Functional depression of H2 histamine receptors in sheep with experimental allergic asthma

Functional depression of Hz histamine receptors in sheep with experimental allergic asthma

Tahir Ahmed, M.D., James P. Krainson, M.D., and Lawrence D. Yerger, B.S.

Miumi Beach, Flu.

We tested the hypothesis that airway hyperresponsiveness to histamine in the allergic sheep is reluted to functional depression of H2 histamine receptors. Thirteen of 32 sheep responded with hronchoconstriction to inhaled Ascaris suum antigen (allergic sheep), and the remainder serbled us controls (nonallergic sheep). In the allergic sheep, 50 and 100 breaths of 5% histamine solution increased mean pulmonary resistance (R,) to 235Yc and 4380/c of baselines, respectively. The corresponding values in nonallergic sheep were 200% and 21 I%, indicating a greater response to the higher dose of histamine in allergic sheep. Selective H,-receptor stimulation with 50 breaths of histamine (pretreatment with the Hz-receptor antagonist metiamide) failed to enhance the effect of histamine in allergic sheep (mean RL increased to 239% of baseline) whererrs it enhanced the histamine response in nonallergic sheep (RI. increased to 438% of baseline). Selective HP-receptor stimulation (pretreatment with the H,-receptor antagonist chlorpheniramine) caused histamine to decrease RI. by 31% in the nonallergic sheep group; it blocked but did not re\serse the histamine effect in the allergic sheep. Similar observations were mude in a different group of animals when selective HI- or selective Hz-receptor stimulation was produced by 100 breaths of histamine. The cutaneous wheal response to intradermal histamine dilutions of 0.0001, 0.001, 0.01, and 1 mglml was similar in both groups. In nonallergic sheep, both chlorpheniramine and metiamide blunted the cutaneous wheal response. In allergic animals, only chlorpheniramine blunted the cutaneous wheal response, whereas metiamide was without effect. We c,onclude that airway hyperresponsiveness to histamine in allergic sheep is related to a functional depression of H2 receptors and that such a defect is observed both in the air\z,ays as well as in the skin. I .I ALLERGY CLIN IMMUNOL 72:3/O-320, 1983.)

Several mechanisms have been proposed for the airway hyperreactivity of bronchial asthma, including diminished beta-adrenergic responsiveness,‘-3 liber- ation of chemical mediators,4 and increased cholinergic responsiveness.” Airway hyperresponsiveness in asthma is seen not only to the specific antigen (“specific reactivity”) but also to other chemical mediators (“nonspecific reactivity”), including acetylcholine,6 prostaglandin FZu,’ and histamine.R

Increased bronchial reactivity to histamine is of

From the Division of Pulmonary Disease, Department of Medicine, Mount Sinai Medical Center, Miami Beach, Fla.

Supported by a grant from Florida Lung Association and National Institutes of Health Young Investigators Grant HL 27798.

Received for publication July 22, 1982. Accepted for publication April 15, 1983. Reprint requests: Tahir Ahmed, M.D., Associate, Division of Fkl-

monary Disease, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140.

310

special interest in view of its role in IgE-mediated allergic asthma. The actions of histamine are mediated by at least two types of receptors.g, lo Histamine receptors that are blocked by pyrilamine (mepyr- amine) have been defined as HI receptors,” whereas the receptors that are blocked by metiamide have been classified as Hz receptors.‘O These receptors have been shown to exist in the airway smooth muscle.“-‘3 Contraction of tracheobronchial smooth muscle is mediated by H, receptors, whereas relaxation is gen- erally mediated by H, receptors.“, I3 Eyre14, I5 and Kleinstiver and Eyre16 reported a preponderance of H, receptors in the tracheal strips, strips of major bronchi, and parenchymal lung strips of sheep, whereas H2 receptors predominated in intermediate-size airways. The administration of selective HI- and Hz- receptor antagonists enabled us to establish the exis- tence of this dual histamine receptor system in the tracheobronchial tree of conscious sheep.‘”

Busse and Sosman” have shown that H2 histamine

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YUMBER 3 l-l2 histamine receptors in experimental asthma 311

Ahhreviutions used R,,: Pulmonary resistance ID: Inner diameter

receptors are decreased in the granulocytes of subjects with bronchial asthma. We therefore hypothesized that the airway hyperresponsiveness to histamine may be related to a functional suppression of Hz receptors, thus enhancing the bronchoconstrictor effect of histamine through unopposed HI-receptor stimulation. We also reasoned that such a defect may also be present in other organs. The purpose of the present investigation was to compare the airway and cutaneous H,- and Hz-receptor function between allergic and nonallergic sheep.

