Density heterogeneity of human lung mast cells

Density heterogeneity mast cells Edward S. Schulman, Robert J. Vigderman,

of human lung

MD, Thomas J. Post, BS, and Pa. BA Philadelphia,

Suspensions of enzymatically dispersed human lung parenchymal mast cells were fractionated according to density by flotation through discontinuous Percoll gradients and examined for their responsiveness to release stimulants and pharmacologic agonists. Mast cells localized to all six density fractions (I-VI) examined; densities varied from specijic gravities of 1.053 gmlml to 1.123 gmlml. Most (67%) lung mast cells localized to fractions III and IV, corresponding to spec$c gravities of I .077 to 1.088 gmlml, respectively. Histamine content increased with density from 2.7 ? 0.3 pg per cell in fraction I to 4.8 -C 0.7 pg per cell in fraction VI (mean 2 SEM; n = 19). Fraction Ill was least responsive to high concentrations of anti-IgE than to any other fractions and, along with fraction IV, the most responsive to ionophore A23187. All fractions released the arachidonate mediators prostaglandin D, and leukotriene C, in response to anti-lgE. In four of eight lungs tested, formyl methionine peptide (iO-” to IO-’ mollL) weakly elicited histamine release (3% to 6%) in fractions I and II cells. Compound 48180 (0.1 to 10 pglml; n = 3) failed to induce histamine release in any fractions. The cyclic adenosine monophosphate-active drugs, isoproterenol (IO-’ mollL), dibutylyl cyclic adenosine monophosphate (3 mmollL), and isobutylmethylxanthine (3 X 10e4 mollL) inhibited anti-IgE-induced histamine release from all fractions equivalently. Dimaprit (3 x lo-’ mollL) and cromolyn sodium (IO-’ -3 x 10-j mollL) failed to signi$cantly inhibit any fraction. Nordihydroguaiarectic acid (3 X IOes mollL; n = 6) markedly inhibited histamine release from fractions III to VI but had no signiJicant effects on fractions I and II mast cells. We conclude that mast cells from human lung are morphologically heterogeneous with respect to density. These density subsets differ functionally in their responsiveness to secretagogues and to nordihydroguaiaretic acid. (J ALLERGY CLIN IMMUNOL I988;82:78-86.)

HLMCs and their mediators play a central role in asthma and other lung hypersensitivity disorders.” 2 Despiteimportant clinical implications, little is known of the morphologic and functional differencesthat exist amonghuman mastcells. In the rodent, two distinct mastcell phenotypeshavebeendefined. The first type, the mucosal mast cell, was originally describedin the rat gut but is nearly identical to T cell factor-dependent hematopoieticcells recently grown in culture.3-‘5Connective tissue mast cells constitute the second type and are most easily studied with the mouse or rat peritoneal mast ce11.4-8 Describeddifferencesbetween From the Division of PulmonaryDisease,Departmentof Medicine, JeffersonMedical College of ThomasJeffersonUniversity, Philadelphia, Pa. Supportedby National Institutes of Health Grant AI-20634. Received for publication July 13, 1987. Accepted for publication Jan. 20, 1988. Reprint requests:Edward S. Schulman,MD, Division of Pulmonary Medicine, HahnemannUniversity Hospital, Broad andVine Sts., Philadelphia, PA 19102.

Abbreviations used

HLMC: PGD,: IBMX: LTC,: NDGA: CAMP: ANOVA: f-met:

Human lung mast cell ProstaglandinD, Isobutylmethylxanthine Leukotriene C, Nordihydroguaiaretic acid Cyclic adenosinemonophosphate Analysis of variance N-formyl-L-methioninyl-L-leucyl-Lphenylalanine PA: Piperazine-N,N’-b&(2-ethanesulfonic acid), 7.6; NACl, 6.4; KCl, 0.37; NaOH lON, 4.2 ml/L; and human serum albumin, 0.03 PAGCM: PA; CaCl, . 2H,O, 0.14 gm/L (1 mmol/L); MgCI, * 6H,O, 0.2 gm/L (1 mmol/L); glucose, 1.0 gm/L TGMD: Tyrode’s buffer with magnesium (0.25 gm/L); gelatin (1 gm/L); deoxyribonuclease(10 mglL) added

