Serum and secretory immunoglobulins of the rat

Immunochemistry. PergamonPress 1970. Vol.7, pp. 453-460. Printed in Great Britain

SERUM AND SECRETORY I M M U N O G L O B U L I N S OF T H E RAT* T. S. BISTANY and T. B. TOMASI, Jr. Immunology and Rheumatic Disease Unit of the Department of Medicine, State University of New York at Buffalo, Buffalo, N.Y. 14215, U.S.A. (Received 8 September 1969) A b s t r a c t - T h e major species of antibody present in the rat external secretions, saliva and colostrum was found to be an intermediate sedimenting (approximately llS) immunoglobulin of rapid mobility apparently analogous to human IgA. Antibody activity was demonstrated by radioimmunoelectrophoresis of colostrum obtained following intramammary injection of bovine serum albumin. Using antisera prepared against partially isolated secretory IgA, serum IgA could be demonstrated. IgA was present in small amounts in serum, and was primarily 7S in sedimentation although higher polymers (17S) were also detected by gel filtration. Serum IgA was significantly more rapid (anodal) in electrophoretic mobility than secretory IgA, and this was not dependent on differences in sialic acid content.

INTRODUCTION The serum immunoglobulins of the rat were first characterized by Arnason [1] who described three arcs on serum immunoelectrophoresis which were designated IgG, IgM, and provisionally, IgA. Subsequent work by Binaghi et al. [2] led to the partial purification of the protein termed IgA by Arnason. The finding of a higher carbohydrate content in this protein was felt to be consistent with the thesis that it was the counterpart of human IgA. Nussenzweig and Binaghi[3] using rat anti-DNP antibodies, described the presence of four immunoglobulins on immunoelectrophoresis, and Bloch, Morse, and Austen [4] reported antibody activity in each of these immunoglobulins on radioimmunoelectrophoresis (RIEP). The immunoelectrophoretic arcs were designated IgGa, IgGb, IgA, and IgM by both groups of investigators. Work in man as well as other species has shown that an intermediate sedimenting primarily 11S immunoglobulin in external secretions is closely related to the IgA class of serum, although the secretory immunoglobulin possesses certain unique characteristics[5]. In this communication we present evidence for the existence of a secretory system in the rat analogous to that in man and in addition the presence of four and probably five immunoglobulins in rat serum. The major secretory immunoglobulin in this species is intermediate in sedimentation and is related to a component which is present in relatively low concentration in serum in both a 7S and polymeric form. *Supported by Research Grant No. 2 RO1 AM 10419 and Training Grant No. 5 TO1 AM 05075. Presented in part at the 53rd Meeting of the Federation of American Societies of Experimental Biology, Atlantic City, New Jersey. 453

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T. S. BISTANY and T. B. TOMASI, Jr. MATERIALS AND METHODS

Colostrum and saliva Rats of the Sprague-Dawley strain were lightly anesthetized with intraperitoneally administered Pentobarbital. Pilocarpine was administered in a dose of 0.1 ml of a 1 per cent solution and whole saliva collected, spun in the Sorvall centrifuge at 15,000 rev/min for ½hr and the supernatant concentrated tenfold. Colostrum was obtained within 48 hr after littering by manual expression from the mammary gland and collection of droplets into capillary pipettes. Colostrum was spun at 40,000 rev/min for two hours, the pellet and supernatant fat discarded and the middle layer was utilized for further fractionation and immunization. Saliva was concentrated tenfold by negative pressure dialysis and centrifuged for ½hr at 15,000 rev/min in a Sorvall centrifuge and the supernatant used for further studies.

Isolation of serum components Serum IgM was isolated by centrifuging approximately 50 ml of pooled rat serum at 40,000 rev/min for 12 hr. The pellet and a few drops above it were passed over Sephadex G-200. The void volume was concentrated and subjected to starch or pevikon block electrophoresis. Serum IgG was isolated utilizing DE-52 chromatography. Rat serum was dialyzed against phosphate buffer, pH 7.5, 0.005 molar and applied to a DE-52 column equilibrated with the same buffer. The initial peak after concentration was found on immunoelectrophoresis "to contain only IgGa. No attempt was made to isolate IgGb, 7Syl, or IgA from rat serum.

