Isolation of haptenic material from ragweed pollen

lmmunochemistry. PergamonPress 1969. Vol.6, pp. 609-619. Printedin Great Britain

I S O L A T I O N OF H A P T E N I C MATERIAL FROM RAGWEED POLLEN* NICOLA A. ATTALLAH and ALEC H. SEHON The Laboratory for Biophysical- and Immuno-chemistry, Department of Chemistry, McGill University, Montreal 2, Quebec, Canada (Received 23 October 1968) A b s t r a c t - A haptenic fraction (A-2RS-I) was isolated from the dialysate of the aqueous

homogenate of short ragweed pollen (WSRH). This fraction was inactive in direct skin and Prausnitz-Kiistner (P-K) passive transfer tests, but inhibited specifically the P-K reactions given by WSRH and reagins to ragweed. It is suggested that fraction A-2RS-I might prove effective in hyposensitization treatment. In recent investigations, Malley et al. [2-4] reported that the dialyzable fraction of the aqueous extract of timothy grass pollen contained low molecular weight materials with haptenic properties, as demonstrated by the ability of this fraction to inhibit some of the precipitin lines between rabbit antibodies to whole timothy pollen extract and the nondialyzable fraction of this extract. However, this material was still able to induce skin reactions on intradermal injection into allergic individuals, or in passive transfer Prausnitz-KiJstner (P-K) tests in normal nonallergic volunteers. As has been shown [5-7], for an inflammatory reaction to occur it is essential that muhimolecular antibody-antigen aggregates be formed in vivo. Therefore, one may infer from these findings of Malley et al. [2-4] that, although their dialyzable fraction contained hapten-like constituents with respect to antibodies elicited in rabbits, some of these materials possessed properties of polyvalent allergens or acquired such properties after intradermal injection into human skin. More recently, Feigen et al. [8] demonstrated that the dialyzable components of ragweed pollen extract had the ability of inhibiting the in vitro anaphylaxis, which was inducible with the whole extract on addition to guinea pig intestinal and cardiac tissues which had been passively sensitized with rabbit antibodies to nondialyzable ragweed preparations, and concluded that their dialysates contained hapten-like components. However, it ought to be stressed that this conclusion, which is similar to that arrived at by Malley et al. [2-4] for their dialyzable fraction of timothy grass pollen, is based on evidence obtained with rabbit antibodies which may have been elicited by antigenic determinants different from those responsible for the induction of reagins in man. In a previous study from this laboratory[9] it was observed that one of the dialyzable fractions of the aqueous ragweed pollen extract (D1) did not elicit skin reactions in the P-K test with sera of non-treated allergic individuals. This *A preliminary report[l] of this study was presented in April 1968 at the Annual Meeting of the American Association of Immunologists, Atlantic City, N.J. 609

IMM Vol. 6 No. 4 - G

610

N.A. ATTALLAH and A. H. SEHON

finding prompted us to re-examine the immunologic behaviour of the components of the dialyzable fraction of ragweed pollen extract, in order to establish if some of these constituents had hapten-like properties with respect to reagins produced in man and, if so, to determine their chemical structure. If this were indeed the case, the intravenous administration of non-toxic univalent haptens to allergic patients would be expected to result in the blocking of all antibody sites[10] and, thus, to lead to protection of the patients by the inhibition of reactions between skin-sensitizing antibodies and the multivalent allergens present in the inhaled pollen. Consequently, in addition to the intrinsic value of this study as regards the elucidation of the chemical nature of the haptenic constituents of ragweed pollen, it can be visualized that, as a result of such investigation, univalent haptens of known composition might be used as desensitizing agents. This article describes the isolation of a fraction from ragweed dialysate which was not skin active even at a concentration of 1 mg/ml when injected into a series of 23 allergic patients, who gave strong skin reactions to the whole extract of ragweed pollen. Moreover, this fraction was shown to possess hapten-like properties inasmuch as it inhibited the P-K tests performed with sera of two ragweed sensitive individuals. MATERIALS AND METHODS The following materials were purchased from the suppliers indicated in brackets: Bio-Gel P-2, 50-100 mesh (Bio-Rad Laboratories, Richmond, Cal.); Amberlite IRC-50 (Rohm and Hass Co., Ltd., Philadelphia, Pa.); Sephadex G-15 (Pharmacia, Upps~la, Sweden); short ragweed pollen (Greer Drug and Chemical Corporation, Lenoir, N.C.).

