Clin. exp. Immunol. (1978) 32, 283-289.

Anaphylactic bronchoconstriction in rats immunized with antigen-coated latex particles R. P. EADY, S. TRIGG & T. S. C. ORR Life Sciences Department, Fisons Pharmaceutical Division, Loughborough, Leicestershire

(Received 28 November 1977) SUMMARY

Studies have been carried out on the immunization of rats with antigen-coated latex particles. These studies confirmed that a significant lung and blood eosinophilia is induced by a repeat intravenous injection of antigen-coated latex particles. Rats treated with antigen-coated latex particles also become sensitive to antigen such that on antigen challenge the animals show bronchoconstriction. Studies show that the bronchoconstriction is specific for antigen, but is unlikely to be mediated by tissue-fixing antibodies. Experiments comparing different immunization schedules involving latex particles indicate a strong association between the level of eosinophilia and degree of antigen-induced bronchoconstriction. These studies suggest that eosinophils may contribute to the anaphylactic bronchoconstriction. INTRODUCTION Allergic reactions, including asthma, are associated with a tissue eosinophilia (Felarca & Lowel, 1971; Turner-Warwick, 1971). Although the function of the eosinophil in these reactions is poorly understood, it has been proposed that the eosinophil exerts a regulatory role (Austen & Orange, 1975). Few models of allergic reactions include eosinophilia, and studies on eosinophils have concentrated on those associated with parasitic infections. Basten & Beeson (1970) showed that the eosinophilia associated with Trichinella infection is under thymus control, and more recently Mahmoud, Warren & Peters (1975) have shown the eosinophil to be the effector cell involved in the antibody-dependent cell-mediated immunity to Schistosoma mansoni. In setting up a model of asthma, we have been interested in methods of inducing lung eosinophilia. Schriber & Zucker-Franklin (1974, 1975) reported that the pulmonary embolization of antigen-coated latex particles induced a blood and lung eosinophilia. These authors also showed that there was a high correlation between the blood eosinophilia and lymphocyte responsiveness to antigen. The purpose of this study was to confirm and extend the observations of Schriber & Zucker-Franklin (1974, 1975), and to determine whether bronchoconstriction could be induced in animals previously treated with antigen-coated latex particles. We have also investigated any possible associations between bronchoconstriction and eosinophilia. MATERIALS AND METHODS Immunization. Sprague-Dawley 200 g female rats (Charles River) were used for these studies. Animals having a blood eosinophil count, before treatment, above 150 per [lI were not included in experiments. Immunization was carried out essentially as described by Schriber & Zucker-Franklin (1974). Latex particles, 45 Jm in diameter (Dow Chemical Co.), were adsorbed with bovine gamma globulin (BGG) Cohn fraction II by incubating particles in a 3 0 mg/ml solution for 1 hr at room temperature. The washed particles (3 x 105 per animal) were injected via the lateral tail vein. Identical booster injections were given 13 days after the primary injection. Correspondence: Dr R. P. Eady, Fisons Pharmaceutical Division, Research and Development Laboratories, Bakewell Road, Loughborough, Leicestershire. 0099-9104/78/0500-0283$02.00 (© 1978 Blackwell Scientific Publications

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Blood cell counts. Venous blood from the lateral tail vein was collected into EDTA tubes (Teklab). The total leucocyte count was determined using a Coulter counter and eosinophils measured using the method of Discombe (1946). Lung lavage cells. Animals were anaesthetized with sodium pentobarbitone (60 mg/kg) and the lungs lavaged, via the trachea, with 10% foetal calf serum in phosphate-buffered saline (15 ml/kg). After 1 min the solution was removed from the lung. A differential cell count was carried out on a concentrated suspension of cells using the method of Kimura, Moritani & Tanyaki (1973). Antibodies. Haemagglutinating antibodies were determined using the tanned red cell technique with the modification of Scott & Gershon (1970), allowing estimation of total and mercaptoethanol-resistant antibody. Skin-fixing antibodies, heatlabile long-term and short-term heat-stable, were measured by the technique of passive cutaneous anaphylaxis (Mota, 1963). Measurement of bronchoconstriction. Animals were anaesthetized with sodium pentobarbitone (60 mg/kg) intravenously. The trachea was cannulated and the rat ventilated with a Palmer miniature Ideal respiratory pump in a closed system. Tracheal pressure was recorded through a side-arm of the cannula connected to a differential air pressure transducer (UPI, Pye Ether Ltd). The respiratory rate was set at 90 strokes per min with a tracheal pressure of 6 cm water (Stotland & Share, 1974). Animals were challenged intravenously with antigen (25 mg/kg) and the subsequent increase in tracheal pressure expressed as a percentage of maximum possible degree of bronchoconstriction (APCB max.), measured by clamping the trachea as described by Stotland & Share (1974).