Immunology Antisera against rat immunoglobulins were prepared in rabbits by injection of antigens in complete Freund's adjuvant into all four footpads. If boosting was necessary, it was done by subcutaneous injection, again using Freund's adjuvant. Rats were immunized by footpad injections with human serum albumin (HSA) in Freund's adjuvant. After 23 days the rats were exsanquinated and the serum de-complemented using a heterologous antigen-antibody system. The rat antiserum was then added to the antigen and the precipitate washed three times with cold saline prior to mixing with complete Freund's adjuvant and injection into rabbits. The antiserum referred to as 'anti L-chain' was an anti-IgG antiserum with both H and L chain activity as evidenced by the reaction of partial identity of IgG with IgA and IgM on Ouchterlony analysis. Absorption of this antiserum with IgG resulted in loss of all activity against IgG, IgA, and IgM. Immunoelectrophoresis was performed by the micro method of Scheidegger [6] with 2 per cent agar in 0.05 M barbital buffer at pH 8.2. Radioimmunoelectrophoresis was done according to the method described by Yagi [7]. Radio labelling of the HSA was accomplished by the technique of Greenwood and Hunter [8]. Forty-two rats immunized with HSA in complete Freund's adjuvant were bled in groups of 7, at 9, 16, 22, 36, and 42 days. These sera were used for radioimmunoelectrophoresis. In some experiments, endotoxin, (Sal-

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monella typhosa, Difco) was administered as an adjuvant [9] in 100 m/xg doses to animals immunized intravenously with HSA or bovine gamma globulin. Starch or pevikon block electrophoresis and DEAE chromatography were performed according to previously described methods [10, 11]. Ultracentrifugation was performed in a Spinco Model E Ultracentrifuge at 52,640 rev/min and 20°C. Density gradients were done using a 10-40 per cent sucrose gradient as described by Kunkel et al. [12]. Purified human IgM and IgG were used as sedimentation markers in some of the gradients.

Dissociation experiments Disulfide bond reduction experiments of serum and salivary immunoglobulins were performed using 0.2 molar fl-mercaptoethanol (BME) for two hours at room temperature in 0" 14 M NaCI. Alkylation was accomplished with a 10 per cent excess of iodacetamide for ten minutes at 4°C. Serum IgM was reduced with 0" 1 molar BME. RESULTS Figure l(a) shows the components designated IgGa, IgGb, 7Syl, and IgM which are all seen on immunoelectrophoresis of normal rat serum developed with an anti-immune precipitate antiserum. IgA is not detected by this antiserum but its position is shown in the composite diagram illustrated in Fig. l(b). IgGa and 7Sy 1 are eluted in the 7S region on Sephadex G-200 chromatography and were shown to have antibody activity by radioimmunoelectrophoresis (Fig. 2(a)). These components may represent subgroups within the major IgG group or separate immunoglobulin classes; further definition requires additional studies on homogeneous preparations. IgGa can be isolated in purified form by DEAE chromatography (pH. 7.5 using 0.005 molar phosphate buffer), is the major immunoglobulin class in rat serum, and appears to be most analogous to human IgG. The immunoglobulin designated IgM isolated as described in the methods section is always contaminated with small amounts of IgA polymers. It is present in the void volume on Sephadex G-200 chromatography, contains L polypeptide chains and antibody activity can be demonstrated by radioimmunoelectrophoresis as early as the fourth day after antigenic stimulation with bovine gamma globulin as antigen and endotoxin as an adjuvant (Fig. 2(b)). Preparations of this immunoglobulin show a single peak in the ultracentrifuge having a sedimentation coefficient of 17S and are dissociated to 7S subunits on treatment with 0.1M BME for two hours at room temperature.

The rat IgA system Whole saliva, when subjected to immunoelectrophoresis and developed with an anti L-chain antiserum, (see methods), gave a single precipitin arc having a mobility similar to that of human IgA (Fig. 3). Fractionation of rat saliva on Sephadex G-200 revealed the L-chain reactive material to be present solely in the void volume as shown in Fig. 4(a). The void volume was shown to be heterogeneous on analytical ultracentrifugation, the fastest sedimenting component having

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T. S. BISTANY and T. B. TOMASI, Jr.

an s rate of 10.9S. Sucrose density gradient ultracentrifugation of this material and subsequent immunological analysis of the fractions using an anti-colostral antiserum (same antiserum as shown in Fig. 3), showed the salivary immunoglobulin to be intermediate in size between human IgG and IgM used as markers in the gradient (Fig. 5). Disulfide bond reduction for two hours at 20°C with 0.2 molar BME and alkylation with iodoacetamide did not change the sedimentation characteristics on repeat density gradient analysis. Immunization with this (o) Blue dextran VoIU volume

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Figs. l(a, b). (a) Normal rat serum (center well) developed with rabbit anti-rat immune ppt. antiserum. Four arcs are demonstrated corresponding to IgGa, IgGb, 7Syl and IgM (labelled in Fig. l(b)). IgA is not detected by this antiserum. Anode is to the right. (b) Schematic diagram of rat immunoglobulin system. IgA is detected by an anti secretory antiserum in 5X concentrated serum (also see Fig. 6).