MATERIALS AND METHODS Animal preparation

Thirty-two nonsedated ewes, weighing 32 to 45 kg, were restrained in the prone position, and their heads were im- mobilized with a sling. After topical anesthesia of the nasal passages with 2% lidocaine solution, an esophageal balloon catheter was introduced through one nostril and placed in the lower esophagus. The animals were intubated with a cuffed nasotracheal tube (ID 7.5 mm) through the other nostril with the guidance of a flexible fiberoptic broncho- scope. All sheep had a chronic carotid arterial loop preparation to facilitate sampling for arterial blood gas composition. Thirteen sheep underwent biweekly inhalation challenge with 1 : 20 dilution of Ascaris ~uum extract until reproducible antigen-induced bronchoconstriction was demonstrated. These sheep were designated allergic. The remain- ing 19 sheep served as nonallergic controls. The sensitization procedure has been published in detail previously.‘“-*I Briefly, sheep with natural cutaneous reaction to 1: 1000 or I : 10,000 dilutions of A. suum extract (Greer Laboratory, Lenoir, N.C.) were actively sensitized by inhalation challenge withA. suum extract. For this purpose, the sheep were intubated with a cuffed nasotracheal tube and inhaled an aerosol of a 1 : 20 dilution of A. suum extract for a period of 20 min. Two to three challenges were necessary to produce the antigen-induced bronchospasm previously described for this animal model.‘X-” Biweekly antigen challenge was continued during the study period to maintain the allergic state.

Experimental techniques

Pulmonary resistance. Pleural pressure was estimated with an esophageal balloon catheter.22 The catheter was so positioned that inspiration produced a negative pressure deflection and cardiogenic oscillations were clearly detectable on the pressure tracings. In this position, which was between 5 and 10 cm from the gastroesophageal junction, the end-expiratory pleural pressure ranged between -2 and

-5 cm H,O in different animals. Lateral pressure in the trachea was measured with a side-hole catheter (ID 2.5 mm) advanced through and positioned distal to the tip of the endotracheal tube. Transpulmonary pressure was defined as the tracheal pressure minus pleural pressure and measured with a differential variable reluctance gauge (type P-7D; Pace Engineering, Hollywood, Calif.). For measurement of RL, the proximal end of the endotracheal tube was con- nected to an 8 L dry spirometer (Electromed Model 780; Ohio Instruments), and the volume signal was electrically differentiated to obtain flow. The signals of flow, volume, and transpulmonary pressure were recorded on an Elec- tronics for Medicine (White Plains, N. Y .) multichannel recorder that was linked to a PDP- I 1 digital computer (Digi- tal Equipment, Maynard, Mass.), which received the on- line signals for the calculation of mean Rr.‘:% Peak flow rates were usually less than 1 Lisec. The pressure transducer- catheter system was dynamically balanced, and no phase shift was detectable between pressure and flow up to a frequency of 6 Hz. RL was averaged from five consecutive breaths free of swallowing artifacts.

Arterial blood composition. For arterial blood gas analysis, 2 ml of blood was withdrawn from the carotid loop by a 22-gauge needle. The blood samples were analyzed at 37” for pH, Pco,, and PO, with a Radiometer (model ABL-I; Radiometer, Copenhagen, Denmark).

Histamine skin test. The sheep were restrained in the prone position, and the wool was shaved along their paravertebral area bilaterally. A 27-gauge needle was used for the intradermal injection of 0.02 ml of decreasing dilutions of histamine in duplicates. Each animal also received 0.02 ml of buffered saline injection as a control. Twenty minutes after the histamine injection, the largest wheal diameter (D1) and the diameter perpendicular to this (D.J were measured. The wheal size was expressed as wheal area (mm*) by the equation:

Area = (Dz + D2)*

4

Experimental design

Airway studies. Seven allergic and six nonallergic sheep comprising this study were studied on six different occasions separated by 3 to 5 days. An additional group of six allergic and seven nonallergic animals was studied on 3 different days to evaluate the effect of selective Hi- and HZ-receptor stimulation with 100 breaths of histamine.