RESULTS Injection of 3 x 105 BGG-coated latex particles intravenously on days 0 and 13 induced a slight increase in the numbers of blood eosinophils on day 5 and a greater eosinophilia on day 18 (Fig. 1). Control animals given latex particles alone, or heat-aggregated BGG, gave only a small or no eosinophilic response. The blood eosinophilia following primary and secondary injections of BGG-coated latex was not associated with changes in total leucocyte count. To investigate the relationship between blood eosinophilia and cell changes in the lung following latex-BGG treatment, the technique of lung lavage was used to obtain a quantitative estimate of lung cells. A primary injection of BGG-coated latex caused an obvious infiltration of mononuclear cells and neutrophils (Fig. 2). Only small changes in lung cells were observed in animals injected with latex alone or heat-aggregated BGG. In contrast, a second injection of latex-BGG (day 13) induced a significant increase in eosinophils 48 hr after injection and 72 hr before the blood eosinophilia (Fig. 2). 600 A

500 400 A

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w0/ 200 100

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10 12 14 16 18 20 22 Days FIG. 1. Blood eosinophil counts after immunization on days 0 and 13 with: (A) 300,000 BGG-coated latex particles; (A) 300,000 particles oflatex alone; (0) 1 0 mg heat-aggregated BGG (n = 6). 4

6

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Bronchoconstriction in latex-treated rats

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Changes in other cell types were similar or less noticeable than those observed following the primary injection of latex-BGG. Control animals given latex alone or heat-aggregated BGG showed small or no changes in eosinophil numbers (Fig. 2).

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FIG. 2. Lung lavage cells after immunization on days 0 and 13 with: (A) 300,000 BGG-coated latex particles; (U) 300,000 particles of latex alone; (0) 1 0 mg heat-aggregated BGG (n = 6). (a) Eosinophils; (b) neutrophils; (c) mononuclear cells.

Antibodies Rats injected on days 0 and 13 with BGG-coated latex particles formed serum haemagglutinating antibodies to BGG. These antibodies rose throughout treatment to give a titre of 1/64 (1/16, mercaptoethanol-resistant) 8 days following the second injection. Similar levels of haemagglutinating antibodies were found in the serum from animals treated with heat-aggregated BGG. However, serum taken on days 5, 12, 14, 18 and 21 from animals injected with latex-BGG (days 0 and 13) showed no detectable levels of 48 hr heat-labile skin-fixing antibody to BGG. Also no 2 hr heat-stable skin-fixing antibody was detected in serum taken on day 21.

R. P. Eady, S. Trigg

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To investigate the possibility that antibodies were being produced locally, in the lung, the supernatant from lung lavages was taken (day 21) for antibody estimation. Lavage supernatants, from animals injected with latex-BGG, and concentrated five-fold by freeze-drying, gave no positive 48 hr PCA reaction, but they did agglutinate BGG-coated sheep red blood cells.

Antigen-induced bronchoconstriction Experiments were carried out to determine whether animals injected on days 0 and 13 with antigencoated latex particles would respond on antigen challenge. Rats treated with latex-BGG were found to show bronchoconstriction when challenged on day 21 with BGG (Table 1). In order to determine the TABLE 1. Bronchoconstriction in rats immunized on days 0 and 13 with 300,000 BGG-coated latex particles, 300,000 particles of latex or 1 0 mg heat-aggregated BGG and challenged on day 21 with 25 mg/kg BGG; human gamma globulin (HGG) or egg albumin (EA)

Immunization Latex-BGG

Latex

Heat-aggregated BGG

Intravenous challenge

No. of responders

BGG HGG EA BGG HGG EA BGG

6/6 0/6 0/6 3/6 3/6 0/6 0/6

HGG EA

2/6 0/6

A PCB max (mean±s.e.m.)

