Induction of IgE antibodies to antigen isolated from tobacco leaves

Induction of IgE Antibodies to Antigen Isolated From Tobacco Leaves and From Cigarette Smoke Condensate Carl G. Becker, MD, Roberto Levi, MD, and James Zavecz, PhD

Neonatal California White rabbits were sensitized with a glycoprotein purified from cured Virginia Bright tobacco leaves. Their serums, but not serums from normal rabbits, were demonstrated by passive cutaneous anaphylaxis technique to contain heat-labile, homocytophilic antibodies to this antigen and to similar material purified from cigarette smoke condensate. Serums containing IgE antibodies to tobacco antigen would not be demonstrated to contain hemagglutinating antibodies to this antigen. These experiments demonstrate that antigen capable of triggering specific IgE-mediated release of inflammatory mediators is present in cigarette smoke. It can be hypothesized that IgE-mediated responses to antigen in cigarette smoke are causally related to the development of vascular injury and of myocardial arrhythmia in hypersensitive smokers. (Am J Pathol 96:249-256, 1979)

IMMEDIATE CUTANEOUS HYPERSENSITIVITY to extracts of tobacco

leaves has been described in smokers and nonsmokers.'-3 Harkavy et al suggested that allergy to constituents of tobacco may underlie the relationship between tobacco smoking and coronary artery disease and peripheral vascular disease.2 Fontana and colleagues demonstrated that 28% of smokers with immediate cutaneous hypersensitivity to tobacco extracts had changes in peripheral circulation when they smoked cigarettes, compared with smokers with negative skin tests.8 Harkavy presented evidence that smokers who were hypersensitive to extracts of tobacco leaves experienced heart attacks at younger ages than did nonhypersensitive smokers who had also suffered heart attacks.4 However, the concept of allergy to tobacco constituents has not been generally accepted, because it was not clearly demonstrated that antigens extractable from cured tobacco leaves were also present in tobacco smoke and capable of eliciting IgE-mediated immediate hypersensitivity reactions. In addition, until recent years preparations of tobacco antigen from either leaves or cigarette smoke condensate may have included noxious materials capable of inducing wheal-and-flare reactions by nonimmunologic mechanisms, since the means for purification were not available. From the Departments of Pathology and Pharmacology, Cornell University Medical College, New York, New York. Supported by grants HL-18828, HL-01803, GM-20091, Fellowship HL-05013 from the National Institutes of Health and by a grant from The Cross Foundation. Accepted for publication March 5, 1979. Address reprint requests to Carl G. Becker, MD, Department of Pathology, The New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, NY 10021. 0002-9440/79/0709-0249$01.00 249

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American Journal of Pathology

The purpose of this communication is to describe experiments in which rabbits were immunized neonatally with glycoprotein antigen purified from cured tobacco leaves. The serums of these rabbits, but not of controls, were found to contain heat-labile, homocytophilic antibodies to antigen purified from cured tobacco leaves or from cigarette smoke condensate. Materials and Methods Antigen Preparation

Tobacco glycoprotein (TGP) was isolated from cured tobacco leaves and from cigarette smoke condensate according to methods described previously.I Cured tobacco leaves were obtained through the courtesy of Dr. Raymond Fagan of The Philip Morris Tobacco Co. Cigarette smoke condensate, produced from University of Kentucky 1Ri cigarettes smoked in a standard manner, was kindly supplied by Dr. M. R. Guerin of Oak Ridge National Laboratories, Oak Ridge, Tennessee, through the courtesy of Dr. Robert C. Hockett of The Council for Tobacco Research-U.S.A., Inc., New York. TGP derived from cured tobacco leaves will be referred to as "TGP-L." TGP derived from cigarette smoke condensate will be referred to as "TGP-CSC." Neonatal Immunization of Rabbits Nine California White rabbits from two litters born to two sets of parents were injected intraperitoneally on the first day of life with 1 mg of TGP-L adsorbed to alum. They received two similar injections at weekly intervals and then five injections at biweekly intervals, essentially according to the technique of Pinckard et al.5 The rabbits were bled on the 87th day and serum was collected. Thereafter, the rabbits

were boosted at monthly intervals by injecting 200 ,ug of TGP-L in alum intracutaneously. Serum samples were collected before each immunization. This continued for approximately 9 months to 1 year. Serum samples were also obtained from unimmunized California White rabbits, and frozen pooled serum from normal rabbits of other varieties was obtained from Pel-Freez Biologicals, Inc., Rogers, Arkansas. All serum samples were stored at -70 C. Passive Cutaneous Anaphylaxis Experiments Serum from rabbits immunized neonatally with TGP-L and from normal rabbits was diluted serially fourfold in phosphate-buffered physiologic saline (PBS). Half the volume of each sample was heated to 56 C for 1 hour. The heated samples were cooled to room temperature. One-tenth milliliter of unheated or heated serum or dilutions thereof was injected intracutaneously into the shaved backs of normal California White rabbits. The sites of injection were 2.4 cm from each other. Seventy-two hours later the prepared rabbits were injected intravenously with 2 ml of a 1.5% solution of Evans Blue in saline. The prepared skin sites were challenged by injection of 0.1 ml of PBS containing 3 jg of either TGP-L or TGP-CSC or 0.1 ml of PBS as control. The sites were observed for leakage of Evans Blue and development of a wheal within 30 minutes after injection of antigen. Examination of Serums for Hemagglutinating Antibodies to TGP Human Type 0, Rh-positive erythrocytes were tanned and coated with TGP-L as described elsewhere.' The capacity to agglutinate these cells by serial dilutions of heatinactivated serums from neonatally immunized rabbits, normal rabbits, and adult rabbits

