Modulation of the IgE antibody response in rats to Kentucky blue grass pollen allergens.

Int. Archs Allergy appl. Immun. 53: 162-173 (1977)

Modulation of the IgE Antibody Response in Rats to Kentucky Blue Grass Pollen Allergens13 A. K. M. Ekramoddoullah, F. T. Kisil and A . H. Sehon MRC Group for Allergy Research, Faculty of Medicine, University of Manitoba, Winnipeg, Man.

Introduction The standard ‘hyposensitization’ treat ment commonly used for allergic conditions, 1 Presented at the American Academy of Al lergy Meeting, San Diego, Calif., 1975, and at the Canadian Federation of Biological Societies Meet ing, Winnipeg, Man., 1975. 2 This work was supported by grants from the Medical Research Council of Canada and the Sell ers Foundation. 3 The capable technical assistance of Tom Cook is gratefully acknowledged. Received: February 19, 1976.

such as rhinitis or extrinsic asthma caused by inhalation of pollens, consists of a series of injections of the aqueous extract of the offending pollen, as introduced by Noon in 1911 [16], It has been generally accepted that this treatment results in the formation of blocking antibodies, which combine with the allergens released from the inhaled pol len. Thus, blocking antibodies are visualized as sequestering the allergens and preventing the reaction of the latter with reaginic anti bodies fixed to the mast cells located in the

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Abstract. The low molecular weight dialyzable fraction (D) prepared from the aqueous extract of Kentucky Blue Grass pollen was shown to suppress the formation of IgE anti bodies in rats immunized with the nondialyzable fraction (R). In an attempt to establish the nature of the constituents responsible for this suppression, D was fractionated by gel filtra tion through Sephadex G-25. The first fraction eluted (D,) elicited skin reactions in rats sensitized with a rat reaginic serum to R and also gave a precipitate with a rabbit antiserum to R. A later fraction (Dm) was devoid of these two properties. To investigate the effects of these fractions on the antibody response, rats received either or Dm, administered in saline, prior to their immunization with R in presence of aluminum hydroxide. Pretreatment with Dj resulted in a reduction of IgE antibody levels as compared with the IgE antibody response in control animals which had received pretreatment only with saline; however, pretreatment with D, did not affect the anti-R-hemagglutinating titers. In contrast, pretreat ment with Dm enhanced both the IgE and the hemagglutinating antibodies. Hence, it is concluded that the composite fraction D contains one group of constituents capable of suppressing and another of enhancing the IgE antibody response.

shock organs. In support of this interpreta tion for the therapeutic value of blocking antibodies, one may quote the observation that transitory improvement for allergic pa tients was achieved by passive immuniza tion with human globulins rich in blocking antibodies [4]. Following the identification of reagins with the IgE class of immunoglobulins [9], quantitative methods were developed to de termine the levels of specific IgE antibodies to the allergen [8, 21]. It was found that the levels of IgE antibodies rose initially after treatment with the aqueous pollen extracts and decreased after prolonged treatment [11]. Although a significant degree of alle viation of the clinical conditions has not been consistently achieved by hyposensitiza tion therapy, this treatment has been shown to result in a decrease of IgE antibody lev els, in an increase of blocking antibody ti ters and in a reduction of the histamine re leased from the allergic patients’ leukocytes on challenge with the allergen [17]. Over the past 65 years there has been lit tle improvement in therapeutic procedures and this lack of progress may in part be due to the lack of pure allergens and in part to the ignorance of the complex mechanism of immunological reactions underlying the reg ulation of IgE antibody formation. The use of allergens complexed to adsorbents, such as aluminum hydroxide or tyrosine [14], for hyposensitization therapy - at one time be lieved to lead to dramatic clinical improve ments - has not resulted in striking break throughs. On the other hand, penicillin al lergy, which is triggered by this chemically well-defined antibiotic, has been managed by inhibiting the reaction between the tissue fixed anti-penicillin reagins and the corre sponding multivalent antigen(s) by the ad

