Allergy induced by environmental agents is. one of the widely prevalent diseases in India and. the common environmental agents of seasonal allergic

Transcription

1 113 D I S C U S S I 0 N Allergy induced by environmental agents is one of the widely prevalent diseases in India and elsewhere in the world. Pollen grains are one of the common environmental agents of seasonal allergic diseases (Bostock, 1819; Schadewaldt, 1967). There is lack of knowledge concerning pollen induced allergy. Frequency and wide spread occurrence of allergic reaction of pollen to human prompted to study the allergenic nature of pollen allergens. Prosopis juliflora (Pj) plants are widely prevalent in arid region of India and allergy induced by it are reported (Shivpuri and Prakash, 1967). Therefo~e, in the present work:it was of main interest ' to purify and to analyse the chemical nature of allergens of Pj pollen and to study allergenic response of it.

2 114 Among the several existing media such as buffered saline, Coca's solution, Glycerine solution, Dextrose solution etc., we used phosphate buffer saline, i because of its simplicity, allergenic specificity and easy to get different allergenic components (Sheldon et al., 1967; Belin, 1972; Viander et!!:.l, 1979; Baldo et nl., 1980, 1982). Extracts of pollen in phosphate buffer saline gave homogeneous mixture. Thus our results showed that Pj pollen allergen would diffuse rapidly from pollen grains during extraction in physiological saline. So far there is no proper chemical method for analysis of allergenic potency of pollens. Several different methods have been evolved from time to time for giving approximate expression of the potency of allergenic extracts. Most of the presently available scientific and commercial extracts had measured. potency of extract according to protein-nitrogen content {PNU) or had stated the weight to volume (w/v) ratio of the extracted ingredients (Schaeffer and Claysisk, 198l,;). Besid es protein and nitrogen, pollen extracts also contain various other chemical constituents such as carbohydrate, phosphate, sialic acid and sulfhydryl

3 115 group which may also be effective allergens. Moreover, there are many conflicting reports in the literature on the chemical nature of pollen allergens. Till date, there is no report regarding the estimation of chemical constituents of Pj pollen. Although there are reports concerning other pollen {King and Norman, 1962; Goldber~ and Kaplan, 1967; Belin, 1972). Protein and nitrogen content were assayed and found to be 8.0 mg/ml and 1.34 mg/ml respectively. This was similar to the earlier reports (Belin, 1972; viander et.!!! 1979). The amount of carbohydrate, phosphate, sulfhydryl group and sialic acid was found 2.1 mg/ml, ' p mole/ml, 8 n mole/ml and 0.2 p mole/ml was similar to ragweed and birch poll~n (King and Norman, 1962; Belin, 1972). The amount of othp.r chemical constituents such as sialic acid and sulfhydryl group from Pj pollen extract differ from the extract of other pollen observed by King and Norman ( 1962). It might be due to different sources of pollen extract and different methods used for extraction. Although the amount of phosphate, sialic acid, sulfhydryl group are comparatively very less than protein, nitrogen and carbohydrate

4 116 content i~ Pj pollen extract, there is possibility that these chemicals might be playing important role in inducing allergy alongwith protein and nitrogen or alone. There are reports which indicate that these. chemicals have some role in inducing allergy {King and Norman, 1962; Berrens rnd Bleumink, 1965). For I example, sul fhydry 1 'is considered to be responsible for stability of allergens. (King and Norman, 1962). Thus, it appears that Pj pollen extracts or other pollen extracts contain multiallergens and might act as multiple allergen system. Since crude allergen extracts used for clinical diagnosis contain non-allergenic materials, occasional irritant components and active allergens,. therefore, it is imperative to purify the active allergenic components from Pj pollen extract. The uses of the purified allergens are important for the standardization of allergenic activity of the pollen crude extract. Thus, physicochemical and immunological characteriza.:;, tion of the individual purified allergen fractions are important because it will give knowledge to the extent the most active fraction represen1sthe original activity in pollen. Therefore, in the present work

