Increased IgE antibody responses in rats exposed to tobacco smoke

Increased IgE antibody responses exposed to tobacco smoke

in rats

0. Zetterstriim, M.D., Ph.D.,* S. L. Nordvall, M.D., Ph.D.,**,***** B. Bjiirkstbn, M.D., Ph.‘D.,****~***** S. Ahlstedt, M.D., Ph.D.,*****~****** and M. Stelander, B.A.*** Stockholm,

Uppsala,

Linkiiping,

and Gothenburg,

Sweden

Raised serum IgE levels were found in a high proportion of rats that had been exposed to tobacco smoke twice daily 5 days a week for 8 wk in a Dontenville-type smoking machine. Levels above 1 nglml of IgE were found in nine of 20 animals exposed to cigarette smoke and in jive of 20 rats exposed to smoke from cigarettes with I .45% phenylmethyloxidiasole added for possible protection against the effects of the smoke. None of the 20 control rats exhibited similarly increased serum IgE. Exposure to tobacco smoke did not signtficantly affect the serum concentrations of IgM and IgG. The development of specijc IgE and IgG antibodies was also injluenced by tobacco smoke exposure. Rats exposed to ovalbumin aerosol developed increased levels of IgG and IgE antibodies, whereas no effect on the development of antibody titers was found in rats immunized by the subcutaneous route. This study demonstrates that exposure to tobacco smoke increases serum IgE levels and enhances sensitization via the airways by a local effect, thus supporting the ’ ‘mucosal theory of atopy.” (.JALLERGYCLINIMMUNOL

75.394-8, 1985.)

Several local adverse reactions in the respiratory tract caused by cigarette smoking have been reported, including hypersecretion of mucus, increased thickness of the epithelium, increased numbers of goblet cells and of cell mitoses, and inhibition of the ciliary movements or even damage and loss of the ciliated cells.’ Several studies have demonstrated an association between smoking and increased serum levels of IgE,‘” although other investigators have not been able to confirm this.’ A relation between smoking and development of specific IgE antibodies to inhaled allergens has also been reported.’ In the present study we have used an animal model to study further the association between smoking and levels of immunoglobulins as well as antibody responses with special reference to IgE antibodies. In addition we studied immunoglobulin levels in rats exposed to smoke from tobacco to which PM0 had been

From the *Departments of Lung Medicine, Stockholm, **Pediatrics and ***Hygiene, University of Uppsala, ****Pediatrics, University of Linkoping, *****Experimental Allergy Research, Phannacia AB, Uppsala, and ******Department of Clinical Immunology, University of Gothenburg, Gothenburg, Sweden. Received for publication Dec. 13, 1983. Accepted for publication Sept. 4, 1984. Reprint requests: Olle Zetterstrom, Dr., Department of Lung Medicine, Karolinska Hospital, S-104 01 Stockholm, Sweden.

594

Abbreviations used PMO: Phenylmethyloxidiazole SPF: Specific pathogen

OA: Ovalbumin SC: Subcutaneously

added. This oxalamine derivate has been proposed to protect the bronchial mucosa from some of the effects of tobacco smoke.‘, * MATERIAL AND METHODS Exposure to cigarette smoke Two types of cigarettes were used. The first type was without filter with a tar content of 25.7 mg and a nicotine

content of 1.67 mg per cigarette. The secondtype also was a nonfilter cigarette with a tar content of 28.7 mg and a nicotine content of 1.45 mg per cigarette. To this second type PM0 was added equal to 1.44% of the weight. The smoking equipment was of Dontenville type“ (type 2, H. Burkwald,

Hamburg,

Germany).

Rats were exposed

to cigarette smoke 5 days per week for 8 wk. After an updosing period, groups of 10 animals were exposed to smoke from 10 cigarettes twice daily for about 10 min.

