Immunology, 1970, 18, 611.

Effects of Pharmacological Inhibition of HistamineSynthesis upon Immunological Reactions

in Guinea-Pigs

J. P. PHAIR, A. J. EISENFELD, R. J. LEVINE AND F. S. KANTOR

Department of Medicine, University of CincinnatiCollege of Medicine, Cincinnati, Ohio and theDepartments of Medicine and Pharmacology,

rale University School of Medicine, New Haven, Connecticut

(Received 1st July 1969)

Summary. Administration of a specific histidine decarboxylase inhibitor,4-bromo-3-hydroxybenzyloxyamine (brocresine) failed to affect PCA or delayedhypersensitivity reactions in guinea-pigs. Skin histamine levels measured follow-ing PCA reactions, when the skin was refractory to a second stimulus showed nosignificant changes in both treated and untreated animals. Triprolidine, anantihistamine, also failed to significantly alter PCA reactivity. These findings maybe interpreted to mean that the operative pool of histamine is very small, its pro-duction unaffected by brocresine and its action protected from the effects of anti-histamines. Alternatively, it seems more likely that histamine plays little role inthe PCA reactions of guinea-pigs.

INTRODUCTION

The union of antigen and anaphylactic antibody in the skin of guinea-pigs induces atransitory period of increased vascular permeability which has been termed 'passivecutaneous anaphylaxis' (PCA) (Ovary, 1958). The release of the chemical mediator,histamine, has been thought to be responsible for this reaction (Lieberman and Ovary,1968). An increase in histamine concentration at the skin site of delayed hypersensitivityreactions (Prochazka-Fisher and Cooke, 1958) and a rise in urinary histamine before andduring graft rejection have also been reported (Moore and Chang, 1968). The develop-ment of inhibitors of specific histidine decarboxylase, the enzyme that catalyses the con-version of histidine to histamine, has provided a new tool with which to study the role ofthis mediator in antibody and cell mediated reactions. One of these, 4-bromo-3-hydroxy-benzyloxyamine (brocresine), has been shown to be a potent inhibitor of histidine decar-boxylase in vitro and in vivo (Levine, Sato and Sjoerdma, 1965).PCA reactions are followed by a period during which the skin at the site of the antigen-

antibody union is refractory to the induction of a second PCA reaction (Ovary, 1958).One possible explanation for this refractory period would be exhaustion of mediators;the return of the activity might then be due to replenishment of mediators such as hista-mine. If depletion of histamine is responsible for the refractory period, inhibition of histi-dine decarboxylase activity by brocresine should result in prolongation of the refractoryperiod.

611

The present experiments were designed to explore the role of histamine as a mediator ofPCA and delayed hypersensitivity reactions in the guinea-pig. Treatment with brocresinedid not prolong the refractory period to PCA elicited with either rabbit antiserum orguinea-pig yG1 antibody. The implication that agents other than histamine mediate theincreased vascular permeability of PCA reactions was supported by the findings that anantihistamine, triprolidine, was ineffective in suppressing the reaction, and that skinhistamine concentrations did not change significantly after PCA reactions. It was alsofound that brocresine administration did not alter either the intensity or the course ofdelayed reactions in actively or passively sensitized guinea-pigs. This finding was inter-preted as evidence against the concept that histamine functions as a mediator of delayedhypersensitivity reactions.

MATERIALS AND METHODS

AnimalsHartley strain male albino guinea-pigs weighing 200-300 g were used for PCA reactions

in this study. Guinea-pigs from the same strain weighing 400-500 g were immunized foruse in delayed hypersensitivity experiments. Recipients of sensitized cells in the transferexperiments were animals from the same breeding stock weighing 200-300 g.

AntigensConjugates of picryl human serum albumin (Pic-HSA) and picryl guinea-pig albumin

(Pic-GPA) were prepared by modification of a method previously described (Eisen, Bel-man and Carsten, 1953) and previously utilized in this laboratory (Bullock and Kantor,1965). Ovalbumin (OVA) was purchased from Worthington Biochemical Corp., Free-hold, New Jersey.

