Regulation of human basophil activation

I. Dissociation of cationic dye binding release in activated human basophils

from histamine

Francis Beauvais, MD, B&atrice Bidet, MD, B6atrice Descours, Corinne HUblot, Claude Burtin, PhD, and Jacques Benveniste, Clamart, France

MD

Human basophil activation was demonstrared by histamine release (HR) and by the decrease oj rhe toluidine blue-positive basophils (TB +). In four experimenral systems, TB + number decreased in the absence of HR II) in basophils from atopic subjects stimulated by allergen concentrarions below the threshold for HR, (21 in basophils sensitized by anti-2.4-dinitrophenyl IgE stimulated by noncovalenrly linked 2.4-dinitrobenzene sulfonic acid-human serum albumin (also. rhe threshold for decrease of TB + required lower concentrations of sensitizing anti-2 ,I-dinitrophenyl IgE than for HR), (3) in low Ca ’ * medium, and (4) in the presence of the Na +IH’ exchanger, monensin. These results suggest that (I) there is a lower threshold for TB * decrease than for HR in allergen concentration, number of membrane IgE molecules, and number of IgE cross-linkings; moreover. external Ca requirement is lower for decrease qf TB’ than for HR and (2) TB- decrease rejlecrs either granule exocytosis or, in the absence of HR. biochemical changes (mosr probably cation exchanges) altering the inreracrion of rhe basic dye with the granules. Thus, moniroring decrease in TB- allows delecrion of basophil activation in rhe absence of HR. (J ALLERGY CLIN IMMJNOL 1991;87:1020-8.)

Since the basophil polymorphonuclear leukocyte is involved in immediate hypersensitivity phenomena, this cell has been extensively studied in vitro as an effector cell in human allergic diseases. ’ The basophil contains granules, the matrix of which is mostly composed of a glycoprotein complex rich in a heparinlike compound.’ This highly sulfated substance exhibits many electronegative charges and is responsible for the metachromatic properties of the basophil granules that become red stained in the presence of blue basic dyes, such as toluidine blue or azur A.’ The granules also contain histamine that is a specific marker of the human basophil granules. Cross-linking of adjacent membrane IgE by specific allergen or antiIgE Ab leads to cell activation and finally to the exo-

From INSERM U 200, Universitk Paris-Sud. Clamart, France. Received for publication April 24. 1990. Revised Nov. 19, 1990. Accepted for publication Nov. 29, 1990. Reprint requests: F. Fkauvais, MD, INSERM U 200. 32 me des Carnets, 92140 Clamart, France. 1/1/2m14

1020

Abbreviations used

DNB: DNP: EDTA: EGTA: TB +: Ab: HR: HSA: Ag:

2,4-Dinitrobenzene sulfonic acid 2,CDinitrophenyl Ethylenediarninetetcetic acid Ethylene glycol-bis-(y-aminoethyl ether) N,N,N’,N’-tetraacetic acid Toluidine blue-positive basophil(s) Antibody Histamine release Human serum albumin Antigen

cytosis of the granule, also named “degranuiation.” The main difficulty encountered in the study of human basophils is their low frequency among the other blood leukocytes (< 1%). Two methods have mainly been described to monitor human basophil degranulation after anti-IgE or allergen-induced cell stimulation: (1) the measure of histamine released in the supematanrl and (2) the count before and after stimulation of the TB-, since degranulated basophils lose their affinity

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Human basophil

for toluidine blue.’ This second method was demonstrated to he correlated with other in vivo and in vitro procedures for the diagnosis of allergy6-lo and with HR when the cells were stimulated with common aeroallergens or ionophore A23 187.” In this article, we describe four experimental conditions in which decrease of TB+ basophils is obtained without concomitant HR. MATERIAL Reagents

AND METHODS

Goat antihuman IgE Fc-specific antiserum containing 1 mg/ml of Ab (Nordic Immunological Laboratories, Tilburg, The Netherlands), toluidine blue 0 for microscopy (Merck FrosstLaboratories, Darmstadt,Germany), EDTANa, and Nh, EGTA, N-2-hydroxyethylpiperazine-N’-2etbanesulfonic acid, monensin, ouabain (Sigma Chemical Co., St. Louis, MO.), allergen extracts, Dermatophagoides pteronyssinus, D. farinae, mixed grasspollens, cat danders, dog danders (Laboratoire des Stallergenes, Joinville-lePont, France), andDNB (EastmanKodak, Rochester,N.Y.) were purchasedas indicated. The anti-DNP IgE (1 mg of Ab per milliliter) was purified from the mouse ascites as described.‘* DNP covalently bound to HSA was prepared exactly as previously described.I3 Isotonic

