Neutrophil chemotactic activity in toluene diisocyanate (TDI)-induced asthma J. Sastre, MD, PhD, D. E. Banks, MD, M. Lopez, MD, H. W. Barkman, and J. E. Salvaggio, MD New Orleans, La.


We quantitated serum neutrophil chemotactic activity (NCA), which is associated with mast sell or basophil activation, to determine if mast cell or basophil mediators are released during bronchoprovocation-inhalation challenge with subirritant levels of toluene diisocyanate (TDI). Four subjects with suspected TDI-induced asthma and four mite-sensitive subjects with asthma who served as a comparison group were studied. NCA was measured in a multiwell, microchemotaxis chamber. Blood samples were collected, and FEV, measurements were performed before challenge and at regular intervals during the subsequent 24 hours. Three oj four workers clinically sensitive to TDI reacted to a subirritant TDI exposure. There was no increase in NCA during placebo challenges. NCA increased in the three TDI-sensitive worker,7 during early and late asthmatic reactions in quantities proportional to the FEV, decline. No increase in NCA was found during TDI exposures in the TDI-negative worker. Gel Jiltration analysis demonstrated the main NCA fraction eluted with macromolecules of an estimated molecular weight >440,000 d&tons. This characteristic is compatible with neutrophil chemotartic factor of basophil or mast cell origin. The kinetics of NCA release were similar in mite- and TDI-induced asthmatic reactions. A high correlation (r = 0.97: p = OLW6) was obtained between the percent decrease in FEV, during early asthmatic reactions and percent increase in NCA. These observations support the hypothesis that activation of mast cells or basophils is associated with TDI-induced early and late asthmatic reaction. (J ALLERGY CLN


TDI is a low MW chemical that can induce asthma in 5% of occupationally exposed individuals. ’ The

Abbreviations used

pathogenesisof TDI-induced asthmaremains unclear, although some investigators have suggestedthat immunologic factors may be involved in its pathophysiology. 2 Evidence for specific IgE antibodies in the pathogenesisof TDI-induced asthmais contradictory. Studies by Karol et a1.3 using RAST with P-tolyl(mono)isocyanatecoupled with human serumalbumin demonstrated specific IgE antibodies in the sera of sensitizedworkers. Other investigatorshave not found this to be a consistent finding.2,4 Butcher et al.’ attempted to measureplasma histamine levels in patients clinically sensitized to TDI but, challenged with subirritant quantities of TDI, re-

TDI: Toluenediisocyanate NCA: Neutrophilchemotacticactivity HMW: High molecularweight EAR: LAR:

Early asthmatic reaction


ppb: Parts per billion

From the Department of Medicine, Tulane University Medical School, New Orleans, La. Received for publication April 29, 1988. Revised Sept. 8, 1989. Accepted for publication Sept. 9, 1989. Reprint requests:Manuel Lopez, MD, Tulane University Medical School, 1430Tulane Ave., Rm 7209, New Orleans, LA 70012.

sulted in no detectableplasma levels. There is, however, indirect evidencethat degranulationof mastcells or basophils occurs after TDI-inhalation challenge of sensitized individuals, since disodium cromoglycate inhibits EAR and dual asthmaticreactionsand steroid pretreatmentsinhibits LAR.“, ’ Steroids inhibit the inflammatory LAR and has been proved to prevent basophil degranulation and subsequentreleaseof pharmacologic mediators. By contrast, mast cell degranulation is not inhibited by steroids.’ In this study we identify NCA of HMW in TDIsensitive workers after specific antigen challenge and compare the kinetics of release in individuals with TDI and mite sensitivity after antigen-inhalation chal-


lenge. 567


566 Sastre et al.

TABLE I. Results of inhalation



% FEV, decline Immediate (%.)


1 2 3 4 5 6 7 8 10 11

35 NR NR 52 30 47 20 27 50 25


Challenge Late (“/.I

44 35 NR NR 29 NR NR NR NR NR

Dose times time+

TDI (10 x 30) TDI (10 x 30) TDI (20 x 240) TDI (10 x 30) Mites Mites Mites Mites Methacholine Methacholine

% NCA change et maximal FEV, decline


159 235 163 204 128 140 95 105


162 174 187 -

NR, No response,no FEV, declineoccurredfrom baseline;-, no declinein FEV,; NCA wasnot measured. NCA wasmeasured every

30 minutesduringplacebochallenge. *Dataexpressedaspartsperbillion timesminutes.

