The pharmacologic modulation of mediator release from human basophils

J. ALLERGYCLIN.IMMU~OL. SEPTEMBER1988

Garriga et al.

nibutions of mast cells in human skin. Ital Gen Rev Dermatol 1959;1:17. 16. Mikh~l GR, Miller-Millinska A. Mast cell ~pulation in human skin. J Invest Dermatol 1964;43:249. 17. Nishioka K, Kobayashi Y, Katayama I, Takijiri C. Mast cell numbersin diffuse scleroderma.Arch Dermatol 1987;123:205. 18. Atkins FM, Clark RAF. Mast cells and fibrosis. Arch Dermatol 1987;123:191. 19. Elias J, Boss E, Kaplan AP. Studies of the cellular infiltrate of chronic idiopathic ~ic~a:.pmminence of T-lymph~ytes,

monocytes, and mast cells. J ALLERGYCLIN IMMUNOL1986; 78:914. 20. Mitchell EB, Crow J, Williams G, Platts-MilIs TAE. Increase in skin mastcells following chronic housedust mite exposure. Br J Dermatol 1986;114:65. 21. Wilkin JK. Quantitative assessmentof alcohol-provokedflushing. Arch Dermatol 1986;122:63. 22. Wilkin JK. Flushing reactions. In: Rook AJ, Muiback HI, eds. Recent advancesin dermatology, ~016. Edinburgh: Churchill Livingstone, 1983:157.

The pharmacologic modulation release from human basophils

of mediator

Jane A. Warner, PhD, Donald W. MacGlashan, Jr., MD, PhD, Stephen P. Peters, MD, PhD,* Anne Kagey-Sobotka, PhD, and Lawrence M. ~i~hten~ejn, MD, PhD** E~~ti~ore, &Id. We have characterized the effects of eight different drugs on the IgE-mediated histamine release (HR) and leukotriene C, (LTCfi)frorra human basophils. Arachidonic acid analogues 5,8,11 eicosatriynoic acid and 5,8,If ,I5 eicosatetraynoic acid inhibit the release of both mediators in the range IO+ to lo-” mollL with almost total ~g0~~to l~%~ inhibition of release at 10” moliL. The inhibition of LTC$ was signi$cantly (p < 0.05) greater than the inhibition of HR only at intermediate (IO-’ to 3 x lo-’ mollL) dosesof the drugs. Two other inhibitors of phospholipase A, (bromophenacyl bromide and phenidone) affected the release of both mediators equally. Two drugs that activate adenylate cyclase (prostaglandin E, and dimaprit) inhibited release in a dose-depe~ent fashion but failed to preferentially affect either HR or LTCa. isoproterenol (IO-” to 10e4moflLJ, a third activator of adenylate cyclase, caused only m~erate (30%) inhibition of HR, even when the reaction was staged, but was slightly (0.1 < p < 0.05) more potent against leukotriene release. Thefinal drug tested was the phosphodiesterase inhibitor, isobutylmethylxanthine, which proved to be an effective (50% to 100%) inhibitor of both mediators in the range IO-S to 10-j mollL. (J ALLERGYCLIN IMMVNOL 1988;82:432-8.)

From the Department of Medicine, Division of Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, Md. Supported by National Institutes of Health Grants AI 20253, AI 20136, and AI 07290. Received for publication Feb. 9, 1987. Accepted for publication March 24, 1988. Reprint requests: Donald W. MacGlashan, Jr., MD, Division of Clinical Immunology, JohnsHopkins University Schoolof Medicine at The Good SamaritanHospital, 5601 Loch Raven Blvd., Baltimore, MD 21239. *Recipient of Clinical Investigator Award HL 01034 from National Heart, Lung, and Blood Institute. Current address: Jefferson Medical College, Pulmonary Division, Room 804, College Building, 102 S. Walnut St., Philadelphia, PA 19107. **Recipient of the Pfizer Biomedical ResearchAward. Publication No. 729 from the O’Neill Laboratories at The Good Samaritan Hospital, Baltimore, MD.