(1 - BTIBC) x 100

solution

The solution for specific metachromaticstaining of basophils contained, per 100 ml, 100 mg of toluidine blue and 280 p.1of acetic acid in 25% ethanol, pH 3.2 to 3.4.5 It was filtered after preparation and kept at room temperature. Human basophil

1021

aliquot of lyrode’s solution. The leukocyte suspensions (1 x lo5 to 2.5 x 10’ leukocytes per cubic millimeter) contained 60% to 70% polymorphonuclear cells and 30% to 40% mononuclearcells. Since platelets contaminatedthe suspension (1 to 2 x 1041mm”), we verified that human platelets did not contain histamine at a detectablelevel with the automated spectrofluorimetric assay described below. Ten microliters of the cell suspensionwas layered on the bottom of a 96-well microtiter plate that contained 10 pl of Ca++ (5 mmol/L, final concentration) and 10 pl of the stimulus (anti-IgE, allergen, and DNB or DNP-HSA) at indicated concentrations(both in Tyrode’s solution). Ca++ was added to the control well in the absenceof anti-IgE antiserum. The plates were covered with adhesive tape to limit the evaporation and then incubated at 37” C for 30 minutes. The toluidine blue-containing staining solution (90 ~1) was added to each well, and the suspensionwas gently but thoroughly mixed. Specifically, red-stainedbasophils were counted under a microscope with a hemocytometer. At the X 250 magnification, the granules were not observed individually, and the whole cell appearedred; <5% of the stainedcells were ambiguous(partially or lightly stained, damagedcells). The scarcity of in-between states in our preparationsagreeswell with other studiesindicating that basophil responseis an all-or-none process.” The percentagesof TB + basophil decreasewere calculated according to the following formula:

solutions

The standardisotonic solution was a modified ‘Qrode’s solution containing (millimolars) NaCI, 140; KCl, 2.6; N2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 10; EDTA-N&,, 2.5; glucose, 5.5; heparin, 5 IU/ml (Choay, Paris, France), pH 7.4. When it was indicated, EDTA was omitted. CaCI, from stock solution (0.2 mol/L) was diluted at appropriate concentrationsin Tyrode’s solution. To dissociatebasophil-boundIgE before passivesensitization, we used a solution containing (millimolars) NaCl, 130; KCI, 5; and lactic acid, 10, pH 3.9.14 Staining

activation

stimulation

and staining5

Venousblood from healthy and allergic donors was collected with anticoagulant (heparin, 1 IU/ml, and EDTAN&/EDTA-Na,, 2.5 mmol/L, final concentrations). Basophil numbersin whole blood rangedfrom 20 to 5O/mm’. Because this number was too low for proper counts on hemocytometer,blood sampleswere allowed to sediment with dextran for 30 to 45 minutes. The leukocyte-rich layer was recovered, twice washed in Tyrode’s solution by centrifugation (200 g for 10 minutes) and resuspendedin an

where BC is the number of basophils in the control well (from 40 to 150) and BT the number in the test well. In the casesin which TB’ basophil decreasewas observedwithout HR (seeRESULTS), we repeatedlycheckedthat basophils were indeed present in the cell suspension and not, for example, adherenton plastic walls. Sameamountsof total histamine were measuredin aliquots from test- or controlcell suspensions. Leukocyte

HR

After the incubation step, 230 pl of Tyrode’s solution containing EDTA was added in each well of the microtiter plate that was centrifuged at 750 g for 10 minutes. The supematants(170 p.1)were addedto equal volume of HClO,, 0.8 N. The releasein microtiter plates was usedfor the first experimentsaimed to comparethreshold and optimal stimulus concentration for TB’ decreaseand HR. Otherwise, HR experiments were performed in plastic tubes. Leukocytes were suspendedat 30 to 40 basophils per cubic millimeter in Tyrode’s solution. The leukocyte-rich cell suspensionswere then aliquoted (400 pl) in plastic tubes containing 50 pl of anti-IgE antiserum, or specific allergen at defined concentrations (or buffer alone as control) and 50 u.1of CaCI, (2 mmol/L, final concentration), or buffer alone. After incubation in a water bath (37” C for 30 minutes), the tubes were centrifuged (700 g for 20 minutes), and 300 pl of each supematantwas added to 300 ~1 of HClO,, 0.8 N. Total histamine content was assessedby