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the two rodent mast cell types include those of morphology (diameter and density),‘, 4. I5 capacity for granules to stain metachromatically after formalin fixation,j. 4 stimulants effective in causing cell activation.‘. ’ ” mediators generated,“, ” nature of proteoglycan,” and pharmacologic responsiveness.7. ’ In humans, at the lung tissue level, we previously observed differences in mast cell responsiveness between passively sensitized and antigen-challenged airway versus parenchymal fragments.‘“, ” At comparable antigen concentrations, parenchymal mast cells released significantly greater histamine and PGD, than airway mast cells. More recently, our studies have focused on mast cell heterogeneity at the cellular level. Subpopulations of enzymatically dispersed HLMC, fractionated by elutriation, were found to vary in diameter from <8 km to > 18 Frn.‘* As previously described in the rodent,3. ‘. a, ” these diameter differences were found to correlate with histamine content and responsiveness to secretory stimulants, as assessed by the release of histamine and PGD,. No significant differences among diameter-separated subpopulations were found in their responsiveness to CAMP-active drugs. In We first discovered marked differences in HLMC densities while Percoll step gradients were being used in our purification procedure. I9 In the mouse mast cell, density serves as a critical marker for differentiation, maturation. and function. 15.‘a In this article, we explore the morphologic and functional differences among human mast cells separated according to density. METHODS Reagents Isoproterenol, IBMX, NDGA, dibutyryl CAMP, cromolyn sodium, chymopapain, elastase type 1, compound 48 180, and f-met-peptide were purchased from Sigma Chemical Co.. St. Louis, MO.; other reagents used include cullagenase (Worthington, Freehold, N.J.), deoxyribonuclease. pronase, calcium ionophore (Calbiochem, San Diego, Calif.), and gelatin (Difco Laboratories, Detroit, Mich.). Dimaprit and rabbit antihuman IgE antibody were provided by M. J. Bell, Smith Kline & French Research. Ltd., Hertfordshire, England, and Dr. Kimishige Ishizaka, Johns Hopkins University, Baltimore, Md., respectively. The antrsera for the PGD, and LTC, radioimmunoassays were provided by Dr. L. Levine (Brandeis University, Waltham. Mass.) and Dr. Anthony Ford-Hutchinson (MerckFrosst Inc.. Montreal, Canada), respectively.

Buffers Four different buffers were used in the study: Tyrode’s buffer. which contains (grams per liter) NaCl, 8.0; KCI.

Density heterogeneity

of lung; (.:a~! cells

79

0.2; NaH?PO,. 0.05; and glucose. 1.0: thy !uffer was titrated to pH 7.2 by the addition of Na&‘G. TGMD, PA. and PAGCM. For overnight cuiture, m;~sr ccl]\ wcrc suspended in RPM1 1640 containing 15 nl~nrnl~l. of iv-:hydroxyethylpiperazine-!V’-2-cthanesulfonrc .tL*rtl. pcntamrtin (.Microbiological Associates. Waikcr
Preparation

of lung suspensions

The methods for dispersing lung mtc if smgle coil subpension have been described in detail.” ‘. Br&Iy, grossly normal lung tissue was dissected from tumor jr) specimens usually removed for bronchogenic carcinoma. ‘The tissue was finely minced and extensively washed m divulent cation-free Tyrode’s buffer. Fragment\ were then twice incubated with a mixture of pronase (2 mg/mlj and chymopapin (0.5 mgiml). After harvesting of freed c-ells, thr residual lung fragments underwent two further digestions with collagenase (1 mgiml) and clastase (10 I!‘rnl).” All recovered cells were washed thoroughly in TGMD and then cultured overnight in RPM1 1640 medium in 100~mm tissue culture plates (Falcon Labware. Oxnard. Calrl’. j at room temperature. The following morning. nonadhercnt cells were harvested from the plates, combined.. and sedimented at 150 g for 8 minutes. it was previously demonstrated that under these conditions. mast cell recovery is c’:rmplete after overnight culture. ‘” To enrich our preparations m mast cells before the density gradient step, irrelevant lung cells were depleted by counter-current centrifugation elutriation As we previously demonstrated. elutriation at !ow buffer flows (I 1.O to 15.5 mllmin) depletes red blood zcli+ and ?>60% of nucleated cells while >97% of mast cells ZI: being prcserved.‘X Therefore. lung cells eluting at flower ot r !5.5 mlimin were combined for density-gradient i’:&ronation. Mast cell and total ccl1 counts were performed w!h dir aician blue stain.“