EFFECT OF HISTAMINE (EXPERIMENT DAY 1, 2, AND 3). After baseline measurements of RL and Pao,, the sheep inhaled 50 breaths of an aerosol of 5% histamine solution produced by a D-30 generator (mass median aerodynamic diameter 1.4 pm; geometric S.D. 1.7 pm)24 with a fluid output of 0.3 mI/min,2Z and the measurements were repeated immediately. The histamine aerosol was delivered with a Harvard Animal respirator at a fixed tidal volume of 500 ml and frequency of 20imin. To establish reproducibility, the response to 50 breaths of histamine was performed on two occasions (experiment day 1 and 2). On day 3, the histamine challenge was performed with 100 breaths.

312 Ahmed et al. J ALLERGY CLIN. IMMUNOL.

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AOO-

iiT z

2 300-

:

; 200-

I*

loo-

I - ALLERGIC

00 BASELINE HISTAMINE HISTAMINE

(501 (lOOi

FIG. 1. Comparative effects of 50 and TOO breaths of histamine (5% solution) on RL in nonallergic sheep and sheep allergic to A. suum antigen. Data are shown as mean; vertical bar represents S.E.M. *Significantly different from baseline p < 0.01. tsignificantly different from nonallergic animals p < 0.05.

MODIFICATION OF HISTAMINE EFFECT BY H, ANTAGONIST (EXPERIMENT DAY 4). After the baseline measurements of RL and Paop, the sheep were pretreated with the H, antagonist chlorpheniramine (Schering, Kenilworth, N. J.), diluted with physiologic saline to a volume of 50 ml. Chlor- pheniramine (2 mg/kg) was infused intravenously over 5 min,‘” and the baseline measurements were repeated 15 min later. Histamine challenge (50 breaths) was then performed, and the measurement was repeated as with histamine alone.

MODIFICATION OF HISTAMINE EFFECT BY H2 ANTAGONIST (EXPERIMENT DAY 5). HZ blockade was achieved with metiamide (3 mg/kg), given as a single intravenous bolus diluted in 10 ml of normal saline.r3 Baseline measurements of RL and Pao, were obtained before and 10 min after metiamide injection. Histamine challenge (50 breaths) was then performed and the measurements repeated as with histamine alone.

MODIFICATION OF HISTAMINE EFFECT BY COMBINED H, AND H2 ANTAGONISTS (EXPERIMENT DAY 6). After obtaining the baseline measurements, the animals were pretreated with both chlorpheniramine and metiamide as described above. Repeat baseline measurements were obtained 10 to 15 min later. Histamine challenge (50 breaths) was then performed, and the measurements were repeated as with histamine alone.

SELECTIVE H,- AND ~~~~~~~~~~ STIMULATION WITH 100 BREATHS OF HISTAMINE. These additional experiments were conducted in a different group of nonallergic animals (n = 7) and allergic animals (n = 6). Each animal was studied on 3 different days. On day 1, a control histamine challenge with 100 breaths was performed, and measurements of RL were obtained before and immediately after the challenge. On days 2 and 3, the histamine challenge

100

80

40

20

-

1-1 NON-ALLERGIC

I - ALLERGIC

0

BASELINE HISTAMINE HISTAMINE PO) ~1OOl

FIG. 2. Comparative effects of 50 and 100 breaths of histamine (5% solution) on arterial p02 in nonallergic and allergic sheep. Data are shown as mean; vertical bars represent SEM. *Significantly different from baseline p < 0.01. tsignificantly different from nonallergic animals p i 0.05.

(100 breaths) was repeated after pretreatment with H, antagonist chlorpheniramine (day 2) and H, antagonist metiamide (day 3). The doses of antagonists and the measurements of RL were obtained in the same sequence as with 50 breaths of histamine (vide supra). No measurements of Pao, were made during these experiments.

Skin tests. Seven allergic sheep that had been used for the airway study were selected for cutaneous testing. The six nonallergic control sheep did not take part in the previous airway study. Each animal was studied on four different occasions separated by 3 to 5 days. On day 1, a baseline histamine skin test was performed with decreasing histamine dilutions of 0.0001, 0.001, 0.01 and 0.1 mglml. The wheal size was determined after 15 min. The sheep were pretreated with chlorpheniramine (2 mg/kg) on day 2, with metiamide (3 mg/kg) on day 3, and with both chlorpheniramine and metiamide on day 4. Histamine skin tests were performed IO to 15 min after the antagonist pretreatment, and the wheal size was determined as without antagonist pretreatment.

