Studies on human gastric mucosal immunoglobulin A. II. Further evidence for the absence of the secretory component from the predominant immunoglobulin A of human gastric mucus

Biochimica et Biophysica Acta, 576 (1979) 9--16 © Elsevier/North-Holland Biomedical Press

BBA 38076

STUDIES ON HUMAN GASTRIC MUCOSAL IMMUNOGLOBULIN A II. FURTHER EVIDENCE FOR THE ABSENCE OF THE SECRETORY COMPONENT FROM THE PREDOMINANT IMMUNOGLOBULIN A OF HUMAN GASTRIC MUCUS MARTHA SPOHN and IAN McCOLL

Department of Surgery, Guy's Hospital Medical School, London SE1 9RT (U.K.). (Received March 29th, 1978) (Revised manuscript received September 15th, 1978)

Key words: Immunoglobulin A; Secretory component; (Gastric mucosa)

Summary In this work fresh human gastric mucosal scrapings were fractionated on an isopycnic CsC1 gradient into three fractions: L1, the top part of the gradient free of immunoglobulin A, immunoglobulin A-positive middle layer L2, and L3 the lower part of the gradient in which again immunoglobulin A was not detected, but which accounted for a large proportion of the total carbohydrate of the original suspension. Layer 2 (L2) was further fractionated by gel chromatography to give an immunogloblin A-enriched fraction $4B/2. The material in this fraction failed to reveal the presence of the secretory component indicating that the predominant gastric mucosal immunoglobulin A is not of the secretory type. In the second part of our work we have attempted to determine the effect of gastric mucosal scrapings on the secretory component of saliva as well as of milk whey. No apparent reduction of the amounts of the secretory component due to the presence of the scrapings was observed. We have also followed the fate of the immunological activity of the secretory component of the gastric° salivary immunoglobulin A-containing mixture throughout the whole fractionation scheme used for the isolation of the original gastric immunoglobulin A-positive fraction S4B/2.The secretory component was detected at all stages of fractionation of the mixture in the immunoglobulin A-containing fractions. These results indicate that, if present, the secretory component can be detected even in the presence of gastric mucosal components. Consequently, it is concluded that our inability to detect the secretory component in suspensions of gastric mucosal scrapings reflects the absence of this component from the tissue in measurable quantities, although appreciable quantities of immunogiobulin A are present.

10 Introduction

Evidence from earlier work [1] suggests the absence of the secretory component from the predominant immunoglobulin A (IgA) molecule of human gastric mucosal scrapings. This observation cannot be explained on the basis of the currently held views on the involvement of the secretory c o m p o n e n t in transepithelial transport of IgA across mucosal tissue [2--4], and could be the result of blocking of the antigenic sites of the secretory c o m p o n e n t by gastric mucoproteins. The experiments reported in this paper were designed to investigate the effect of gastric mucoproteins on the secretory component. Preliminary separation of an IgA-enriched fraction from gastric mucoproteins was achieved by 2-fold fractionation of gastric mucosal scrapings on an isopycnic CsCI gradient. The IgA positive layers were then subjected to further separation by gel chromatography on Sephadex G-200, followed by Sepharose 4B. The purified, IgA-containing fraction $4B/2 was investigated. In this second part of our work we have mixed original gastric mucosal scrapings suspension with a crude salivary secretory IgA-containing fraction, and also with secretory c o m p o n e n t positive human milk whey. The effect of gastric mucosal scrapings on the secretory c o m p o n e n t of saliva and of milk whey was examined. Materials and Methods Unless otherwise stated the buffer used throughout this work was 0.1 M TrisHC1 (pH 8.3) containing 5 mM EDTA. Materials. In this study we have used scrapings of fresh human mucosal tissue, taken from the antrum of the stomach during gastrectomy for duodenal ulcer. Three such samples were processed individually, one of which was also used for mixing with salivary IgA-contaning fraction and with milk whey. The washing and scraping of gastric tissues was performed within 10 min of resection. Before scraping the tissues were thoroughly washed with physiological saline to remove loosely adhering luminal material. Fresh scrapings were homogenised in an MSE homogeniser, 30 ml buffer per g wet scrapings being used to give gastric mucosal suspension. Isolation of crude salivary secretory IgA [raction. 15 ml fresh saliva were mixed with an equal volume of buffer and the mixture was centrifuged for 5 min at 500 × g. The supernatant was decanted, the sediment resuspended in buffer and again centrifuged. The combined supernatants were dialysed against distilled water and lyophilised. Preparation of human milk whey. Fresh human milk (3 days post partum) was centrifuged for 1 h at 35 000 × g (15°C). The fatty layer was removed, the supernatant adjusted to pH 4.6 with dilute acetic acid and the resulting precipitate removed by centrifugation at room temperature for 15 min at 1500 × g. The supernatant was adjusted to pH 8.5 with 1 M NaOH, dialysed again.st distilled water and lyophilised. CsCl gradient fractionation. For CsC1 gradient fractionation 40 mg of the lyophilised salivary material were mixed with 24 ml of the original gastric mucosal suspension. Solid CsC1 was added to both the gastric suspension and