432

The role of the basophil in allergic diseasehasbeen appreciated for many years,‘,’ and HR to both IgEmediatedand other stimuli has been extensively characterized.3** Recently, interest has focused on LTC,, an oxidative metabolite of AA released by the basoPhil. This is partly becauseof its potent pharmacologic actions’. 6 and also becauseit appearsto be the principal AA metabolite in this cell. LTC, is a potent bronehoconstrictor’ and has long been implicated in the pa~ophysiol~y of asthma’ Ex~~rnen~ with an in vivo model of allergic rhinitis have demonstrated that LTC, is present during both the immediate responseto antigen and also in the late-phasereaction.g There is now circumstantial evidence linking the basophil with the initiation of the late-phasereaction.‘0 The basophil appearspartially responsiblefor the IgE-

VOiUME NUMBER

82 3. PART 1

Abbre~li~t~~~~s used AA: BPB: ETI: ETYA: HR: IBMX: ID,,: LTC,, LTD,, LTE,: LTC,R: PGE,: HPLC: PIPES: PAGC: RIA: CAMP:

A~~hidonic acid Bromophenacyl bromide 5,8,11 eicosatriynoic acid 5,8, I I,15 eicosatetraynoicacid Histamine release isobutylmethylx~~ine Dose of drug required to inhibit mediator release50% Leukotrienes C,, D,, and I$ LTC, release ProstaglandinE, High-performance liquid chromat~aphy P~~razine-~,~‘-bis-2-ethanesulfonic acid PIPES-albumin-glucosebuffer Radioimmunoassay Cyclic adenosinemonophosphate

dependentreleaseof mediators that either directly or indirectly lead to the recruitment of granulocytes and relocation of the reaction. The central role of the basophil and the wide-ranging actions of LTC, mean that it is of critical importanceto understandthe mechanisms by which leukotriene is modulated and the effects of various drugs on HR and LTC,R. We have recently characterized LTC,R from both pure and impure basophil p~p~ations, investigating calcium and temperaturedependenceand the kinetics of release.‘,‘I We have also demonstratedthat similar amountsof LTC, were releasedby purified and impure cells, indicating that the only cell to releasesubstantial amounts of LTC, in impure preparations was the basophil. Metabolism of LTC, to LTD, and LTE4 was negligible in vitro unless high concentrations (>20 X lo6 total cells per milliliter) of impure cells were usaL The IgE-mediated HR and LTC,R appear to be closely linked with similar dose-responsecurves and requirements for release,’ despite the fact that the release of preformed mediators is constrained by the amounts stored within the granules, whereas LTC,R is limited only by the availability of AA and the enzymesinvolved. Although we previously believedthat leader release and HR were tightly coupled,“, I2 a number of recent studiesclearly suggestthat @etwo mediators can be independently regulated.13.” It is becoming apparent that some drugs are more potent inhibitors of leukotriene release.For example, steroids are able to completely inhibit leukotriene release” from human basophils while they are causing only