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ministration of an appropriate univalent penicillin derivative, benzylpenicilloylformyl-L-lysine [6]. Similarly, it was dem onstrated earlier in this laboratory that the aqueous extract of ragweed pollen con tained hapten-like constituents capable of inhibiting the Prausnitz-Kiistner reaction, which was inducible with the high molecular weight multivalent components of ragweed pollen [1]. Unfortunately, however, due to the unavailability of haptenic materials from pollen in sufficient amounts, this tech nique of inhibition of allergic reactions by saturating IgE antibodies on target tissues with the hapten-like components present in pollens has not been exploited on a wide scale. Nevertheless, the use of heteroge neous, hapten-like dialyzable fractions for the treatment of grass-sensitive patients has been claimed to result in clinical improve ment and in reduction of the IgE antibody levels [12]. In addition, the peripheral leu kocytes of these patients did not undergo transformation when these cells were stimu lated in vitro with the antigen [12], A simi lar observation was made in another study in which it was demonstrated that the leuko cytes obtained from an allergic patient un derwent transformation in vitro in the pres ence of the antigen, but this effect could be blocked by prior incubation of the cells with a dialyzable preparation from an aqueous extract of the pollen [18]. The purpose of the present study was to establish if the different components present in the dialyzable fraction of the aqueous ex tract of Kentucky Blue Grass (KBG) pollen, which is responsible for a large incidence of respiratory allergies in the Prairies and else where, were capable of modulating the for mation of IgE antibodies to the allergens of KBG. Using a rat model system for reagins


Ekramoddoullah/Kisil/Sehon


to KBG pollen, it was demonstrated that these dialyzable components contained, in deed, at least two major groups of immunologically active constituents, i.e. one of these specifically suppressed the IgE antibody re sponse and the other specifically enhanced this response.

Materials and Methods Animals Inbred adult male Wistar-Furth rats weighing 225-275 g (ARS Sprague-Dawley, Madison, Wise.) were used for the production of reaginic antibod ies. For passive cutaneous anaphylaxis (PCA) as says, random bred hooded rats (North American Laboratory Supply Co., Gunton, Man., Canada) were used. Preparation of Dialyzable and Nondialyzable Fractions of Pollen An aqueous extract of KBG pollen (HollisterStier Laboratory, Mississauga, Ont., Canada) was prepared according to the procedure previously established in this laboratory [7j. The aqueous ex tract was dialyzed through Visking tubing, No. 20 Dialysis (Union Carbide Canada Limited, Lindsay, Ont., Canada) against distilled water for 24 h at 4 °C; the dialyzable components are referred to as dialyzatc or D. To free the nondialyzable fraction of low molecular weight constituents, it was sub jected to dialysis for an additional 24 h; the nondi alyzable components are referred to as the retén tate or R. Both preparations, R and D, were lyophilized and stored in the dry state. Chromatography Gel filtration was carried out according to the conditions described in the caption for figure 1. Sephadex G-25 (particle size 20-80 /t) Pharma cia, Uppsala, Sweden) was equilibrated with wa ter. The absorbance of the eluates was measured at 280 nm with the Perkin-Elmer Model 139 UV-V1S Spectrophotometer. Induction of IgE Antibody Formation For the production of reaginic antibodies, rats

were injected intraperitoneally with a mixture of the appropriate antigen, in different concentra tions, dissolved in saline and a suspension of 30 mg aluminum hydroxide (Amphojel, Wyeth Ltd., Toronto, Ont., Canada). These animals were bled from the orbital sinus at weekly intervals and their sera were used for the titration of IgE anti bodies. Measurement of Serum IgE Antibody Levels by PCA Hooded rats were passively sensitized by intradermal injections of volumes of 50 ftl of the se rially diluted reaginic antibody-containing sera. At least two animals were sensitized with each scrum diluted identically. The PCA reactions were elicit ed 48 h later by challenging these sensitized ani mals with an intravenous injection of 1 ml of a solution containing the antigen (1 mg) and Evans blue dye (0.5%>) (Matheson, Coleman & Bell, Norwood, Ohio). The end point of the titration was expressed as the reciprocal of the highest di lution of the reaginic serum capable of sensitizing a skin site so as to give a PCA reaction of 5 mm in diameter or greater. PCA titers of the reaginic sera of a given group of animals are expressed as the geometric mean. Passive Hemagglutination (HA) The levels of other classes of antibodies, in ad dition to reagins, in the sera of immunized rats were determined by the passive HA procedure, us ing sheep red blood cells (SRBC) to which R was coupled by cross-linking with glutaraldchyde as described by Avrameas et al. [2]. To monitor the sensitivity of the HA analysis, a rabbit antiserum containing precipitating antibodies to R was em ployed as the reference antiserum. The reciprocal of the HA titer obtained with this antiserum was 2560. Rabbit Antiserum to R New Zealand white rabbits were immunized by i.d. injections, into multiple sites, of a total vol ume of 0.4 ml of an emulsion prepared from 0.2 ml of R (5 mg/ml saline) and 0.2 ml Freund’s complete adjuvant (FCA) (Difco Laboratories, Detroit, Mich.). 2 weeks later and at weekly inter vals thereafter for a period of 3 weeks, the ani mals received intramuscular injections of the


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Table I. Variability of the IgE antibody response Experi ment No.