Animals

and sampling

SPF Wistar rats (Mollergaard, Denmark) were used for the immunization experiments, and non-SPF hybrid rats

IgE antibody

VOLUME 75 NUMBER 5

responses

595

(BN x Wi/Fu) (Prof. 0. Sjogren, University of Lund, Lund, Sweden) were used to analyze the effect of smoking on serum IgE levels. They had accessto food and water. All the animals in the different treatment groups were kept in the same room in the animal house during the treatment period. Blood samples were obtained from the tail of unanesthetized animals before and every 2 wk during the treatment period. The final bleeding was made 3 to 5 days after the end of the smoking period. The sera were stored at - 20” C until it was analyzed. Immunizations In one set of experiments, groups of six to 10 SPFWistar rats were immunized with OA (OA, Sigma Chemical Co., St. Louis, MO.) daily either as aerosol or SC. Aerosol immunizations were done in the morning simultaneously with smoke exposure. The subcutaneous immunizations were done immediately after the smoke exposure. The sensitization protocols have been described in detail elsewhere.‘“, I’ Briefly, the rats were exposed to 0.1% aerosol of OA in water for 30 min or received 100 ng of OA in 0.25 ml physiologic saline SC.The antigen was administered without the use of any adjuvant daily for 2 wk (Monday through Friday) in two periods with a 4-week interval.

0.5-0.991

0.1-0.49

i

Iq+ z 1

0

Antibody

determinations

Total serum IgE and IgE directed against OA were determined in duplicate according to the PRIST and RAST principles, respectively, as describedpreviously,“. ” by use of radiolabeled anti-rat IgE (T. Karlsson, Biocell AB, Uppsala, Sweden).

The accuracyof the methods was in our hands ? 14%. The levels of IgM and IgG were determined in duplicate with single radial diffusion according to the principle describedby Mancini et al. I2The specificIgG antibodies were

recorded in duplicate with ELISA as previously described.‘” ” Statistical

methods

Student’s t test for unpaired observations was used to compare the levels of antibodies in the different treatment groups. To compare the prevalence of raised antibody levels in the various groups of animals, the chi-square test was used. RESULTS

In the pretreatment serum samples of IgE levels above, the detection limit of 0.1 rig/ml was demonstrated in 25 of 60 rats. None of the animals had an IgE concentration above 1 rig/ml. In the control rats the IgE values remained low throughout the study period. Eleven of 20 rats had IgE levels below 0.1 rig/ml; the other nine demonstrated values between 0.1 and 0.9 rig/ml (Fig. 1). After 28 days of exposure to cigarette smoke, one of 20 rats demonstrated a markedly increased IgE

14

26

42

, days 60

FIG. 1. Serum IgE levels in rats. Control rats not exposed to tobacco smoke (+ ), closed circles denote animals exposed to tobacco smoke alone, and open circles indicate animals exposed to smoke from cigarettes with PM0 added.

level. After 42 days three rats had increased IgE levels, and by 60 days an IgE level of 1 rig/ml or more was found in six animals, whereas seven animals lacked demonstrable serum IgE. The prevalence of elevated IgE and the geometric mean of serum IgE concentrations in this group differed significantly from those of the control group (p < 0.05). After exposure to cigarette smoke containing PMO, one rat had an increased IgE concentration after 42 days. After 60 days five of the rats had IgE levels equal to or above 1 rig/ml, and only in six of the 20 rats, no IgE could be demonstrated. The prevalence of increased values and the geometric mean of the IgE levels differed significantly from those of the control group (p < 0.05) but not from the animals exposed to cigarette smoke without PMO. The IgG levels varied with the age of the animals with a tendency to increased levels toward the end of the study period. The differences between groups were, however, not statistically significant. Similarly, there were no significant differences in IgM levels between the groups of animals or over time (data not shown). Sensitization via aerosol induced more pronounced

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et al.

J. ALLERGY CLIN. IMMUNOL. MAY 1985

RAST

IgE antibodies. % of T

test control

IOT

% of total serum serum

9 I

d A

-

1 Subcutaneously

IgG,

Elisa 4

0 + p -

aerosol

Immunization

titer

300. 200-

1 0

l

0

;

:

loo-

500 (2Od

-+I++

@SubXl;aerosol

: +

8

+

0 -&is

IgG, ELISA titer

la-# r

immunization

FIG. 2. A, IgE antibodies. 6, IgG antibodies. Antibodies to OA in rats immunized with aerosol or SC. Control rats not exposed to tobacco smoke ( + 1, closed circles denote animals exposed to tobacco smoke alone, and open circles indicate animals exposed to smoke from cigarettes with PM0 added.