50% to 70% inhibition of HR.“’ Sterrtiils tire ;:iso IO times more potent inhibitors of lc~k~~~r~~~~e i-&ease from basophils. Similarly, CAMP ~~~~~~i~rs more cffectively inhibit leukotriene releasefrom human lung parenchymalmast cells. I’ The following studies were designed to examine whether such diftcrentiai inhibition was a general feature of all dru:~ thar inhibit basophil secretion and whether any ~l~ch~i~ist~~ conclusions could be made con~~~i~n~ the secretor) events. We focusedon two principal c&~scsot‘&ugs: (I) those known to inhibit the mctaboli\m r)i iliA at various steps and (2) those that eievatc:ir~rracellular levels of CAMP. Material. The foftowing materials were purchascd: PIPES, IBMX. isoproterenol, and phenidone(Sigma Chemical Co., St. Louis, MO.); crystallized human serumalbumin (Miles Laboratories.Elkhart, Ind. 1;Percoll (Pbarmacia.Piscataway, N.Y.): 3H-LTC, (New England Nuclear. Boston, Mass.); BPB (Atdrich Chemical Co.. ~fi~~l~~~kee” Wis.r; and PGE, (Upjohn Co., Kalamazoo. Mich. i LTC, was the generous gift of Dr. J, Rokach (Merck Frosst Laboratories, Quebec, Canada): dimaprit was provided by Dr. M. E. Parsons(Smith Kline B French, Werrs. U.K. 1; ET1 was provided by Dr. 5. ~~rn~r~~~rn i Karolinska Institute, St~~olrn, Sweden); ETYA wus provided by Dr. W. E. Scott (Hoffman-LaRocbe Inc., Nmley, N.J.): and LTC, antibody was provided by Dr. E, Haye\ (Merck Institute, Ranway, N .J.)” 3ufler.s. PAG contained 25 mmol:L of PIPES. 110 mmot/L of NaCf, 5 mmoi of KCI. 0.003% of human serum albumin, and 0.1% glucose. PAG ~uppI~m~nred with 1 mmol/L of CaCI, and 1 mmolii.. of M@. was used during the reteaseexperiments. Cell isolation. Venousblood, collected from normal and atopic volunteers who had given informed consent. was anticoagulated with ethylenedi~juetetraa~~ti~ acid, dituted with normal saline, and fra~t~oilated over Percolf (density 1.080). The mononuclearceil layer containing t% to 5% basophils was collected, washed onct with salineethylenediaminetetraaceticacid, and twice with PAG Basophil purification. Basophils were p&tied according to the methodof MacGiashanand Lichtenstein. I’ The purity ranged from 37% to 61% (mean 44 z 7%; n -r 7,. Challenge. Washedcells were resuspendedin PAG supplementedwith 1 mmol/L of CaCI, and I mmol il.. of MgCI, to elicit a final concentration of 5 x IO’ basophils per mitliliter. The cells were preincubated with either the drug or a buffer control for 10 minutes and then challenged with 0.05 p,g!mI of anti-lgE or the appropriate antigen. Since anti-IgE is known to differ from antigen&z~~~mu~ation in severalrespects,’ ” we first comparedthe effect of the drugs on mediator release inhibited by both stimuli. WC were unable to detectany differences in either the control release of mediators or the inhibition curves obtained; therefore. the results have been combined. The release process was halted after 45 minutes by centrifugati~~n.and the super-

434

Warner

J. ALLERGY

et al.

1.-o

CLIN. IMMUNOL. SEPTEMBER 1988

100

Concentrative of ETI (plvl)

L

E! 3

100

80

100

80 80 40 20 lcz

; 380 !%“,, 5340 c E $

O1.0

O 1.0

’ 10

10

100

100

Concentration of ETYA (ptvl) FIG. 1. Dose-response curves to ETI (upper panel) and ETYA (lower panel). Basophils (n = 9) were incubated (IO minutes at 37” C) with the drug and challenged with either 0.05 wg/ml antiIgE (n = 7) or a suboptimal dose of antigen (n = 2) and the percent inhibition of HR (0) (control HR 37 2 4%) and LTC,R to) (control LTCIR 153 f 29 ng per lo6 cells) was calculated. Similar results were obtained when purified basophils (47 r 3%; n = 3) were pretreated with both ETI and ETYA, demonstrated in inset to the right. Control HR in these cells was 30 -+ 2%. whereas LTC4R was 101 a 32 ng per IO6 cells.

natant was recovered. Aliquots were removedfor histamine analysis and LTC, RIA. Histamine assay. HR was estimatedaccording to the automatedfluorometric method of Siraganian.“’ The drugs did not fluoresce, nor did they affect the fluorescenceof known amounts of histamine. LTC, RZA. LTC,R was measured according to the method of Hayes et al.*’ The antibody displayed some crossreactivity with LTD, (55%) and LTE, (12%); cross-reactivity with other eicosanoidswas negligible (
solved in a mixture of 0.1% acetate buffer, pH 4.6 per methanol (2: 1 vol/vol). HPLC was performed on a Beckmanmodel (BeckmanInstruments Inc., Irvine, Calif.) 324 mol/L liquid chromatographwith an Ultrasil-ODS (C,,) column and precolumn. This solvent systemclearly resolves the leukotrienesz3while both ET1 and ETYA are retained on the column. The prostaglandins, including PGEl, are eluted before the leukotrienes. The HPLC solvents were removed underreducedpressure,and the LTC, wasmeasuredby RIA. We found a similar degreeof inhibition of LTC,R as compared to that obtained by RIA alone, and therefore, these values are included in the calculation of mean inhibition of release. Statistical analysis. The differences in the percent inhibition of HR and LTCR were compared with Student’s paired t test. The slopes of the inhibition curves obtained for ET1 and ETYA were compared with the analysis of variance.