45-6±9-6 7-9±6-5 13-3±6-7

3-6±1-7 -

specificity of this bronchoconstriction, rats treated on days 0 and 13 with BGG-coated latex particles, latex alone or heat-aggregated BGG were challenged on day 21 with BGG, human gamma globulin or egg albumin. Anaphylactic bronchoconstriction was only consistently observed in animals treated with BGG-coated latex particles and challenged with BGG (Table 1). A time-course study showed that this bronchoconstriction was optimum when animals were challenged 6 to 8 days after the second injection of latex-BGG. Because some animals treated with latex particles alone also responded to antigen challenge (Table 2), it was possible that injections of latex particles induced a state of bronchial 'hyperreactivity'. To investigate this possibility, the 5-hydroxytryptamine response in rats injected with latex-BGG, latex alone or heat-aggregated BGG (days 0 and 13) was measured 7 days after the second injection. No TABLE 2. Blood eosinophil counts (day 18) and bronchoconstriction following BGG (25 mg/kg) challenge (day 21) in animals immunized on days 0 and 13 with 300,000 antigen-coated latex particles, 300,000 particles of latex or 1 0 mg heat-aggregated BGG (n = 8)

First injection

Latex-BGG Latex-BGG Latex-BGG Latex Latex-BGG Heat-aggregated BGG

Second injection

Latex-BGG Latex-bovine serum albumin Latex Latex Heat-aggregated BGG Latex-BGG

Blood eosinophil (cells per ,ul) (mean± s.e.m.)

A PCB max.

(mean±s.e.m.)

348±21 188±8 177±8 178±9

23±9 11

222±24

26±4

192±13

0

51±8 15±9

Bronchoconstriction in latex-treated rats

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difference was found in the dose-response curve (Fig. 3) to 5-hydroxytryptamine, indicating that immunization with antigen-coated latex particles does not induce a state of bronchial 'hyperreactivity' to this mediator. A

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FIG. 3. 5-Hydroxytryptamine-induced bronchoconstriction in animals immunized on days 0 and 13 with: (A) 300,000 BGG-coated latex particles; (A) 300,000 particles alone; (0) 1.0 mg heat-aggregated BGG (n 8).

Specificity ofthe eosinophilia and antigen-induced bronchoconstriction Using the information obtained from previous experiments on the requirement for two injections of BGG-coated latex particles and the observed time-course of eosinophilia, we investigated the relationship of eosinophilia and bronchoconstriction following BGG challenge. In animals treated with latex-BGG (day 0)/latex-BGG (day 13),an obvious eosinophilia was accompanied by a significant degree ofbronchoconstriction (Table 2). In rats treated with latex-BGG (day 0)/latex-bovine serum albumin (day 13) there was a significant reduction in eosinophilia (P< 0001, Student's t-test) and bronchoconstriction (P< 0-01) (Table 2). In all other schedules used, i.e. latex-BGG (day 0)/latex (day 13); latex (day 0)/latex (day 13); latex-BGG (day 0)/heat-aggregated BGG (day 13); heat-aggregated BGG (day 0)/latex-BGG (day 13), there was a low level of blood eosinophilia (P< 0.01) and a low level of BGG-induced bronchoconstriction (P < 0 05). These results indicate that in this system bronchoconstriction is associated with a high level of eosinophilia. DISCUSSION We have confirmed the findings of Schriber & Zucker-Franklin (1974) in that the repeat injection of antigen-coated latex particles in rats induces a blood eosinophilia. The eosinophilia observed in our experiments was not as great as that reported by Schriber & Zucker-Franklin (1974), probably due to the use of BGG as antigen rather than human gamma globulin. The technique of lung lavage gave quantitative results which were in general agreement with the histological study of Schriber & Zucker-Franklin (1974) and ourselves (unpublished). These results showed that a lung eosinophilia is observed 48- hr following a repeat injection of latex-BGG and 72 hr before the blood eosinophilia. Schriber & ZuckerFranklin (1974) also described a mononuclear cell infiltrate, however, our results indicate that the mononuclear cell infiltrate is small compared to the eosinophilia and similar to that observed following the primary injection of latex-BGG. As also shown by Schriber & Zucker-Franklin (1975), the antigenspecific antibodies associated with immunization with antigen-coated latex particles are haemagglutinating and not reaginic antibodies. Huggins & Brostoff (1975), using the technique of nasal washing,