Maximum PCA titer1 primary response2

Days after PCA titer1 primary secondary immuni response3 zation when maximum titer was detected

1 2 3 4 5 6 7 8 9

13.9 9.5 6.1 4.3 4 40.3 1.8 8 4.5

21 35 28 56 28 42 14 14 35

Average titer

10.2

ND 19 17.4 19 13.4 ND ND 39.3 ND 21.6

ND = Not determined 1 All PCA titers are expressed as the geometric mean calculated from the PCA titers which were individually determined for each serum of a group of animals composed of 10 rats. 2 Wistar-Furth rats immunized with a dose of 100 us of R (see text) in Amphojel. 3 The rats were reimmunized with R 3 months after the first immunization. The PCA titers were deter mined for the sera obtained 7 days following the second immunization.

emulsified solution containing R. The animals were bled 1 week after the last immunization. Statistical Analysis To establish the statistical significance of the differences in the PCA and HA titers observed be tween the experimental and control groups, the Mann-Whitney U tests were employed [13].

Results A single dose of 100 /zg of R elicited an IgE antibody response to the allergens pre

sent in the high molecular weight fraction of the aqueous extract of KBG pollen. 7 days after immunization with R, serum IgE anti body could be rarely detected, but by the tenth day the antibody activity usually ap peared in circulation. The time required to observe the maximum primary IgE antibody response varied, i.e. the maximum IgE anti body titer was seen as early as 14 days or as late as 56 days after immunization (table I). Serum IgE antibodies were still detected even 3 months after immunization. Reim munization of animals after 3 months with 100 fig of R resulted in an elevated IgE an tibody response and the maximum titer was obtained within 1 week after secondary im munization. The reaginic antibodies were identified as belonging exclusively to the IgE class of immunoglobulins on the basis of the follow ing criteria: (i) PCA reactions were elicited 48 h after the passive sensitization of rats with the reaginic sera; (ii) these PCA reac tions were abolished by heating the reaginic sera at a temperature of 56 °C for a period of 4 h prior to use for passive sensitization of rats, and (iii) the PCA activity of the rat reaginic sera was removed by coprecipita tion (which was kindly performed by Dr. D. H. Conrad of this Department) of the IgE immunoglobulins with a rabbit antiserum specific for a rat myeloma IgE [3], followed by the addition of a goat antiserum to rabbit gammaglobulins [5]. Although inbred rats were employed, the magnitude of the primary IgE antibody response was not uniform, i.e. the maximum IgE antibody titer, expressed as a geometric mean for any one group of animals, ranged from 1.8 to 40.3 and the average geometric mean was 10 (table I). Similarly, the magni tude of the secondary IgE response ranged


Modulation of Reagin Formation

from 13.4 to 39.3 and the average geome tric mean was 21.6. For statistically mean ingful experiments, a sufficiently large batch of animals matched with respect to their birth date was obtained from the supplier and randomly divided into groups of 10 rats each; one of these groups served as the con trol group for the whole batch. Effect on Antibody Formation of Ad ministration of Dialyzate Rats were pretreated with D prior to their immunization with R. For each rat this pretreatment consisted of five daily i.p. dos es each of 50 mg of D in saline. This daily dose of 50 mg of D had been determined as the minimum amount of D capable of sup pressing the formation of IgE antibodies to R. The control group of rats received five daily injections of saline only. On the sixth day both groups were immunized with 100 //g of R in Amphojel. Following immu nization the sera were collected at weekly intervals for 8 weeks. The PCA titers of sera from the treated group were consistently lower than the PCA titers of sera from the control group. Fol lowing the decline of the primary IgE re sponse, which required about 3 months, the treated on control groups received a second series of five daily i.p. injections of 50 mg of D and saline, respectively, and were then reimmunized with 100 fig in R in Amphojel. As a result of the second pretreatment with D, within a week following reimmuni zation, only a barely detectable IgE anti body response was detected (GM value of 0.1) in contrast to the control group which showed a typical secondary antibody re sponse with elevated IgE antibody levels (GM of titers was 16; these differences were significant, p = 0.002).