RESULTS AND DISCUSSION ETI, an AA aualogue,was originally claimed to be a specific inhibitor of the Slipoxygenase pathway.% However, more recent studies have demonstrated it to

VOLUME 82 NUMBER 3, PART 1

Mediator release from ~saphi~s

435

FIG. 2. Dose-response curves to BP6 kft w) and phenidone (right panel). Basophils were pretreated (10 minutes at 37” C) with different concentrations of BPB (n = 3) or phenidone In = 4) and then challenged with 0.05 pglml of anti-IgE. The results are expressed as percent inhibition of mediator release (control HI? was 44 f 8% and control LTC,R was 103 * 40 ng per 1D6 ceils for BPB, whereas control HR was 41 f 8%, and control LTCJ? was 132 ~fr 17 ng per IO6 cells for phenidone). The inhibition of HR is presented by the filled circles (01, and the inhibition of LTC:R, by the open circles (0).

TABLE I. A comparison Drug

ET1 ETYA BPB Phenidone PGE, Dimaprit Isoproterenol IBMX

of the dose of drug required ID, (LTC.1 (mol/L)

4.3 x 1o-6 2.9 x IO-” 8.1 x 10 ’ 5.3 x 1o-4 2.8 x 10m6 3.0 x 10m6 >10-4 1.0 x 1o-5

to inhibit

the HR and LTC,R by 50%

ID, (histamine) tmol/L) 3.1 x 7.1 x 2.6 x 7.6 x 1.0 x 3.8 x >I0 5.8 x

lo-* lo-’ 10 IO-’ IO-’ 1O.-h .a IO-‘

IDS k#iaWdw/ Iclgl’l%, 7.2 24.5 3.2 1.3 2.7 1.3 NA

NA

NA = not available

be less specific; in human lung mast cells, it inhibits

the releaseof all AA metabolitesand also affectsHR.*’ ETYA, another AA analogue, is known to be a relatively nonspecific inhibitor, affecting both cyclooxygenaseand lipoxygenase pathways by preventing AA releasefrom the phospholipids.26 The data in Fig. 1 illustrate that both drugs are potent inhibitors of basophil LTC,R and HR. The two drugs sharemany of the samecharacteristics, including almost complete inhibition of all mediator release at the highest doses tested. The drug doses required to provide ID,, for each mediator are presented in Table I. In eachcase,the ID,, for HR is approximately tenfold greaterthan the IDsofor the LTGR, indicating that the LTC.,Rprocessis more sensitive to modulation by these two drugs. Analysis of the inhibition curves for histamine and LTC, demonstratedthat they also had different slopes over the range 3 to 30 X 10e6

rather than simply demonstrating pa&lel displacement. Student’s paired t test revealed significant differences at the intermediate (1 to 3 X lo-’ mol/L) doses of the drug (p < 0.05) with the maximal inhibition of HR and leukotriene releasebeing identical at the higher concentrations. These results have been confirmed in purified cells (average purity 47 -t 3%; n = 3) and the sameinhibition curves were obtained (see insert, Fig. 1). We were unable to find any evidence that either ET1 or ETYA catalyzed the conversion of LTC, to LTD, or Lm, which, beca~~-e of the reduced cross-reactivity of our antibody with these products (55% and 126, respectively), could be interpreted as inhibition of LTC,R. Basopbils were preincubated with drug and challenged with 0.1 bg/ml of anti-IgE in the presenceof 3W-LX,. Aliquots were removed at 0, 5, 15, and 45 minutes, and the lipid was extracted. HPLC analysis revealed