MATERIAL AND METHODS Subjects Four patients referred to Tulane Medical Center for evaluation of TDI-induced asthma were studied. As a comparison group, four other mite-sensitive, methacholine-reactive patients with asthma and with demonstrableIgE-mediated reactivity to Dermatophagides antigen were selected. All subjectsgave informed consentfor inhalation challenge and serologic studies.

Blood samples Five milliliters of venous blood was withdrawn through an indwelling catheter, placed in a glass tube (without anticoagulants), and allowed to clot at 4” C. Serum was separatedby centrifugation at 1000g for 15 minutes, aspirated, and stored at -20” C. For NCA quantitation, sera were inactivated by heating at 56” C for 30 minutes before gel filtration or dilution in medium 199 (Microbiological Associates, Inc., Bethesda,Md.).



Neutrophils were obtained from peripheral whole blood of normal volunteers. Heparinized blood was sedimentedin dextran for 45 minutes at 37” C. Leukocyte-rich plasmawas removed and centrifuged at 400 g for 10 minutes. The cell pellet was washed first in distilled water to lyze red blood cells and then in medium 199. Cell counts were adjustedto 1 x 106/n& and 50 ~1 was placed in the upper compartment of a 48-well microchemotaxis assembly chamber9 (Neuroprobe, Bethesda, Md.) in which the upper and lower compartments were separated by a 3 km polyvinylpyrrolidone-free polycarbonate filter (Nuclepore Corp., Pleasanton,Calif.). Dilutions of serum (1: 5) or gel filtration fractions (seebelow) wre placedin the lower wells. The chamber was incubated for 60 minutes at 37” C in

humidified air with 5% CO,. After incubation, the filters were fixed and stained. The number of neutrophils that had migrated through the filter was determined microscopically. Four high-power fields per well were read. Because polyvinylpyrrolidonefree polycarbonate filters were used, cells adhered to the membranesurfacein one optical plane for counting.“’ DiffQuik jet (American Scientific Products, McGraw Park, Ill.) was usedfor fixing and staining. NCA was expressedas the ratio of the mean number of neutrophils migrated per field in the postchallengesampleto the mean number of neutrophils per field of the prechallenge sample times 100. All samples were assayedin duplicate, and values were reported asthe averageof the two samples.Duplicate samples varied 20% of baseline with the development of an acute episode of asthma was consideredas a positive reaction. Mite and mthacholine challenges. Mite and methacholine challengeswere performedby a modification according to the method of Chai et al.” Briefly, aerosols were generated by a DeVilbiss No. 46 nebulizer (Devilbiss Co., Somerset, Pa.) attached to a Rosenthal-Frenchdosimeter (Laboratory for Applied Immunology, Inc., Baltimore, Md.). Aerosols were delivered for 0.6 secondsat 20 psi of pressureduring a slow inspiratory capacity maneuver. Patients received five inhalations at IO-minute intervals of serially increasingdoses(tenfold) of mite extract, Defarinae (Hollister-Stier Laboratories, Spokane, Wash.), starting

with a concentration tenfold higher than the end point skin test titration or methacholinedilutions varying from 0.03 to 25 mg;ml in fivefold increments;FEV, was measuredwith the above-noted bellows spirometer. After baseline FEV, measurementswere made, the maximal FEV, was determined from the three expiratory maneuvers.5 minutes after each incremental dose of methacholine or mite extract. br both types of challenge, bronchial responsivenesswas reported FEV,.

as the dose that provoked

a 20% decrement


RESULTS TDt challenges No patient had a decreasein FEV, or an increase in NCA during the placebo challenge. Patient I demonstrated a 33% fall in FEV, immediately after completing TDI exposureof 10 ppb for 30 minutes and a late FEV, maximal decline of 44% at 3 hours. Patient 2 developeda 33% FEV, decline 21/2 hours after TDI exposure at IO ppb. Subject 3 did not react after an exposure of TDI at 20 ppb for 4 hours. Patient 4 demonstratedan immediatedrop of 52% in FEV, after TDI exposure of 10 ppb for 15 minutes. NC4 was measured in at least five samplesduring challenge and up to 24 hours after placebo and TDI challenges. During placebo challenge, there was no evidence of increased chemotactic activity in any of the patients, and the neutrophil counts did not vary >8% among

the samplesin each patient. Subjects with a positive TDI challenge demonstratedan increasein NCA during the EAR and LAR (Table I; Fig. 1). NCA returned to baseline 24 hours after challenge. No increase in



Sastre et al.