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TABLE I. Basophil numbers after exposition

to coded anti-lgE Experiment

Anti-IgE

1

()rg/ml)

0 0 0.1 I

10

129.0 117.3 113.0 52.3 82.3

k _’ !r + ?r

No.

_--.. .-.-

2

2.6* 10.9 14.7 3.8 0.6

141.6 129.0 30.3 19.3 36.0

3

-t 13.6 +

10.8

+ 7.6 2 4.9 !. 2.0

40.3 36.3 26.0 9.3 43.7

Data representthe means -t I SD of triplicates of numbersof actually counted basophils. *Tubes containing anti-IgE at indicated concentrations or Tyrode’s solution alone as control observer and then assayedfor TB * basophil decrease

adding HCIO,, 0.8 N. to an equal volume of uncentrifuged cell suspension. After centrifugation, the HCIO, extracts were kept at 4” C until histamine determination. Spontaneous release was < 10% of the total histamine content and was subtractedfrom all values to elicit net percent of release. The supematantswere analyzed for histamine with an automated spectrofluorometric assay. “. ”

Passive sensitiiation

of basophils

The sensitization was performed as previously described.” Briefly, lactic acid-treated (5 minutes, 4” C) leukocyte suspensions” were incubated at 37” C with 20 pg/ml of monoclonal anti-DNP IgE for 2 hours. In some experiments, lactic-acid treatment was omitted and led to the same results. After centrifugation and washes, the leukocytes were stimulated as described above, and HR and numbers of TB A were assessed.

Statistical

CLIN. IMMUNOL WAY 199:

studies

The Willcoxon’s test for paired variates was used for the comparison of threshold and optimal concentrations for HR and TB’ basophil decrease. However, in a first step, we selected experiments positive for both HR and decrease of TB’ basophils. We thus had to decide for each individual test if the TB ’ basophil decrease or the HR in the presence of stimulus was significantly different from that of the controls. For HR. a 10% threshold was determined as being significantly greater than background after examining these data. The 30% threshold for TB’ decrease was chosen as previously determined.“. ” According to the Poisson’s law, a 30% difference was statistically significant (p < 0.05) if >30 basophils were counted in the controls. Moreover, we required two consecutive points statistically different from the control lo declare a test positive (p < 0.0025). Finally, our experience for this test indicates that TB’ basophil decrease with serial allergen or anti-IgE concentrations fits into a regular dose-response curve, generally a bell-shaped curve, and not scatteredpoints. For the other experiments. the Student’s r test for paired variates was used. The statistic tests were done with Statgraphics software (STSC Inc., Rockville, Md.).

k t -c i t

4

4.0 7.4 2.6 2.5 4.7

were randomly

34.3 37.5 14.3 s.0 28.6

*. 1: * +

11.3 .i.s 3.x IO

t: 6.1 _-

coded by an independent

RESULTS Counting of TB+ Since the interpretation of the results was highly dependent on the counts of basophils stained with the

toluidine blue method, we performed blind experiments. The triplicates of defined anti-IgE concentrations (from 0.1 to 10 pg/ml) or diluting buffer were randomly coded by an independent technician. The leukocytes were incubated (30 minutes at 37” C) in the presence of the different dilutions and. after toluidine blue staining, the TB - basophils were counted by another technician. The results in Table I of four independent experiments indicate that these data obtained with this method were sufficiently reproducible and accurate to allow additional studies. The multiple regression analysis indicated a correlation coefficient. r = 0.98, between the triplicates. Attergen concemtration for optimal threshold HR and TB+ decreese

and

Blood was obtained from normal subjects (N = 12) or from patients allergic to common allergens (D. pteronyssinus, N = 23; D. farinae, N = 3: cat dander, N = 12; dog dander, N = 3; and grass pollen, N = 14). We selected the samples exhibiting leukocyte HR > 10% and TB + decrease >30% after specific allergen or anti-IgE stimulation and compared threshold and optimal concentrations for HR and TB’ decrease (for threshold determination, see METHODS). Optimal concentration was defined as the concentration eliciting the maximal effect. With IgE-dependent stimuli, a bell-shaped curve was generally obtained, and thus, optimal concentration was the concentration eliciting the peak of HR and/or of TB + decrease. When the maximal concentration was obtained with the upper concentration (100 pg/ml), then this concentration was plotted. Since no significant differences either in optimal or in threshold con-