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Figs. 2(a, b). (a) The wells contain pooled sera from separate groups of seven rats immunized with a single footpad injection of HSA and bled after 22 days (top well), and 42 days (bottom well). The immunoelectrophoretic arcs were developed with an anti-immune precipitate antiserum and the immunizing antigen was then diffused from the opposite trough. Labelling is present in the IgGa, 7S71, and IgM arcs at 22 days but by 42 days, IgM activity is no longer detectable. (b) T h e well contains pooled serum from rats immunized intravenously with BGG and endotoxin as adjuvant and bled four days later. T h e antiserum is the same as in Fig. 2(a). T h e major arc showing activity is IgM but faint labelling is also seen in IgG.

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Fig. 3. Center well contains concentrated whole rat saliva. T h e u p p e r trough contains an anti IgGa antiserum known to have both H and L chain activity; the lower trough contains an anti-whole colostral antiserum.

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Fig. 4. Sephadex G-200 chromatograms of rat serum, saliva, and colostrum showing the distributions of IgG and IgA. In Fig. 4(a), 7S IgG as a marker was eluted in the second peak. intermediate sedimenting material produced an antiserum which detected more than one component in saliva but which after absorption with IgGa to remove Lchain activity, delineated a single fast moving arc in concentrated whole rat serum as shown in Fig. 6. This immunoglobulin is designated serum IgA. When reacted with whole colostrum, or with the void volume from a Sephadex G-200 fractionation of colostrum, this antiserum produced a single arc in the y region as demonstrated in the bottom of Fig. 6. Note that the electrophoretic mobilities of the colostral and salivary (See also Fig. 3) proteins are more cathodal than that of their serum counterpart. Ouchterlony analysis of serum and whole colostrum using the absorbed anti-salivary antiserum demonstrated immunologic identity between the serum and secretory IgA molecules (Fig. 7). The immunological relationship between IgGa and the colostral IgA are shown in Fig. 8. Antibody activity in colostral IgA was demonstrated by RIEP (Fig. 9). In this experiment, two pregnant rats were immunized with BSA in tissues adjacent to the mammary glands two weeks prior to littering. On radioimmunoelectrophoresis antibody activity was found in both the IgA and IgG arcs. In colostrum, IgA was found only in the Sephadex G-200 void volume (Fig. 4(b)), but on sucrose density gradient analysis of the void volume, IgA determinants were present in both the rapidly (17-19S) and intermediately (11S) sedimenting regions of the gradient. The distribution of IgA in serum on Sephadex G-200 chromatography is illustrated in Fig. 4(c). Experiments done with fresh rat serum revealed IgA determinants both in the void volume and in the 7S region of the eluates. How-

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T.S. BISTANY and T. B. TOMASI, Jr.

Density gradient Human y G Human 7"M Rat salivary 7"A

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Fig. 5. Void volume from whole saliva chromatographed on Sephadex G-200 (see Fig. 4(a)) mixed with human 7S IgG and 19S IgM markers and applied to a 10-40 per cent sucrose density gradient. The distribution of the salivary IgA and sedimentation markers in the gradient fractions were determined immunologically using specific antisera. ever, no intermediate sedimenting IgA was detected in serum. It appears, therefore, that normal rat serum does contain significant amounts of higher polymers of IgA in addition to the major 7S form; the relative amounts of IgA in the two peaks was not, however, accurately quantitated. DISCUSSION The results of the experiments with the anti-immune precipitate and antiIgM antisera are similar to those of Bloch, Morse, and Austen [4]. However, the immunoglobulin called IgA by these investigators is probably the component designated 71 by Banovitz and Ishizaka[13] and 7S71 in this communication. This protein was present only in the 7S region on G-200 filtration of serum and was not found in large amounts in colostrum or saliva. The immunoglobulin described here as IgM appears to be analogous to the 19S IgM of other species. The IgM in non-immune serum is found exclusively in the void volume on Sephadex G-200 chromatography, but using serum from animals immunized intravenously with endotoxin, IgM determinants were also present in small amounts in the 7S region of the gradient. The possibility exists that these 7S IgM determinants represent in vitro degradation of 19S IgM. However, in view of their occurrence in the serum from freshly bled animals and the

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Fig. 6. T h e u p p e r well contains five tbld concentrated rat serum, the lower well, whole colostrum. T h e antiserum is an anti salivary IgA antiserum absorbed with IgG.