The effect of pretreatment with D on the formation of other classes of antibodies, in addition to those of the IgE class, was eval uated in terms of HA titers. Following the first injection of R, only very low HA titers (GM titer of 1-1.5) could be detected in the sera of either the treated or the control groups. It was, therefore, concluded that the formation of antibodies associated with im munoglobulin classes other than IgE was only minimal. On the other hand, reimmun ization of both the experimental and control animals resulted in significant HA titers, i.e. the GM values for treated and control groups were 320 and 233, respectively (p>0.2). On the basis of the results of this series of experiments, it was concluded that treat ment of rats with D prior to the immuniza tions with R resulted in the suppression of the IgE antibody response, but that this treatment had no significant effect on the formation of other classes of antibodies. Suppression by D of an Ongoing IgE Antibody Response The possible suppression of an ongoing IgE antibody response by the administration of D was investigated. Rats were first im munized with R and daily treatment with D or saline (as described in the preceeding section) was given on days 11-15 following administration of R, by which time IgE an tibody formation had been established. In comparison with the PCA titers of the sera of the control group which had been first immunized and then given only saline on days 11-15, the PCA titers of the sera of the test group progressively dropped to val ues well below those of the control group. However, the significant suppression of the formation of IgE antibodies was observed


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only 8 weeks after the treatment with D (p = 0.02). Specificity of the Suppression The specificity of the suppression of anti-R IgE antibody formation by D was es tablished as follows. A group of rats was first treated with D according to the proto col already described. Another group of rats was treated only with saline. Thereafter, both groups of rats were immunized with the reténtate prepared from the aqueous ex tract of ragweed pollen and mixed with Amphojel. It was found that treatment with D, prepared from the KBG pollen extract, did not suppress the formation of IgE antibod ies to the ragweed reténtate and, therefore, one may conclude that suppression of anti-R IgE antibodies was indeed specific. Fractionation of D To facilitate identification of factor(s) re sponsible for the suppression of IgE anti body formation, D was subjected to gel fil tration on Sephadex G-25. Fraction D was thus resolved into at least nine components (fig. 1). The allergenic activity of these frac tions was evaluated in terms of their ability to elicit PCA reactions in rats passively sen sitized with reaginic serum to R. The PCA titer of the particular reaginic serum, used in this experiment, was 8 when elicited with a dose of 1 mg of R. An equal PCA reac tion was obtained with this serum on chal lenge with 10, 1 and 2 mg of D, D, and Dn, respectively. The chromatographic fraction eluted after Dn, which had the highest optical density, did not elicit PCA reactions even at a dose as high as 20 mg. Moreover, all subsequent fractions were in capable of eliciting a PCA reaction. Conse quently, all fractions eluted after DM were

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pooled and combined into one fraction, re ferred to as Dn„ which was inactive in PCA tests. The fact that the whole dialyzate (i.e. D) and its two subfractions (i.e. D, and D,,) could elicit PCA reactions in rats sensitized with reaginic serum prepared against R in dicated that D, as well as Dt and Dn, shared at least some of the allergenic deter minants) of R. In addition, the antigenic relationship between D, and R was estab lished by immunodiffusion analysis with a rabbit antiserum to R. A precipitin band was obtained indicating that D, possessed at least an antigenic determinant common to R. Identification of the Subfraction(s) of D Responsible for (i) Suppression of the IgE Antibody Response and (ii) Enhancement of the IgE Antibody Respone To establish if the skin-active or the skininactive fractions of D were responsible for the observed suppression of the IgE an tibody formation, the experimental protocol in which rats were pretreated prior to their immunization with R was used. For this purpose, D, was selected on the basis of its skin activity and D,H was considered as representative of all skin-inactive fractions. Thus, groups of 10 rats each were treated separately with D, or Dm prior to their immunization with R in Amphojel. Treat ment of these rats consisted of five daily i.p. injections each containing 1.8 mg of D, or 24 mg of Dn[ dissolved in saline; these doses were calculated on the basis of the following considerations: (a) suppression of IgE antibody formation had been achieved with administrations of a daily dose of 50 mg of D per rat; (b) the total number of daily doses of D for administration to 10




Fig. I. Elution profile of D chromatographed on Sephadex G-25 fine. A volume of 15 ml con taining 2.5 g of D in water was applied on a col umn 5X90 cm; distilled water of a flow rate of 60 ml/h was used as the eluant. Fractions desig nated as Di, Dn and Dm were pooled. Symbols in parentheses indicate PCA reactions, if any, elic ited in rats passively sensitized with a reaginic se rum-containing antibodies to R and challenged in travenously with the pooled fractions; (+ + ) high ly skin-active, (+ ) moderately skin-active, (-) not skin-active.