J. ALLERGY CLIN. IMM~NOL. SEPTEMBER 1988

438 Warner et al.

gj 8

0 lo4

C 8 I

10-g

I

lo4

pGE1P4

20-

0

10-7

I

lo6 Dimaprit(M)

.-l lo’5

FIG. 3. Effect of PGE, (left panel) and dimaprit (right panel) on mediator release. The anti-IgE induced HR (e) (control HR 37 2 2%) and LTC,R (0) (control LTC,R 166 rt 55 ng per 10’ cells) were inhibited when basophils (n = 3) were pretreated (IO minutes at 37” C) with PGE, or dimaprit.

FIQ. 4. Dose-response curves to isoproterenol (left panel) and IBMX (right panel). Basophils (n = 7) were exposed to isoproterenol in the absence of calcium, washed, and challenged with either 0.05 pg/ml of anti-IgE (n = 5) or a suboptimal dose of the appropriate antigen (n = 2), and the percent inhibition of HR (e) (control HR 42 -e 8%) and LTC,R (0) (control LTC,R 223 r 83 ng per IO6 cells) was calculated. Cells In = 3) were treated with IBMX (10 minutes at 37” C) before anti-IgE (0.05 *g/ml) challenge, and the percent inhibition of the mediators was calculated. Control HR (0) was 28 2 4%, and control LTCAR(0) was 86 d 11 ng per lo6 cells.

no catabolism of the exogenous LTC, (data not presented). Two other drugs that m~ulate AA metabolism, BPB and phenidone, were also tested. These drugs inhibit phospholipaseAZ, the enzyme that cleavesAA from the phospholipid membranes, reducing the amount of AA available to the S-lipoxygenase enzyme.*’ BPB was active in the range 1W8 to 10m6 mol/L (Fig. 2), causing maximum inhibition of both mediators at a concentration of 10m6mol/L. The two inhibition curves did not differ significantly at any point, suggestingthat BPB was not exerting its effect solely on the 54ipoxygenase pathway but had more general antireleaseproperties. Phenidoneproved to be

a poor inhibitor of mediator releaseunless millimolar con~n~tions were used. This makes it difficult to interpret results, since such high concentrations of drug may affect the cells in a myriad of ways and are unlikely to reflect the physiologic relevance of the drug. However, both mediators were inhibited eqmlly, there being no statistical difference between either the i~ibi~on curves or the ID= values for histaine and LX,. The secondclass of drug we examined were those that increase the intracellular levels of CAMP, either by activating adenylate cyclase or by inhibiting the action of phosphodiesterase.Dimaprit and PGE, both proved to be potent inhibitors of mediatorrelease( Fig.