AntiseraRabbit and guinea-pig antisera were obtained from animals immunized with the

antigens emulsified in Freund's complete adjuvant (Difco Laboratory, Detroit, Michigan)as previously described (Bullock and Kantor, 1965). The rabbit anti-Pic-HSA and anti-OVA produced positive PCA reactions when diluted 1: 1600 and 1: 6400, respectively.The guinea-pig anti-Pic-GPA produced a positive bluing reaction (see below) at a dilutionof 1: 12, 800. Guinea-pig yG1 was prepared from serum of animals immunized with OVAemulsified in Freund's complete adjuvant. This serum was harvested 21 days after im-munization. The y-G1 anti-OVA was separated from (NH4) 2SO4 precipitated y-globulinby starch block electrophoresis. It was identified as a fast moving fraction of y-globulin byimmunoelectrophoresis and did not cause lysis of red cells tanned with OVA in the pres-ence of guinea-pig complement (Bloch, Kourilsky, Ovary and Benacerraf, 1963). Thefraction selected for these experiments gave PCA reactions at a dilution of 1 : 200.

DrugsBrocresine was kindly supplied by Lederle Research Laboratories. The drug was

dissolved in isotonic saline to a concentration of 100 mg/ml and administered intraperi-toneally to guinea-pigs. Triprolidine (Burroughs Wellcome and Co.) was commerciallypurchased and dissolved in saline to a concentration of 1 mg/ml. Doses and concentrationsof drugs were calculated for their free bases.

612 1. P. Phair et al.

Pharmacological Inhibition of Histamine Synthesis 613

Determination of histamine concentrationHistamine was assayed by a modification (Levine and Senay, 1968) of the fluorometric

method (Shore, Burkhalter and Cohn, 1959). Each assay was done in duplicate.

PCA reactionsOvary's method (Ovary, 1958) of performing PCA reaction was utilized throughout

with rabbit antisera and guinea-pig yG1 antibody. In all experiments 0 1 ml of the antiserawas injected intradermally 4 hours before challenge. In the first experiments rabbit anti-Pic-HSA (1: 4000) was first injected intradermally into sites on the right flank. Animalswere challenged with 250 4ug of Pic-HSA in saline intravenously. The reaction sites weremarked and reinjected 1, 4, 20 and 24 hours later with rabbit anti-OVA (diluted 1: 400).At the time of reinjection, control sites on the opposite flank were injected with rabbitanti-OVA at the same concentrations. Five, 9, 24 and 48 hours following the initialreaction 250 ug of OVA in 1 ml of 1 per cent Evans blue dye was administered intra-venously. Perpendicular diameters of the bluing reaction were measured and averaged.This experiment was repeated utilizing guinea-pig anti-Pic-GPA (1: 100) to elicit thefirst reaction and guinea-pig y-Gl anti-OVA (1: 100) in the second.Animals treated with brocresine received the following dose schedules: 25, 50 and 100

mg/kg, administered at 08.00, 13.00 and 18.00 hours, for a total of six doses. At doses of150 mg/kg or greater the drug was toxic to guinea-pigs.The effect of triprolidine treatment upon PCA reactions induced with guinea-pig

yG1 anti-OVA and rabbit anti-OVA in varying concentrations was determined. Thirtyminutes before challenge with OVA the animals received triprolidine 0-8 mg/kg intra-peritoneally and at the time of challenge they received 0-2 mg/kg intravenously. Com-parisons were made between reactions elicited with yG1 and rabbit anti-OVA in treatedand untreated animals. To establish the efficacy of the model, the effect of pretreatmentwith triprolidine upon vascular permeability induced by the intradermal injection of hista-mine (General Biochemicals, Chargin Falls, Ohio) was determined.

Skin histamine concentration at sites of PCA reactions induced with guinea-pig yG1anti-OVA (diluted 1: 50) was measured in animals treated with brocresine and in un-treated controls. Histamine concentration was also assayed in control sites which had beeninjected on the opposite flank with normal guinea-pig serum. The treatment group re-ceived 50 mg/kg of brocresine intraperitoneally every 8 hours for a total of six doses.Both treated and untreated animals were divided into four subgroups killed at 30 minutes,3 hours and 24 hours following challenge with OVA. At the time ofsacrifice the two con-trol and the two challenged sites were excised and histamine concentration was determined.

Delayed reactionsBrocresine, 50 mg/kg every 12 hours, was administered to guinea-pigs for 48 hours

prior to immunization with 100 Mug Pic-GPA in Freund's adjuvant. Treatment was con-tinued until the 5th day after immunization when the animals were challenged with 30ug Pic-GPA in 0 1 ml saline intradermally. Reactions were read at 4 and 24 hours follow-ing challenge. Two perpendicular diameters of the reaction site were measured andaveraged; reactions less than 4 x 4 mm were regarded as negative.