Fig. 2. Effect of pretreatment with Di on an tibody formation in rats immunized with R. Each rat received 5 daily i.p. doses of 1.8 mg of Dp The control group received only saline. On the 6th day (indicated as day 0) each rat was immunized with 100 iig of R in Amphojel. Treated group, solid bars; control group, open bars.

rats was 50 which amounted to 2.5 g of D; (c) chromatography of 2.5 g of D yielded 0.09 g of D, and 1.2 g of Dm; these amounts were divided into 50 doses for each fraction, i.e. 1.8 mg of D,/rat/day and 24 mg of Dm/rat/day. The control group of rats received i.p, injections of saline for the same period. On the 6th day, both groups were immunized with 100 jug of R in the presence of Amphojel. The formation of IgE antibodies was followed at regular in tervals by determining the PCA titers of the sera of these animals. (1) The results presented in figure 2 il lustrate the effect on the formation of IgE antibodies to R by the treatment with frac tion D,. The treated group showed consis tently lower PCA titers compared to the control. The differences in PCA titers be

tween the treated and control groups are statistically significant with the exception of the difference in titers of sera obtained on the first bleeding after immunization. The HA titers, on the other hand, were not sig nificantly different between the treated and control groups indicating that D, did not af fect the formation of the classes of antibod ies other than IgE. (2) By contrast to pretreatment with D,, the pretreatment with Din resulted in sig nificantly higher IgE antibody levels, by comparison with the reaginic titers of the control group (fig. 3), for a period of 1 month following immunization. After this period, the PCA titers of the sera of the treated group were still consistently higher than those of the control groups, but the dif ferences were no longer significant.


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169

Days After Immunization

Fig. 3. Effect of pretreatment with Dm on antibody formation in rats immunized with R. Each rat received 5 daily i.p. doses of 24 mg of Dur- The control group received only saline. On the 6th day (indicated as day 0) each rat was im munized with 100 fig of R in Amphojel. Treated group, solid bars; control group, open bars.

The HA titers of the sera of the group treated with DU| remained significantly higher than the corresponding titers of the sera of the control group after reaching the highest levels (fig. 3). These experiments demonstrated that treatment of rats with Dm prior to their immunization with R enhanced the forma tion of both IgE and other classes of anti bodies. Specificity of the Enhancement The protocol used previously to evaluate the specificity of suppression of the IgE an tibody formation was again employed to test

for the specificity of the enhancement. It was found that the titers of IgE antibody in rats pretreated with DIU and then immu nized with the reténtate prepared from the aqueous extract of ragweed pollen were comparable to the reaginic antibody titers of animals which had received saline prior to their immunization with the ragweed retén tate. Consequently, it was concluded that enhancement of anti-R IgE antibodies was indeed specific for the KBG allergens. Evaluation of the Immunogenic Proper ties of D and its Subfractions The immunogenic properties of D were evaluated in terms of its ability to induce an IgE antibody response. For this aspect of the study, the magnitude of the IgE re sponse was related to that obtained by im


DAYS AFTER IMMUNIZATION

Fig. 4. IgE antibody response in Wistar-Furth rats following immunization with R, D, D] and Dm- The doses used in this experiment were de termined according to procedures described in the text. • = D (10 mg); O = R (0.1 mg); ▲ = Di (0.329 mg); ■ = Dm (6.75 mg).

Ekramoddoul lah/Kisil/Sehon

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each rat of the corresponding two groups of animals. For reasons already discussed, the PCA titers of the sera of these animals were determined using R as the challenging anti gen. Immunization of rats with D, in the presence of Amphojel induced an IgE anti body response comparable to that induced by unfractionated D. On the other hand, none of the sera from the group of rats im munized with Dm had any PCA activity as demonstrated by the lack of any reaction on challenge with either R or Dm; one may therefore infer that Dm did not in duce an IgE antibody response. No HA activity could be detected in any of the sera of animals immunized with D, or Dni and only low HA titers of the ord er of 1.2-1.6 (GM) were detected in sera of animals immunized with D or R; these titers were not considered to be significant. In view of the observations that Din was not immunogenic, it was concluded that the ability of D to induce the formation of IgE antibodies could be accounted for by the immunogenic component(s) present in D,. Moreover, the relatively large dose of 10 mg of D in comparison with the dose of 0.32 mg of D, required to induce IgE anti body formation, could be explained by the fact that the bulk of D was composed of the nonimmunogenic material, Dai.