5 117 crude extract of P j pollen has been purified and chemical and immunological properties of purified fraction (Pj pollen) have been analysed. On Sephadex G-100 the pollen protein appeared to be separated mainly by simple gel filtration. Both proteins and considerable amount of other water soluble P j pollen components such as carbohydrate and pigment were retarded by adsorption and were eluted near bed volume. Gel filtration result showed crude pollen extract contained six different fractions. Fractions molecular weight varied from 81,000 to 13,000 daltons. Our present study is very similar to that of gel filtration observation of birch pollen (Belin, 1972; Viander et al., 1979) and of ragweed pollen extract (King et al., 1964; King et al., :1967). But fractionate obtained by gel filtration pollen extract fro~ other different so,urces, viz. Kentucky (Ekramoddoullah ~.! al., 1977, 1980, 1981, 1982; Chakrabarty et!!!, 1981); Timothy (Puttonen ~!! 1982) is somewhat comparable to our study. Our gel filtration results differed from the report of Vela et ~! ( 1982) who observed only four fractions in Olea europea; Russian thistle where two glycoprotein (RT 1 and RT 2 ) were

6 118 isolated by Shafiee ~!l.! (1981) and Ja.pane se cedar observed by Yasueda et!!..! (1983) am fractionate SBP-a 1 and SBP-a 2 allergens. Fractions obtained from gel filtration were further purified and characterized by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Six bands of P j pollen crude extract were obtained by polyacrylamide ge 1 electrophoresis. Each pooled gel fraction containing the allergens of Pj pollen were separated by polyacrylamide gel electrophoresis and it was found that each fraction contained single band. was homogenous. Thus, it appears that each ge 1 fraction Our result suggested that PAGE of crude extract contained six allergenic band> Wl'lS similar to the PAGE observed in birch pollen (Underdow'1J._(:,oGd+nl~"l'l~: 1966; Belin, 1972; Viander et al., 1979). Further, ' -- the Single band obtained by PAGE from each gel fraction revealed the purity of the gel filtered pooled fractions. Sodium dodecyl sulphate-polyacrylmide gel electrophoresis ( SDS-PAGE) were performed to determine and confirm the molecular weight and subunits of

7 119 crude alle'rgen P j extract and fractions obtai ned by gel fi 1 t ration. Crude Pj pollen extract was electroseparated by SDS polyacrylamide gel electrophoresis and six protein bands were obtained. Molecular weight of each band' separated from crude P j extract by SDS- / PAGE varied from 81,000 to 13,000 daltons. All the six bands obtained from SDS-PAGE ;were stained with PAS (Periodic Acid Schifi'1reagent). But only one band showed positive stain with PAS, whose molecular weight was 20,000. This suggests that this molecular weight allergen is glycoprotein. Pooled gel fractions containing the Pj allergens were separated and single band was obtained. This revealed that pooled gel fraction does not possess different protein sub-units. The molecular weight of different bands obtained by SDS-PAGE which varied from dalton 81,000 to 13,000 are identical to the molecular '"eights determined by gel filtration in this study. The molecular weigh~determined in this study were similar to the reports in"kentucky blue grass ddoullah e t a l., 1981, of!molecular weights determined an~ rye grass pollens (Ekramoi 1982). I!

8 120 It.appears that the chemical nature of Pj pollen extract particularly mol. wt. and protein subunits studied in this work showed resemblances with the pollen extract from different sources, viz. Birch, Kentucky bluegrass, ragweed, rye grass etc. and suggest that chemical properties of pollen grains are identical. Overall results of purification of Pj pollen extract by 'all three methods used in this sttrly {gel filtration, PAGE, SDS-PAGE) are similar and furtber indicates tmt a considerable extent of purification of major allergens of pollen extract separated at gel filtration level itself. To further characterize the Pj pollen allergens, DEAE cellulose chromatography was performed. Nine fractions were obtained by DEAE cellulose chromatography. Our present finding is similar to the fin:ling in birch pollen (Belin, 1972). It appears that the nine fractions resulted in DEAE cellulose I chromatography further extend in the separation of allergenic components of ~j pollen extract which is evident from the separation of extra three fractions. Thus, extra fractions might be due to separation of allergenic components from non-allergenic components.