500 -








300 loo-

1;; ---- *--







100 !


1 20

FIG. 2. Biogel, 1.5 m gel filtration chromatographic analysis of NCA in serum from subject 1 before and during EAR and LAR after TDI challenge. The elution profile of MW markers 2000 kd (blue dextran), 440 kd (apoferritin), 150 kd (human IgG), and 40 kd (albumin) are indicated; chemotactic activity is expressed as the number of neutrophils per four high-power field (HPF). NCA of the buffer (- - - -1.



1 40






I 80


, 100


FIG. 3. Biogel, 1.5 m gel filtration chromatographic analysis of NCA in serum from a mite-sensitive subject with asthma (subject 5) before and during EAR and LAR after mite challenge. The elution profile of MW markers 2000 kd (blue dextran), 440 kd (apoferritin), 150 kd (human IgG), and 40 kd (albumin) are indicated; chemotactic activity is expressed as the number of neutrophils per four highpower field (HPF). NCA of the buffer (- - - -).

NCA was found in subject 3 who did not react to TDI exposure. Prechallengeand postchallengepeaksof NCA from patients 1, 2, and 4 were subjected to gel filtration. Peaksof NCA from patient 1 are representativeand are illustrated in Fig. 2. In sera obtained at early and late-phasepostchallenge, the maximal peak of NCA was found in fractions corresponding to molecules with an MW >500,000 daltons. An additional slight increase of NCA was noted in sera obtained at early and late-phasepostchallengein the region of very low MW eluate. Sera obtained at prechallenge did not demonstrateappreciableNCA of either HMW or low MW eluate. Results of checkerboard analysis with peaks of NCA from sera and column fractions demonstrated a gradient-induced directional activity, indicating chemotactic rather than chemokinetic activity.

from subjects with EAR and LAR, maximal NCA was eluted together with molecules having an MW >400,000 daltons with another small peak in the very low MW eluate (Fig. 3). No significant NCA level was detectedin serafrom patients after prechallenge. In two patients, NCA was measuredduring methacholine challenge. The patients had a drop in FEV, of 50% and 25%, respectively. NCA did not change from baseline during the decline of FEV, or in five samplesobtained within 24 hours of challenge test. A high direct correlation, r = 0.97 @ = 0.0006), was found between percent decreasein FEV, during mite and TDI challengesand percent increaseof NCA (Fig. 4). However, during LAR, the correlation was inverse, although it was not statistically significant (r = 0.99; p = 0.097).

Mite and methacholine challenge Three patients demonstratedan EAR and one patient demonstrated a dual reaction after mite challenge. NCA increased during the decline of FEV, in EAR and LAR. Prechallenge and postchallenge NCA peaks were subjected to gel filtration. In sera

DlSCUSSK3N Our data indicate that a rise in serum NCA accompanies the EAR and LAR induced in TDI-sensitive subjectswith asthmaafter challenge. The NCA-level increasesparallel the decline in FEV, after TDI challenge. Partial purification demonstratedthat the NCA