Preparation of ragweeddialysate Fifty grams of pollen were defatted with ether in a Soxhlet extractor for 24 hr. The defatted pollen was then suspended in water* and homogenized with a Virtis-45 homogenizer at 100,000 rev/min for 3 min in an ice bath; extraction of this suspension in water was continued by stirring for 48 hr at 4°C. The insoluble portion was removed by filtration; the filtrate was centrifuged and the supernate (WSRH) lyophilized. The lyophilized material was dissolved in a minimum amount of water and dialyzed against four changes of water for 48 hr. The dialysates were pooled, freeze-dried and stored at 4°C in evacuated vials. The nondialyzable material was freeze-dried and stored in a similar manner for other investigations.

Chromatography In all steps involving column chromatography, elution was carried out with water at 4°C, with the exception of gel filtration through Sephadex G-15 (column 1.1 x 110 cm; flow rate 15 ml/hr) which was performed at room temperature. The flow rate for the Bio-Gel P-2 and Amberlite IRC-50 columns (2"5 X 45 cm and 2"5 x 33 cm, respectively) was about 20 ml/hr. *Glass redistilled water was used throughout this study.

Isolation of Haptenic Material from Ragweed Pollen

611

Methods of analysis Microzone electrophoresis was performed in vernonal buffer, pH 8.6 and ionic strength 0-075, for 40 min at 250 V, with the cell Model R-101, Beckman Instruments Inc. After electrophoresis the cellulose acetate membrane was dried and stained by spraying with 0.5% ninhydrin in n-butanol. The sedimentation coefficients were measured in the analytical Spinco Model E ultracentrifuge using a synthetic boundary cell at 25°C. The concentration of the samples was l0 mg/ml of saline. The ultra-violet spectra were determined with a double beam recording Perkin-Elmer, Model 350, spectrophotometer or with a recording Beckman-DB spectrophotometer. For amino acid analysis the sample (2 mg) was hydrolyzed with 2 ml of triple distilled 5.7 N HCI in sealed tubes under nitrogen for 20 hr at ll0°C. At the end of the hydrolysis, HC1 was removed in a vacuum desiccator containing NaOH pellets. The hydrolysate was dissolved in water and freeze-dried; to completely remove any traces of HCI this process was repeated three times. The amino acid composition of the hydrolysate was determined according to Spackman, Stein and Moore [11] with the Beckman Model 120B amino acid analyzer.

Immunologicprocedures Precipitating antisera were elicited in rabbits by three intracutaneous injections (on both sides of the abdominal region) of WSRH (10 mg in 0.5 ml saline) in complete Fruend's adjuvant (0.5 ml) at weekly intervals. The animals were bled 1 week after the last injection. Attempts were also made to induce production of antibodies to the 'haptenic fraction' (described in the following section) in rabbits according to the above schedule. For immunodiffusion analysis, the procedure described by Ouchterlony[12] was adapted; the WSRH antigen and the dialysate fractions were placed in the peripheral wells and the rabbit antiserum to WSRH in the central well. Each fraction including WSRH was dissolved in saline and tested at a concentration of 5 mg, 10 mg and 20 mg/ml. To test the ability of the fractions to inhibit precipitin bands, the agar plate was saturated with the haptenic fraction for a period of over six hours, at a concentration of 20 mg/ml. Subsequently, a mixture consisting of the rabbit antiserum to WSRH and the haptenic fraction (at three final concentrations of 5 mg, 10 mg and 20 mg/ml) was placed in the central well and the WSRH and the dialysate fractions at the above concentrations were added to the outer wells. P-K tests were performed in the backs of normal nonallergic volunteers who had neither a history of allergy, nor gave a skin reaction when challenged directly with either WSRH or the haptenic fraction. Sites were sensitized intracutaneously with 0.05 ml of a suitably diluted reaginic serum and were challenged 24 hr later with 0.025 ml of the fraction to be tested for skin activity. The skin reactions were read 15 rain after challenge. For testing the capacity of a fraction to inhibit the P-K reaction, the reaginic serum at an appropriate dilution was incubated with that fraction (at several concentrations from 0.001 mg/ml to 5.0 mg/ml) for 1 hr at 37°C, followed by a further incubation for about 18 hr at room temperature before sensitization of skin sites. After 24 hr the same

