Characterization of mononuclear cell-derived histamine release enhancing factor

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22. Dusbabek F. Dynamics and structure of mixed populations of Dermatophagoides farinae and D. pteronyssinus. Ret Adv Acarology 1979;2:173-7. 23. Korsgaard J. Mite asthma and residency: a case-control study on the impact of exposure to house dust mites in dwellings. Am Rev Respir Dis 1983;128:231-5. 24. Tovey ER, Chapman MD, Wells CW, Piatts-Mills TAE. The distribution of dust mite allergen in the houses of patients with asthma. Am Rev Respir Dis 1981;124:630-5. 25. Lind P, Lewenstein H. Identification of allergens in Dermatophagoides pteronyssinus mite body extract by crossed ra-

allergens

in Baltimore

area

dioimmunoelectrophoresis with two different antibody pools. Stand J Immunol 1983;17:263-73. 26. Lind P, Weeke B, I.&wenstein H. A reference allergen preparation of the house dust mite D. pteronyssinus, produced from whole mite culture-a part of the DAS 76 study. Comparison with allergen preparations from other raw materials. Allergy 1984;39:259-74. 27. Schwartz B, Lind P. Immunochemical methods for investigations of allergic patients’ environment: results of elimination treatment of the allergen source [Abstract]. J ALLERGY CLIN IMMUNOL

1984;73:156.

Characterization of miononuclesr cdl-derived histamine release enhancing factor Robert E. Younger, M.D., Henry G. Herrod, M.D., Phillip L. Lieberman, M.D., William R. Valenski, B.S., Robert L. Trouy, MS., and Lloyd V. Crawford, M.D. Memphis and Chattanooga, Tenn. Histaminereleaseenhancing factor (HREF) is a product of phytohemagglutinin-stimulated mononuclearcellsthat substantiallyaugmentsin vitro IgE-mediatedbasophilhistaminerelease. Thefactor is stableat 56” C and hasa molecularweightin the 10,000to 30,000dalton range. The magnitudeof HREF activity produced is dependenton the concentrationof mononuclear cellsculturedand thefinal concentrationof HREF during basophilchallenge.The HREF phenomenoncould not be attributed to phytohemagglutinin,a- or y-inteeeron, arachidonicacid metabolites,or interleukin-I or 2. HREF appearsto be a uniquecytokineof potentialimportancein the immunologyof inflammatoryand atopicprocesses.(J ALLERGY CLIN hlMUNOL

1987;80:547-57.)

The ability of mast cells and basophils to synthesize and release chemical mediators is an integral component of the host inflammatory response. Major advances have been made in our understanding of degranulation mechanisms that operate in these cells and of agents, both endogenous and pharmacologic, that influence or trigger degranulation. The importance of mononuclear cells, particularly T-lymphocytes, in the initiation and modulation of various immune responses is well established,’ but only recently have

From the Section of Allergy-Immunology, Department of Pediatrics, University of Tennessee Centerfor the HealthSciences, LeBonheur Children’s Medical Center, and the Department of Medicine, Baptist Memorial Hospital, Memphis, Term. Received for publication Aug. 5, 1985. Accepted for publication March24, 1987. Reprint requests: Robert E. Younger, M.D., University of Tennessee Center for the Health Sciences, Chattanooga Unit, 960 East Third St., Suite 306, Chattanooga, TN 37403.

-I

Abbreviationsused HREF: Histaminereleaseenhancingfactor DHRF: Direct histamine-releasing factor II-I: Intrinsic histamine PBMC: Peripheralbloodmononuclear cells CM: Conditionedmedia HR: Histaminerelease PIPES: Piperazine-N,N’-bis(2-ethanesulfonic acid) RIA: Radioimmunoassay MW: Molecularweight IL- 1, IL-2: Interleukin-1and2 PHA: Phytohemagglutinin LT: Lymphocytetransformation

there been studies describing mononuclear cell products that may directly induce HR from basophilP or augment IgE-mediated degranulation.5 Among the factors described that may enhance mediator release 547

548

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Younger et al.