85 3

generatedby thesechallengesreside in an HMW (440 kd) fraction of the serum. NCA generated in mitesensitive subjects with asthma after mite challenges demonstratedsimilar MW. Thus, NCA HMW appears to be a mediator common to mite- and TDI-induced EAR and LAR. Similar results have been reported during cold challenge in cold urticaria and exercise challenge-induced asthma.‘3-‘8Although NCA activity paralleled the increase in airway obstruction observed during EAR (Fig. l), it is not possible to ascribe a direct role to these mediators in causing bronchoconstriction. The increase in mediator concentration correlates closely, however, with the changesin airway obstruction causedby mite and TDI challenge, suggesting a direct association between these events. Similar results have been reported by Atkins et al. I4 The pathophysiologic mechanism for this pattern of release is still not known. Of interest is that in contrast to EAR, during LAR there was an inverse relation between NCA and decline in FEV,. However, because of the small number of LARs, this correlation was not statistically significant. In two patients, NCA measuredduring bronchoconstriction induced by methacholine was not increased.This suggeststhat bronchoconstriction per se does not trigger NCA HMW release as previously suggestedby other investigators.I9 Mast cells and basophils’” are the likely source of NCA HMW. The appearanceof this mediator in serum coincides with the appearanceof additional mediators, such as histamine and eosinophil chemotactic factor, after cold challenge in cold urticaria and in antigeninhalation challenge.I.‘. ” A similar heat-stableNCA HMW hasalso beenidentified from purified peritoneal rat mast cells after stimulation with rabbit antirat F(ab), antiserum.*’ A recent report by Buchanan et al.” suggeststhat blood mononuclearcells may also produce NCA of different molecular sizes. This does not mean, however, that specific IgE must be present to cause NCA HMW release, since NCA HMW has been recognized in non-IgE-mediated asthmatic reactions, such as aspirin-induced asthma,23exercise,” or fog-induced asthma.24 The small peak of NCA found in the very low MW eluate has also been described by Metzger et al.” It wasbelievedto representleukotriene Bq, a potent heatstable chemotactic factor from activated mast cells, macrophages, and platelets. The fact that steroids inhibit the LAR but not the EAR in subjects with TDI-induced asthma6suggests that basophils, which are steroid sensitive,’ may be implicated in the LAR. Coversely, mast cells (disodium cromoglycatesensitive) have a predominantrole in EAR. Indeed, basophils have been demonstrated




in TDi asthma

t. 22 3c s c Em ‘“’ _ SE 1;; P,:I



FIG. 4. Correlation between NCA and FEV, d&Me in EAR (open circles) and LAR (closed circl&] efter TDI or mite challenge. The correlation coefficients {r/ were determined by linear regression analysis.

to releasemediatorsin the late phaseof allergic rhinitis without participation of mastcells.” In support of this suggestion is the finding that a factor derived from stimulated neutrophils released histamine from basophils and tissue mast cells in vitro.26 Conversely, mast cells from lung ftagments challenged with antiIgE demonstratedno further release of histamine or NCA for as long as 12 hours.” Butcher et aL5did not demonstratea rise in plasma histamine levels after positive challenge with TDI. This finding may be explained by the fact that they measured histamine after the onset of bronchoconstriction or becausethe technique may not have been sufficiently sensitive. Our current findings representthe first reported evidencethat releaseof NCA is associatedwith the asthmatic reactions of TDI-sensitive patients. These current findings further indicate that the kinetics of release and partial purification of NCA in these subjects are similar to those found in mite-induced asthmatic reactions. Thus, activation of mast cells and basophils appearsto be an important event in the asthmatic reactions of TDI-sensitive patients with asthma. RERRENCES 1. Butcher BT, Jones RN, O’Neil CE, et al. Lon.@tudinal study of workers employed in the manufacture of toluene diisocyanate. Am Rev Respir Dis 1977;116:41l. 2. Bernstein IL. Isocyanate-induced pulmonary di.seases:a current wrspective. J ALLERGY CLIN IMMUNOL 1982;70:24. 3. Karol MH, Sanberg T, Riley EJ, Alaire Y. Longitudinal study of tolyl-reactive IgE antibodies in workers hypersensitive to TDI. J Occup Med 1979;21:354. 4. Danks JM, Cmmmwell D, Buckingham JA, Newman Taylor AJ, Davies RJ. Toluene-diisocyanate-induced asthma: evaluation of antibodies in the serum of affected workers, against



Sastre et al.