Transfer reactionsIn guinea-pigs used as donors of sensitized cells, a sterile peritonitis was provoked by

intraperitoneal injection of beef heart infusion broth containing 10 per cent proteosepeptone (Difco Laboratories, Detroit, Michigan). Seventy-two hours later the animalswere killed and peritoneum was rinsed with 50 ml of tissue culture medium 199 (BaltimoreBiologic Laboratories, Baltimore, Maryland). Cells were washed in Medium 199, counted,and the final volume was adjusted so that the recipients received approximately 1-5 x 10'cells intravenously. Forty-eight hours before recipients received the sensitized cells, therapywith brocresine, 50 mg/kg every 12 hours was initiated. The guinea-pigs were challengedwith 30 pg of Pic-GPA intradermally on the day of the cell transfer. The longest diameterof the reactions and its perpendicular were measured and averaged 24 hours after chal-lenge.

RESULTS

EFFECT OF BROCRESINE TREATMENT

Following PCA reactions, elicited with rabbit antisera, the reactivity of the site inuntreated animals diminished greatly as seen in Fig. 1. Five and 9 hours after the initialchallenge the average diameter of a reaction was 28 and 45 per cent of reactions elicitedat control sites. At 24 hours reactivity was only slightly decreased and by 48 hours thepreviously challenged sites were 89 per cent as reactive as control sites. Treated animals,as shown in Table 1, showed no appreciable prolongation of unresponsiveness to a secondchallenge on any of the three dose schedules of brocresine. The diameters of the reactionselicited at previously challenged sites 48 hours later were either equal to that seen at con-trol sites or slightly smaller but on average they were equal to the untreated group.

This experiment was repeated using guinea-pig anti-Pic-GPA to elicit the first reactionand guinea-pig yG1 anti-OVA for the second challenge. Again the diameters of the reactionsin animals treated with the histidine decarboxylase inhibitor (50 mg/kg) were similar tothe untreated animals (Table 2).

100 _

80

60C

C

40a.

20

5 9 24 48Hours after initial PCA reaction

FIG. 1. Skin reactivity expressed as percentage of the size of reactions occurring at unchallenged sites5, 9, 24 and 48 hours following initial PCA reaction.

614 J. P. Phair et al.

Pharmacological Inhibition of Histamine SynthesisTABLE 1

EFFECT OF BROCRESINE ON PASSIVE CUTANEOUS ANAPHYLAXIS*

Average diameter of reactionTreatment Time of No. ofGroup rechallenge guinea- Control Previously

(hours) pigs site challenged site

None 5 12 14-2+0-16 3-8+ 1.0None 48 15 13-4+ 1*3 12-0+0-8925 mg/kg 48 4 15-3+ 13 15*5+2*350 mg/kg 48 8 13-8+1-0 11-2+0-6100mg/kg 48 4 12-2+3-3 11-8+3-1

*Rabbit antisera used to elicit skin reactions.Diameter expressed as mm+ SEM.

TABLE 2EFFECT OF BROCRESINE UPON yGj MEDIATED PASSIVE CUTANEOUS ANAPHYLAXIS

Average diameterTreatment Time of No. ofGroup rechallenge guinea- Control Previously

(hours) pigs site challenged site

None 4 4 10-5+0 7 22+0-3None 48 4 112+2-3 9-5+ 1.650 mg/kg 48 4 9-1+2-2 6-6+ 17

Diameter expressed as mm+ SEM.

615

EFFECT OF TRIPROLIDINE UPON PCA REACTIONS

Administration of the anti-histamine did not markedly suppress PCA reactions elicitedat sites injected with yG, antibody varying in dilution from 1: 100 to 1: 25, or hetero-logous (rabbit) antisera, but did reduce the vascular permeability induced by intradermalinjection of histamine (Table 3).

ASSAY OF SKIN HISTAMINE (Table 4)

The concentration of histamine at control sites injected with normal guinea-pig seraremained relatively stable throughout the 24 hours ofthe experiment and was not changedby treatment with brocresine. All of the challenged sites differed insignificantly from theirmatched controls in histamine concentration (less than 15 per cent) except the untreatedgroup biopsied 30 minutes after challenge. These sites had a significantly (P < 0.05) lowerhistamine concentration than the unchallenged site when matched pairs were compared.The mean value of the group as a whole did not vary significantly from the mean of thecontrol group, however. Three hours following the reaction, at a time when sites arerefractory to a second challenge, the histamine concentration was similar to that found at24 hours when the skin was regaining reactivity.