TABLE 1. Products

in CM affecting

HR

CLIN. IMMUNOL. OCTOBER 1987

-.---..--. % HR

Mononuclear cell donor

1

2 3 4 5 6 7 8 Mean 2 SD

IH

DHRF

6.2 21.8 8.7 7.7 7.8 9.0 2.5 4.1 9?6

0.2 0 5.4 0 2.0 2.9 2.8 7.9 3c3

are interferon,6 prostaglandin,,‘7 Shydroxyeicosatetraenoic acid, and HPETE,8 and a mononuclear cellderived, releaseLnhancing factor.5 These observations have suggested a role for mononuclear cell products in the regulation and activation of histamine-containing inflammatory effector cells. An appreciation of the function of these modulators in the inflammatory mediator-release mechanism may be important for further understanding of the ontogeny, expression, and therapy of inflammatory and atopic processes. The present study was performed to investigate the ability of stimulated mononuclear cells to produce both a DHRF and an HRRF. Specifically, in a series of in vitro experiments, we attempted to characterize the production of these factors and to delineate their effect on basophil HR.

The followed were purchased:PIPES, 30% humanserum albumin,dextran, andethylenediaminetetraacetic acid from SigmaChemicalCo., St. Louis, MO.; RPM1 1640, N-2-hydroxyethylpiperazine-N’-2-ethanesuifonic acid buffer, penicillin, stmptomyinsulfate, and L-glutaminefrom M. A. Bioproducts,Walkersvile,Md.; PHA-P from Difco Laboratories Inc., Detroit, Mich.; interleulcin-1 and 2 (ultrapure)from Genzyme,Boston,Mass.; y-interferon (1.8 x 106 U/ml) and a.-interferon (106 U/ml) from Meloy Laboratories,Springfield, Va.; IMRX y-interferon RIA kit from Centocor, M&em, Pa.; methyl-‘H&ymidine (2 CilmmollL), Tom ResearchPrc&tcts International Corp., Mt. Prospect,JR.; Ficoll from Pharma&, Piscataway, N. J.; Hypaquefrom Winthrop-BreonLaboratories, New York, N. Y.; 2,5-3H-histamine dihydrochloride(33 Ci/mm~i/L)from AmerahamCorp., Arlington Heights,111.; “C-methyl-S-adenosylmethionefrom ICN Pharmaceuticals, Irvine, Calif.; l&hem, saxlDiego,calif.; and purified to >90% specificity)from Tago Inc., Burlmgame,Calif.

Anti-lgE

14.6 7.0 11.1 17.3 19.8 24.3 21.s 17 k 6

HREF

% Enhancement

8.1 10.4 15.4 10.2 13.5

47 149 139 70 6X

26.6 24.8 16 t 7

106 115 99 rt- 36

QuantitativeRIAs for anaphylatoxiccomplementcomponentsC3a, C4a, andCSawereperformedby Dr. Robert Wyatt. QuantitativeIgE wasmeasured with a standardRIA. af PBMC were separatedfrom fresh heparinizedvenous bloodprovidedby eight healthyadultvohmteersin a series of experimentswith a modificationof the Ficoll-Hypaque bouyant density-gradientmethodof Boyum.9The atopic statusof thedonorswasnot evahtsited. PBMC werewashed threetimesin phosphate-buffered salinewith heat-activated 2%fetal bovineserum.Exceptwhere it is statedotherwise, 5 x 105 PBMC per milliliter were cultured at 37” C and5% CO, in RPMJ1640mediacontainingN-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid buffer, penicillin, 100 U/ml, streptomycin, 100 rig/ml, and r.-glutamine, 2 mmol/L, but withoutfetal bovineserum.ThePBMC were culturedin the presenceof PHA, 100pg/ml, for 4 hours, thenwashedtwice with media,andreculturedfor 20 hours in mediawithout PHA. At the end of the cuhttre period, supernatants were harvested,and CM were assayed@r DHRF activity, HRBF activity, andJH In selectedexperiments,PBMC were culturedwith varying concentrations of PHA for the entire 24-hourperiod. GM that were nat usedimmediatelywerestoredfrozenat - 25”C. LT studies wereperformedasdescribedelsewhere.‘0