a tolye-mono-isocyanateprotein conjugate. Clin Allergy 1981; 11:161. 5. Butcher BT, Karr RM, O’Neil CE, et al. Inhalation challenge and pharmacologic studies of toluene diisocyanate (TDI)sensitive workers. J ALLERGYCLM IMMUNOL1979;64:146. 6. Fabbri LM, Chiesura-Corona P, Dal Vecchio L, et al. Prednisoneinhibits late asthmatic reactions and the associated increase in airway responsivenessinduced by toluenediisocyanate in sensitized subjects. Am Rev Respir Dis 1985;132:1010. 7. MacClashan DW, Schleimer R, PetersSP, et al. Comparitive studies of human basophils and mast cells. Fed Proc 1983; 42:2504. 8. ScheleimerRP, SchulmanES, MacGlashamDW, et al. Effects of dexametasoneon mediator release from human lung fragments and purified human lung mast cells. J Clin Invest 1983;71:1830. 9. Falk W, Foodwin RH, LeonardEJ. A 48-well microchemotaxis assembly for rapid and accurate measurementof leukocyte migration. J Immunol Methods 1980;33:239. 10. Harvath L, Falk W, Leonard EJ. Rapid quantitation of neutrophil chemotaxis: use of a polyvinylpyrrolidone-free polycarbonatemembranein a multiwell assembly.J Immunol Methods 1981;37:39. 11. Barkman HW, Banks DE. Bronchoprovocation testing in occupational asthma. Folia Allergol Immunol Clin 1985;32:45. 12. Chai H, Farr RS, Froelich LA, et al. Standardizationof bronchial inhalation challenges procedures. J ALLERGYCLIN IMMIJNOL1975;56:323. 13. WassermanSI, Soter NA, Center DM, Austen KF. Cold urticaria: recognition and characterization of neutrophil chemotactic factor, which appearsin serumduring experimental cold challenge. J Clin Invest 1977;60:180. 14. Atkins PC, Norman M, Weiner M, Zweimann B. Releaseof neutrophil chemotactic activity during immediate hypersensitivity reactions in humans. Ann Intern Med 1977;86:415. 15. Lee TH, Nagy L, Nagakura T, Walport MJ, Kay AB. Identification and partial characterization of an exercise-induced chemotactic factor bronchial asthma. 3 Clin Invest 1982;



in allergen-inducedlate-phaseasthmaticreactions. J ALLERGY CLINIMMUNOL1984;74:49. 17. Metzger WJ, Richerson HB, WassermanSI. Generation and partial purification of eosinophil chemotacticactivity and neutrophil chemotactic activity during early and late-phaseasthmatic response.J ALLERGYCLINIMMUNOL1986;78:282. 18. Howarth PH, Durham SR, Lee TH, Kay AB, Church MK, Holgate ST. Influence of albuterol, cromolyn sodium, and ipratropium bromide on airways and circulating mediator responsesto allergen bronchial provocation in asthma. Am Rev Respir Dis 1985;132:986. 19. Orr TSC, Elliot EV, Altounyon REC, Stem MA. Modulation of releaseof neutrophil chemotacticfactor (NCF). Clin Allergy 1980;10:491. 20. Akiyama K, PruzanskyJJ, Ricketti AJ, GreenbergerPA, Patterson R. Neutrophil chemotactic activity released in supernatantsof human leukocytes challenged with antigensor antihuman IgE. J Lab Clin Med 1984;104:391. 21. Center DM, Soter NA, WassermanSI, Austen KF. Characterization of neutrophil specific chemotactic activity from rat mast cells. Am Rev Respir Dis 1980;121(suppl):61. 22. BuchananD, Fitzharris P, Cromwell 0, Kay AB. Neutrophil chemotacticactivity from cultured blood mononuclearcells in acute severe asthma [Abstract]. J ALLERGYCLM IMMUNOL 1987;79:15. 23. Hollingsworth HM, Dowing ET, Braman SS, Glassroth J, Binder R, Center DM. Identification and characterization of neutrophil chemotacticactivity in aspirin-inducedasthma.Am Rev Respir Dis 1984;130:373. 24. Shaw RJ, Anderson SD, Durham SR, et al. Mediators of hypersensitivity and “fog’‘-induced asthma. Allergy 1985;40:48. 25. Naclerio RM, Proud D, Togias AG, et al. Inflammatory mediators in late antigen-induced rhinitis. N Engl J Med 1985;313:65. 26. Kelly MT, Martin RR, White A. Mediatorsof histaminerelease from human platelets, lymphocytes, and granulccytes. J Clin Invest 1971;50:1044. 27. O’Driscoll BRC, Lee TH, Cromwell 0, Kay AB. Immunologic release of neutmphil chemotactic activity from human lung tissues. J ALLERGYCLIN IMMUNOL1983;72:695.

16. Durham SR, Lee TH, Cromwell 0, et al. Immunologic studies


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Neutrophil chemotactic activity in toluene diisocyanate (TDI)-induced asthma.

We quantitated serum neutrophil chemotactic activity (NCA), which is associated with mast cell or basophil activation, to determine if mast cell or ba...
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