612

N.A. ATTALLAH and A. H. SEHON

sites were challenged with WSRH at a concentration of 10/.tg/ml. The P-K tests and the inhibition of the P-K reaction were performed in the back of the same volunteer. All reaginic sera and fractions were sterilized by passage through millipore membranes. RESULTS AND DISCUSSION

Preparation and properties of the haptenicfraction In Fig. 1 is given the flowsheet for the preparation of the haptenic fraction. Two grams of the dialysate, dissolved in 3 ml water, was passed through the POLLEN (IOOg) I Defatted I Homogenized 8 extracted witn water I WSRH Ii L y o l ~ i l i z e d j

Ololysed

!

I

Non,dialysable Residue

Dialysate (10-15 g) J

Fract'ion A

Chromatography Bio-Gel P-2 Pink' bend

Fracti°n B

J IRC-50 I

I

Fraction A-I

Fraction A-2 (2-3g) I Rechromatography IRC-50 I Froction A-2R (I.O-I.5¢J)

• (A-I)

I G-15 I A- 2RS-I

|

!

!

(A-2RS-H)

(A- 2RS-II~

(A-ZRS-IV)

(0.8 - 1.2g)

Fig. 1. Flow sheet for the preparation of fraction A-2RS-I. column of Bio-Gel P-2 resulting in two heterogeneous fractions A and B separated always by a pink band (Fig. 2); these fractions were pooled separately and freeze-dried. Since the ultraviolet spectra of the components present in all individual tubes constituting fraction A showed the highest absorption at 257 m/x, the absorbance of all the chromatographic fractions was measured at this wavelength. Further chromatography of fraction A (400 mg in 2 ml) on Amberlite IRC-50 led to the separation of two major fractions A-1 and A-2 (Fig. 3). When fraction A-2 was rechromatographed on Amberlite IRC-50, it was well separated from any contaminating fraction A-1 (Fig. 4); the second peak eluted on rechromatography will be referred to as A-2R. In further rechromatography of fraction A-2R, on the standard IRC-50 column, no components with properties of fraction A-1 were detected and the material was eluted as a sharp, but unsymmetrical peak, suggesting that it contained more than one component. This

Isolation of Haptenic Material from Ragweed Pollen

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Column

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20

30

40

50

60

70

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120

130

140

150

160

number

Fig. 2. Chromatography of ragweed dialysate on Bio-Gel P-2.

1.8 1.6 1.4 1.2 ~" 1.0 od

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N . A . A T T A L L A H and A. H. SEHON 2.0 1.8 1.6 1.4 I= 1.2 p,.. u')

d d

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0.6

(A-I)

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16

20

24

28

32

36

40

44

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52

TUBE NUMBER

Fig. 4. Rechromatography of fraction A-2 on Amberlite IRC-50.

was clearly revealed by gel filtration of fraction A-2R (100 mg) on Sephadex G-15 which led to further subfractionation into four components referred to as A-2RS-I to A-2RS-IV in order of their elution from the column (Fig. 5). The first fraction A-2RS-I was colourless and represented, on a weight basis, about 88 percent of A-2R. Therefore, one may conclude that the disproportionately high absorbance of the other three fractions, which were present in trace amounts, was somewhat misleading being due to pigmented materials associated with these fractions. Similarly on microzone electrophoresis fraction A-2R was 0.5

= 0.4 0.3 d

O.2 0.1 0

I

20

30

40

50

Tube number

Fig. 5. Chromatography of fraction A-2R on Sephadex G-15.