Statistical analysis

Data were expressed as mean ? S.D. For the control histamine challenges, the means within the two groups were analyzed by a paired t test, whereas the means between the two groups were compared by an unpaired t test. The means within the different groups after various antagonist pre- treatments were analyzed by analysis of variance (block design), and a Newman-Keuls test was used for pairwise comparison of means. A two-way analysis of variance was performed for analyzing and comparing skin test results of normal and allergic groups at various histamine dilutions

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TABLE I. Comparative effect of histamine (100 breaths of 5% solution) on RL and PaO, in nonallergic

and allergic sheep

Baseline

RL (cm t+OlL/sec)

After histamine Baseline

Pa02 (mm Hg)

After histamine

Nonallergic

Allergic

p value

1.42 p < 0.005 3.00 98.0 p < 0.005 61.6 (0.33) (1.12) (11.2) (17.2) 1.13 p < 0.005 4.95 92.0 p < 0.0001 44.8

(0.38) (1.9) (9.3) (4.9) N.S. co.05 N.S. co.05

Values are mean with S.D. in parentheses.

without and with antihistamine pretreatment and completed with a Newman-Keuls test for pairwise comparison of means .26

RESULTS

The mean baseline values for RL and Pao, were comparable on different experimental days, and there were no significant differences within each group or between the allergic and nonallergic groups.

Effect of combined HI- and Hz-receptor stimulation

On two different occasions, inhalation of 50 breaths of 5% histamine had highly reproducible effects on RL in individual sheep. Fig. 1 shows the changes in mean RL. With 50 breaths of histamine, mean RL increased to 200% and 235% of baseline in nonallergic and allergic animals, respectively @ < 0.01). With 100 breaths of histamine, mean RL increased to 211% of baseline in the nonallergic group (p < 0.005) and to 438% of baseline in the allergic group (p < 0.005) (Fig. 1 and Table I). The increase in RI> after 100 breaths of histamine was significantly greater in the allergic than in the nonallergic group (p < 0.05).

As shown in Fig. 2, with 50 breaths of histamine the mean S.D. Pao, decreased from 95 (6) and 92 (9) mm Hg to 63 (11) and 57 (14) mm Hg in nonallergic and allergic animals, respectively (p < 0.01). With 100 breaths of histamine in nonallergic animals, mean Pao, decreased to 61.6 (17.2) mm Hg (p < O.OOS), whereas in allergic animals, it decreased to 44.8 (4.9) mm Hg (p < 0.0001) (Fig. 2 and Table I). With 100 breaths of histamine, the decrease in Pao, in the allergic group was significantly greater than in the nonallergic group (p < 0.05).

Effect of selective receptor stimulation (50 breaths of histamine)

H2 receptor. Chlorpheniramine pretreatment by itself increased mean RL to 182% and 175% of baseline

in nonallergic and allergic animals, respectively (p < 0.05) (Fig. 3). In nonallergic sheep, the subsequent histamine challenge decreased mean RL by 3 1% from the postchlorpheniramine value @ < O.OS), whereas in allergic sheep, mean RL decreased by only 11% (P = NS) (Fig. 3 and 4).

In both nonallergic and allergic groups, chlorpheniramine pretreatment partly protected against the histamine induced decrease in Pao,. Mean S.D. Paoz decreased to 70 (6) mm Hg and 70 (8) mm Hg (p < 0.05) in the nonallergic and allergic groups, respectively (Fig. 5). No significant difference between the two groups was observed.

HI receptor. The Hz antagonist metiamide by itself had no significant effect on mean RL in either group (Fig. 6). In the nonallergic sheep, the subsequent histamine challenge increased mean RL to 438% of the postmetiamide value (p < 0.01) (Figs. 4 and 6); this was significantly greater than the increase to 200% of baseline with histamine alone (p < 0.01). In the allergic sheep, metiamide pretreatment failed to enhance the effect of histamine on mean RL, which increased to 239% of baseline @ < 0.001) (Figs. 4 and 6). This was not significantly different from 235% of baseline increase observed with histamine alone (p = NS).