IgE antibody responses to OA in animals exposed to smoke than in the similarly immunized controls not exposed (Fig. 2, A). Addition of PM0 to the cigarettes inhibited this enhancing effect of tobacco smoke on the development of IgE antibodies. In the SCsensitized animals there were no differences in antibody responses between rats exposed to tobacco smoke than in unexposed rats. Raised IgG antibody levels to OA were only detected in immunized animals (Fig. 2, B). The levels of these antibodies were higher in rats exposed to tobacco smoke than in unexposed animals (p < 0.05). There was no significant difference between animals exposed to tobacco smoke and smoke with PM0 with regard to number of animals with increased IgG antibody levels or with regard to antibody levels. An overall good, although not complete, correlation was found between IgG and IgE antibody levels to OA in the individual animals (Fig. 3). DISCUSSION

In a high proportion of the rats exposed to cigarette smoke, elevated IgE levels were found. This effect was not observed immediately but appeared after 6 to 8 wk, at which time 68% of the rats exposed to smoke

FIG. 3. Relation between IgG and IgE antibodies against OA in rats immunized via aerosol. A, aerosol smoke immunization; A, smoke immunization SC; V, aerosol smoke immunization plus PMO; V, smoke immunization SC plus PMO; n , aerosol no smoking immunization; O, no smoking immunization SC.

with or without PM0 had detectable S-IgE, 30% of them with levels of 1 rig/ml or more. In the unexposed rats 45% had detectable S-IgE, but none had a concentration of 1 rig/ml or more. Since the experimental conditions were similar for the rats except for the exposure to tobacco smoke, the observed IgE elevations were most probably caused by tobacco smoke exposure. The influence of individual genetic factors was minimized by the use of inbred animals in each experiment. In spite of this, the distribution of IgE levels ranging from normal in some animals to high in other animals is similar to that which is observed in humans.5 Thus, as in human studies, our data demonstrate that the effect of smoking on immunoglobulin levels is somewhat selective, i.e., not being present in all exposed individuals. This may possibly be related to varying sensitivity of the airways to smoke as Bahna et a1.3 have reported higher IgE levels in moderate smokers than in heavy smokers. Raised IgE levels are strongly associated with atopic disease in humans.” The pathogenesis of atopy is unknown, but properties of the airway mucosa are probably important. Some diseases affecting the mucosa are associated with a high prevalence of IgE-mediated sensitization (the mucosal theory of

IgE antibody

VOLUME 75 NUMBER 5

atopy). 14-17Mucosal injury and inflammation induced by smoke may explain the effect on IgE level and antibody production in our study. There are accumulating experimental’8~ I9 as well as clinical data*’ that exposure to cigarette smoke increases mucosal permeability. The finding that smoke-exposed rats were more easily sensitized against OA in aerosol than similarly exposed control rats unexposed to smoke may be a consequence of increased antigen penetration. Exposure to air pollutants (0, and SO,) in mice have also been demonstrated to result in an increased sensitization when antigen was administered via an aerosol.” These findings may be explained similarly. By use of this model with aerosol sensitization, we have regularly found IgE antibody formation” and no evidence of tolerance induction as reported by others.‘* The most probable explanation for this discrepancy is different protocols used for the immunization. In studies of sensitization after respiratory virus infections, Frick and coworkers23. 24reported enhanced risks for atopic disease and development of specific IgE after viral infections of the respiratory tract. The results were interpreted as caused by a general effect on the immune system. A general effect appears less likely in our study, since there was no difference in antibody levels between smoke-exposed rats and rats unexposed to smoke after immunization SC. Addition of PM0 to the cigarettes had no certain preventive effect on the development of raised concentrations of total IgE even though the levels after 60 days were somewhat lower. However, in the aerosol-immunized rats the addition of PM0 appeared to protect against the adjuvant effect of tobacco smoke on the development of specific antibodies. This would support previous indications that PM0 may afford at least some protection against tobacco smoke-induced damage of the mucous membranes.7,8 In those animals that responded both with IgG and IgE antibodies, there was a correlation between the levels in the individual animals. This agrees with the findings in studies of human antibody responses to other antigens. 25.26The correlation was, however, not total. As illustrated in Fig. 3, a few rats had IgE antibody responses without any detectable IgG antibodies. These findings are interesting in light of a recent report by Francus et a1.27 who, by use of a different immunization protocol, found that a tobacco glycoprotein elicited a long-lasting IgE antibody response in mice without inducing any detectable hemagglutinating antibodies. This could possibly indicate that tobacco smoke may have a somewhat selective effect on the IgE antibody production. In conclusion the study demonstrates that tobacco smoke may increase IgE antibody levels. The ex-