Venousblood leukocyteswere isolated by dextrosedextransedimentation with ~y~~~~~~~c acid andpreparedwith PIPESbuffer aspreviouslydescribed.” Approximately3 million leukocytesin 0.35 ml suspension and the internalstandard>H-histaratine were addedto duphcatesetsof I.5 ml screwcapt&es conrainingHKl~1of buffer, media,or the CM to be tested.PTPBSbuffer containingalbumin.30 mg/dl, Ga (1 mmol/L), and Mg (OS mmol/L) wasusedin all histamine-releasing experiments. Unlessit is otherwisenoted,tettkocytesfr r * wereusedduringeachseriesof e+pq+e.n c. ,f& c4]*~ tiple CM for a particularcw solutionswere incubatedfor 30 minutesin a 33”.C water

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enhancing

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949

00

3

50

1 t! E 40 .E (P 2 30 I E 0) 2

Anti-IgE

(22% HR)

20

0” 10

L

DHRF (8% HR)

CM

Intrinsic

CM

Histamine

Anti-IgE

Basophil

CM+Anti-IgE

Challange

FIG. 1. IH content of CM from PBMC of subject 8 and subsequent basophil HR attributed DHRF, antAgE, and HREF. HREF percent HR represents 115% enhancement of anti-IgE-induced HR.

bath. Subsequently, 50 p,l of anti-IgE in a concentration selectedto producesuboptimalbasophilHR of 15%to 30% wasaddedto solutionsbeingtestedfor HREF activity and to the appropriatecontrols. An equal volume of PIPES buffer wasaddedto solutionsbeingtestedfor DHRFactivity eliciting a final volumeof 0.5 ml. The mixtureswerereincubatedat 37” C for 30 minutes.Histamine-releasing reactionswereterminatedby harvestingthe supematants after centrifugationat 220 x g and4” C. Histaminecontentwas consideredas the amountliberatedduring 3Ominuteincubationat 90’ C. Eachfinalcell-freesupernatant wastransferredto another1.5 ml screwcaptubefor histamineassay. Measurement of III in the CM wasperformedwith volumesof CM andbuffer equivalentto thoseusedduringthe histamine-releasing experimentsthat werebeingperformed concurrently.For easeof interpretation,the IH contentof the CM is expressed asthe equivalentpercentof total histaminecontentin eachexperiment.Varying concentrations of purifieda- andy-interferon, IL-l, andIL-2 weretested for DHRF andHREF activity by substitutingthesefor the CM in the experimentaldesign. The histaminecontentof cell-freesupematants wasassayedwith a modificationof the doubleisotoperadioenzymatic techniqueof Beaven et al.” as previously described.“‘,*I PercentHR wascalculatedwith the formula:

% HR = ExperimentalHR - Spontaneous HR x 100 Total HR

to

The percentHR attributedto the HREF effect on basophils during subsequent anti-IgE challengewascalculated astheamountof HR thatcouldnot beattributedto thedirect releasingactivity of the anti-IgE aloneor to the CM alone (DHRF andIH). Percentenhancement of anti-&E-induced HR represents the percentincreaseattributedto the HREF phenomenon. Themagnitudeof HREFeffectwasdependent in part on the magnitudeof basophilresponseto IgEmediatedstimulation.Therefore,data representingHREF activity are expressedas percent enhancementof IgEmediatedHRor asbothpercentenhancement andthepercent releaseattributableto HREF. In all experiments,thetestsolutionsfor spontaneous HR were identicalto reactionsolutionsexcept for the substitution of buffer or mediain placeof anti-IgEor CM. Spontaneousreleasedid not exceed5%. Data arc presentedas meank standarddeviation. Statistical significancewas evaluatedwith the student’st test.