9 121 Testing of allergenic activity of Pj pollen we.s performed by.!.!:! vivo skin test and in vitro histamine release test. In addition to crude pollen extract, purified and chemically characterized pollen fractions were used for testing allergenic activity and immune responses in guinea pigs. The guinea pigs were choosen as experimental model in this study because antibodies formation in it are similar to great extent I. to human beings, for studying the regulating mechanisms involved in the IgE or IgG antibodies production and finally its proneness to s~nsitization (Chase, 1941; Bloch, 1931; Storck, 1962; Tada, 1975; Tamura and Ishisaka, 1978). In this study a quantitative skin test (intracutaneous) was performed in the sensitized guinea pigs after giving challenging dose. Different doses of Pj pollen extract were administered for different period for the sensitization of animals. Challenging dose I (0.5 )lg/ml) was given in different sensitized guinea pigs. For different periods early skin response (development of wheal-flare) and late response (erythemaredness) were measured. 15 days and 45 days sensitized guinea pigs with 50 pg/ml pollen extract resul~ed

10 122. in maximum early and late response after giving challenging dose, whereas 7 days sensitized guinea pigs with same amou~t of pollen extract (50 Jig/ml) did not give similar response. In 7 days sensitized animals, 25 pg/ml pollen extract gave maximum early and late response. In contrast, the guinea pigs sensitized with higher and lower doses (100 pg/ml, 5 pg/ml and 1.5 pg/ml) for different days (7, 15 and 45 days) showed poor response. This finding is comparable to earlier findings (Voorhorst and Krieken, 1973; Solley et al., 1976). Thus, it seems that both th~ responses (early and late) were typical dermal manifestation induced by pollen allergens might be due to the interaction of ~llergens with specific mast cells fixed antibodi~s (Ellis, 1979; Turkeltanb et ~!, 1982). It is thought that IgE or IgG)'l antibodies are responsible for the inter.action with antigens in guinea pig mast cells. It was observed in the present work that late responses (erythemaredness) which appeared after the disappearance of primary dermal manifestation of early response (whealflare) at the same site are thought to be due to the infilteration of inflammatory cells (Solley et al., 1976; Ellis, 1979; Gleich et al., 1982; Creticos et.!!.! 1984).

11 123 The most significant finding that emerged from the present study was shifting of optimum sensitizing dose from 25 )lg/ml as observed on 7 days to 50 pg/ml on 15 days and 45 days. It could be due to the elicitation of reaginic antibody profile which was maximuo on 15 days. After that, reaginic antibody response was decreased, due to elicitation of blocking antibodies, i.e. IgG. This shifting in dose might be due to the elevation of IgG type of immunoglobulin compared to IgE or IgGY:1 (reaginic antibodies)(ishizaka and Okudaira, 1972; Andersson, 1981). The higher concentration of 100 pg/ml which did not react sharply, might be due to higher dose which inhibited IgE or IgGtl by elicitation of IgG bl6cking antibodies. Lower dose was not sufficient to produce reaginic antibodies. Thus, our results favour the earlier concept that the suppression of reaginic antibodies, may be regula ted by IgG anti bodies through a feedback inhibition mechanism (TadaC:'-/):c, 197~; ~. ~""-~... Ishizaka an:l Ishizaka, 1978). Our results sug~ested a~optimum sensitizing dose of skin reaction test was some--where middle of high and low sensitizing doses (50 pg/ml). This

12 124 differenca might be due to the presence of allergen specific suppressor T cells (Kishimoto et ~, 1976; Tamura and Ishisaka, 1978). According to their theories, large dosesof antigen (allergen) used for sensitization might activate antigen specific suppressor T cells, which we'i!2-responsible for early termination of the response (Fig. 3 9 ). Thus, it is important to consider the factors involved in controlling IgG synthesis. It could only be solved by availability of IgG myelomas and development of monoclonal antibodies to. IgG for further research work. The atopic skin test was considered an exact and reliable test (Wiseman, 1951). The great majority of skin tests were performed in primitive ways, poorly defined and scatter in the measurements (lrtdrajana et al., 1971 ). Erythema has advantage over wheal for measurement of allergenic potency near the end point. The skin reaction grading system was significantly correlated (Norman e t al., 1973). To further confirm the allergenic activity of P j pollen extract and to correlate with reproducible quantity skin test, we had carried out in yitro histamine release test induced with Pj pollen allergens.