Discussion In the present study it was shown that an IgE antibody response could be induced in rats by immunization with the nondialyzable components of the aqueous extract of KBG pollen using Al(OH)s as the adjuvant. The maximal primary reaginic antibody response


munization with the optimal dose of 100 ,«g of R, which had been determined previously as the dose capable of eliciting the highest IgE response. Three groups of 10 rats each were im munized with D at 0.1, 1 and 10 mg of D, respectively, in the presence of Amphojel, as previously described for immunization with R. These animals were bled at regular intervals and the IgE antibody titers of the sera were established by PCA. The highest IgE antibody response (PCA titer with a GM value of 8) was obtained in the group of animals immunized with a dose of 10 mg of D (fig. 4); only a minimal IgE antibody response was observed with a dose of 1 mg of D (the highest GM of PCA titers was 1.4) whereas no IgE antibodies could be detect ed over a period of 5 weeks following im munization with 0.1 mg of D (not shown in fig. 4). It should be pointed out that R was used as the challenging antigen to elicit the PCA reactions rather than D, since the lat ter elicited only at best smaller reactions than R. Two conclusions were drawn from the results of this series of experiments: the dialysate when administered in presence of Amphojel could induce the formation of IgE antibodies and these antibodies reacted also with R. Thus, the previous inference (see p. 167) that D and R share a common allergenic determinant(s) was reaffirmed. From the chromatogram of D, it was de duced that 10 mg of D corresponded to 0.329 mg of D, and 6.75 mg of Din. Hence, to establish if either of these two subfractions of D had the capacity to induce IgE antibody formation and since 10 mg of D was capable of eliciting an IgE response, 0.329 mg of Dt and 6.7 mg of DriI were separately mixed with Amphojel and, re spectively, used for the immunization of

was observed on immunization with R at a dose of 100 f/g. Low levels of the reaginic antibody could still be detected in the sera of these animals 3 months after the initial immunization. After this period of time, reimmunization resulted in an earlier ap pearance of IgE antibodies, i.e. within 1 week, and the antibody titers were higher than in the primary response (table I). How ever, this secondary IgE antibody response was short lived. This animal model system was used to investigate the ability of the dialyzable com ponents to modulate the formation of IgE antibodies to the reteníate of KBG pollen. Suppression of the IgE antibody response to R was achieved when the dialyzable compo nents of the aqueous extract of the pollen were administered to the animals prior to immunization with R. The degree of sup pression was more apparent if treatment with D was repeated just prior to the second immunization with R. On the other hand, omission of the pretreatment with the dialyzate prior to the second immunization with R resulted in a characteristic secondary an tibody response. Thus, it would appear that the immunosuppression of IgE antibody formation required repeated injections with D. The suppression of an ongoing IgE anti body response by the administration of the dialyzate was also effective, but a significant suppression occurred only 1.5 months after the treatment. Hence, we may conclude that once the reaginic response has been estab lished, its abrogation by treatment with D was not as readily achieved as the suppres sion of the induction of this response. In view of the fact that D possessed al lergenic activity, it could be suggested that suppression of IgE antibody formation was simply due to the neutralization of the re-

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agins by the allergenic constituents of D. If this had been the case, it would be expected that the PCA titers of the sera obtained from animals with an ongoing IgE antibody response would be lower after treatment with D. In actual fact, the sera of such ani mals obtained 3 days after the last treatment with D had PCA titers higher than those of the control groups. Therefore, the suppres sion observed cannot be attributed to neu tralization of the IgE antibodies by D. Gel filtration studies revealed that D was composed of a heterogeneous mixture of components. Moreover, the relative yields of D„ D„ and D,n were found to vary for each chromatographic separation of D. The aver age yield of D, and Dm was 3.3 and 67.5%, respectively. Only the chromatographic frac tion containing the larger molecular size components, i.e. D„ had the ability to sup press the formation of IgE antibodies to R, without significantly affecting the formation of antibodies detectable by hemagglutina tion. It should also be noted that Dj pos sessed essentially all of the allergenic and antigenic activity of the unfractionated di alyzate. Therefore, we may propose that the components responsible for these properties possessed at least two allergenic and/or two antigenic sites on the same molecule capa ble of reacting with the homologous anti bodies to form multimolecular aggregates as detected by PCA and by precipitin reactions. Although it has not as yet been determined if the allergenic components detected in the dialyzate are distinct products synthesized during the reproductive processes of the grass pollen or if they represent degradative products of the larger size components pre sent in R, it has nevertheless been conclu sively established that D, and R share com mon allergenic properties. Thus, IgE anti