Similarly, selective H, stimulation enhanced the effect of histamine on Paoz only in nonallergic sheep; mean Pao, decreased to 44.6 (5.6) mm Hg (p < 0.001) (Pig. 5), which was significantly lower than 63 (11) mm Hg observed with histamine alone (p < 0.05). In the allergic sheep, selective H, stimulation failed to enhance the decrease in Pao,; mean Pao, decreased to 54 (10) mm Hg (p < O.OOS), which was not different from 57 (14) mm Hg observed with histamine alone (p = NS).

Modification of histamine effect by combined HI and H2 antagonists

In both the nonallergic and allergic groups, combined pretreatment with chlorpheniramine and meti-

314 Ahmed et al. J. ALLERGY CLIN. IMMUNOL.

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4oo _ NON-ALLERGK

O- BASELINE CHLORPHENIRAMINE HISTAMINE BASELINE CHLORPHENlkAMlNE HISTAMME

FIG. 3. Comparative effects of selective HP-receptor stimulation (H, blockade) on RL in nonallergic and allergic sheep. Data are shown as mean: vertical line represents S.E.M. *Significantly different from baseline p < 0.05.

amide caused a significant increase in mean RL. How- ever, the effects of subsequent histamine challenge were completely blocked (Fig. 7).

Selective HI- and Hz-receptor stimulation (100 breaths of histamine)

The results of these additional experiments are shown in Table II. With 100 breaths of histamine alone, the increase in mean RL was significantly greater in the allergic group (360%) than in the nonallergic group (230%). Although in the nonallergic group, selective H, stimulation (metiamide pretreatment) enhanced the effects of histamine, in the allergic group, metiamide failed to enhance the effects of histamine. Similarly, selective H, stimulation (chlorpheniramine pretreatment) caused a significant decrease in RL in the nonallergic group, whereas no such decrease in the allergic group was observed.

Skin tests

In nonallergic animals, the mean S.D. wheal sizes caused by intradermal histamine dilutions of 0.0001, 0.001, 0.01 and 0.1 mg/ml were 41 (ll), 72 (19), 134 (27), and 235 (56) mm2, respectively, whereas in allergic animals the mean wheal sizes produced by the same dilutions were 31 (7), 59 (9), 107 (22), and 220 (52) mm’, respectively (Fig. 8). No significant difference was observed between the two groups. In both groups, the H1 antagonist chlorpheniramine shifted the histamine dose-response curve to the right, with a significant decrease in wheal size at all histamine concentrations (p < 0.05). In the nonallergic animals, the H2 antagonist metiamide also shifted the histamine dose-response curve to the right and significantly decreased the histamine-induced wheal size at all concentrations (p < 0.05). However, in the allergic

animals, metiamide failed to decrease the histamine- induced wheal size except at the highest concentration of 0.1 mg/ml; otherwise, the dose-response curve was not different from that of histamine alone.

DISCUSSION

Among the various proposed mechanisms of airway hyperreactivity in bronchial asthma, the p-adrenergic blockade theory of Szantivanyi’ and cholinergic hyperresponsiveness” have received the most attention. However, no unifying hypothesis explains all the experimental observations. Bronchoconstric- tion in asthma may develop as a result of many stimuli, including “specific ” allergens and various “nonspecific ” mediators. Histamine is perhaps the most important mediator released during airway anaphylaxis in allergic sheepls and perhaps in other species, with both direct and indirect effects. The results of the present investigation suggest that in sheep allergic to A. suum antigen, airway hyperreactivity to inhaled histamine is related to functional depression of Hz histamine receptors and that this defect is paralleled by the absence of cutaneous HZ-receptor response.

Airway hyperresponsiveness to histamine

A characteristic feature of patients with bronchial asthma is the presence of bronchial reactivity in response to nonspecific stimuli, including cholinergic agents and histamine. 27 To serve as an accurate physiologic and immunologic analogue of human asthma, an animal model of this disease should exhibit this characteristic feature of increased airway reactivity.