13 C3? lt. f/ 0 0 '( e s s Antigen (allergen) + Histamine \ \ 1.Sensitisation by antibody (recognition of target cell) Cells B cells \ 2 I.. \. nteract1on w1th (---...triggering of \ \ \ antigen target cell) Genes 3. Histocompatibility F 1g.. 39 : Postulated Hechanism 0f Irnmunological Triggering of Mast Cells ~nd Suggested Hypothetical Role of IgG in Immunopathways.

14 126 Reference external and internal histamine was assayed for standardization and found that net fluore~ scence was directly proportional to the concentrations of reference histamine. Thus, it showed that sensitivity of leukocyte cell populations vnried in terms of quantity of histamine (reference i.e., standard). A dose dependent histamine relerse was assayed with the interaction of leukocytes and different concentrations of Pj pollen extract. Present result indicated the release of histamine was directly propor- I tional to the concentration of pollen extract. 5.0 x 10-1 pg/ml and 5.0 x 10-2 pg/ml of allergens of Pj released detectable quantities of histamine from mast do nor cells. This result is similar to earlier study in which histamine was assayed in man and guinea pig leukocytes (Lichtenstein and Osler, 1964; May et al., 1970; Lichtenstein et al., 1978; Andersson, 1981). The findings that histamine released by sensitized leukocytes could be due to cell fixed reaginic antibodies through the interactiori of their antigen binding sites with the allergen or by interactio~of their Fe portion of antibody (Ishizaka et g., 1969;. Stanworth,

15 ; 4<;yQ:~ et al., 1984). The time course of histamine release by Pj allergen peaked after 25 min i nc u b a t i on. T hi s f i nd i ng i s s i m i 1 a r to ear 1 i e r f i nd - ings (Lichtenstein and Osler, 196Lq Tanizaki et al., 1984). Thus, it appears that 25 min is the optimum time for optimum release of histamine in vitro. It was further observed that maximum histamine was released on 15 days sensitized guinea pigs followed by 45 anl 7 days sensitized guinea pigs with all this used concentrations of P j pollen ( 10-1 to 10-4 )lg/ml). This result is similar to findings observed by Lett-Brown et ~ (1981) observed in ova. lbumin induced histamine release from guinea pig leukocytes. Here shifting of histamine release in sensitized guinea pigsfor different periods remain to be understood. However, the difference in histamine rel~ase at different period of sensitization of guinea pigsis thought to be due to different sensitivity of peripheral blood derived leukocytes (Lett Brown et!!.! 1981). Other explanation for this difference is suggested that histamine release from basophil:~ might be mediated via homocytotropic antibodies bound to the cell surface, blocking antibodies, i.e., IgG and suppressor T cells (Andersson, 1981; Tamura and Ishizaka, 1978; Kishimoto et ~., 1976).

16 128 Since estimation of allergenic activity by skin tests is limited due to obvious drawbacks of a practical and methodological nature (King and Norman, 1962; Marsh et ~, 1966; Aas and Jebsen, 1967), in vitro histamine release method has also been studied in the present work for the identification and quantitation of allergens of fractionated Pj pollen allergen. From the present study it was found that allergenic molecules often have molecular weight varied between 20,000-49,000 daltons. From the results obtained by prick test and histamine release test it was also found that protein fraction of 20,000 molecular weight obtained from gel filtration, i.e., peak E has major allergenic activity followed by peak C. For instance, prick test with peak E fraction gave maximum dev~lopment of wheal and similarly maximum histamine was observed with the 3ame fraction (E). Res~ other fractions {A, B, D, F) observed by gel filtration sho,ve.d insignificant allergenic ac ti vi ty. This result is si milfl r to the previous observation in birch, kentucky, ragweed, and alder pollen {King and Norman, 1962; Belin, 1972; Ekramoddulah et al., 1977; Viander, 1979). ---