Density

gradient

Lung cells were fractionated by flotation through discontinuous Percoll (Pharmacia Fine Chemicals, Piscataway. N.J. j layers as previously described.‘” ” Approximately 2 x IO’ nucleated lung cells were suspended 111 1 .if ml of a 100% Percoll solution (nine parts concentrated PercoIl to one part tenfold-concentrated Hanks’ balanced salt solution. neutral pH) in a 12 X 75mm polystyrene tube. The JO@% Percoll cell suspension was then overlayered wjth 0.8 ml aliquots of 80%, 70%. 60%. 50%. and 40%’ PercoIl, made by diluting the 100% Percoll with 1 x Hank:%‘ balanced salt solution. Densities were designated by sp p. grams per milliliter, as measured with a hydrometer. After ccntrifugation at 400 g for 12 minutes, cells at each interface were collected and washed twice in TGMD buffer. Cells collected from the 40% Percoll interface were designated traction J (sp g, 1.053 gmiml); 40% to 500/c, fraction I1 L! .(I63 gm/ml); 50% to 60%, fraction 111(1.077 gmimlr: 60% to 70%. fraction IV (1.088 gm/ml); 70% to 8O%, f?actioR \

J. ALLERGY

80 Schulman et al.

TABLE I. Density distribution Fraction

of HLMCs (n = 19) % Of total HLMC

Density (gmlml)

No.

1.053 1.063 1.077 1.088 1.100 1.123

I

II III IV V VI

4.4 14.5 37.8 28.8

-c -c f 2

0.8 2.4 3.4 2.5

11.1 2 1.7 3.3 -+ 0.9

of anti-IgE-induced histamine release from HLMC density fractions Fraction

r

a

SEM

b

SEM

I

0.993 0.999 0.998 0.956 0.994 0.983

0.732” 0.675” 0.588 0.716* 0.920t 0.945t

0.053 0.019 0.027 0.109 0.034 0.049

0.585$ 0.569$ 0.5404 0.453 0.3948 0.3428

0.039 0.014 0.020

IV V VI

Histamine content (pglMC1

2.7 3.0 3.2 4.2 4.4 4.8

Arachidonate

0.081 0.025 0.037

r = Correlation coefficient for linear regression analysis; a = intercept;b = slopeof log-logregressionof net histamine releaseversusan&IgE concentration (microgrampermilliliter). *, t, $, 5 Fractionsnotatedwith similar symbolsarestatistically indistinguishablewith respectto slopeor intercept. (1.100 gm/ml); and 80% to 1008, fraction VI (1.123 gm/ml).

Histamine-release

-c 2 -c -c 5 _’

0.3 0.4 0.3 0.4 0.4 0.7

% HLMC purity

4.7 9.8 28.5 53.2 58.9 46.9

r 1.1 r 2.6 t 5.0 t 5.5 r 6.2 _c 7.1

Corp. (T-town, N.Y.); the assay is sensitive to 500 pg/ml of histamine. Histamine releasewas expressedas the net histamine releasedinto the supematantdivided by the total histamine content X 100%.

TABLE II. Comparison

II III

CLIN. IMMUNOL. JULY 1988

assay

Duplicate aliquots of 10 to 20 x 10’ mast cells from each density fraction were challenged with release stimulants for 20 minutes at 37” C. All reactions were performed in 96-well microtiter plates in a final volume of 0.2 ml of PAGCM. Microtiter plates were incubated at 37” C in an atmosphereof 95% air and 5% C02. Release stimulants were prewarmed to 37” C before their addition. During challenges, microtiter plates sat on a 37“ C slide warmer. After challenges, plates were immediately returned to the incubator. At the end of incubation, plates were removed and spun at 400 g for 5 minutes, and supematantswere removed for determination of histamine release. Spontaneousreleaseof histaminewas assessedby addition of buffer instead of stimulants and was always <5% of cellular histamine. The total histamine content of mast cells was determined by lysis in 2% perchloric acid, In pharmacologic studies, agonistswere added 15 minutes before addition of submaximal anti-IgE concentrations (as predeterminedon anti-IgE dose-responsecurves of each lung mast cell preparation). Histamine measurementswere performed by the automatedfluorometric method of Technicon Instruments

radioimmunoassays

Radioimmunoassaysfor PGD, and LTC4, the principal cyclooxygenaseand lipoxygenaseproducts, respectively, of HLMC,24-*6were performedon unextractedsupematants,as previously described.16.Z4The PGD, and LTC, assaysare sensitive to 0.1 rig/ml.