In the present investigation, we showed that sheep sensitized with an aerosol of A. suum antigen demonstrate increased bronchial reactivity to inhaled hista-

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iii Z 3 % 4 m 100

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FIG. 4. Comparative effect of histamine (50 breaths) on RL in nonallergic and allergic animals, without and with antihistamine pretreatment. Histamine alone (open bars); histamine followed by metiamide pretreatment (closed bars); histamine after chlorpheniramine pretreatment (hatchedbars). Data are shown as mean with SE. *Signif- icantly different from histamine alone p < 0.05. tsignifi- canny different from the nonallergic group p < 0.05.

mine vs. nonsensitized controls as related to the dose of histamine administered. Although 50 breaths of5% histamine failed to differentiate between the two groups, with 100 breaths of histamine a much greater increase in RL was observed in the allergic animals (438%) as compared to the nonallergic animals (211%). Similarly, a much greater decrease in Pao, was observed in the allergic group, i.e., 47 mm Hg vs. 36 mm Hg in the nonallergic group. To avoid the potential problem of tachyphylaxis associated with multiple doses of histamine bronchoprovocation, we gave histamine in a single-dose fashion on two different days, i.e., 50 and 100 breaths of histamine. The increase in RL at a given dose was determined as a measure of “reactivity. ” These findings are consistent with the previously published reports from our laboratory, in which allergic sheep showed enhanced bronchial reactivity to larger doses of carbacholzO and methacholine,” thus further validating the experimental sheep asthma model. I8

Patterson et alszs by using rhesus monkeys allergic to A. suum antigen have shown that animals with bronchial reactivity to inhaled antigen demonstrated a lowered threshold to carbachol. Sensitized guinea pigs did not demonstrate enhanced bronchial reactivity to histamine before antigen challenge, although they temporarily exhibit enhanced reactivity after sub- lethal inhalations of antigen.30 Others, however, have failed to demonstrate enhanced airway reactivity to histamine and methacholine in dogs and monkeys

r” t t z I

0 HISTAMINE

- METIAMIDE + HISTAMINE

= CHLORPHENIRAMINE + HISTAMINE

FIG. 5. Comparative effects of histamine (50 breaths) on arterial p0, in nonallergic and allergic sheep, without and with antihistamine pretreatment. Histamine alone (open bars); histamine after metiamide pretreatment (closed bars); histamine after chlorpheniramine pretreatment (hatchedbars). Data are shown as mean with SE. *Signif- icantly different from histamine alone. tsignificantly different from the nonallergic group.

sensitized to A. suum antigen.31, 32 Species differences, variation in the distribution of airway histamine receptors, specific experimental design, and particularly doses of histamine or carbachol administered are some of the factors responsible for this variable response.

There are obvious differences between human asthma and the experimental asthma. At least in the sheep model, hyperreactivity is only detectable at higher doses of histamine. Similar observations have been made with carbachol and methacholine.20* 28 This is in contrast to human allergic asthma in which hyperreactive subjects are easily separated at lower doses of agonists. We have no explanation for this repeatedly observed phenomenon.

For the control experiments, only 100 breaths of histamine were used to demonstrate airway hyperreactivity to histamine in the allergic group. In subsequent experiments with the antagonists, 50 breaths of histamine were administered to avoid the theoreti- cal possibility of receptor saturation, so that enhancement or absence of enhancement could be demonstrated with selective H, stimulation. However, similar observations were made in a different group of animals when selective Hi- and selective HZ-receptor stimulation (antagonist pretreatment) was achieved with 100 breaths of histamine.

Suppression of airway Hz receptors

Our investigations indicate that airway hyperreactivity to inhaled histamine in sheep with experimental allergic asthma is related to functional suppression of H&istamine receptors. This conclusion is based on


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500 - NON-ALLERGIC ALLERGIC

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0 , I I I I I BASELINE METIAMIDE HISTAMINE BASELINE METIAMIDE HISTAMINE

FIG. 6. Comparative effects of selective H,-receptor stimulation (H, blockade) on RL in nonallergic and allergic sheep. Data are shown as mean; vertical bars represent S.E.M. *Significantly different from baseline. tsignificantly different from allergic group.

0 I I I I I I

SAsELlrE cnloRmEN HISTAWJNE MSELINE CWLOEPHEN HISTAMINE

MEllLE +

MEMMIOE

FIG. 7. Modification of histamine (50 breaths) effects on RL by combined H, and Hz-receptor blockade in nonallergic and allergic sheep. Data are shown as mean with S.E.M. shown as vertical bars. *Significantly different from baseline.

the absence of Hz-mediated effects of histamine in the airways of sheep allergic to A. suum antigen. Airway effects of histamine are such that stimulation of HI receptors causes bronchoconstriction whereas stimulation of Hz receptors mediates bronchodilatation13; the net response is thus a composite of these two opposing effects. Therefore selective stimulation of HI receptors with histamine in the presence of Hz blockade (metiamide pretreatment) will produce enhanced bronchoconstrictor response. Similarly, selective stimulation of H, receptors with histamine, in the presence of H, blockade (chlorpheniramine pretreatment) will produce a bronchodilator response.‘3 In the present investigation, enhancement of bronchoconstrictor response (selective H, stimulation) and presence of bronchodilator response to 50 and 100 breaths of histamine (selective H, stimulation) in

control animals were evidence for the presence of functional airway H, receptors, whereas absence of such responses strongly suggested functional suppression of Hz receptors in sheep with airways hyperresponsiveness to A. suum antigen.