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Human basophil

87 5

Optimal concentration 0.1

1

10

0.017

0.31

5.5

O.Ol&ml O.OOlj4g/ml

for histamine

0

*

activation

release 100 100

(0) w

00

0

0

g

Htt

00

l a.e 0

me

“08 --

Threshold concentration

for histamine

release

O.OlW/ml

0.1

1

10

100

(0)

O.OOlW/ml

0.017

0.31

5.5

100

(*I

FIG. 1. Optimal and threshold concentrations for human basophil HR and TB+ decrease. Human basophils from allergic and normal donors were incubated at 37” C for 30 minutes with defined concentrations of allergens or antiAgE, respectively; anti-lgE (0); allergens (e).

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J. ALLERGY

DNB or DNP-HSA

LLlN.

tMMUNClL MAY 199:

(pg/ml)

FIG. 2. HR and TB’ decrease in IgE-sensitized basophils induced by DNP covalently linked to HSA (DNP-HSA) or not (DNB). A, HR after incubation with hapten alone (0); hapten mixed with the carrier (HSA at 0.1%) immediately before addition to the cell suspension (0); covalently linked to HSA (0). B, Measure of TB’ decrease after incubation with hapten alone (0); hapten mixed with the carrier (HSA at 0.1%) immediately before addition to the cell suspension (0); covalently linked to HSA (0); after 60 minutes of incubation in EDTA (2.3 mmol:L) without Ca’ I addition and stimulated with hapten mixed with HSA (ii); presented as mean 2 SEM (n = 5).

centrations for HR and TB + decrease existed between the different allergens, the results were pooled. As illustrated in Fig. 1, the plots of optimal concentrations were not equally distributed along the left diagonal, thus indicating that optimal TB + decrease required significantly less antigen (or anti-IgE in normal donors) than optimal HR (Wilcoxon’s test, p < 0.001). Among the 67 tests, 22% had both maximal effect (for HR and TB - decrease) at the same concentrations, and 68% required about 18-fold to 5800fold less allergen or anti-IgE for optimal TB + decrease than for HR. We then plotted the minimal concentration necessary to observe HR >lO% and decrease of TB >30% (Fig. 1, borzom). TB + decrease could be obtained with lower concentrations than for HR (Wilcoxon’s test, p < 0.001). Among the 67 tests, the threshold was at the same concentration for both HR and TB’ decrease in 25% of the cases. In contrast, 66% exhibited TB + decrease at concentration in which HR was not yet obtained, and threshold for TB + decrease required from 18-fold to lOO,OOO-fold lower Ag concentration than for HR. The TB * decrease was not an unspecific protein effect since (1) nonrelated antiserums did not induce TB’ decrease (results not l

presented) and (2) specific allergens did not induce TB” decrease in leukocyte suspensions from nonallergic patients (results not presented). It should be noted that the choice of thresholds for HR (10%) and decrease of TB’ (30%) favored the HR. In spite of this finding, TB’ decrease appeared to be more sensitive than HR. IgE-se@tized

covalentiy

basepMl activation by hapten linked or mot to carrier

Anti-DNP IgE-sensitized basophils were incubated with defined concentrations of DNP covalently linked to HSA (DNP,-HSA) or with noncovalently linked DNB (defined DNB concentrations extemporaneously added to HSA, 0.1%). With the latter stimulus, only decrease of TB + was observed and could be blocked by 60-minute preincubation with 2.3 mmol/L ot EDTA (without external Ca’ ‘). By contrast. with covalently linked DNP-HSA, both HR and decrease of TB + were obtained (Fig. 2). DNB alone, in the absence of HSA, did not induce either TB + decrease or HR (Figs. 2 and 3). Addition of HSA (0.1%) after 30 minutes of incubation with DNB alone led to TB + decrease but not to HR. The decrease of TB + required IgE cross-linkings because,