EFFECT ON DELAYED HYPERSENSITIVITY REACTION

Challenge of actively immunized animals elicited a positive reaction at 24 hours in allof the sixteen controls and sixteen brocresine treated animals. The average reaction in the

616 J. P. Phair et al.TABLE 3

EFFECT OF TRIPROLIDINE UPON VASCULAR PERMEABILITY INDUCED BY HISTAMINE, HOMOLOGOUSY1 ANTI-OVA, AND HETEROLOGOUS RABBIT ANTI-OVA ANTIsERuM

Treated* Untreatedaverage averagediameter diameter

Histamine concentration30jg 2+0-7 (4) 5+2-1 (4)

600 ug 3+5-7 (3) 50±0 0 (2)tHomologous antibody

1:25 12±0-96 (14) 13+0-11 (12)1:50 101+19(14) 11-5+0.64(12)1:100 7-3+ 1 4 (14) 8-5± 1*1 (12)

Heterologous anti-serum1:6400 9l1+0-7 (7) 8+ 1-8 (8)1:3200 11+2'1 (7) 10*6+1±1 (8)

Diameter expressed as mm+ SEM. Number of animals in parentheses.* 1 0 mg of Triprolidine before challenge.t An additional animal died in histamine shock.

two groups was 11-4 and 12 6 mm in diameter, respectively. The possibility that circulat-ing antibody may have contributed to the 24-hour skin reaction in actively immunizedanimals prompted an assessment of the effect of brocresine upon the transfer reaction.Six of seven recipients of sensitized cells used as control animals developed a positivereaction at 24 hours. The average diameter of these reactions was 8-6 mm. In sevenof eight brocresine-treated recipients challenge elicited a positive reaction, and theaverage diameter of these reactions was 9-6 mm. Reactions in treated and untreatedanimals were apparent during the first 6 hours following challenge, reached a maximumdiameter at 24 hours, and developed the same intensity of erythema and induration.

TABLE 4SKIN HISTAMINE CONCENTRATION*

Treated with brocresine UntreatedTime

following Challenge Control Challenge Controlchallenge site site site site

30 minutes 2 03+0 26 2.29+0 27 1-50+0 44 2-16+0 223 hours 2.17+0.34 2 43+0 27 211+0.17 2 27+0 433 hours 2-58+ 0-26 2-79+0 40 2 09+0 23 2-31+ 0 26

*Each value represents the mean histamine concentration at skin sites. Histamine is ex-pressed as pug/g of tissue+ SEM.

DISCUSSION

The present studies provide evidence against the hypothesis that histamine is either thesole or even a major mediator of PCA reactions elicited with heterologous rabbit antiseraor homologous yG1 antibody in the guinea-pig. However, as discussed below, these resultsmust be interpreted cautiously.Much of current understanding of histamine biosynthesis and the effects of histidine

decarboxylase inhibitors is derived from studies done in the rat. Histamine exists in rattissues in at least three pools which turnover at different rates (Levine et al., 1965). Acute

Pharmacological Inhibition of Histamine Synthesis 617

administration of histidine decarboxylase inhibitors results in depletion of histamine onlyfrom the rapidly turning over pools. This depletion can be demonstrated only when sucha pool forms a substantial fraction of total histamine content of the tissue being assayed(Levine et al., 1965). In the skin of rat as in other species, histamine concentration correlateswith the presence of mast cells (Riley, 1959). This mast cell pool turns over very slowly;its half-life was estimated as approximately 7 weeks (Schayer and Ganley, 1959). Brockle-hurst, Humphrey and Perry (1955) have reported that virtually complete depletion ofmast cell histamine by Compound 48/80 did not suppress PCA reactions in the rat. Whilethis finding would seem to exclude the mast cell pool of histamine as a mediator of PCAreactions, it is possible that these reactions occur during a period of accelerated histaminesynthesis. Release of histamine from many tissues, including skin, is following by a markedincrease in histidine decarboxylase activity (Schayer, Rothschild and Brozony, 1959).