17 129 AlL the 9 fractions obtained by DEAE cellulose chromatography were tested for allergenicity by prick test. Only two f.ractionsiii (45%) and IV (25%) showed allergenic activity as observed in gel filtration allergenicity test whereas rest of the fractions (I, II, V to IX) showed insignificant allergenic effect. Another significant finding emerged in this work that only coloured fraction obtained by both gel filtration and ;r>eae cellulose chromatography gave maximum allergenic a cti vi ty. Although it appears from the present work that intensity of allergenicity was same in coloured (45%) and non-coloured (25%), fractions obtained by both gel filtration and DEAE cellulose chromatography. Since maximum allergenic activity was observed in coloured fraction, hence the. major allergen fraction appears to be glycoprotein in nature. Be cause of the prese nee of sugar residues, gl ycosidically linked to the protein framework of the molecule are not only considered to provide the stability of structure but it is also thought to stimulate reagenic synthesis in cell. That is why the major allergens observed in this study which is glycoprotein is maximum allergenic.

18 130 Immunological responses of allergens of Pj were assayed by gel diffusion test, immunoelectrophoresis, Radioallergosorbent Test (RAST) and passive cutaneous anaphylaxis (PCA) tests. Gel diffusion test was used for the assessment of allergenic activity. Different concentrations of Pj pollen nllergen were quantitified by gel diffusion. Different fractions (Peak A, B, C, D, E and F) obtained from gel filtration are used in immunodiffusion analyses to give precipitation lines towards the antiserum. It was observed that fraction E band was more thick and smrp;,.comparis.on to other fractions. The localization of the precipitates associated with allergenic activity might be due to the lol\'er molecular weight of the allergens present in the gel fractions. It is known that the quantitative relations in the antigen composition of the extract influ~nce the position of the lines in the precipitation spectrum. Hence it indicates tmt allergenic antigens are of lower molecular weight than the non-allergenic antigens of Pj gel "( fraction (Augustin, 1959; A}le:::::sman ~ al., 1963; Belin, 1972). This reveals that peak E is most allergenic compared to other large molecular weight allergen.

19 131 For qunlitative and quantitative analys~s of P j pollen allergens eros s,, over immunoel ectrophoresis and two dimensional crossover immunoelectrophoresi~ were carried out. Different fractions of Pj pollen allergens were cross-electrophoresed against guinea pig P j antiserum. It was found that fraction E band among other (A, B, C, D, F) bands was highly condensed and sharp followed by C, D, B, F and A. This clearly indicated that band E is most allergenic. Two dimensional cross over electrophoresis (Laurell, 1965) was performed}-quali tative and quantitative allergenic activity of the Pj pollen extract and different fractions obta1ned by gel filtration. Analysis of different components indicated that Pj extract contains at least two major allergens out of ' seven allergens differed with respect to their electrical charge but antigenically similar. These findings are comparable to the findings of Belin (1972), Johnson and Marsh (1965) and Augustin (1959) in case of birch and grass extracts respectively. A preferential binding of different penkswith guinea pigsantiserum obtained by gel filtration revealed that peak E band was strong and sharp compared to others. This

20 132 indicates that the major part of the allergens in the whole extract are of fraction E, followed by fraction C and D'. Our results sho\ved similarity with the observations of Viander ~ ~ (1979) in the case of birch ' pollen allergen obtained by gel filtration. In vitro Rndioallergosorbent Test (RAST) and in vivo passive cutaneous anaphylaxis (PCA) elicitation and inhibition tests were performed in this study to test the chemical properties of allergens in Pj pollen extract and the nature of reaginic antibodies had taken part in guinea pigs allergy. RAST was performed to test the allergenic activity of different doses of Pj pollen crude extract by the incorporation of radio-labelled anti-ige with the prior incubation of guinea pig Pj antiserum lvith the paper disc (Whatman filter-i). It was ob<1erved that allergen~c activities, i.e., binding of anti-ige varied according to the concentrations. (0.01 pg to 1 pg) of Pj pollen extract. RAST inhibition was also performed to verify the preferential binding of the guinea pig Pj antiserum neutralized allergens to the paper disc after the incorporation of labelled anti-ige antibodies.