RESULTS Relation of IH to direct histamine-releasing factor Mononuclear cell-derived CM initially appeared to produce significant direct HR from basophils. However, the major portion of this was due to histamine presentin the CM before basophil challenge. This IH

550

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J. ALLERGY

CLIN. IMMUNOL. OCTOBER 1987

12aDHRF

42

o

z

101

;

16.

.-2 E Q b .I

12-

2 a3

r 44%

enhancement

82

+ 22%

84. 0

2

15

24

72

12Dtmwrs

ffi. 2. The 120-hour time course of mononuclear cell-derived DHRF, HREF, and IH. Each of the four symbols indicates CM from one of four mononuclear cell donors. The CM wgre harvaead ePter varying periods of PBMC culture. Results represent mean + SD percent HR and par&r& enhancemt&t caused by HREF in separate experiments with basophik from one of the subjm.

was spontaneously release during culture by basophils contaminating the Ficoll-Hypaque-isolated cell preparations. In one experiment, Ficoll-Hypaque-isolated PBMC were further separated by plastic adherence, followed by T-rosette bouyant density separation. I3 The nonadherent, T cell-depleted preparation consisted of 12% basophils and produced the greatest IH content (39%). The nonadherent, T cell-enriched population contained no detectable basophils and produced no II-I. Unfractionated PBMC contain 2 + 2% basophils that produced IH content of 9 k 6%. Table I presents the results of experiments with CM from cultures of PBMC from eight donors. In CM of six of the eight donors, IH accounted for most of what might have been considered I3HRP activity. Seven of the CM were also assayed for the ability to augment antS&induced HR. Significant HREF activity was deteckd in each case, generally doubling the magnitude of an&IgILinduced release. Cells from subject 8 gbia&u& h&large ISLES and ISIR@ activities.

With data from this single experiment, Fig. 1 gaphically illustrates our ~~~ and &&kgs. l-imesrsurrrar& PBMC from four of the subjoets were cukmd in the presence of PI-IA for 4 hours, wasbed, and, &en cultured for up to 5 days. As i&&rat& fin Pig. 2, significant DHIZF activity was detected itl tlib CM from only one of the subjects at 15 hours. This p&red at 72 hours of culture. HREF activity was d&nonoccurredat 1Shours III increased ~8th

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60

3 z d

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551

76 f 31 %enhancement

93f26%

50 HREF

2

40

E (R bi

30

DHRF and IH

E E5 b

20 Anti-IgE (f4W

10

i

ii

1’0

Duration FIG. 3. The 24-hour time represent mean f SD for

course percent

of PBMC Culture

of HREF production HR during separate

peaked after 10 hours with 21 + 6% HR. This represented 93 k 26% enhancement of the response to anti-IgE. Effect of varyins concentrations and PHA on HREF production

24 hours

of PWIC

PBMC from three subjects were cultured at varying concentrations for 20 hours after PHA stimulation. Combined DHRF and IH content of the CM decreased significantly when the mononuclear cell number was reduced from 5.0 to 1.25 million cells per milliliter (Fig. 4). HREF activity was similar at both these cell concentrations and then fell significantly by use of 300,000 cells per milliliter. Culture media containing PHA, 100 pg/ml, were assayed repeatedly for HREF activity. With a 1: 5 final dilution of the media during basophil challenges, the final PHA concentration of 20 kg/ml failed to significantly enhance anti-IgE-induced HR. However, a small but consistent direct histamine-releasing effect was detected (1.3 2 l.O%, N = 8). Two experimental designs were used to investigate further the possible effect of PI-IA itself on HREF activity. Mononuclear cells from three subjects were cultured for 24 or 120 hours in the presence of varying concentrations of PHA. Cells cultured in the presence of 100 kg/ml

by mononuclear experiments.