ll

the gastric and salivary mixture (0.425 g CsCl/ml suspension). The mixtures were centrifuged for 72 h at 5°C, 40 000 rev./min (105 000 ,~g) in a Beckman L65 ultracentrifuge, angle rotor 40. 1-ml layers were collected. All layers were examined by immunodiffusion against anti-lgA (a-chain) and anti-secretory component sera. In the case of the gastric sample the distribution of hexose and of protein between the layers were also determined. The CsCI layers were combined to give L1, the top part of the gradient free of IgA; the IgA-containing middle layer L2; and L3, the bottom part of the gradient which was again free of IgA but which did contain a large proportion of the total hexose of the original suspension. The specific gravity of the IgA-containing layers, L2 from the original gastric mucosal suspension and L2 from the mixture of gastric and salivary material, was then adjusted to 1.3 g/ml with solid CsCl, and the mixtures again centrifuged as above. The resulting IgA positive layers L2 and (gastric and gastric-salivary) were dialysed against distilled water, lyophilised, and the material subjected to gel chromatography on Sephadex G-200, followed by Sepharose 4B. Gel chromatography. The material from L2 {gastricand gastric salivary) was fractionated by column chromatography on Sephadex G-200, followed by Sepharose 4B as described [1] to give purified IgA positive fractions $4B/2 and (gastric and gastric-salivary) which were dialysed, lyophilised and used for analysis. Immunodiffusion. Routine double diffusion was carried out on 1% Agar (DIFCO Noble) gels in 0.1 M Tris-HCl buffer. The anti-lgA (a~hain) and antisecretory component sera were from Hoechst Pharmaceuticals. For quantitative immunodiffusion gels were prepared by mixing 20 ml of a warm (40°C) solution of 1% Agar with 0.3 ml anti-secretory component serum. 10-,,I samples were used per well. SDS electrophoresis of the reduced samples was carried out as described by Weber and Osborn [6]. Protein bands were located with Coomassie Blue.

Analytical methods Carbohydrate analysis. The phenol/H2SO~ method of Dubois et al. [7] was used.

Protein. Protein distribution on the. gradients was determined essentially by the method of Lowry et al. [8] adapted for the use on a Technicon autoanalyser by the manufacturers. Results and Discussion .i

CsCI isopycnic gradient fractionation of human gastric mucosal scrapings separates an IgA-containing fraction from the bulk of gastric mucoproteins [5]. A typical tracing of the distribution of total hexose and of protein on the gradient is shown in Fig. 1, together with thb relative' position of IgA. Second fractionation of the IgA positive layer gave an IgA~ontaining fraction L2 which accounted for between 4 and 7% of the total hexose of the original scrapings, indicating almost complete removal of gastric mucoproteins. Gel chromatography on Sephadex G-200 did not reveal any differences

12 CsCI GRADIENT FRACTIONATION 1 ----~ Hexose 0~g/ml) Protein (pg/0. lint) L1

L2

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V o l . f r o m top of tube (ml)

Fig. 1. ( A ) D i s t r i b u t i o n of total h e x o s e and of proteLn o n f r a c t i o n a t i o n of the original gastric m u c o s a l s u s p e n s i o n o n CsC1 gradient. T h e p o s i t i o n w h e r e IgA can be d e t e c t e d is indicated. ( B ) T h i s s h o w s d o u b l e d i f f u s i o n pattern o f layers (gastric s a m p l e ) L 2 ( w e l l 6 ) and (gastric-salivs-~T m i x t u r e ) L 2 (well 7) and m i l k w h e (well 5) against anti-secretory c o m p o n e n t serum ( w e l l s 1 - - 4 ) and anti-IgA (s-chain) serum (wells 8--11).