Statistical methods Data were expressedas the mean 2 SEM. In experiments designed to detect possible differences in immunologic responsivenessamong density populations, data from anti-IgE dose-responsestudies were log-log transformed, and the resultant lines were analyzed with a two-way ANOVA, with dose of anti-IgE and density fraction as the two variables examined. A log-log transformation was used becauseit resulted in the highest correlation coefficients between anti-IgE dose and response.Points of the anti-IgE dose-responsecurve used for the transformation were of suboptimal or optimal, but not supraoptimal, release. Significant effects of both dose and cell type were noted. This permitted a regression analysis of dose for each cell type. The resultant regressions,representingthe different density fractions, were statistically compared with the NeumanKeuls’ method used for testing for differences between the slopes and intercepts. The fewer studies performed with ionophore precluded a detailed analysis with linear repssion. Instead, a least-significant difference test was performed.” In pharmacologic studies, significance of preferential effects of drugs on individual density fractions was analyzedwith a one-way repetitive ANOVA and a NewmanKeuls’ test. Significance in all testswas taken atp < 0.05.2’

RESULTS Mast cell-density distribution and histamine content In 19 individual human lungs examined, mast cells were found at every density tested, from 1.053 to 1.123 gm/ ml (Table I), with most mast cells localizing

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Density heterogeneity

of rung :nzsr cells

81

Anti-IgE (I~~/ITI)

exdl

-

L Fractmn

I

cer’%ty

1053

n

1.063

m

E

P

m

1.077

l.oea

1.m

I.123

“imwe

F-atoll Fratm r)em,ty

__I __..

I !.053

n / 063

m

Ip

P

pI

1077

r088

/ 100

! 123

FIG. 1. A, Anti-IgE-induced histamine release from HLMCsof eight individual lungs fractionated by discontinuous Percoll step gradients. Fractions of increasing density are numbered I to VI. Density is expressed as gram per milliliter. Maximum release for all fractions was attained at 8 to 18 Pg/mJ of anti-IgE. Brackets illustrate the mean f SEM. B. lonophore A231Wincluced histamine release from HLMCs of three individual human lungs.

in the density rangesof 1.063 to 1.088 gmlml. Fraction III, representinga sp g of 1.077 gm/ ml, retained the highest percentageof HLMC (37.8 + 3.4%). The content of histamine per mast cell increasedas a function of density, 2.7 k 0.3 pg per mastcell in fraction I to 4.8 + 0.7 pg per mast cell in fraction VI cells. As indicated by their lower purities, low-density mast cells of fractions I and II tended to localize with most human lung cell contaminants. Mast cells of highest purities were found in the density range of 1.088 to 1.100 gm/ml, as previously reported.” Wi~mine

&ease from density fractions

Anti-ZgE. The percent of total cellular histamine releasedin responseto rabbit antihuman IgE challenge for the six density fractions is illustrated in Fig. 1, A (n = eight lungs). Although it is not presented,spontaneous histamine release for all fractions in all experiments ranged from 1% to 5%. Maximum histamine release from each fraction was obtained at a concentration of 18 p,g/ml for this antihuman IgE preparation. At the two highest anti-IgE concentrations tested, fraction III containedthe least responsive mast cells. To better define potential differences in the immunologic responsivenessamong fractions, a detailed analysisof anti-IgE dose-responsecurves was undertaken with a linear regression analysis. The re-