Less complete information is available on histamine biosynthesis and the effects ofhistidine decarboxylase inhibitors in the guinea-pig. Using a method that does not dis-tinguish between specific and non-specific decarboxylases, Schayer and Ganley (1959)reported very low histidine decarboxylase activity in guinea-pig skin. In this laboratory,using a sensitive method for assay of the specific histidine decarboxylase (Levine andWatts, 1966), no activity has been detected (unpublished). However, the finding of highconcentrations of histamine in a tissue is strong circumstantial evidence that the tissue iscapable of histamine synthesis. In the unlikely event that histamine biosynthesis in theguinea-pig is catalyzed by a non-specific aromatic L-amino acid decarboxylase, brocresinewould still be expected to decrease histamine synthesis since it is also a potent inhibitorof these enzymes (Levine et al., 1965). We were unable to study histidine decarboxylaseinhibition directly owing to inability to detect the enzyme. However, since brocresineinhibits all mammalian enzymes known to be capable of histidine decarboxylation in vitro,we assumed that it would inhibit this process in guinea-pig skin.The finding that histamine levels in guinea-pig skin changed insignificantly after ad-

ministration of brocresine indicates that most of the histamine in guinea-pig skin turnsover slowly. The lack of significant reduction in skin histamine content following PCAreactions may indicate that histamine, if involved in mediating PCA reactions, is from apool that forms a very small fraction of the total skin histamine. The alternate explana-tion may be that a substantial amount of histamine released by PCA reactions is re-plenished rapidly by synthesis ofnew histamine. This possibility is ruled out by the findingthat PCA reactions elicited during brocresine administration also failed to lower histaminelevels.The possible involvement of a very small rapidly turning over pool of histamine in

control of the microcirculation is discussed in a recent review article (Kahlson and Rosen-gren, 1968). These pools, termed 'nascent' or 'induced' histamine by their proponents,purportedly are released and exert their effects immediately upon synthesis. The findingsthat, during administration of brocresine, PCA reactions could be elicited and that re-fractoriness to PCA was not prolonged provides evidence against involvement of a smallrapidly turning over pool of histamine.The lack of significant change in concentration of skin histamine found in these ex-

periments is in contrast to previous studies which reported a 50 per cent decrease in skinhistamine concentration at the site of a PCA reaction in guinea-pigs, and a small transientfall in the histamine content of adjacent but unchallenged skin of experimental animals(Inderbitzen, 1961). In these earlier studies, histamine concentration was determined by

618 J. P. Phair et al.

bioassay; control values were much higher than those in the present experiments whichagree with values reported by other investigators (Riley, 1959; Brocklehurst et al., 1955).The failure of the anti-histamine, triprolidine, to inhibit PCA reactions in guinea-pigs

is not of itself conclusive evidence that histamine does not mediate these reactions. Ananalagous situation is the failure of anti-histamines to block stimulation of gastric acidsecretion by histamine (Kay, 1953). However, in these experiments pretreatment with thedrug did suppress the effects of the intradermal injection of histamine. Another possibleexplanation of the failure of triprolidine is that the serum used was too potent and thereactions produced too intense for attenuation to be appreciated. The yG1 anti-OVA pro-duced PCA consistently at a dilution of 1: 100, and occasionally at 1: 200 in untreatedanimals. Triprolidine treated animals also consistently manifested PCA reactions at sitesinjected with yG1 anti-OVA diluted 1: 100. Thus a minimal PCA reaction was notappreciably altered by triprolidine administration.Our results, while at variance with those reported in guinea-pigs by others (Lieberman

et al., 1968; Movat, DiLorenzo, Taichman, Bergin and Stein, 1967) perhaps are explainedby the recent report that guinea-pig yG1 will release slow reacting substance (SRS-A) fromguinea-pig lung slices (Stechschulte, Austin and Bloch, 1967). SRS-A may be a primemediator of PCA reactions in guinea-pig skin and is uneffected by the action of anti-histamines.

Administration of brocresine did not effect cell mediated delayed reactions suggestingthat histamine is not involved in the mechanisms of delayed hypersensitivity. Previousinvestigators (Prochazka-Fisher and Cooke, 1958) believe the change in histamine, fol-lowing induction of allergic or toxic dermatitis, represents reparative events in the guinea-pig skin. The changes in histamine (Prochazka-Fisher et al., 1958; Inderbitzen, 1961)and histamine-forming capacity, at sites of delayed reactions, and the rise in urinaryhistamine preceeding skin graft rejection (Moore and Chang, 1968) reported by others,may be therefore associated rather than causal alterations.

ACKNOWLEDGMENTS

This work was supported in part by United States Public Health Service Grants Al271, AI 06706, GM 13016 and CA 08341. Dr Kantor is a Research Career DevelopmentAwardee, United States Public Health Service, National Institute ofAllergy and InfectiousDiseases, National Institutes of Health. The excellent technical assistance of Miss DorisWatts and Miss Periann Wood is gratefully acknowledged.

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