cells

(N = 4). Results

of PHA demonstrated peak HREF activity in 24-hour cultures and maximum LT at 120 hours (Fig. 5). As the PHA concentration was increased, both the stimulation index for LT and the HREF activity significantly decreased, whereas combined DHRF activity and IH content increased slightly with increasing PHA concentration. In the second group of experiments, CM containing PI-IA, 100 pg/ml, were passed over a thyroglobulin column. l4 This procedure effectively removed the PHA as demonstrated by subsequent failure of the tbyroglobulin-treated CM to stimulate LT (stimulation index 1.2 versus 462 in PHA-containing medium). However, the HREF activity of the thyroglobulintreated CM was completely intact (62 rt 14% enhancement after thyroglobulin treatment versus 48 + 15% enhancement without thyroglobulin treatment). Effect of varying concentrations of CM on DHRF and HREF activii CM from cells of three different subjects were used to evaluate tbe effect of varying the CM concentration during HR experiments. As illustrated in Fig. 6, the magnitude of enhancing activity and the combined DHRF activity and IH content were dependent on the final concentration of the CM during basophil chal-

552

J. ALCERGY

Younger et al.

CLIN. iM44UNOL. OCTOBER 1987

0.3

5.0

1.25

c

of &ilawbaucW

C*l)g

( x 1tWmf)

FIG. 4. The effect of varying concentrations of FfMC from three sub&&. DHRF activity and IW are combined (closed circk$; HREF activity is expressed as percent enhancement of antMgEinduced kfR lopen circles~. Results represent mean + SD for sepwatstw~itients with base&ii from a single donor.

lenges. DHRP and IH content appeanxl to diminish proprtionately with the reduction in final concentration of the CM &&xise-enhancing activity was similar with the sta&rd final dilution of 1: 5 and the lower coneen@ation of 1: 20 and then fell progressively with f&t&r dilution of the CM. n Pooled CM from multiple subjects were passed through difknxrt ultra f&ration membranes (Amicon membranes: PM-30, PM-lo, and UMO5, Amicon Corp., Lexington, Mass.) and then evaluated for HREF activity. In three separate experiments, components of the CM that passed &rough the 30,000 MW 6&r retained HREF activity. No enhancing acti~ity was present in filtrates < lO,soO MW. IH was still, in filtrates that passed through 500 MW filters. DHRF activity was not great enough to assess MW

range.

In three different experiments, pooled CM were diMed tenfold with buffer, placed in dialysis tubing with 12 to 14,atW NW pore filtration, and concentrated with a vaenum dialysis. The concentrate was then dia&ed approxhte original volume. IH was in the di&y& mmt%fe, btC= a&iv-&y E-C&&XI intact, yi&ing 92 +

14% enhancement compamd to the 85 r- 36% enhancement of the CM from the ~gi~-~~ its& contained signiPcant III (10 k 2%).

r-interferosr and IL-l and IL-2 were

Monon&ear ceils for varying periods Each CM were wsw well as HWP a&&y. As ikkatr~$ in activity ~a~ with IxitoEe &an 15 trim. In con-t, y-interferon content hoursintwooftiKCMarmdat24~l~ho~sin eachoftbeothertwo was at least one tim

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Anti-IgE

PHA Concentration FIG. 5. Effect

of PHA concentration 120-hour LT assay. Results represent from one donor.