sultant lines allowed a comparison of both the slopes

and interceptsof all fractions (Table II). This analysis demonstratedthat, with respectto both slope and intercept, fraction I was indistinguishable from fraction II, and fraction V was indistinguishable from fraction VI. However, fractions I and II were significantly different in both respects(p < 0.05) from fractions V and VI, suggesting two functionally distinct populations. Although fraction III had the same slope as fractions I and II, the intercept was significantly (p < 0.05) lower. The slope of fraction IV was significantly different (p < 0.05) from all other fractions, being intermediate between fractions I and II and V and VI; the intercept, however, was indistinguishable from fractions I and II. By this analysis, therefore, fractions III and IV were functionally intermediate between the extreme densities. In three experiments, we examined the capacity of density-separated fractions to release the nonpreformed arachidonate products, PGD2 and LTCI, in responseto anti-IgE. All fractions generatedboth mediators, andwe could not identify exclusive generation of an arachidonateproduct by any fraction. For example, anti-IgE challenge of the lowest density mast cells (fraction I) generated36.2 + 25.6 ngi106 mast cells of LTC, and 206.1 2 44.7 ngi 10’ mast cells of PGD,; high-density fraction V mast cells generated 34.1 t 13.5 ng/ lo6 mast cells and 146.2 +- 16.7 ng/ lo6 mast cells of these mediators. respectively.

82

Schulman

J. ALLERGY CLIN. IMMUNOL.

et al.

(n=4)

FIG. 2. The effects of isoproterenol, dibutyryl CAMP, and IBMX on submaximal anti-IgE-induced histamine release from mast cells of different densities. Control anti-IgE-induced histamine release is illustrated on the far /efL Percent inhibition of control release resulting from drug preincubations is illustrated on the right ordinate. Density is expressed as gram per milliliter. The results are the mean 2 SEM for four individual human lung experiments.

ZonophoreA23187. In contrastto anti-IgE-induced release, where fraction III was least responsive, this fraction, along with fraction IV, was significantly more responsive (p < 0.05; least-significant difference test, 27) to ionophore than any other fractions of higher or lower density (Fig. 1, B); this heightened responsivenessis most pronounced at the lowest ionophore concentrations. Similar to our findings with anti-IgE, ionophore failed to elicit selectivegeneration of arachidonate products from any density fraction (data not presented). F-met peptide and 48180. F-met peptide and compound 48/80, activators of the human basophi128 and rodent connective tissue mast ce1L5,29respectively, were examined for their selectivity in triggering histamine release from different density mast cells. In four of eight lungs tested, f-met peptide ( 10m4to 10m6 mol/L) induced 3% to 6% histamine releasein fractions I or II. Otherwise, this stimulus failed to elicit responses. In three experiments, compound 48/80

failed to induce histamine release in any density fraction. Pharmacologic agonists. The effects of isoproterenol, dibutyryl CAMP, and the phosphodiesteraseinhibitor, IBMX, all drugs that increase intracellular CAMP, were examined for selectivity of inhibitory effects on density fractions from four lungs (Fig.. 2). Drugs were usedat predeterminedmaximal inhibitory concentrationson submaximal (70% to 90% of maximum) anti-IgE-induced histamine release. All drugs appearedto selectively inhibit histamine releasefrom fractions III and IV more effectively (40% to 64% inhibition) than from other fractions (27% to 46% inhibition). However, when these fractions were analyzed statistically (repetitive one-way analysisof variance), they were not different from any other fractions in their drug sensitivities. The histamine H,-receptor agonist, Dimaprit, which inhibits human basophil histamine release by a CAMP-dependentmechanism,30 failed to significantly inhibit any HLMC density frac-