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have shown local production of specific IgE, in the absence of serum IgE, in a group of patients with allergic rhinitis. However, we were unable to detect skin-fixing antibodies in lung lavage supernatants from rats injected with latex-BGG, indicating that in this system there is no evidence of local production of IgE. As well as inducing an eosinophilia, immunization with antigen-coated latex particles results in a sensitivity to antigen such that on antigen challenge animals show bronchoconstriction. Other models of anaphylactic bronchoconstriction, using rats immunized with egg albumin and B. pertussis (Stotland & Share, 1974), or rats infected with N. brasiliensis (Church, Collier & James, 1972), have been associated with specific IgE antibodies. In our system, IgE antibodies are unlikely to be involved. Also haemagglutinating antibodies do not appear to be directly responsible for the bronchoconstriction induced in rats immunized with latex-BGG, as similar levels of antibodies are found in rats treated with heat-aggregated BGG, which does not induce bronchoconstriction on antigen challenge. Latex particles, of diameter 45 pm, lodge in the lung (Schriber & Zucker-Franklin, 1974) and it was possible that injection of latex particles induces a state of bronchial 'hyperreactivity' (Benson, 1975). However, this is unlikely, as the animals' response to 5-hydroxytryptamine, the major mediator responsible for anaphylactic bronchoconstriction in the rat (Farmer et al., 1975) is unaffected by immunization with antigen-coated latex particles. Experiments comparing various immunization schedules involving latex particles showed that both the eosinophilia and antigen-induced bronchoconstriction were highly dependent on primary and secondary injections of BGG-coated latex. These results suggest that the lung eosinophilia caused by the trapping of antigen-coated latex particles is closely associated with bronchial sensitivity to antigen. Asthma is defined as an increase of airways resistance, which is completely or partially reversible either spontaneously or as a result of treatment (Turner-Warwick, 1971). Patients with this type of disorder fit into two categories. The most easily distinguishable group are extrinsic asthmatics, in which specific reaginic antibodies react with external antigens, causing mediator release from sensitized cells. The second group of patients, intrinsic asthmatics, do not seem to be hypersensitive to inhaled or ingested substances and reaginic antibodies do not appear to be associated with this disorder. Common to both groups of patients is a blood or sputum eosinophilia (Turner-Warwick, 1971; Scadding, 1971). It has been proposed that eosinophils are attracted to the site of an allergic reaction by the mediator eosinophil chemotactic factor of anaphylaxis, released by mast cells (Kay & Austen, 1971). It has further been suggested that histaminase (Zeiger & Cotten, 1977) and arylsulphatase (Wasserman, Goetzl & Austen, 1975) released from eosinophils could inactivate mediators released by mast cells (Austen & Orange, 1975). The eosinophils can contribute to an inflammatory response. Eosinophils constitute a major cellular component of schistosome egg granulomas (James & Colley, 1975), and recently Butterworth et al. (1977) showed that eosinophils can damage Schistosomula in the presence of specific antibody. Also, Spry & Tai (1976) have shown that patients with Lofflers endocarditis have eosinophils which contain vacuoles and are partially degranulated. These authors suggest that circulating eosinophil granule products can damage the heart. In our model, antigen-induced bronchconstriction appears to be associated with eosinophilia. Although further work will be necessary to show whether eosinophils are involved in the bronchoconstriction, we would like to suggest that the lung eosinophilia observed in asthmatics is contributing to the asthmatic syndrome. We should like to thank P. Sheard and Patsy Riley for helpful advice and Marilyn Mather and Linda Lee for excellent technical assistance.

REFERENCES AusTEN, K.F. & ORANGE, R.P. (1975) Bronchial asthma: the possible role of the chemical mediators of immediate hypersensitivity in the pathogenesis of subacute chronic disease. Am. Rev. resp. Dis. 112, 423.

BASTEN, A. & BEESON, P.B. (1970) Mechanism of eosinophilia. II. Role of the lymphocyte.Z. exp. Med. 131, 1288. BENSON, M.K. (1975) Bronchial hyper-reactivity. Brit. J. Dis. Chest, 69, 227.