between the elution patterns of the two samples, gastric sample and gastric and salivary mixture, but on immunodiffusion the IgA positive, excluded fraction 1 from gastric-salivary mixture was also positive for the secretory component. In the gastric sample the component was not detected. Sepharose 4B chromatography of the IgA positive, excluded fraction 1 from Sephadex G-200 resulted in IgA-enriched fractions (gastric sample)S4B/2 and (gastric-salivary mixture)S4B/2 which were not purified further (Fig. 2). The gastric fraction $4B/2 accounted for not more than 7% of the hexose content of the original suspension. Analytical CsC1 gradient ultracentrifugation of the material in the gastric

13 SEPHARO~E 4B SEPARATIONOF IgA+ ve FRACTION FROM SEPHADEX G200 COLUMN S4B/1 ~

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~ 0.3 ~ o.~.

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20 I 30 Vo

40

50

60

70

80

90 100

Volume (ml)

Fi~. 2. This shows double diffusion pattern o f fractions (gastric sample)S4B/2 ( w e l / 2 ) a n d (gastrlc-salivary m lxture)S4 B/2 (we/l 3) against anti-secretory c o m p o n e n t serum (well 1) and anti-lgA (~-cha/n) serum (well 4).

fraction $4B/2, carried out in a two-place analytical rotor for 72 h at 20°C (56 000 rev./min) in a Beckman Ld5 ultracentrifuge, suggests homogeneity of the sample. SDS electrophoresis of the reduced material from the gastric fraction $4B/2 confirms the absence of the secretory component from this fraction. When compared with the electrophoretic pattern of human colostral IgA the band corresponding to the secretory component could not be demonstrated in the

14

Fig. 3. SDS gel e l e c t r o p h o r e s i s p a t t e r n of gastric m u c o s a l f r a c t i o n (gastric s a m p l e ) S 4 B / 2 (A), compared w i t h a n e q u i v a l e n t f r a c t i o n f r o m h u m a n c o l o s t r u m (B). H = h e a v y c h a i n ; L = light c h a i n ; SC = s e c r e t o r y component.

gastric fraction (Fig. 3). There is a second protein band in the heavy chain region in the gastric fraction which has so far not been identified.

Immunodiffusion (1) Double diffusion. We have examined the following gastric mucosal samples in serial dilutions of 1 to 128, by double diffusion against anti-secretory c o m p o n e n t and anti-IgA (s-chain) sera: (a) Original gastric mucosal scrapings suspensions, containing approx. 33 mg wet scrapings]ml. (b) CsCl layers L1 and L3 (gastric scrapings) in concentrations of 20 mg lyophilised solid/ml. (c) CsC1 layers L2 (gastric sample and gastric-salivary mixture) in concentrations of 10 mg lyophilised solid/ml. (d) The finally purified" fractions $4B/2 (gastric sample and gastric-salivary mixture) in concentrations of 10 mg lyophilised solid]ml.

15 TABLE I D I A M E T E R S I Z E D OF I M M U N O P R E C I P I T I N R I N G S A R I S I N G F R O M D I F F U S I O N O F S A L I V A R Y SUSPENSION AND OF MILK WHEY MIXED WITH G A S T R I C MUCOSAL SCRAPINGS. AND ALSO WITH BUFFER Results. (in r a m ) are f r o m p h o t o s r a p h l c en]ar1~ements of the o r / ~ n a l i m m u n o d i f f u ~ o n pl~tes. Sample

Diameter (ram)

SaUvary | ~ I p e n l i o n . u n d i l u t e d Salivary mulpenllon m i x e d with | a l t r l c ~ r a p i n ~ Salivary ~ u ~ e n ~ o n + b u f f e r (I : I , v/v)

25.0 20.0 20.0

.

Milk M~k Milk Milk Milk

whey. undiluted whey mixed with ~ t ~ c ~ r a p ~ w h e y • b u f f e r (1 : 1. v/v) whey mixed wi~t~c~rapin~(l w h e y + b u f f e r (I : 2, v / v ) .