Changes in Pao, correlated with the changes in Rr. Development of hypoxemia with histamine is related to ventilation-perfusion inequality. Although we did not measure N2 clearance delay in the present investigation, the fall in Pao, in normal animals correlated well with our previously reported observations regard- ing N, clearance delay. l3 Selective H, stimulation (metiamide pretreatment) enhanced the histamine-induced decrease in Pao, in normal animals (Pao2 45 mm Hg), whereas no such enhancement was seen in the allergic animals (Pao, 54 mm Hg). We interpreted these changes as enhancement of peripheral airway

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TABLE II. Comparative effects of histamine (100 breaths) RL (cm I-WI-h4 without and with

antihistamine pretreatment in allergic and nonallergic animals

Baseline After

antagonist Histamine

.4llergic t------------p < 0.005 -I

Control (n = 6) 1.08 * 0.31 3.89 k 1.22* Metiamide pretreatment t-----------p < 6.05’ (selective H, stimulation) 0.82 ‘- 0.12 1.31 ? 0.32 3.84 2 0.95* (n = 5) l-p < 0.05-l t--p < 0.05w Chlorpheniramine pretreatment I--p < 0.05 PI (selective H, stimulation) 0.85 +- 0.24 1.76 k 0.34 1.67 ir 0.56* (n = 5) t------p < O.Ol+ +--p = NSW

Nonallergic

I p < 0.005F Control (n = 7) 1.02 2 0.36 2.35 t 0.89 Metiamide pretreatment 1-p <0.05~’ (selective HI stimulation) 0.92 +- 0.22 1.47 k 0.65 10.7 k 5.4

(n = 7) l-----p < 0.05* t-----p < 0.05----l Chlorpheniramine pretreatment -P= NS-1 (selective H2 stimulation) 0.96 ? 0.24 2.2 + 0.92 0.90 * 0.20 (n = 7) Vp < 0.05+ ‘7-p < 0.05-i

Values are mean 2 S.D. *Significantly different from the nonallergic group (p < 0.05)

constriction (with selective H, stimulation) in normal animals and absence of such enhancement in the allergic sheep as indirect evidence of functional depression of H2 receptors in the peripheral airways.

Observations of Abraham et al.lg from our laboratory are consistent with this concept. These investigators showed that in allergic sheep the H, antagonist metiamide failed to enhance the antigen- induced bronchoconstriction. Similar observations have been made by others in sensitized dogs33, 34 and guinea pigs. 35 In the latter studies 33-35 cimetidine and metiamide failed to enhance he antigen-induced bronchoconstriction. Others have shown in guinea pigs that metiamide did not enhance the antigen-induced bronchospasm, whereas cimetidine did but only at high doses. 36 In contrast to these investigations I97 33, 36 Drazen et al.37 and Advenier et al.38 en- hanc:d the antigen-induced anaphylactic response in guinea pigs with metiamide and burimamide but not with cimetidine.37 This variability might be related to mode of action and dose of these compounds37 or to variation in the immunologic and experimental techniques. However, in none of these studies was the effect of sensitization on the H, and Hz-receptor function investigated.

In human subjects with allergic asthma, a temporary enhancement of bronchial reactivity to histamine after antigen exposure, lasting many days, has been

observed. A similar temporary enhancement of airway reactivity to histamine lasting up to 1 day has also been observed in allergic sheep.3s The Hz receptors that were functionally depressed before antigen challenge did not explain this enhancement.3g How- ever, to avoid any such potential problems related to antigen challenge, no experiments were performed for 5 days after the inhalation challenge with antigen.