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Human basophil

-DNB

+DNB

+DNB

FIG. 3. HSA plus DNB-induced TB+ decrease in anti-DNP IgE-sensitized leukocytes. Leukocyte suspensions sensitized (+/gE) or not (-IgEl by anti-DNP IgE were preincubated with 100 pglml of DNB (37” C for 30 minutes) or diluting buffer alone. After extensive washings, HSA (0.1%) or diluting buffer alone was added, and the incubation was continued for 30 minutes. The TB’ (Cl) were then counted, and histamine was measured in the supernatants (!Zl); presented as mean 2 SEM (n = 5).

in the absenceof anti-DNP IgE, albumin did not decreasethe number of TB + from DNB-pretreatedleukocytes. This effect occurred only if the basophils were previously sensitized with anti-DNP IgE (Fig. 3). Thus, no significant decreaseof TB+ ( - 2.6% + 4.2%; N = 3) was observed if basophils (without anti-DNP IgE sensitization and without acid lactic treatment)were incubatedsuccessivelywith DNB and HSA, thus indicating that only DNB (in the presence of HSA) interacting with specific anti-DNP IgE was able to activate the cell. Moreover, we checked that addition of an unrelated hapten (benzylpenicillenic acid) in the presenceor absenceof HSA was without effect on TB’ decrease(data not presented). Leukocyte suspensionswere sensitized with decreasing concentrations of anti-DNP IgE from 10 to 0.01 pg/ml. After cells were washed,they were challenged with DNP,-HSA (from 0.01 to 10 pg/ml) or diluting buffer alone and incubated at 37“ C for 30 minutes. Histamine was then measuredin the supernatantsand the TB + were counted after toluidine blue staining. Results in Fig. 4 indicate that, at optimal stimulus concentration, significant basophil activation could be detectedwith the toluidine blue methoddown to 0.1 kg/ml of anti-DNP IgE (p < 0.05; Student’s r test). Significant releaseof histamine was obtained

activation

1025

0.01

0.1

Anti-DNP

1

5

IgE (pg/ml)

FIG. 4. TB+ decrease and HR in leukocytes sensitized with decreasing concentrations of anti-DNP IgE. Leukocyte suspensions were sensitized with defined concentrations of anti-DNP IgE. After washes, the leukocytes were incubated with DNP-HSA at 0.01 to 10 pg/ml. TB+ decrease and HR were then measured. Results are presented as mean ? SEM (n = 4).

up to 3 pg/ml of anti-DNP IgE. Release was still observed, but not statistically significant, becauseof greatvariation, at 1 pg / ml in two of four experiments. At 0.1 pg/ml, HR was near zero. We noted a slight shift in optimal stimulus concentration from 0.1 pg/ml of DNP,-HSA with 10 pg/ml anti-DNP IgE toward 1 pg/ml of DNP,,-HSA with 0.1 pg/ml antiDNP IgE. Extracellular Ca+ + dependency anti-IgE-induced TB+ decrease and HR

of

Leukocyteswere incubatedwith anti-IgE antiserum ( 1 pg / ml) in the presenceof increasingconcentrations of Ca’ + added in the EDTA-free Tyrode’s solution. HR, as expected, increased with the extracellular Ca++ concentrations up to 10 mmol/L (Fig. 5) to plateau or even decreasethereafter (results not presented). In contrast, the TB+ decreasewas independent of the Ca’ + addedand reachedsimilar values at any concentration of Ca++ or even in its absence. Ca’ + (10 mmol/L) in the absenceof anti-IgE did not induce HR or TB+ decrease. When EDTA (5 mmol/L) was added 15 secondsbefore the incubation period to the cells in the “no Ca++” tubes, the TB’ decreasewas not modified (from 5 1.3% + 5.0% to

1026

Beauvais

et al.

CK

a.-cl

.i ALLERGY

100

CLlk

I

IMMUNOL. MAY 1991

1

4 -

^ n- 800 0 0

-WV

.