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Fig. 7. Ouchterlony analysis of serum and colostrum. The upper left well contains normal rat serum, the well on the right, whole delipidated colostrum. The lower well contains absorbed (with IgG) anti salivary IgA antiserum. Fig. 8. Upper left well, serum IgGa. Upper right well, colostral IgA obtained from the void volume from Sephadex G-P.00 chromatography. Lower well, anti salivary IgA, unabsorbed. The reaction with IgG is due to the presence of anti L-chain antibodies in this antiserum. The specific H-chain determinants of the colostral lgA are shown by the single spur.

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Fig. 9. Radioimmunoelectrophoresis of rat colostrum. T h e wells contain colostrum from two rats immunized with BSA subcutaneously in proximity to the m a m m a r y glands. T h e antiserum is an unabsorbed anti salivary, IgA antiserum containing L-chain as well as a chain antibodies.

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recent descriptions of similar components in human serum [14], it is also possible that a low molecular weight IgM antibody may be elaborated in this species as part of a normal immune response. Evidence that the protein designated IgA is an immunoglobulin is furnished by the presence of L-chain antigenic determinants, by the reaction of partial identity with IgGa, and by the presence of antibody activity as shown by RIEP with colostrum. Although not accurately quantitated, IgA appears to be in higher concentration in colostrum than serum. Serial dilutions of serum and subsequent Ouchterlony analysis with an anti IgA antiserum showed activity to a 1:8 dilution, whereas colostrum reacted to a dilution of 1 : 256. IgA was the only immunoglobulin found in saliva except for trace amounts of IgG detected by Ouchterlony analysis of ten times concentrated samples. O f considerable interest is the consistently slower mobility of secretory IgA (both salivary and colostral) as compared to serum IgA. The reason for this difference in electrophoretic mobility is at present unknown. Treatment with neuraminidase did not alter the mobility of serum IgA sufficiently to explain the observed mobility differences as being due to a higher sialic acid content of serum IgA. By Ouchterlony analysis, newborn rat serum did not contain IgA in animals that have not nursed. However, IgG is present in the serum of unsuckled animals, presumably because of selective placental transport. In adult animals, IgA is also found in bile and in small amounts in urine, in addition to the secretions described above. It should also be mentioned that on IEP, IgA appears to be the only immunoglobulin present in concentrated rat bile. Preliminary immunofluorescent studies with an anti-IgA antiserum in this laboratory have shown that the majority of the immunoglobulin producing cells in the lamina propria of the gut are of the IgA type. This finding is similar to that previously described for man [15] and rabbit[16]. In other areas, such as the rat spleen, IgA type cells are found in relatively small quantities as compared to the total number of immunoglobulin producing cells. The identification of an immunoglobulin as IgA in animal species is sometimes difficult. Electrophoretic mobility, carbohydrate content, precipitability with zinc, etc. are by no means absolute criteria. The finding that IgA is the predominant immunoglobulin in certain external secretions, such as saliva and colostrum [5], has been useful in identifying an immunoglobulin as belonging to the IgA class. A secretory system similar to that of the human has been previously reported in the monkey[17], rabbit[18], dog[19, 20], and more recently in the mouse [21]. The mouse IgA system appears to be quite similar in many respects to that found in the rat. However, in the mouse significant amounts of IgG were found in saliva and some intermediate size IgA polymers were present in serum[21]. It should be emphasized that the major immunoglobulin present in secretions is not always IgA as illustrated by studies on the sheep and cow, [23], and horse[24] in which the predominant species is a 7Syl related to IgG. However, small amounts of IgA may also be present in the secretions of the sheep and cow [25, 26, 30]. One of the best criteria for identifying an immunoglobulin as IgA has recently