21 133 The main allergenic fraction E gave maximum RAST inhibition (58%/1 pg) whereas other fractions (C, D, B, A and F) inhibition w~t-y'. insignificant. This suggests thftt fraction E is major all~rgen. It was observed that RAST inhibition was dependent on the amount of gel fraction antigen (allergen) of Pj used for neutralization of guinea pig Pj antiserum. For instance, incorporation of labelled anti-lge antibody to guinea pig Pj antiserum neutralized. with 1 pg (highest amount) of ge 1 fraction antigen was minimum (i.e., maximum RAST inhibition). Whereas, guinea pig Pj antiserum showed more incorporation of labelled anti IgE antibody (i.e., less RAST inhibition). Thus, it appears that RAST inhibition (i.e., incorporntion of lgE to Pj antigen) directly depends on the amounts or concentration of Pj antigen (allergen) used. Therefore, present observation suggests that IgE or IgE like antibodies seem to be responsible for P~ allergen (antigen) induced allergy in guinea pigs. Thus, another significant finding emerged by RAST inhibition test which gave clue regarding the type of antibodies found in guinea pig atopic diseases. Our result is similar to the observation

22 13~ of birch pollen (Viander et ~., 1979); timothy pollen (Puttonen ~ ~., 1982) and olea pollen (Vela eta}., 198 ) which suggests that glycoprotein having moleculnr weight approximately 20,000 are main allergens of pollen extract. Our result is also in agreement with the previous reports which also suggest that IgE or IgE like reaginic antibody is responsible for atopic diseases in guinea pigs (Catty, 1959; Parish, 1970; Dob~;on e t a 1, ; P e ri n i a nl Hot o, ). In~~ passive cutaneous anaphylaxis (PCA) elicitation and inhibition tests had been performed to test the allergenic activity of Pj pollen extract and the frncti.ons obtained by gel filtration. PCA elicitation test was observed in vivo after the binding of antigen, i.e., Pj allergen extract with the d i f f e rent se ns i t i zed do s e s ( 1 : 3 : 5 : 1 0 ) of g u i n e a pig Pj antiserum. Result indicated that PCA reaction varied.. according to the concentrations of guinea pig Pj antiserum. It might be binding of antigen and antibody in( 't-hu.i guinea pigs,,.._caused capillary leakage and bluing area was observed (Ovnry, 1964)'. PCA inhibition test showed that guinea pig antiserum neutralized with higher dose (1 mg/ml) of all gel

23 135 fractions.completely inhibited PCA induced lesion whereas lowest dose (15 pg/ml) of all gel fractions (A, B, C, D and F) except fraction E, did not inhibit the PCA reaction. Lowest dose or' fraction E of gel fraction, a glycoprotein, having molecular weight 20,000 dalton, inhibited the PCA induced lesion. This could be suggested that the gel fraction E is major allergen of Pj pollen extract. Our result is similar to the observation made by Attallah and Sehon (1969) in case of ragweed pollen; Halley et!u_. ( 1964) in timothy pollen and Chakrabarty et al. (1980) in kentucky blue grass pollen extract who also showed fraction. hewing molecular weight a.pproximntely 20,000 dalton (glycqprotein) w~s most allergic. The significant finding emerged from PCA inhibition in which it was found that lo~ molecular weight (20,000 dalton) of pollen extrnct had t~ ability to inhibit in vivo anaphylaxis might be due to some 'chemical' which neutrrlized tie guinea pig antiserum. Thus, it is oonsidered that inhibition of PCA is mediated via chemica]'!, called 'haptene' like substances which is supposed to neutralize the guinea pig antiserum with gel fraction allergens (Malley~!&., 1964;

24 136 Attallah and Sehon, 1969; Chakrabarty et al., 1980). Our RAST study also showed i-nhibition of IgE incorporation to guinea pig~ P j antiserum neutralized with gel fractions. This RAST inhibition might be mediated via 1 haptene 1 like substances.