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Density

1

tion in these same four studies. Cromolyn sodium, a putative mast cell stabilizer, was devoid of inhibitory activity in all density fractions (n = 3); this applied to both histamine and LTC, release. NDGA. We next examined the effects of NDGA, an antioxidant and inhibitor of the lipoxygenase pathway of arachidonatemetabolism, on mast cells from six lung preparations. At the concentration selected for these studies (3 x 10m5mol/L), NDGA was our most potent (>98%) and consistent inhibitor of HLMC LTC, release (concentration of drug required to produce 50% inhibition, lo-’ mol/L) and serves as our laboratory standard against which pharmacologic inhibitors of HLMC lipoxygenaseare compared, Although anti-IgE-induced histamine release from fractions III to VI was significantly inhibited (49% to 80% inhibition) by this agent, fractions I and II were not responsive (Fig. 3). A higher concentration of NDGA (10-j mol/L) was also tested against these fractions but was toxic (>90% histamine release in the presenceof drug alone). DISCUSSION The demonstrationof at least two functionally distinct mast cell classesin rodents has suggestedthat similar observations in human organ systemswould have important clinical implications. Despite recent studiesdemonstratingdistinct mastcell populations at the histochemical level in human gut and nose,“, 32 no information exists as to functional differences amongthesepopulations. With techniquesfor the dispersion of lung parenchymainto a single cell suspension, we now recognize fundamental differences in HLMC morphology: those of diameter and density. Diameter and density characteristics serve as major markers of differentiation and functional differences among mastcells in rodent systems3.4,‘, “. ” and have served as our starting point for the dissection of functional attributes of HLMC subpopulations.” The current studies were designed to examine the characteristicsof a finite numberof mastcell fractions, each containing sufficient numbers of cells for study and representing the extremes of cellular densities. The experiments demonstratethat HLMC are heterogeneous with respect to density (1.053 to 1.123 gm/ ml), histamine content, and mediator release in responseto various secretagogues.Although the cellular histamine content of mast cells separatedaccording to diameter varies from 2.5 to 10 pg per mast cell,” the range in mast cells separatedaccording to density is narrower, 2.7 to 4.8 pg per mast cell. Most lung mast cells are found in density fractions III and IV (densities of 1.077 and 1.088 gm/ml) and possess

heterogeneity

of iuty

was? celfs

a3

T

Percoll

Froctm

I

II

RI

m

3ensl:v

I053

I.063

1.077

1088

~830

1 I23

FIG. 3. Effects of NDGA (3 x 10e5 moliL) on anti-IgEinduced histamine release from HLMCs of different densities. Results are the mean f SEM for six individual human lung experiments. *p < 0.05.

a histamine content of 3.2 to 4.2 pg per mast cell. These same density fractions are of special interest becausethey comprise the fractions most frequently used to study “purified” HLMC behavior’“, “, 14-“; therefore, such studies probably reflect the cellular biology of most HLMC. Differences in functional responsivenessattend the physical separations.In order to test for these differences,anti-IgE dose-responsedata were log-log transformed, and the resultant straight lines representing the various fractions were analyzed by linear regression. With these methods, two functionally distinct subpopulationswere defined. The first subpopulation is comprised of the least densecells (fractions I and II), and the second subpopulation is from the most densecells (fractions V and VI); the lines representing fractions III and IV share characteristics of the extremes (Table II). Interestingly, fractions III and IV tendedto be the leastresponsiveto anti-IgE (especially at the higher concentrations tested) and the most responsive to the calcium ionophore A231K7. The reasons for the functional differences among mast cell subpopulations are not known. Although they likely represent differences in releasibility, we cannot exclude the potential modutating effects of secondary mediators released from contaminating cells. Like-

84 Schulman et al.

wise, the decreasedresponsivenessof fractions I and II cells, which were the least pure (Table I) to low concentrations of ionophore (0.01 and 0.1 kg/ml), could represent distribution of this stimulus to contaminating cells and a lack of its availability to mast cells. Although the human basophil stimulus f-met peptide2’fails to induce histamine releasefrom whole lung mast cell suspensions,small amounts of release are observed in fractions I and II, raising the possibility of a responding population within low density mast cell fractions. Since basophils have not been found in lung suspensions,“3 20they do not represent this responding population. The rat mast cell secretagogue 48 / 8053*. 29is inactive in all fractions, suggesting an important interspeciesdifference in mast cell behavior. The importance of species specificity is also apparent when the results of the present study are comparedto the density heterogeneityreported in rhesus monkey lung-lavage mast cells.33 Met&amide gradient-separatedmonkey mast cells of highest density were highest in histamine content but were the least responsive to an anti-IgE stimulus; HLMC of highest density are amongthe most responsiveto antiIgE. The use of metrizamide versus Percoll as the separationmedia in the two studies must also be consideredin interpreting thesecontrasting results.34,35In particular, metrizamide is known to produce tonicity changesthat may affect cell function. Our interest in examining mast cell subsetsfor differencesin their oxidative metabolism of arachidonic acid stemsfrom studies in the rodent. Rat connective tissue mast cells are very denseand selectively generate PGD, in preference to LTC, (ratio >40: 1). Mouse “mucosal” mast cells are less dense and selectively releaseLTC, in preferenceto PGD, (ratio of 25 : 1).‘2,I3 Such preferential generation of arachidonate metabolitesis of potential biologic importancebecause of evidence for their different pharmacologic actions (e.g., vasoactivity).12The fact that HLMCs also generatethese metabolites as their principal arachidonate products has been well documented. We also previously demonstratedthat in our lung cell suspension system, most, if not al1 the PGD, and LTC, generatedafter an IgE-mediated stimulus, is derived from the HLMC.17s21,25,26.36Therefore, despite the differences in mast cell purity among fractions (Table II), the present studies demonstratethat, on a per cell basis, mast cells of all densities generate both arachidonate products in roughly equivalent amounts. Therefore, on the basis of density, we could not identify preferential generationof either metabolite by any human mast cell fraction.