460

T. S. BISTANY and T. B. TOMASI, Jr.

been e m p h a s i z e d by V a e r m a n and H e r e m a n s [20], and involved cross reactions with certain antisera specific for h u m a n a chains. However, in o u r experience, although such cross reactions are f o u n d fairly regularly with o t h e r m a m m a l i a n species, especially the dog, they have not been n o t e d with rat serum. In point o f fact, the cross reactions o f all the rat i m m u n o g l o b u l i n classes with anti h u m a n immunoglobulin antisera are extremely p o o r [27]. In addition to man, evidence has been p r e s e n t e d for the existence o f a secretory 'piece' in the rabbit[28], dog[19], monkey[17], mouse[21], a n d p e r h a p s in the sheep a n d cow [29, 26]. In this study, a rat secretory 'piece' could not definitely be identified. However, only two antisera were e x a m i n e d a n d in view o f the low immunoge{aicity o f the secretory 'piece' o f o t h e r species its existence has by no means been excluded in the rat. Note added in proof'. Nash et al. have recently reported similar findings with the rat immunoglobulin system including large numbers of IgA cells in the intestinal lamina propria of the rat [31].

REFERENCES 1. Arnason B. G., de Vaux St.-Cyr C. and Relyveld E. H., Int. Arch~Allergy 25, 206 (1964). 2. Binaghi R. A., and Sarondon de Merlo E., Int. ArchsAllergy 30, 589 (1966). 3. Nussenzweig V. and Binaghi R. A., Int. ArchsAUergy 27, 355 (1965). 4. Bloch K.J., Morse H. E. and Austen K. F.,J. Immun. 101,650 (1968). 5. Tomasi T. B., Jr., Tan E. M., Solomon H. and Prendergast R. A.,J. exp. Med. 121, 101 (1965). 6. Scheidegger J. J., Int. Arch~ Allergy 7, 103 (1955). 7. Yagi Y., Maier P. and Pressman D. J., Immunology 89,736 (1962). 8. Greenwood F. C. and Hunter W. M., Biochem.J. 89, 114 (1963). 9. Pierce C. W., Lab. Invest. 16, 768 (1967). 10. Kunkel H. G. and Trautman R., Electrophoresis: Theory, Methods and Applications, p. 279. Academic Press (1960). 11. Sober H. A., Gutter F. J., Wychkoff M. M. and Peterson E. A., J. Am. chem. Soc. 78, 756 (1956). 12. Kunkel H. G., Rockey J. H. and Tomasi T. B., Jr., In Immunochemical Approaches to Problems in Microbiology. Rutgers University Press, New Brunswick (1961). 13. Banovitz J. and Ishizaka K., Proc. Soc. exp. Biol. Med. 125, 78 (1967). 14. StoboJ. and Tomasi T. B.,Jr.J. clin. invest. 46, 1329 (1967). 15. Crabbe P. A., Carbonara A. O. and HeremansJ. F., Lab. Invest. 14, 235 (1965). 16. Crandall R. B., Cebra J. j. and Crandall C. A., Immunology 12,147 (1967). 17. Tourville D. R., Adler R. H., Bienenstock J. and Tomasi T. B., Jr.,J. exp. Med. 129, 411 (1969). 18. CebraJ.J. and Robbins J. B.,J. Immun. 97, 12 (1966). 19. Johnson J. S. and VaughanJ. H.,J. Immun. 98, 923 (1967). 20. VaermanJ. P. and HeremansJ. F., Immunochemistry 5, 425 (1968). 21. Asofsky R. and Hylton M. B., Fed. Proc. 27, 617 (1968). 22. FaheyJ. L., Wunderlich J. and Mishell R.J. exp. Med. 120, 223 (1964). 23. Sullivan A. L. and Tomasi T. B.,Jr, Clin. Res. 12, 463 (1964). 24. Genco R., Yecies L. and Karush F. Immunoglobulins of Equine, Colostrum and Parotid Fluid. 46th Mtg. Int. Assn. for Dental Research, p. 176 (1968). 25. Heimer R., Clark L. G. and Maurer P. H., ,4rchs Biochem. Biophys. 131, 9 (1969). 26. ButlerJ. E., Coulson E.J. and Groves M. L., Fed. Proc. 27, 617 (1968). 27. Mehta P. D. and Tomasi T. B., Jr., Fed. Proc. 28, 820 (1969). 28. CebraJ. J. and Small P. A., Biochemistry 6, 503 (1967). 29. Heimer R., Clark L. G. and Maurer P. H., Fed. Proc. 127, 617 (1968). 30. MachJ. P., PahudJ.J. and Isliker H., Nature, Lond. 223, 952 (1969). 31. Nash D. R., Vaerman J. P., Bazin H. and Heremans J. F.,J. Immun. 103, 145 (1969).