VOLUME NUMBER

Mediator release from basophils

82 3. PART 1

3). Both were active at concentrationsof 10m6to 10 -5 IT ,‘L, affecting HR and leukotriene releaseequally. PGE, was perhaps the most effective, with total inhibition of LTC,R at the highest doses, whereasonly a maximum of 80% of the HR was abolished. The H, antagonist, dimaprit, displayed maximum inhibition of mediator release at 10e5 moi/L, both inhibition curves reaching a plateau at higher concentrations. The two curves were statistically indistinguishable, as were the ID,, values. The experiments with dimaprit were repeatedin purified cells (averagepurity 41 t 2%; n = 2) yielding similar results. Isoproterenol is known to be a poor inhibitor of mediator release unless tin reaction is “staged,” that is, unless the cells are exposed to the drug in the absence of calcium, washed briefly to remove the drug, and then challenged in the presenceof exogenous calcium.28 To obtain significant inhibition of LTC,R, we used a stagedreaction, since the presence of isoproterenol during the release process was not effective in inhibiting mediator release (data not presented). Even in the staged reaction, HR was only inhibited 30% at 10e4 mol/L of isoproterenol (Fig. 4). The drug wasonly moderatelymore effective in blocking the LTC4R, although this failed to reach statistical significance (0.1 < p < 0.05). The final drug examined was the methylxanthine, IBMX,29 which proved to be an effective inhibitor of both mediators over the range 10e5 to lo-” mol/L. The maximum inhibition of LTC,R was obtained at lo-“ moli L, at which concentration LTC,R was completely abolished. Once again, the drug inhibited both mediators equally well with no statistical difference in either theinhibition curves or the ID,, values. These results were repeatedwith purified basophils (n = 2) (data not presented). These results demonstratethat not al1drugs differentially inhibit leukotriene releaseand HR. Phenidone and BPB were found to be equally potent inhibitors of leukotriene releaseand HR, as were the agentsthat causedelevations of CAMP. Our previous human lung mast cell studies suggestedthat elevations in CAMP had multiple effects on the secretory apparatus but most effectively modulated AA generation or metabolism.” The steroid studies in the basophil suggest that a similar distinctive modulation is possible in this cell.“~ I6 Although there is a hint that PGE, is a more potent leukotriene-releaseinhibitor, we were surprised to find that, in general, the CAMP agonists studied here were essentially equally effective inhibitors of leukotriene release and HR from human basophils. Although the mechanisms of CAMP action are currently under investigation, we do not yet know which aspectof the activation cascadeis selectively sensitive

437

to elevations in CAMP in the mast cell. These data once againhighlight the evolving idea that the basophil and mast cell secretorymechanismsare not similar in their details. It is perhapsnot surprising that ET1and ETYA were more potent inhibitors of leukotriene release, since these drugs were designed to specifically alter the metabolism of AA through the Iipogenasepathway. In previous studies we were surprised to find that drugs that inhibited AA metabolism also led to the inhibition of degranulation.30.31 Such experiments led to the hypothesis that degranulation was closely coupled to AA metabolism. Now, however, several studies clearly decouple thesetwo processes,and the new data provide further support for separatelycontrolled pathways. REFERENCES 1. Lichtenstein LM, Osler AG. Studies on the mechanism of ~y~~~nsitivi~ phenomena. IX. Histamine release from human leukocytesby ragweedantigen. J Exp Med 1964;120:507. 2. MacGlashanDW Jr, Peters SP, WarnerJA, Lichtenstein LM. Characteristicsof basophil sulphidopeptidelenkotriene release: releasability defined as the ability to respondto dimeric crosslinks. J Immunol 1986;136:2231. 3. Marone G, Kagey-Sob&a A, Lichtenstein LM. IgE-mediated histamine from human baosphils: differences between antigen E and anti-IgE induced secretion. Int Arch Allergy Appl Immunol 1983;65:339. 4. Siraganian RP, Hook WA. Mechanisms of histamine release by formyl-methionine containing peptides. J Immunol 1977; 119:2078. 5. Biscaard H, Kristensson J, SandergaardJ. The effect of leukotriene C, and D4 on cutaneousbiood flow in humans.Prostaglandins 1982;23:797. 6. Maron Z, Shellhamer JH, Bach MK, Morton DR, Kalner M. Slow-reacting substances,leukotrienesC, and D,, increasethe releaseof mucusfrom human airways in vitro. Am Rev Respir Dis 1982;126:449. 7. Dahlen S-E, Hedqvist P, HammarstromS, SamuelssonB. Leukotrienes are potent constrictors of human bronchi. Nature 1984;288:484. 8. Orange RP, Austen K.F. Slow-reacting substanceof anaphylaxis. Adv Immunol 1%9;10:105. 9. Creticos PS, PetersSP, Adkinson NF Jr, Naclerio RM, Hayes EC, Norman PS, Lichtenstein LM. Peptide leukotriene release after antigen challenge in patients sensitive to ragweed. N Engl J Med 1%9;310:1626. 10. Naclerio Rm, Togias A, proud D, Kagey-SobotkaA, Adkinson NF Jr, Plaut M, Norman PS, Lichtenstein LM. Inflammatory mediators in late antigen-induced rhinitis. N Engl J Med 1985;313:65. 11. Warner JA, MacGlashan DW Jr, Proud D, Lichtenstein LM. Mediator release from human basophils [Abstract]. Ann AlIergy 1985;55:275. 12. Peters SP, Seigel MI, Kagey-Sobotka A, Lichtenstein LM. Lipoxygenase products modulate histamine release in human basophils. Nature 1981;292:455. 13. Razin E, Marx G. Thrombin-induceddegranulationof cultured bone marrow-derived mast cells. J Immunol 1984;133:3282. 14. Schulman ES, Post TJ, Henson PM, Gicias PC. Differential