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enhancing fraction DIU were not skin-ac tive, antigenic or immunogenic, it can be postulated that these component(s) lacked the haptenic moieties present in D[ or R which are capable of interacting with B lym phocytes. On the other hand, it may be sug gested that DIU possesses determinants capable of interacting with T lymphocytes as demonstrated by the fact that animals primed with Din had an enhanced IgE an tibody response after immunization with R. The mechanisms responsible for the modulation of the IgE antibody response to R by the subfractions of D remain to be elu cidated. The isolation and characterization of the active component(s) responsible for the modulation of the IgE antibody re sponse is expected to pave the way towards the development of new experimental ap proaches which, in turn, may lead to the de velopment of an effective therapeutic proce dure for the suppression of IgE antibodies mediating common forms of allergy in man.

References 1 Attallah, N. A. and Sehon, A. H.: Isolation of haptenic material from ragweed pollen. Immunochemistry 6: 609-619 (1969). 2 Avrameas, S.; Tandon, B., and Cleniton, S.: Glutaraldehyde, cyanuric chloride and tetraazitized 0-dianisidine as coupling reagents in the passive hemagglutination test. Immunochemistry 6 :67-76 (1969). 3 Bazin, H.; Beckers, A., and Querinjean, P.: Three classes and four (sub) classes of rat im munoglobulins: IgM, IgA, IgE and IgG„ IsG2a, lgG2t„ IgG2C. Eur. J. Immunol. 4: 44-48 (1974). 4 Bernton, H. S.; Chambers, D. C., and Querry, M. W.: Therapy of ragweed pollenosis with blocking antibody. J. Allergy 33: 356-364 (1962). 5 Conrad, D. H. and Froese, A.: Characteriza


bodies elicited by immunization with D or D, cross-reacted with R and, conversely, IgE antibodies elicited by immunization with R cross-reacted with D or D,. The pretreatment of animals with the nonimmunogenic and nonskin active frac tion Dm prior to immunization with R markedly enhanced the formation of IgE antibodies. This enhancing property of Dn[ was further substantiated by the con sistent observations that all animals which had received the pretreatment always pro duced IgE antibodies, whereas at least one animal of the control group consistently failed to produce IgE antibodies following immunization with R. At the present time, only a hypothesis as to the nature of these components can be offered. It is proposed that D, possesses both the haptenic and carrier determinants capable of interacting with B and T lympho cytes, respectively, and that thus the appro priate cell cooperation for initiation of anti body production can be brought about [15]. In view of the findings of Tada et al. [19] and of Ishizaka et al. [10] that formation of IgE antibodies could be suppressed by the generation of carrier-specific suppressor T cells, it can be visualized that the suppres sion observed in the present experiments was also due to the generation of such cells by pretreatment with D,. It is known that the synthesis of IgE an tibodies in the rat can be regulated by other classes of antibodies [20]. However, in view of the coexistence of both hemagglutinating antibody activity and IgE antibodies (e.g. following reimmunization with R) the pres ence of other classes of antibodies detected in sera cannot account for the observed sup pression of IgE antibody formation. Since the component(s) present in the

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tion of the target cell receptor for IgE. II. Po lyacrylamide gel analysis of the surface IgE re ceptor from rat mast cells and rat basophilic leukemia cells. J. Imrnun. 116: 319-326 (1976). DeWcck, A. L. and Girard, J. P.: Specific inhi bition of allergic reactions to penicillin in man by a monovalent hapten. II. Clinical studies. Int. Archs Allergy, appl. Immun. 42: 798-815 (1972). Ekramoddoullah, A. K. M. and Sehon, A. H.: Studies on the allergens in Timothy grass (phleum pratense) pollen. Int. Archs Allergy appl. Immun. (to be published). Gleich, G. J. and Jones, R. T.: Measurement of IgE antibodies by the radioallergosorbent test. II. Analysis of quantitative relationships in the test. J. Allergy 55: 346-357 (1975). Ishizaka, K.; Ishizaka, T., and Hornbook, M. M.: Physico-chemical properties of human reaginic antibody. IV. Presence of a unique im munoglobulin as a carrier of reaginic activity. J. Immun. 97: 75-85 (1966). Ishizaka, K.; Okudaira, H„ and King, T. P.: Immunogenic properties of modified antigen E. II. Ability of urea-denatured antigen and «-polypeptide chain to prime T cells specific for antigen E. J. Immun. 114: 110-115 (1975). Levy, D. A.; Lichtenstein, L. M.; Goldstein, E. O., and Ishizaka, K.: Immunological and cellu lar changes accompanying the therapy of pol len allergy. J. clin. Invest. 50: 360-369 (1971). Malley, A.; Wilson, B. J.; Barret, M., and Perl man, F.: The site of action of antigen D im munotherapy. J. Allergy 48: 267-275 (1971). Mann, H. B. and Whitney, D. R.: On a test of whether one of two random variables is sto chastically larger than the other. Ann. math. Statist. 18: 50-60 (1947). Miller, A. C. M. L. and Tees, E. C.: A meta bolizable adjuvant. Clinical trial of grass pol len-tyrosine adsorbate. Clin. Allergy 4: 49-55 (1974).