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immunized with TGP as described previously was measured using Microtiter U-plates (Cook Laboratory Products, Alexandria, Va.)

Results Passive Cutaneous Anaphylaxis Assays

TGP-L and TGP-CSC elicited immediate cutaneous hypersensitivity reactions in skin sites in normal rabbits sensitized 72 hours previously with serum from rabbits immunized neonatally with TGP-L. Heating these serums at 56 C for 1 hour abolished their capacity to sensitize the skin of normal rabbits in dilutions greater than 1:1. The titer of heat-labile homocytophilic antibodies to TGP in serum of rabbits immunized neonatally ranged between 1: 16 and 1: 256, with a mean titer of 1:64. Heatlabile, homocytophilic antibodies to TGP were not demonstrable in the serum of normal California White rabbits or in the pooled serum of normal rabbits of other varieties. An example of such an experiment is shown in Figure 1. The skin in Columns A and D, Rows 1-4, was injected with serial fourfold dilutions of heat-inactivated serum from a rabbit neonatally sensitized with TGP-L. The skin in Columns B and C, Rows 1-4, was injected with serial fourfold dilutions of serum from the same rabbit, not heat-inactivated. The skin in Columns B and C, Rows 5 and 6, was injected with serial fourfold dilutions of normal rabbit serum. The skin in Columns A and D, Rows 5 and 6, was injected with serial fourfold dilutions of heat-inactivated normal rabbit serum. Seventy-two hours later the rabbit was injected intravenously with Evans Blue, and prepared sites in Columns A and B were each challenged with 0.1 ml of PBS containing 3 ,ug of TGP-L. Prepared sites in Columns C and D were each challenged with 0.1 ml of PBS containing 3 jig of TGP-CSC. This photograph was taken 30 minutes after antigenic challenge. It can be seen that TGP-L and TGP-CSC both can stimulate immediate cutaneous hypersensitivity reactions, evidenced by leakage of Evans Blue at sites (Columns B and C, Rows 1-4) prepared by injection of serum from a rabbit immunized neonatally with TGP-L. Heating this serum at 56 C inhibits this reaction (Columns A and D, Rows 1-4). The positive response in Column A, Row 1, and Column D, Row 1, may be due to either the presence of homocytophilic IgG antibodies directed against TGP or incomplete inactivation of IgE when neat serum is heated at 56 C. Neither TGP-L nor TGP-CSC induced vascular leakage at sites previously injected with normal rabbit serum before (Columns B and C, Rows 5 and 6) or after (Columns A and D, Rows 5 and 6) heating.6

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Hemagglutination Assays

Human Type 0, Rh-positive erythrocytes, tanned and coated with TGP-L, were agglutinated by serums from adult rabbits immunized with TGP-L to serum dilutions of 1:256 to 1:1024. These cells were not agglutinated by serums from rabbits immunized neonatally with TGP-L, even after repeated booster immunizations; they were also not agglutinated by normal rabbit serums. Tanned cells not coated with TGP-L served as controls and were not agglutinated by any of the rabbit serums described above. Discussion

Our experiments demonstrate that rabbits immunized neonatally with a glycoprotein purified from flue-cured tobacco leaves preferentially synthesize heat-labile, homocytophilic, hence IgE, antibodies to this antigen and essentially reproduce observations of Pinckard et al, who induced preferential synthesis of IgE antibodies to bovine albumin in the neonatal rabbit.5 They further demonstrate that antigen purified by the same methods from cigarette smoke condensate can trigger IgE-mediated anaphylactic responses and provide unequivocal evidence that intact allergen is present in cigarette smoke.6 In this connection, Lehrer et al recently demonstrated components in tobacco smoke that stimulated production of specific, precipitating antibodies in rabbits.7 These observations may provide some insight into the pathogenic mechanisms underlying the association between cigarette smoking and cardiovascular and chronic obstructive pulmonary disease. It has been demonstrated that deposition of immune complexes in the walls of blood vessels is dependent on the IgE-mediated release of vasoactive amines from basophiles and mast cells and, indirectly, from blood platelets.8'9 It has also been demonstrated that immune complex-mediated injury to arteries acts synergistically with modest hyperlipidemia to induce atheroarteriosclerosis in coronary and other arteries.10 It is entirely conceivable that repetitive antigenic stimulation in the allergic smoker might potentiate vascular injury in either the systemic or pulmonary vasculature. In this connection, it is important to bear in mind that approximately one quarter to one third of humans, smokers and nonsmokers, exhibit immediate cutaneous hypersensitivity reactions when injected intracutaneously with TGP-L.1 It has also been demonstrated in this laboratory that tobacco glycoprotein purified from either cured tobacco leaves (TGP-L) or from cigarette smoke condensate (TGP-CSC) can activate factor XII-dependent pathways in human plasma in vitro, activating the intrinsic coagulation path-