.I. ALLERGYCLIN.I~MUNOL. SEPTEMBER1998

438 Warner et al. effects of complement peptides, C5a and C5a des Arg on human basophil and lung mast cell release. J Clin Invest 1988;81:918. 15. Schleimer RP, Lichtenstein LM, Gillespie E. Inhibition of basophil histamine releaseby anti-inflammatory steroids. Nature 1981;292:454. 16. Schleimer RP, Davidson DA, Peters SP, Lichtenstein LM. Inhibition of human basophil leukotriene release by antiinflammatory steroids. Int Arch Allergy Appl Immunol 1985; 77:241. 17. Peachell PT, MacGlashanDW Jr, Lichtenstein LM, Schleimer

RP. Regulation of humanbasophil and lung mast cell function by cyclic adenosinemonophosphate.J 1mmuno11988;140:571. 18. MacGlashanDW Jr, Lichtenstein LM. The purification of human basophils. J Immunol i980;124:2519. 19. MacGlashan DW Jr, Schleimer RP, Lichtenstein LM. Qualitative differences between dimeric and trimeric stimulation of basophils. J Immunol 1983;130:4. 20. Siraganian RP. An automatedcontinuous flow system for the extraction and fluorometric analysis of histamine. Anal Biothem 1974;57:383. 21. Hayes EC, Lombardo DC, Girard Y. Measuring Ieukotrienes of slow-reacting substanceof ~aphyl~is: d~elopment of a specific mdio~munoassay. J Immunol 1983;131:429. 22. Rouzer CA, Scott WA, Hamill AL, Cohn ZA. Synthesis of leukotriene C, and the other arachidonic acid metabolites by mouse pulmonary macmphages.J Exp Med 1982;155:720. 23. Lewis RA, Austen KF, DrazenJM, Clark DA, Marfat A, Corey EJ. Slow-reacting substanceof anaphylaxis: identification of

24.

25.

26.

27. 28.

29.

30.

3 1.

leukohiene C and D from human and rat sources.Proc Nat1 Acad Sci USA 1980;77:310. Hamm~s~om S. Selective inhibition of platelet n-8 lipoxygenaseby 5,8,11-eicosatriynoic acid. Biochim Biophys Acta 1979;487:517. PetersSP, MacGlashanDW Jr, Schleimer RP, Hayes EC, Adkinson NF Jr, Lichtenstein LM. The pharmacological modulation of mediator releaseof arachidonicacid metabolitesfrom purified human lung mast cells. Am Rev Respir Dis 1985; 132367. Hamberg M, Samuelsson B. Prostaglanclmendoperoxides: novel transformationsof arachidonic acid in human platelets. Proc Nat1Acad Sci USA 1974;71:3400. Volverk JJ, PetersonWA, deHaasGH. Histidine at the active site of phospholipaseAZ. Biochemistry 1974;13:1446. Lichtenstein LM, DeBemado R. The immediate allergic response:in vitro action of cyclic AMP-active and other drugs on the two stages of histamine release. J Immunol 1971; 107:1131. Lichtenstein LM, Margolis SM. Histamine release in vitro: inhibition by catecholamines and methylxanthines. Science 1968;161:192. MaroneG, Kagey-Sob&a A, Li~htenstein LM. Effect of arachidonic acid metabolites on ~tigen-educe histamine releasefrom humanbasophilsin vitro. J hnmunoll979;123:166. Marone G, Hammarstrom S, Lichtenstein LM. An inhibitor of lipoxygenase inhibits histamine release from human basophils. Clin Immunol Immunopathol 1980;17:117.

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