.

.

(I : I, v/v)

(1 : 1. v / v ) : 2, v/v) .

.

.

.

.

.

.

34.5 28.0 28.0 24.0 24.0

.

Results of double diffusion failed to reveal the presence of the secretory c o m p o n e n t in any of the fractions arising from the o~ginal gastric mucosal scrapings suspension, while demonstrating its presence in corresponding fractions from the gastric-salivarymixture. (2) Quantitative immunodiffusion. The effect of gastric mucosal constituents on the amount of the secretory component present in the salivary suspension and in milk whey was examined by mixing the salivary fraction and a suspension of milk whey with the original gastric mucosal suspension, and quantitative immunodiffusion of the mixtures into anti-secretory component serumcontaining gels. The diameters of the resulting immunoprecipitin rings did not differ from those of rings arising from the salivary and milk whey suspensions diluted with the corresponding volumes of buffer {Table I). This observation indicates that, under the conditions of our experiment, gastric mucosal scrapings have no effect on the secretory component associated with either saliva or milk whey. General conclusions

In the work reported here we have separated an IgA-enriched fraction from gastric mucoproteins by isopycnic CsCI gradient fractionation, combined with gel chromatography. Investigations of the purified fraction failed to reveal the presence of the secretory c o m p o n e n t in associations with gastric mucosal IgA. In order to ensure that our observations were not the result of destruction of the antigenic sites of the secretory c o m p o n e n t during isolation we have mixed salivary secretory IgA fraction, and also milk whey, with the original gastric mucosal suspension. The gastric-salivary mixture was subjected to the same fractionation procedure used for the purification of gastric mucosal IgA. The secretory c o m p o n e n t was detected in all IgA positive fractions from the mixture. Quantitative immunodiffusion of the gastric-salivary and gastric-milk whey mixtures showed no effect of gastric mucosal components on the amount of the secretory c o m p o n e n t present in the original salivary and milk whey suspen-

16 sions. These findings suggest that our inability to detect the secretory component in gastric mucosal scrapings reflects the absence, in measurable quantities, of this c o m p o n e n t from gastric mucus, although appreciable quantities of IgA of the non-secretory type are present. It should be pointed o u t that quantitation of individual Components of gastric mucus are difficult. In general, glycoprotein components of the secretions are n o t easily solubilised and can only be investigated after degradation. Moreover, the ability of these mucoproteins to adhere to non-covalently b o u n d proteins, and the difficulty of separation of such molecules are well recognised [9,10]. Consequently, we could only investigate the solubilised components of gastric mucus and we found IgA of the non-secretory t y p e to be one of them. We are, however, unable to affirm complete absence of the secretory component or of secretory IgA from the insoluble components of gastric mucus which are associated with CsC1 layer L1 in the form of a crust on top of the gradient, and in layer L3 as a gel pellet. Acknowledgements We wish to thank Mr. Simon Haskoll for technical assistance. We are indebted to the Hayward Trust for the grant which made this work possible. References I 2 3 4 5 6 7

SPohn, M. and McColl, I. (1979) Biochim. Biophys. Acta 576, 1 ~ 8 Brandtzaeg, P. (1973) Ann. I m m u n o l , (Inst. Pasteur) 124C, 417--438 Porter, P. (1973) Vet. Res. 9 2 , 6 5 8 - - 6 6 4 Porter, P. (1973) J. I m m u n o l . 2 4 , 1 6 3 - - 1 7 6 Spohn, M. and McColi, I. (1977) Biochem. Biophys. Kes. Commun. 7 9 , 8 3 7 - - 8 4 2 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 44 06--4412 Dubois, M., GiBes, K.A., Hamilton, J.K., Regens, R.A. and Smith, F. (1956) Anal. Chem. 28, 350--356 8 Lowry, O.H., Rosebrough, N.J., Faxr, A.L. and Randall, l~.J. (1951) J. Biol. Chem. 193, 265--275 9 Strakey, B.J., Snaxy, D., A11en, A. (1974) Biochem. J. 1 4 1 , 6 3 3 - - 6 3 9 10 Snaxy, D., Allen, A. and Pain, R.D. (1974) Biochem. J. 1 4 1 , 6 4 1 - - 6 4 6