6O

Isolation of Haptenic Material from Ragweed Pollen

615

resolved into four ninhydrin stainable bands which had migrated to the cathode. The first fraction A-2RS-I, as will be shown below, possessed hapten-like properties with respect to reagins produced by ragweed sensitive individuals and was, therefore, investigated in more detail than the other materials. The ultraviolet spectrum of the haptenic fraction A-2RS-I, exhibiting maximum absorption at 257 m/~, was similar to that of phenylalanine (Fig. 6). Moreover, amino acid analysis of this fraction revealed the presence of phenylalanine and aspartic acid in relatively large amounts, the occurrence of the 2.0

t.O ~: hi (J Z

< O0 n,'

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I 200 220

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I

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240

260

280

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340

360

WAVELENGTH mp

Fig. 6. Ultraviolet spectrum of fraction A-2RS-I at a concentration of 3 mg/ml. latter accounting probably at least in part for the cathodic migration of the components of fraction A-2R. No particular significance can be attached to the amino acid composition of A-2RS-I since the monodispersity of this material has not been proven at this stage of the investigation. All the fractions when analyzed qualitatively were shown to contain carbohydrate materials[13]; previously the sugars in the dialyzable components of the aqueous ragweed pollen extract were identified as glucose, galactose and arabinose [9]. The apparent sedimentation coefficients of fractions A, A-2R and A-2RS-I were in the range of 0.3-0.7S. All ultracentrifugal patterns consisted of single symmetrical broad peaks which suggested that these preparations were heterogeneous (Fig. 7) and, therefore, it was not considered appropriate to correct the sedimentation coefficients for standard conditions.

Immunological characterization of thefractions On immunodiffusion fractions A-1 and A formed with rabbit antisera to WSRH one and two precipitin bands, respectively, at 10 mg/ml, whereas fraction B or the pink band did not form any bands. On the other hand, all these fractions (A, A-I, B and the pink band) were skin active by P - K tests at 1 mg/ml. On immunodiffusion fractions A-2R and A-2RS-I did not form precipitin bands even at 20 mg/ml, nor did these two fractions inhibit the precipitin bands

616

N . A . ATTALLAH and A. H. SEHON

f o r m e d with W S R H o r with fractions A a n d A-1. Only fraction A - 2 R S - I was not skin active (Table 1) either by P - K tests (using two sera o f t r e a t e d r a g w e e d sensitive individual which h a d P - K titers o f the o r d e r o f 1200) at 10 m g / m l , o r by direct skin tests in a series o f 23 patients at 1 mg/ml. M o r e o v e r , the experiTable 1. Skin activity of the ragweed fractions* Fraction

Concentration/zg/ml 10,000

WSRH Dialysate Pink band Fraction A B A-1 A-2RS-1

5000

1000

2+ 2+ 2+

1+ 1+ 1+

2+

1+

2+ .

.

.

1+ .

100 4+ . . . . . .

. . . . . .

10

1

1.0

3+ . . . . . .

3+ . . . . .

1+

Controlt site

*Skin sites sensitized with ragweed allergic serum (1:400 dilution). tNonsensitized sites challenged with the same fractions at 1000 and 5000t~g/ml. m e n t s designed to establish the capacity o f this fraction to inhibit the P - K reaction (Table 2) d e m o n s t r a t e d that the d e g r e e o f inhibition increased with increasing concentration o f A - 2 R S - I a n d that c o m p l e t e inhibition was achieved with A - 2 R S - I at a concentration o f 5 mg/ml. T o test if the inhibition o f the P - K reaction with A - 2 R S - I was specific for the r a g w e e d system, the e x p e r i m e n t illustrated in T a b l e 3 was p e r f o r m e d using the s e r u m f r o m an individual allergic to t i m o t h y grass pollen as an internal control a n d the water soluble extract o f this grass pollen (WSG) for challenge. T h u s , it Table 2. Determination of the inhibitory capacity of fraction A-2RS-I in P-K reactior~s Concentration of fraction A-2RS-I in serum* (mg/ml)

