeragenoid preparations to block the response. Theaction of the antitoxin is clear; it would appear from thiswork (seen also the following paper) that the toxoidcompetes with the toxin for cell receptor sites and therebyblocks its ability to stimulate cyclase.

It is perhaps curious that these two reactions shouldhave such identical inhibition profiles. Wefeel this simi-larity is merely a reflection of the fact that both reactionsare basically secretory in nature. This argument has beenwell-aired with respect to the IgE-mediated release ofhistamine (3): a similar conclusion with respect to thecell-mediated response appears to be tenable, based onthe similarity in inhibition profiles. Further study is re-quired to support this concept. Also to be establishedare the substance or substances "secreted" when sensi-tized lymphocytes are triggered by contact with homolo-gous target cells, and how these act to kill the targetcell. Wedo not doubt, however, that substantial differ-ences in the mechanisms of the two reactions will cometo light as they are further studied.

Finally, there is the question of how the cholera en-terotoxin stimulates adenyl cyclase and why so muchtime is required. Experimental approaches to this prob-lem are considered in the following report (17).

ACKNOWLEDGMENTSThis work was supported by grants A17290, A18290,HL09964, and A110280 from the National Institutes ofHealth.

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696 L. M. Lichtenstein, C. S. Henney, H. R. Bourne, and W. B. Greenough, III

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