60-

I “0 Lb0 H M

O-4

i,/ T 0

0’ 1 1 0.1

0.2

Ca2+

I 0.5

added

1

2

5

10

(ml@

RG. 5. Effect of extracellular Ca” on anti-IgE-induced human TB’ decrease and HR. Human leukocytes were incubated at 37” C with anti-IgE (1 pglml) or medium alone in the presence of defined Ca’ + amounts added in the medium. After 30 minutes, the percentages of the TB’ decrease with 1 pg/ml anti-IgE (0) and the release of histamine (0) were measured. As control, the TB+ decrease (B) and the release of histamine (0) were measured in the absence of anti-IgE. In parallel, the decrease of TB+ was studied after preincubation with EDTA for 60 minutes at 20” C (A). Results are presented as means 2 SEM (n = 3).

53.7% + 6.1%; means ? SEM; n = 3). By contrast, the prolonged preincubation for 60 minutes with EDTA (2.3 mmol/L) at 20” C led to a strong inhibition of the decrease of TB‘ that was restored after the addition of Ca’ + , 5 mmol/L (Fig. 3). Closely similar results were obtained with EGTA (data not presented). Effect of monendn

on human

basophits

The sodium ionophore, monensin ( 10 pmol/L), was added to the leukocyte suspension at 37” C, and the basophil counts were performed at different time intervals. Decrease of TB’ was observed with a maximal effect at 1 minute, reaching up to -80% and then decreased with time (Fig. 6). To inhibit the Na’ / K +-adenosine triphosphatase, which could buffer the uptake of Na+, we added ouabain (50 Fmol/L) for 5 minutes before the incubation with monensin. In the presence of ouabain, monensin reached the same maximal values and remained at a plateau (Fig. 6). The TB + decrease induced by monensin was specific of extracellular Na’, since removal of Na+ from extracellular medium and replacement by the Na’ substitute, N-methylglucamine, abolished the effect. In the presence or absence of ouabain, no HR was detected after a 30-minute incubation with monensin .

MscussJm We observed that allergens or anti-IgE antiserum induced TB’ decrease at lower concentrations than for HR for both maximal and threshold effects. In the last case, this indicates that decrease of TB * induced by low allergen concentrations can occur without exocytosis. When basophils were passively sensitized with DNP-specific IgE, DNP covalently linked to HSA induced both HR and TB’ decrease, whereas hapten alone (DNB) was inefficient. Addition of noncovalently linked DNB followed by HSA led to TB + decrease but not to HR, whereas DNB alone induced no effect whatsoever. These data most probably indicate that decrease of TB + and HR both require IgE cross-linking. However, an antigen configuration able to induce a certain degree of cross-linking sufficient for TB + decrease was unable to trigger HR. Many studies pointed out the importance of Ca * * in HR from mast cells” or human basophils.” Indeed, in the absence of extracellular Ca’ + , &E-mediated HR was abolished, as expected, but not TB + decrease, even when EDTA was added to the medium 15 seconds before anti-IgE. The strong inhibition of the decrease of TB + after prolonged incubation with EDTA (or EGTA) and restoration with Ca’ + most probably indicates that intraceIlular Ca’ + is necessary for this phenomenon.

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Human basophil

A $

activation

1027

80

: -iti '

60

Lc 0 a4

+ P

40 20 0 0

10

20

Time (min)

lo I 1w 30 MON OUA MON 0: A

FIG. 6. Effect of monensin on human basophils. The leukocyte suspension was incubated in a water bath at 37” C with monensin at 10 pmol/L in the presence of 50 pmol/L of ouabain (e) or absence (0); ouabain alone (A) or diluting buffer alone (0). To check the specificity of the monensin effect, Na+ was replaced by N-methylglucamine (A). At the indicated times, aliquots of 10 )LI were harvested and immediately stained with the toluidine blue solution. After 30 minutes the tubes were centrifuged, and histamine was measured in the supernatants (means f SEM; n = 3).