Reaction on challenge with WSRH (10/~g/ml)

None 0.001 0.01

3+ 343+

0.1 1

2+ ±

5 Control sites sensitized with Saline Normal human serum Normal human serum + fraction A-2RS-I (5 mg/ml) *Ragweed allergic serum (1:400) was incubated with fraction A-2RS-I for sensitization of sites.

617

Isolation of Haptenic Material from Ragweed Pollen Table 3. Specific inhibition of the P-K reaction with fraction A-2RS-I Serum* from individuals allergic to Grass Grass Ragweed Ragweed Ragweed and Grasst Ragweed and Grass Saline

Fraction A-2RS-I incubated with allergic sera None 5 mg/ml None 5 mg/ml None 5 mg/ml

Reaction on challenge with WSRH WSG (10/zg/ml) (100/zg/ml) 1+ -3+ -3+ --

3+ 3+ --3+ 3+

None

*The final dilutions for the grass and ragweed allergic sera were 1 : 100 and 1:400 respectively. tSites sensitized with a mixture of two sera i.e. one from ragweed allergic individual and the other from grass allergic individual. was shown that (i) fraction A-2RS-I, even at 5 mg/ml, did not inhibit the P-K reactions of the timothy grass system; (ii) sites sensitized with a mixture, containing reagins to both grass and ragweed pollens and A-2RS-I at 5 mg/ml,did give P-K reactions on challenge with WSG but not with WSRH. Furthermore, the small skin reaction (1+) in column 3 of Table 3, observed on injection of WSRH into sites sensitized with reaginic serum to grass, indicated that this serum contained also some reagins to ragweed; however, as seen in the same column, this reaction was inhibited by fraction A-2RS-I, this being further evidence for the specificity of the inhibitory capacity of this haptenic fraction for the ragweed system. It must be stressed that owing to the inherent danger of transferring to volunteers, on P-K testing, the virus responsible for homologous serum hepatitis, only two 'pedigree' sera from ragweed sensitive individuals were used throughout this study. Therefore, one may only conclude at this stage that while fraction A-2RS-I contained all the hapten(s) necessary for neutralizing all the reagin(s) of these two sera, other allergic sera may possess additional reagins to additional haptens [ 14]. Obviously, before a final answer regarding the maximum number of haptenic determinants present in WSRH can be provided, the inhibitory capacity of A-2RS-I ought to be established, by P-K tests, using a larger number of sera from ragweed allergic individuals. King and Norman[15] have demonstrated that traces of enzymes were present in ragweed pollen extracts. Moreover, Meacock, Freedman and Sehon [9] have shown by cross-neutralization experiments that all the allergenic and antigenic determinants of the dialyzable fractions of the aqueous extract of ragweed pollen (WSR) were present in the nondialyzable components of WSR. Thus, it is possible to postulate that the haptenic fraction, which may occur in a free state or bound to other high molecular weight components of ragweed pollen, may originate from these components by enzymatic or other chemical degradation processes. The findings of this study are in agreement with the observations of Malley