J. ALLERGY CLIN. IMMUNOL. JULY 1988

To examine potential parallels further with the rodent system, we have begun to examine HLMC with respect to histochemical heterogeneity.37We have found that most HLMCs (90%) in whole lung suspension, and in all subsequentlyderived density fractions, lose their detectability with toluidine blue staining after formalin fixation, and, in this respect, would correspondto the mucosal mast cells of rodents. The percentageof mast cells demonstrating formalin sensitivity is highest (95%) in the least densecells (fraction I) and remains at 70% in the most dense(fraction VI) cells. The lack of selectivity of arachidonatemetabolite releasefrom these fractions suggeststhat the histochemical differences among human mast cells may not parallel the functional differences similarly defined in rodent mast cells.37 Recent studies in a subhuman primate also failed to find functional differences between mast cell populations defined primarily by their tinctorial properties.38 One of the most compelling reasonsto define mast cell heterogeneity is to discern the pharmacologic properties of subclasses.Drugs that inhibit mast cells subserving one role, or alternatively residing in one target organ (e.g., stomachor lung), need to be identified to properly target therapies. In the presentstudy, no significant differences could be identified among fractions in their responsivenessto antiallergic drugs that elevate intracellular CAMP, a finding similar to diameter-separatedmast cells.” Dimaprit, also a CAMP-activedrug, but acting through a histamine H,membrane receptor,30fails to inhibit any fraction, strongly suggesting that HLMC mediator release, in contrast to human basophil mediator release, is not modulated by histamine. We are also unable to identify a respondersubsetof any density that is targeted by cromolyn sodium, a putative HLMC “stabilizer.” Perhapsthe most interesting pharmacologicfinding is the selective effect of the antioxidant-lipoxygenase inhibitor NDGA on HLMC density fractions. This drug has proved to be our most consistent and potent (>98% inhibition) inhibitor of HLMC LTC, release and serves as a laboratory standard. Although the histamine release inhibitory effects of NDGA and other 5lipoxygenase inhibitors are believed to involve failure to generate a putative proinflammatory 5-lipoxygenase pathway product requisite for histamine release,39this mechanismis not consistent with prior studies in the rat mucosal mast cellI or with the present human mast cell data. Histamine release in fractions I and II was unaffected in the presence of profound lipoxygenase pathway inhibition. Clearly, detailed studies on the effects of other pharmacologic inhibitors of the cyclooxygenaseor lipoxygenasepath-

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ways of arachidonate metabolismon low-density mast cells are needed to elucidate further the “atypical” responsiveness of these cells. In summary, we have described mast cell subsets derived on the basis of their density differences. These subsets demonstrate differences in their histamine content and responsiveness to anti-IgE and calcium ionopbore A23187. Most importantly, however, are

their differencesin responsivenessto the lipoxygenase inhibitor, antioxidant drug, NDGA. Further understanding of HLMC heterogeneity will be crucial in providing a more rational basis for therapies of mast cell-mediated lung disorders. We thank Drs. Benjamin Bachrach, Hong Choi, Herbert Cohn, Harry S. Cooper, John Decker, Demonic DeLaurentis, Charles Fineberg, Robert McCairns, Melvin J. Moses, Arthur Patchevsky,Francis E. Rosato, and Henry Stofmanfor providing lung specimens,Drs. Rodney Young and Anatole Besarabfor assistancein statistical analysis, and Drs. StephenP. Petersand JamesE. Fish for reviewing the manuscript. REFERENCES