It is difficult to extrapolate these findings to human subjects, partly because of the inherent differences between the clinical asthma and animal models of asthma and partly because of conflicting data in humans. In humans with oral cimetidine as the Hz antagonist, protection, enhancement, and no effect against histamine-induced bronchoconstriction have been found.40-42 The reason for these conflicting findings in human subjects is not clear; variation in dose of the antagonist, route of administration, and use of cimetidine rather than metiamide may be some of the factors responsible. An unexpected finding in a study by Nathan et al. 42 was that effects of a combination of chlorpheniramine and metiamide did not differ from placebo. Similarly, Eiser et a1.40 observed that cimetidine, like chlorpheniramine, partly protected against histamine-induced bronchoconstriction. This might have been related to an HI-blocking effect of this drug. In our laboratory, we have observed that cimetidine at higher doses blocked the histamine-in-


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- HISTAMINE

--- METIAMIDE t HISTAMINE

‘***’ CHLORPHENIRAMINE t HISTAMINE

,001 .Ol .l .OOOl ,001

HISTAMINE CONCENTRATION

Ifw/mU

.Ol

FIG. 8. Modification of histamine skin test response by H, and H,-receptor antagonists in nonallergic and allergic sheep. Data are shown as mean with S.E.M. shown as vertical bars.

duced bronchoconstriction in sheep, whereas enhancement was seen at smaller doses,4” showing thereby that this drug acts like an H, antagonist at lower doses and like an H, antagonist at higher doses.

Suppression of cutaneous Hz receptors

In the present investigation, we also found that HZ- receptor-mediated responses in the skin of allergic sheep were also suppressed. Although, in normal sheep, the H2 antagonist metiamide partly suppressed the histamine skin wheal as indicated by a blunting of dose-response curve; in allergic sheep, metiamide failed to alter the histamine wheal size, suggesting suppression of cutaneous H, receptors. The H, antagonist chlorpheniramine partly suppressed the histamine skin wheal in both nonallergic and allergic sheep. The skin response to histamine correlated well with the airway studies and suggested that in the experimental sheep asthma model, suppression of H, response is not limited to the airways but also found in the skin. This extrapulmonary (cutaneous) defect in H, receptors is consistent with the observation of Busse and Sosman17 who found depression of HZ receptors on polymorphonuclear neutrophils of subjects with asthma.

The skin wheal-and-flare response to an injection of intradermal histamine involves local alteration in capillary permeability and vasodilatation. Both H, and H2 receptors seemed to be involved in this response.44-47 In monkeys46 and guinea pigs,4s H1 antagonist partly blunted the response, whereas Hz an-

tagonist alone had no significant effect. However, in combination, H2 antagonist potentiated the suppression achieved by H, antagonist. Similar findings were reported in normal humans.44 In contrast, Marks and Greaves47 showed that the histamine-induced wheal- and-flare response in nonatopic individuals could be modified by HZ-receptor antagonist alone, whereas Nathan et al.42 were unable to modify the histamine skin response with cimetidine in subjects with bronchial asthma. The findings of Marks and Greaves47 and Nathan et aL4* are consistent with our results in normal and allergic sheep, respectively, and support the concept that in allergic asthma skin H, receptors are also suppressed.

Hz-receptor suppression and airway hyperreactivity

H, receptors have a modulating role in the media- tion of airway responses to histamine. By causing bronchodilation, stimulation of H2 receptors opposes HI-receptor-mediated bronchoconstriction. If, in allergic asthma, H2 receptors are suppressed, unopposed HI-receptors stimulation would occur, causing enhanced bronchoconstriction and thus producing airway hyperresponsiveness to histamine. However, it is important to note that H,-receptor suppression does not explain the nonspecific bronchial hyperreactivity to other agents.

Both chlorpheniramine and metiamide caused an increase in RL. This is in contrast to the findings of Popa, who observed bronchodilatation after intrave-

VOLUME 72


nous administration of chlorpheniramine in human subjects. The discrepancy between our findings and those reported by Popad may be related to a smaller dose of chlorpheniramine used by the latter investi- gator (total dose 10 mg). Hawkin?‘” reported that smaller doses of antihistamine may relax bronchial smooth muscle, whereas larger doses cause constriction of bronchial smooth muscle in vitro. In our experiments, the increases in Rr, caused by chlorpheniramine and metiamide were blocked by atropine.

We thank Dr. John Paul of Smith, Kline & French Lab-

oratories for supplying us with generous quantities of metiamide and Mrs. Esther Ferguson for her devoted assis- tance in the typing of this manuscript

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Functional depression of H2 histamine receptors in sheep with experimental allergic asthma