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way, fibrinolysis, and the generation of bradykinin. " These observations suggest the possibility that thrombogenic and phlogogenic factors resulting from activation of factor XII-dependent pathways might act synergistically with IgE-mediated release of vasoactive amines to induce vascular injury in the allergic smoker. Allergy to constituents of tobacco smoke may also affect the heart directly. Capurro and Levi demonstrated that when hearts isolated from guinea pigs sensitized with antipenicilloyl antibodies were challenged with benzyl penicilloyl protein conjugates a variety of changes, including sinus tachycardia, atrioventricular conduction block, increased ventricular automaticity, coronary constriction, and histamine release, were observed.12 When similarly sensitized guinea pigs were challenged in vivo, ventricular fibrillation was the terminal event in almost half of anaphylactic deaths. Levi and colleagues demonstrated that cardiac and pulmonary anaphylaxis can be induced in rabbits and guinea pigs sensitized with TGP-L and challenged with either TGP-L or TGP-CSC." It can be hypothesized from these observations that cigarette smokers who die suddenly of myocardial arrhythmias may do so as a consequence of IgEmediated hypersensitivity to antigenic constituents of cigarette smoke. Finally, the fact that rabbits neonatally sensitized with TGP-L and then repeatedly boosted with this antigen did not produce hemagglutinating antibodies to TGP raises the possibility that children born of smoking mothers may be extremely hypersensitive to tobacco antigen, because their capacity to produce blocking antibodies has been suppressed. References 1.

2. 3. 4.

5. 6. 7. 8.

Becker CG, Dubin T, Wiedemann HP: Hypersensitivity to tobacco antigen. Proc Nati Acad Sci USA 73:1712-1716, 1976 Harkavy J, Hebald S, Silbert S: Tobacco sensitiveness in thromboangiitis obliterans. Proc Soc Exp Biol Med 30:104-107, 1932 Fontana VJ, Redisch W, Nemir RL, Smith MK, De Crinis K, Sulzberger MB: Studies in tobacco hypersensitivity. III. Reactions to skin tests and peripheral vascular responses. J Allergy 30:241-249, 1959 Harkavy J: Vascular Allergy and Its Systemic Manifestations. Washington, DC, Butterworths, 1953, pp 139-141 Pinckard RM, Halonen M, Meng AL: Preferential expression of antibovine serum albumin IgE homocytotropic antibody synthesis and anaphylactic sensitivity in the neonatal rabbit. J Allergy Clin Immunol 49:301-310, 1972 Becker CG, Levi R, Zavecz JH: Induction of IgE antibodies to antigen isolated from tobacco leaves and from cigarette smoke condensate (abstr). Circulation 56 (Suppl 6):169, 1977 Lehrer SB, Wilson MR, Salvaggio JE: Immunogenic properties of tobacco smoke. J Allergy Clin Immunol 62:368-370, 1978 Henson PM, Cochrane CG: Acute immune complex disease in rabbits: The role of complement and of a leukocyte-dependent release of vasoactive amines from platelets. J Exp Med 133:554-571, 1971

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9. Benveniste J, Henson PM, Cochrane CG: Leukocyte-dependent histamine release from rabbit platelets: The role of IgE, basophils and a platelet-activating factor. J Exp Med 136:1356-1377, 1972 10. Minick CR, Murphy GE: Experimental induction of atheroarteriosclerosis by the synergy of allergic injury to arteries and lipid-rich diet. Am J Pathol 73:265-300, 1973

11. Becker CG, Dubin T: Activation of Factor XII by tobacco glycoprotein. J Exp Med 146:457-467, 1977

12. Capurro N, Levi R: The heart as a target organ in systemic allergic reactions: Comparison of cardiac anaphylaxis in vivo and in vitro. Circ Res 36:520-528, 1975 13. Levi R, Zaveez JH, Holland B, Becker CG: Cardiac and pulmonary anaphylaxis induced by tobacco glycoprotein (TGP) (abstr). Fed Proc 37:590, 1978

Acknowledgments We gratefully acknowledge the technical assistance of Miss Arlene Hardy, Mr. Richard Terek, and Mrs. Nancy Van Hamont and the help of Miss Gioconda Carrozza in preparation of the manuscript.

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