(pg/mU

on HREF activity from 24-hour mean f SD of three separate

activity with 74% enhancement of anti-IgE-induced release compared to 78% enhancement with untreated CM. RIAs of pooled CM revealed no detectable IgE or anaphylatoxic complement components. In a single experiment, PBMC were cultured in the presence of indomethacin, 1 u.g/ml, and PHA, indomethacin alone, or PHA alone. CM containing both indomethacin and PHA produced 140% enhancement of IgE-mediated HR compared to 116% enhancement with CM containing PHA alone. No HREF activity was detected in the CM containing indomethacin alone. Effect of varying the timing of basophil exposure to HREF The effect of altering the time of basophil exposure to HREF in relation to anti-IgE challenge was evaluated in four experiments. Representative findings from studies with granulocytes from one of the donors are presented in Table III. Cells exposed to pooled CM 7M to 90 minutes before anti-IgE challenge demonstrated similar HREF effect. Cells exposed to CM 7% to 90 minutes after anti-IgE challenge also demonstrated similar enhancement of HR. Whether exposure of basophils to HREF occurred before

PBMC cultures (N = 31 and experiments with basophils

or after anti-IgE challenge, the magnitude of histamine-releasing enhancement was nearly equivalent (40 + 5% versus 41 ? 9% enhancement). All histamine-releasing reactions were incubated for 30 minutes in the presence of both HREF and anti-IgE. DlSCUSSlON The findings of this study confirm earlier studies that stimulated mononuclear cells are capable of producing a factor that significantly augments basophil responsiveness during IgE-mediated degranulation. Our observations also point out the potential for confusion when cytokines are being evaluated for direct histamine-releasing activity. The IH present in supernatants of mononuclear cell cultures must be either removed or accounted for during histamine-releasing experiments. Thueson et al.’ used dialysis treatment of CM to remove IH and subsequently detected significant direct histamine-releasing activity. Other investigators who have identified putative histaminereleasing or enhancing factors have not clearly indicated whether this IH was taken into consideration.3* 5 These experiments did not firmly establish whether HREF is separable from DHRF. Although the MW

554

J. ALLERGY

Younger et al.

16

CLIN. IMMUNOL. OCTOBER 1937

T

1

Final

Dilution Of Co#%Wionsd During Basophit kxposure

FIG. 6. Effect of varying CM concentrations during bawphi! cballengas. HREF activity is illustrated as percent enhancement of anti-lgE-induced HR (open circled. DHRF activity and IH are combined (closed circles). Resutts represent mean f SO of three separate experiments.

TAB&E

II. Release-enhancing

Jnta&ron Y

a

Positive control Pooled CM

effects of potential

(tJ/mtl 1000 500 100 loo0 500 100

mediators

)#lf?F%HR 2.2 0.6 2.9 2.1 2.2 0

5 +2 2 +-+

0.2 0.1 0.2 0.2 0.1 0.1

of HREF activity KS

IL- 1

IL-2

W/d) 10 1.0 0.1 500 50 5 1

0.2 0 0 0.1 0.4 1.9 2.2

It It ” L I c -t

0.5 0.5 0.6 1.1 0.9 1.4 1.0

16.4 2 0.4

range of 10,ooO to 30,000 dakons is shan?d by HREF in our studies and the DHRF reported by other investigators , I6 other findings suggest that they are separate factors. In sh&es by Thueson et al. ,* basophil challenges were performed with CM at 75% of the original coslcenbraton and induced up to 70% MR. With CM at 2U% of Eheoriginal conce&mk3n, DWRF activity in our experiments was of lesser magnitude and inconsist~tly present. The releaselenhancing effect of HI%EF, howeva, was consistently present and easily detected even with conce&rations of CM at 5% and less during basophil-challenge experiments. HREF activity peaked in super~~&~~B from mononucIear cells after 10 to 15 hours of cu&re it’d di-

minished in CM of cul&res past this period. In our system, both II-I aad IX-&F cor&ued to increase with prolonged incubation time. The HR enhancing factor deacrikd, by Banzai et aL5 is clearly a diffknt SUB%FZUKX thaw &at which we have i ~r~~~~w~l~,~ arKi was itive (56’ C for 30 m&n@@. In adrliticm, this fa@or p&ma& a *l&i HR (15 rt 4%) eompar+ to tk d observed.