1. Austen KF, Orange RP. Bronchial asthma: the possible role of the chemical mediatorsof immediate hypersensitivity in the pathogenesisof subacutechronic disease.Am Rev Respir Dis 1974;112:423. 2. SchulmanES. The role of mast cell-derived mediatorsin airway hyperresponsiveness.Chest 1986;90:578. 3. Enerback L. Mast cells in rat gastrointestinal mucosa. 1. Effects of fixation. Acta Path01Microbial Stand 1966;66:289. 4. Enerback L. Mast cells in rat gastrointestinalmucosa.2. Dyebinding and metachromaticproperties. Acta Path01Microbial Stand 1966:66:303. 5. EnerbackL. Mast cells in rat gastrointestinalmucosa.3. Reactivity towardscompound48180. Acta Path01Microbial Stand 1966;66:313. 6. Befus AD, PearceFL, Gauldie J, Horsewood P, Bienenstock J. Mucosal mast cells. 1. Isolation and functional characteristics of rat intestinal mast cells. J Immunol 1982;128:2475. 7. PearceFL, Befus AD, Gauldie J, BienenstockJ. Mucosal mast cells. 2. Effects of antiallergic compoundson histamine secretion by isolated intestinal mast cells. J Immunol 1982; 128:2481. 8. BienenstockJ, Befus AD, PearceF, Denburg J, GoodacreR. Mast cell heterogeneity:derivation andfunction, with emphasis on the intestine [Postgraduatecourse]. J ALLERGY CLIN iMMLNOL 1982;70:407.

9. Mayrhofer G. Tbymus-dependent and thymus-independent subpopulations of intestinal intraepithelial lymphocytes: a granular subpopulation of probable bone marrow origin and relationship to mucosal mast cells. Blood 1980;55:532. 10. Guy-Grand D, Dy M, Luffau G, Vassalli P. Gut mucosalmast cells: origin, traffic. and differentiation. J Exp Med 1984; 160:12. 11. Srendi B, FriedmanMM, Bland CE, Metcalfe DD. Ultmstrucrural, biochemical, and functional characteristicsof histaminecontaining cells cloned from mouse bone marrow: tentative identification as mucosalmastcells. J Immunol 1983;131:915.

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of lung fnast cells

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Relation between frequency of asthma and IgE antibody levels against Oermatophagoides farinae and total serum IgE levels in schoolchildren Masanao Shibasaki, MD, Kimio Tajima, MD, Akihiro Morikawa, MD, Masato Mitsuhashi, MD, Ryo Sumazaki, MD, and Kenichi Tokuyama, MD Tsukuba and Maebashi, Japan The relation between the frequency of wheezing illness and IgE antibody levels against Derrnatophagoides farinae (Of) and total IgE levels was examined in 457 randomly selected schoolchildren. From the response to the ATS-DID-78-C respiratory symptoms questionnaire, 14 subjects (3.1%) were found to have asthma syndrome (recurrent episodes of attacks of shortness of breath with wheezing) and 17 subjects (3.7%), wheezing syndrome (only wheezing). The percentage of the asthma syndrome increased with increasing levels of Df-specific IgE, and there was an intimate correlation between the percentage of asthma syndrome and Df-specific IgE levels (r = 0.97; p < O.OOl), whereas such association was not found between the two (r = -0.19; p > 0.5). Similar relations were found between the frequencies of the spectfic syndromes and total IgE levels. There was a signi$cant correlation between total IgE levels and Df-spectfic IgE levels in the total population (r = 0.7; p < 0.001). Theseresults suggest that allergic reaction greatly contributes to the development of asthma in children. (J ALLERGYCLIN IMMUNOL 198&82.&j-94.)

From the Departmentof Pediatrics, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan, and Department of Pediatrics, Gunma University School of Medicine, Gunma, Japan. Received for publication July 7, 1987. Accepted for publication Jan. 20, 1988. Reprint requests: Masanao Shibasaki, MD, Department of Pediatrics, Institute of Clinical Medicine, University of Tsukuba, Ibaraki-ken 305, Japan.

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Asthma is a complex disorder characterizedby increased reactivity of the airways and involving the autonomic nervous system, as well as immunologic, infectious, biochemical, endocrine, and psychologic factors.’ The allergic reaction is believed frequently to play a major role in the pathogenesisof asthmain children becausehigh levels of specific IgE antibodies