Bronchoconstriction in latex-treated rats BUTTERWORTH, A.E., DAVID, JR., FRANKS, D., MAHMOuD, A.A.F., DAVID, P.H., STURROCK, R.F. & HOUBA, V.

(1977) Antibody-dependant eosinophil-mediated damage to "1Cr-labelled Schistosomula of Schistosoma mansoni: damage by purified eosinophils.J. exp. Med. 145, 136. CHURCH, M.K., COLLIER, H.O.J. & JAMES, G.W.L. (1972) The inhibition by dexamethasone and disodium cromoglycate of anaphylactic bronchoconstriction in the rat. Brit.3. Pharmac. 46,56. DISCOMBE, G. (1946) Criteria of eosinophilia. Lancet, i, 195. FARMER, J.B., RICHARDS, I.M., SHEARD, P. & WOODS, A.M. (1975) Mediators of passive lung anaphylaxis in the rat. Brit.]. Pharmac. 55, 57. FELARCA, A.B. & LOWELL, F.C. (1971) The accumulation of eosinophils and basophils at the skin sites as related to intensity of skin reactivity and symptoms in atopic disease.J7. Allergy clin. Immunol. 48, 125. HUGGINS, K.G. & BROSTOFF, J. (1975) Local production of specific IgE antibodies in allergic-rhinitis patients with negative skin tests. Lancet, ii, 148. JAMES, S.L. & COLLEY, D.G. (1975) Eosinophils and immune mechanisms: Production of the lymphokine eosinophil stimulation promotor (ESP) in vitro by isolated intact granulomas. J. reticuloendothol. Soc. 18, 283. KAY, A.B. & AUSTEN, K.F. (1971) The IgE-mediated release of an eosinophil leucocyte chemotactic factor from human lung.]. Immunol. 107,899. KIMURA, I., MORITANI, Y. & TANYAKI, Y. (1973) Basophils in bronchial asthma with reference to reagin-type allergy. Clin. Allergy 3, 195. MAHMOUD, A.A.F., WARREN, K.S. & PETERS, P.A. (1975)

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A role for the eosinophil in acquired resistance to Schistosoma mansoni infection as determined by antieosinophil serum. J. exp. Med. 142, 805. MOTA, I. (1963) Passive cutaneous anaphylaxis induced with mast cell sensitizing antibody. The role of histamine and 5-hydroxytryptamine. Life Sci. 12,917. SCADDING, J.G. (1971) Eosinophilic infiltrations of the lungs in asthmatics. Proc. R. Soc. Med. 64,381. SCHRIBER, R.A. & ZUCKER-FRANKLIN, D. (1974) A method for the induction of blood eosinophilia with simple protein antigens. Cell. Immunol. 14,470. SCHRIBER, R.A. & ZUCKER-FRANKLIN, D. (1975) Induction of blood eosinophilia by pulmonary embolization of antigen-coated particles: the relationship to cell-mediated immunity.]. Immunol. 114, 1348. SCOTT, D.W. & GERSHON, R.K. (1970) Determination of total and mercaptoethanol-resistant antibody in the same serum sample. Clin. exp. Immunol. 6, 313. SPRY, C.J.F. & TAI, P.C. (1976) Studies on blood eosinophils. II. Patients with Loffler's cardiomyopathy. Clin. exp. Immunol. 24,423. STOTLAND, L.M. & SHARE, N.N. (1974) Active-bronchial anaphylaxis in the rat. Can.J. Pharmacol. 52, 1114. TURNER-WARWICK, M. (1971) Provoking factors in asthma. Brit.]. Dis. Chest, 65, 1. WASSERMAN, S.I., GOETZL, E.J. & AUSTEN, K.F. (1975)

Inactivation of slow reacting substance of anaphylaxis by human eosinophil arylsulphatase.J. Immunol. 114, 645. ZEIGER, R.S. & COLTEN, H.R. (1977) Histaminase release from human eosinophils. J. Immunol. 118, 540.

Anaphylactic bronchoconstriction in rats immunized with antigen-coated latex particles.

Clin. exp. Immunol. (1978) 32, 283-289. Anaphylactic bronchoconstriction in rats immunized with antigen-coated latex particles R. P. EADY, S. TRIGG &...
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