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Mononuclear

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555

101 k44% 62+22% 02+39%

enhancement

47226%

2

15

Duration

24

72

of Mononuclear

120 hours

Cell Culture

FIG. 7. HREF activity (open circles) and gamma interferon concentration (closed circles) in CM from mononuclear cells of four subjects cultured for varying time periods. Results represent mean f SD in four separate experiments with basophils from one of the subjects. HREF activity is illustrated as percent HR and percent enhancement of anti-IgE-induced HR.

to stimulate mononuclear cells. Although the MW range of HREF is compatible with the interleukins and interferons, purified preparations of these substances studied over a broad concentration range produced no I-IREF-like responses. Unlike a recent study by Subramanian and Bray,17 we could not demonstrate that IL-l produced either direct HR or had an enhancing effect. The reason for this difference is unclear, although it might be related to the source of the IL- 1 or to the fact that only about 50% of their patients demonstrated this effect. Ida et a1.6reported in 1977 that leukocytes stimulated with virus during 24-hour culture produced supematants containing interferon that also enhanced IgE-mediated HR. Possibly both interferon and HREF were present in these CM produced with viral stimulation. Our studies demonstrated that both products are present in CM from PHA-stimulated mononuclear cells but that peak levels appear at different culture times. IH from the cell preparations may also have contributed to their findings. The possibility that HREF activity may be due to an arachidonic acid metabolite was reasonably excluded by studies of the HREF MW range. Additionally, indomethacin added to the PBMC culture in a concentration reported to inhibit pros-

TABLE Ill. Effect of altering basophil Anti-IgE

exposure challenge

the time of to HREF in relation to After anti-IgE

Before Anti-IgE Min

% Enh

7.5

15 30 60 90

Mean + SD Min

= minutes:

45 31 44 41 41 40 + 5

hJlin

% Enh

7.5

48 36 25 47 49 41 k 9

15 30 60 90

Enh = enhancement.

taglandin synthesis” did not diminish production of HREF. The mononuclear cell-derived factor that we studied in this article appears to be a unique cytokine that may be of potential significance during inflammatory responses that involve degranulation of histaminecontaining cells. T-lymphocytes are probably important in the production of HREF because the magnitude of activity produced is dependent on the presence of

556

J. ALLERGY

Younger et al.

PHA, a known T cell stimulant, and correlates with LT. However, other cells may be involved becausea mixed population was studied. An obvious issuewhen cytokines generatedin vitro are discussedis what role, if they play any role, do cytokines play in vivo. Although the effect of HREF in vivo is not known, severalpossibilitiesare apparent. The time courseof HREF production is suggestiveof involvement during the pathogenesisof late-phaseallergic reactions. Although the causeof thesereactions is not completely understood, there is evidence that after the initial IgE-mediated degranulation of mast cells, there is a secondand more protracted mastcell degranulation that occurs during the late-phasereaction .I9 It has also been reported that antigens that produce immediate hypersensitivity reactions can elicit cell-mediated responses.*‘) Therefore, the dualphase allergic reaction could involve an immediate IgE-mediated degranulation responsecoincident with stimulation of local mononuclear cells to produce HREF. The onset of HREF production, which increasessignificantly from 2 to 10 hours after stimulation, would induce further mediator releaseand enhanceresponsivenessto subsequentantigen exposure. A role for mononuclear cell responsesin the pathogenesisof late-phasereactions may partially explain the effectiveness of prior corticosteroid treatment in blocking their development,21.22 sincesteroidsareparticularly effective at altering T cell responses.The presenceand severity of late-phaseasthmaticreactions are known to correlate with overall diseaseseverity in these atopic individuals.23 The propensity with which an individual produces or respondsto endogenous HREF may contribute to this association. Another possiblerole for HREF is in the modulation and activation of inflammatory effector cells participating in a regulatory feedback system. Within this network, products from mononuclear cells could be capable of enhancing HR via HREF or of directly inducing degranulation through the elaboration of DHRF. The end product, histamine, could inhibit cellmediated responsesvia histamine-inducedsuppressor factort4 resulting in reduced synthesisof HREF and DHRF. This feedback system would also influence related immune responses,including lymphocyte production of chemotactic factors and control of immunoglobulin synthesis.25.26 Perturbations in the proposed feedback network could be operating in the ontogeny and expressionof atopic disease.Successful immunotherapy may represent, in part, a therapeutic manipulation of specific mononuclear cells responsible for influencing the reactivity of histaminecontaining cells. The potential significance of the inflammatory cy-