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15 Mitchison, N. A.; Rajewsky, K., and Taylor, R. B.: Co-operation of antigenic determinants and of cells in the induction of antibodies; in Stcrzl and Riha Developmental aspects of anti body formation and structure, vol. 2, pp. 547-561 (Adademic Press, New York 1970). 16 Noon, L.: Prophylactic inoculation against hayfever. Lancet i: 1572-1573 (1911). 17 Pruzansky, J. J. and Patterson, R.: Histamine release from leukocytes of hypersensitive indi viduals. II. Reduced sensitivity of leukocytes after injection therapy. J. Allergy 39: 44-50 (1967). 18 Romagnani, S.; Biliotti, G.; Passaleva, A., and Ricci, M.: In vitro lymphocyte response to pol len extract constituents in grass pollen-sensi tive individuals. Int. Archs Allergy appl. Im mun. 44: 40-50 (1973). 19 Tada, T.; Okumura, K., and Taniguchi, M.: Reaginic antibody formation in the rat. Regu latory effect of soluble carrier-specific T cell factors on hapten-specific reagin production. Proc. 8th Congr. Int. Ass. Allergol. Int. Cong. Series 323: 472-480 (1974). 20 Tada, T. and Okumura, K.: Regulation of homocytotropic antibody formation in the rat. I. Feed-back regulation by passive administrat ed antibody. J. Immun. 106: 1002-1011 (1971). 21 Wide, L.; Bennich. H., and Johansson, S. G. O.: Diagnosis of allergy by an in vitro test for allergen antibody. Lancet ii: 1105-1107 (1967).

Correspondence to: Dr. A. Sehon, Department of Immunology, University of Manitoba, 730 William Avenue, Winnipeg, Man. R3E OW3 (Canada)



Examination of microheterogeneity in grass pollen allergens.

Quantitation of IgE antibody specific for ragweed and grass allergens: binding of radiolabeled allergens by solid-phase bound IgE.

Variability of crossreactivity of IgE antibodies to group I and V allergens in eight grass pollen species.

A single mouse monoclonal antibody, E58 modulates multiple IgE epitopes on group 1 cedar pollen allergens.

IgE antibody to cat allergens in an allergic population.

Grass pollen allergens globally: the contribution of subtropical grasses to burden of allergic respiratory diseases.

Association between a low IgE response to Phl p 5 and absence of asthma in patients with grass pollen allergy.

Evolution pathways of IgE responses to grass and mite allergens throughout childhood.

B cell-intrinsic MyD88 signaling is essential for IgE responses in lungs exposed to pollen allergens.

II is increased following grass pollen immunotherapy.

Specific IgE recognition of pollen allergens from subtropic grasses in patients from the subtropics.

Allergens. Heterogeneity of molecular sensitization profiles in grass pollen allergy--implications for immunotherapy?

High-density IgE recognition of the major grass pollen allergen Phl p 1 revealed with single-chain IgE antibody fragments obtained by combinatorial cloning.

Rapid extraction of grass pollen allergens and separation of their active fractions.

High-resolution crystal structure and IgE recognition of the major grass pollen allergen Phl p 3.

Comparative and Evolutionary Analysis of Grass Pollen Allergens Using Brachypodium distachyon as a Model System.

Magnetic enzyme immunoassay of anti-grass pollen specific-IgE in human sera.

Induction of IgE antibodies in mice with recombinant grass pollen antigens.

Cypress (Cupressus sempervirens) pollen allergens: identification by protein blotting and improved detection of specific IgE antibodies.

Multiple independent IgE epitopes on the highly allergenic grass pollen allergen Phl p 5.

Influence of seasonal exposure to grass pollen on local and peripheral blood IgE repertoires in patients with allergic rhinitis.

High correlation of specific IgE sensitization between birch pollen, soy and apple allergens indicates pollen-food allergy syndrome among birch pollen allergic patients in northern China.

Cellular mechanisms of IgE antibody response.

Antigens and allergens in birch pollen extract.