618

N . A . ATTALLAH and A. H. SEHON

et al. [2-4] and Feigen et al. [8], only inasmuch as all these investigations have d e m o n s t r a t e d the presence o f hapten-like c o m p o n e n t s in the dialysates o f aqueous extracts o f ragweed and timothy grass pollens. However, it o u g h t to be pointed out that while the low molecular weight fraction o f Malley et al. [2-4], i.e. their fraction D, inhibited the precipitin reactions obtained with rabbit antisera to WSG, it was skin active in both direct skin and P - K tests. By contrast, while fraction A - 2 R S - I was not skin active by both direct skin and P - K tests, it did inhibit specifically the P - K reactions o f the ragweed system, thus behaving as a true h a p t e n with respect to reagins. F u r t h e r m o r e , the fact that fraction A - 2 R S - I did not give or inhibit the precipitin reaction is considered as suggestive evidence that (at least some o f ) t h e d e t e r m i n a n t groups o f WSRH which were allergenic in man were not antigenic in rabbits. Hence, it would a p p e a r that fallacious conclusions r e g a r d i n g the allergenicity o f a given pollen preparation may be arrived at o n the basis o f the immunological manifestations obtained with antibodies p r o d u c e d in a species different f r o m man. Recently, in a n o t h e r study in this laboratory[16] Malley's fraction D was shown to consist o f at least seven electrophoretic components. It can be visualized that, as in the present study, some o f these c o m p o n e n t s might be true haptens with respect to reagins to grass, while o t h e r c o m p o n e n t s might have been responsible for the skin activity o f his fraction D. T h e r e f o r e , it is not surprising that hyposensitization t r e a t m e n t o f grass sensitive patients with fraction D resulted in a reduction o f the reagin titer, as c o m p a r e d to the unchanged titer o f reagins observed on desensitization with WSG[17]. O n the o t h e r hand, the effectiveness o f fraction D in stimulating the p r o d u c t i o n o f blocking antibodies was lower than that o f WSG. Experiments are in progress in this laboratory to establish the desensitizing capacity o f the haptenic fraction A - 2 R S - I o n t r e a t m e n t o f ragweed sensitive individuals with it, and o f its effect on the p r o d u c t i o n o f reaginic and blocking antibodies. Acknowledgements- This investigation was supported by grants from the Medical Research Council of Canada, Ottawa, Ontario, the Life Insurance Medical Research Fund (U.S.A.) and the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. The authors acknowledge their gratitude to Dr. S. O. Freedman for supply of the two allergic sera and for the passive transfer tests, to Dr. H. Z. Hollinger for the direct skin tests, and to Dr. L. Gyenes for many helpful suggestions and criticisms.

1. 2. 3. 4. 5. 6. 7. 8. 9.

REFERENCES Attallah N. A. and Sehon A. H., Fedn Proc. Fedn Am. Socs exp. Biol. 27, 368 (1968). Malley A. and Campbell D. H., FednProc. FednAm. Socs exp.. Biol.22,560 (1963). Malley A., Campbell D. H. and Heimlich E. M.,J. I nmun.~, 420 (1964). Malley A., Saha A. and Campbell D. H., Immunochemistryl, 237 (1964). Farah F. S., Kern M. and Eisen H. N.,J. exp. Med. 112, 1911 (1960). Ishizaka K., Ishizaka T. and BanovitzJ.,J. Immun. 94, 824 (1965). Frick O. L., A. Mtg Am. Acad. Allergy San Francisco, Calif. (1964). Feigen :G. A., Sanz E., Meyers R. L. and Campbell D. H., Int. Archs Allergy appl. Immun.32, 174 (1967). Meacock S. C. R., Freedman S. O. and Sehon A. H.,J. Allergy85, 43 (1964).

Isolation of Haptenic Material from Ragweed Pollen 10. 11. 12. 13. 14. 15. 16. 17.

619

Parker C. W., Fedn Proc. Fedn Am. Socs exp. Biol. 24, 51 (1965). Spackman D. H., Stein W. H. and Moore S., Analyt. Chem. 30, 1190 (1958). Ouchterlony O., Progress inAUergy, Vol. 5, p. 1. Karger, Basel (1958). Feigl F., Spot Tests in Organic Analysis, p. 426. Elsevier, Amsterdam (1960). Kisil F. T., Gyenes L. and Sehon A. H., Proc. Can. FednBiol. Soc. 2, Abs. No. 422 (1968). King T. P. and Norman P. S.,Biochemistry 1,709 (1962). Ekramoddoullah A. K. M., Ph.D. Thesis, McGill University (1968). Perlman F. and Malley A.,A. MtgAm. Acad. Allergy, Boston, Mass. (1968).