CLIN. IMMUNCL. OCTOBER 1987

tokines HREF and DHRF underscoresthe importance of further investigations that are designedto evaluate the cells involved in synthesis, the mechanismof eifects on basophilsand possibly mast cells, the infiuence of pharmacologic agents in these cytologic and biochemical interactions, and the in vivo correlations of these findings.

I.

2.

3.

4.

5.

6.

1.

8. 9. 10. 11. 12.

13. 14. 15. 16.

17.

Reinherz EL, Schlossman SF. Regulation of the immune response-inducer and suppressor T-lymphocyte subsetsin human beings. N Engl .I Med 1980;303:370. Thueson DO, Speck LS. Let&Brown MA, et al. Histaminereleasing activity (HRA). I. Production by mitogm- or antigenstimulated human mononuclear cells. J lmmunol 1979:123: 626. Alum R, Roznieki J, Salmaj K. A mononuclear cell-derived histamine-releasing factor (HRF) in asthmatic patients: histamine release from basophiis in vitro. Ann Allergy 1984; 53%. Kaplan AP, Haak-Frendscho M, Fauci A, et al. Histaminereleasing factor from activated human mononuclear cells. J Immunol 1985; 135:2027. Bamzai AK, Kretschmer RR. Enhancement of antigen-induced leukocyte histamine release by a mononuclear cell-derived factor. J ALLERGY CLIN IMMUNOL 1978;62:137. Ida S, Hooks JJ, Siraganian RP, et al. Enhancement of IgEmediated histamine release from human batqbils by viruses: role of interferon. J Exp Med 1977;14.5:892. Peters SP, Schleimer RP, Kagey-Sobotka A, et al. The role of prostaglandin D, in IgE-mediated reactions in man. Tmns Assot Am Physicians 1982;45:221. Peters SP, Siegel MI, Sobotka AK, et al. Lipoxygenasg products modulate b&amine release in human basopW$. N+%uxe 1981;292:455. Boyum A. Isolation of mononuclear cells and gmnutoCytes from human blood. &and J Clin Lab Invest 19&@1:77. Sanal SD, Buckley RH. Antibody-dependent cellular cytotoxicity in primary immunodeS&ncy disease and with normal leukocyte subpopulations. J Clin Invest 1978;61:1. Younger RE, Herrod HG, Lieberman PL, et al. Characteristics of diatrizoate-induced basophil histamine release. J ALLI?RGY CLIN IMMUNOI<1986;77:94. Beaven M, Jacobsen S, Harakova Z. Modification of the enzymatic isotopic assayof histamine and its app&x&on to measurement of histamine in tissues, serum, and urine. Cfin Chii Acta 1972;37:91. Gerber PS, Herrod HG. 5’-Nucleotidase activity in subjects with abnormal lymphocyte function. J Clin Itnmunol 1983: 3:222. Fagnani R, Bratz JA. Removal of ~yto~~u~~n from conditioned media by af6nity chrortmtog@q. J I-1 Methods 1980,33:313. Bonnard GD, Yasaka K, Jacobson D. Ligand-actiT cell growth-factor induced proliferation: absorptio@ of ‘I’ cell growth factor by activated T cells. J Immurtol 1939;123:27#4. Lett-Brown MA, Thueson W, Plank DE, et al. Histaminereleasing activity. V. Characterization and puriticat~~ using high-performance liquid chromatography. Cell lm&mol 1984 87:445. Subramanian N, Bray MA. Interteruki~l releases histamine

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80 4

Mononuclear

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