Taken together, these data obtained with three different systems with IgE-stimulated basophils are in favor of the existence of two types of TB’ decrease (i.e., loss of staining in the presenceof TB): (1) classic TB + decreaseaccompanyingexocytosis and (2) TB + decreasewithout exocytosis. It remains puzzling that TB+ decreasecould be observed without release of histamine. Metachromaticcoloration by toluidine blue is believed to be characteristic of polyanions, especially of acidic proteoglycans.” The TB+ decrease could be interpreted as a neutralization of the electronegative charges of the matrix by cations, thus preventing the additional fixation of toluidine blue. Another possibility is that toluidine blue, asa weak base, accumulates in acidic compartments, such as granules, as observed with acridine orange.22Thus, decrease of pH gradient between granule and cytosol would inhibit metachromaticstaining. The monensin experimentsare a clear exampleof a situation in which loss of the metachromaticstaining occurs without HR in cells still bearing their granules,(andtheir histamine content), as demonstratedby the quick reappearance of the staining in the absenceof ouabain. This finding also strongly suggeststhat increase of cell Na+ concentration and/or alcalinization is able to inhibit toluidine blue fixation in the absenceof exocytosis. However, in IgE-mediated stimulation, the origin and

the nature of the cations capable of preventing toluidine blue accumulation in granulesremain to be studied. Along this line, we recently observed, with the patch-clamptechnique, the opening of cation channels and Na’ entry after stimulation of human basophils with anti-IgE, even in the absenceof extracellular Ca++ (manuscript submitted). Until the present time, HR was the main marker for basophil activation. In the sameexperimental conditions as in the present study (low Ag concentration or low Ca++ medium), Bochner et al .23demonstrated that IgE-mediated adhesivity of basophilsto endothelial cells via the CD18 Ag was still observed in the absence of HR. Indeed, events other than HR are important in the basophil pathophysiology, such as chemotaxisand adhesivity. Theseprocessescould occur with stimuli “weaker” than stimuli necessaryfor exocytosis, particularly in drug allergy in which HR is seldom found in vitro (often becauseof the lack of covalently bound Ags), whereasTB + decreaseis well correlatedwith clinical symptoms’,7.24or, perhaps,at a distance from the inflammation site, in which the Ag concentration is vanishing. In addition, we recently observed that IgG4-mediated basophil activation was detectedmore frequently with the toluidine blue method than with HR, particularly at low stimulus concentration.25

1028

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et al.

In conclusion, exocytosis appears to be a late event in human basophil activation corresponding to full cell activation. Measure of TB + decrease has a lower threshold of detection than histamine. It could represent a simple method to demonstrate not only full basophil stimulation (then associated with HR) but also weak stimulation related either to the cell status or experimental (and may be pathophysiologic) conditions. REFERENCES I. lshizaka T, De Bernard0 R, Tomioka H. Identification of basophil granulccyte as a site of allergic histamine release. J Jmmunol 1972;108:1C@O-8. 2. Rothenberg E, Cautield JP. Austen KF, et al. Biochemical and morphological characterization of basophilic leukocytes from two patients with myelogenous leukemia. J lmmunol 1987; 138:2616-25. 3. De Robertis E. Nowinski W. Saez F. Cell biology. 5th cd. New York: WB Sanders, chap 7, 1970. 4. Levy DA, Osler AC. Studies on the mechanisms of hypersensitivity phenomena. XIV. Passive sensitization in vitro of human leukocytes to ragweed-pollen antigen. J Jmmunol 1966: 97:203-I 1. 5. Benvenistc J. The human degranulation test as an in vitro method for the diagnosis of allergy. Clin Allergy 198l:l I: Ill 6. Yeung Laiwah AC. Pate1 KR, Seenan AK. Galloway E. McCulloch W. Evaluation of the commercially available Basokit as a test of immediate hypersensitivity in hay fever sufferers. Clin Allergy 1984;14:571-9. 7. Camussi G, Tetta C, Coda R, Benveniste J. Release of plateletactivating factor (PAF) in human pathology. I. Evidence for the occurrence of basophil degranulation and PAF release in SLE. Lab Invest 1981;44:241-5 I. 8. Aoki H, Vellieux P, Theobald-Segalen C. et al. Clinical evaluation of the human basophil degranulation test in grass pollen allergic patients. Ann Allergy 1986;57:319-20. 9. Camussi G. Tetta C. Benveniste J. Detection of basophil sensitization by IgE antibodies to nuclear antigens in connective tissue diseases. Int Arch Allergy Appl lmmunol 1982;69:35862. 10. Pirotzky E. Hieblot C, Benveniste 1. Laurent J. Noirot C. Lagrue G. Basophil sensitisation in idiopathic nephrotic syndrome. Lancet 1982;1:358-61.

J. ALLERGY

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IMMCINOL MAY 1991

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