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panding use of tobacco and increased exposure to other air pollutants may partly explain the suggested increase in the prevalence of atopic diseases in many countries. REFERENCES JH: Tobacco smoking. In Petersdorf RG, Adams 1. Holbrook RD, Braunwald E, et al., editors: Harrison’s principles of internal medicine. New York, 1983, McGraw-Hill Book Co, p 1302 2. Gerrard JW, Heiner DC, Ko CG, Mink J, Meyers A, Dosman JA: lmmunoglobulin levels in smokers and nonsmokers. Ann Allergy 44:261, 1980 E pattern 3. Bahna SL, Heiner DC, Myhre BA: Immunoglobulin in cigarette smokers. Allergy 3857, 1983 4. Burrows B, Halonen M, Barbee RA, Lebowitz MD: The relationship of serum immunoglobulin E to cigarette smoking. Am Rev Respir Dis 124523, 1981 5. Zetterstrom 0, Osterman K, Machado L, Johansson SGO: Another smoking hazard: raised serum IgE concentration and increased risk of occupational allergy. Br Med J 283: 1215, 1981 6. Hlllgren R, Nou E: Smoking and circulating IgE in bronchial carcinoma. Acta Med Stand 211:269, 1982 T: Inhibition of the ciliostatic effect of cigarette 7. Dalhamn smoke by oxolamine citrate. Am Rev Respir Dis 94:799, 1966 8. Jones R, Bolduc P, Resid L: Protection of rat bronchial epithelium against tobacco smoke. Br Med J 2:142, 1972 9. Dontenwill W, Chevalier HJ, Harke HP, Lafreuz U, Reckzeh G, Schneider B: Investigations on the effects of chronic cigarette smoke inhalation in Syrian golden hamsters. J Nat1 Cancer Inst .51:1781, 1973 IO. Ahlstedt S, Bjorksten B: Specific antibody responses in rats and mice after daily immunization without adjuvant. Int Arch Allergy Appl Immunol 71:293, 1983 11. Ahlstedt S, Bjiirksten B, Nygren H, Smedegard G: Induction of humoral immunity and pulmonary mast cells in mice and rats after immunization with aerosolized antigen. Immunology 48:247, 1983 12. Mancini G, Carbonara AO, Hercmans IF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235, 1965 0, Johansson SGO: IgE concentrations measured 13. Zetterstrom by PRIST in serum of healthy adults and in patients with respiratory allergy. A diagnostic approach. Allergy 36:537, 1981 14. Leskowitz S, Salvaggio JE, Schwartz HJ: An hypothesis for the development of atopic allergy in man. Clin Allergy 2.237, 1972 15. Vanselow NA, Yamate M, Adams MS, Callies Q: The increased prevalence of allergic disease in anhidrotic congenital ectodermal dysplasia. J ALLERGY 45:302, 1970 16. Warren CPW, Tai E, Batten JC, Hutchcroft BJ, Pepys J: Cystic fibrosis-immunological reactions to A. fimigatus and common allergens. Clin Allergy 1: 1, 1975 17. Warner JO, Norman AP, Sothill JF: Cystic fibrosis heterozygosity in the pathogenesis of allergy. Lancet 1:990, 1976 18. Simani IAS, moue S, Hogg JC: Penetration of the respiratory epithelium of guinea pigs following exposure to cigarette smoke. Lab Invest 31:75, 1974 19. Hulbert WC, Walker DC, Jackson A. Hogg JC: Airway permeability to horseradish peroxidase in guinea pigs: the repair phase after injury by cigarette smoke. Am Rev Respir Dis 123:320, 1981

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