64

EARL DIXON, JR.

Acknowlegement--This investigation was supported by General Research Support Grant No. 42510, National Institutes of Health, U.S. Public Health Service. REFERENCES

BARTLETTG. R. (1970) Patterns of phosphate compounds in red cells of man and animals. In Red Cell Metabolism and Functions (Edited by BREWER G. J.), pp. 245256. Plenum Press, New York. BREWER G. J. (1966) Erythrocyte metabolism and function: hexokinase inhibition by 2,3-diphosphoglycerate and interaction with ATP and Mg ~+. Biochim. biophys. Acta 192, 157-161. CHAPMAN R. G., HENNESSEYM. A., WALTERSDOLPHA. M., HUENNEKENN F. N., & GABRIO B. W. (1962) Erythrocyte metabolism--V. Levels of glycolytic enzymes and regulation of gylcolysis. J. Clin. Invest. 41, 1249-1256. D~XON E., MCMANUS T. J. & TOSTESOND. C. (1966) Glycolytic enzymes in pig and sheep red cells (an abstract) Fedn Proc. Fedn Am. Socs exp. Biol. 25, 641. GERBER G., SCHULTZE J. • RAPOPORT S. M. (1970) Occurrence and function of high K m hexokinase in immature red blood cells. Fur. J. Biochem. 17, 445-449. GROSSBAROL. & SCr~IMKER. T. (1966) Multiple forms of rat tissue hexokinase. J. biol. Chem. 241, 3546-3560. KIM H. D. & MCMANUS T. J. (1971) Studies on the energy metabolism of pig red cells--1. The limiting role of membrane permeability in glycolysis. Biochim. biophys. Acta 230, I-11. MCMANUS T. J. (1967) Comparative biology of red cells. Fedn. Proc. Fedn Am. Sots exp. Biol. 26, 1821-1826. MCMANUS T. J. & KIM H. D. (1968) Energy metabolism in the pig red cell. In Stroffwechsel und Membrane Permeability yon Erythrocyten und Thrombocyten (Edited by DEUTSCHE., GERLACH E. & MOSER K.), pp. 43-49. George Thieme Verlag, Stuttgart.

PARRY M. & WALKER D. G. (1966) Purification and properties of adenosine-5'-triphosphate-D-glucose-6phosphotransferase from rat liver. Biochem. J. 99, 266-274. PONCE J., ROTH S. & HARKNESS D. R. (1971) Kinetic studies on the inhibition of glycolytic kinases of human erythrocytes by 2,3-diphosphoglyceric acid. Biochim. biophys. Acta 250, 63-74. RAPOPORT S. (1966) The regulation of glycolysis in mammalian erythrocytes. In Essays in Biochemistry (Edited by CAMPBELLP. N. & GREENVILLEG. D.), Vol. 4, 69-103. Academic Press, London. ROSE I. A. & O'CONNELLE. L. (1964) The role of glucose6-phosphate in the regulation of glucose metabolism in human erythrocytes. J. biol. Chem. 239, 12-17. Rose I. A., WARMSJ. V. B. & O'CoNNELL E. L. (1964) Role of inorganic phosphate in stimulating the glucose utilization of human red blood cells. Biochem. biophys. Res. Commun. 15, 33-37. SALAS J., SALAS M., VINVELA E. & SOLS A. 0965) Glucokinase of rabbit livers. J. biol. Chem. 240, 1014-1020. SRIVASTAVAS. K. & BEUTLER E. (1972) The effects of normal red cell constituents on the activities of red cell enzymes. Archs Biochem. Biophys. 146, 249-255. DE VERDIERC. H. & GARBYL. (1965) Glucose metabolism in normal erythrocytes II. Factors influencing the hexokinase step. Scand. J. Hematol. 2, 305-317. WIDDAS W. F. (1955) Hexose permeability of foetal erythrocytes. J. Physiol., Lond. 127, 318-327. Key word index--Erythrocyte metabolism; red cell hexokinase; isoenzymes of hexokinase; pig; Sus scrofa.

Comp. Biochem. Physiol., 1975, Vol. 5011,pp. 65 to 70. Pergamon Press. Printed in Great Britain

HUMORAL ANTIBODIES TO SOLUBLE ANTIGENS IN LARVAE OF XENOPUS LAEVIS RICHARD D. JURD, SHEILA M. LUTHER-DAVIES AND GEORGE T. STEVENSON Tenovus Research Laboratory, Southampton General Hospital, Southampton SO9 4XY, England (Received 29 November 1973) Abstract--1. Larvae (tadpoles) of Xenopus iaevis produce precipitating humoral antibodies to both "Mum-precipitated" human immunoglobulin light chains and to Limulus haemocyanin. 2. Most of the larval antibody is in the 19 S serum protein fraction, but a significant proportion is in the 7 S protein fraction. 3. Immunoclectrophoresis of the larval antibody shows that Xenopus tadpoles are capable of synthesizing both IgM and IgG antibodies. 4. The phylogenetic significance of these findings is discussed.

INTRODUCTION

elicited in the tadpole of this phylogenetically primitive anuran amphibian (Noble, 1931), and to investigate the classes of Ig involved.

HUMORAL antibody responses in the adult South African clawed toad, Xenopus laevis (Daudin), to both particulate and soluble antigens have been observed by numerous workers (Lykakis & Cox, 1968; Lykakis, 1969; Marchalonis et al., 1970; Hadji-Azimi, 1971; Manning & Turner, 1972; Jurd & Stevenson, 1973; Turner, 1973; Turner & Manning, 1973; Yamaguchi et aL, 1973). There is an initial 19 S response, later supplemented by a 7 S response (Hadji-Azimi, 1971); these two immunoglobulin (Ig) populations have antigenically distinct heavy chains (Jurd & Stevenson, 1973) and can therefore be regarded as analogous to the mammalian classes IgM and IgG, respectively. Production of humoral antibody in Xenopus larvae (tadpoles) has not been reported. However, they have well characterized lymphomyeloid tissue identifiable in several of their organs from stages 42 to 44 (Nieuwkoop & Faber, 1967) of ontogenesis (Manning & Horton, 1969), and have been observed to reject allografts of skin (Horton, 1969; Horton & Manning, 1972). A potential to secrete antibody is suggested by the appearance in the larvae of lymphocytic surface Igs (du Pasquier et aL, 1972) which in mammals signify the B (antibody-secreting) cell lines (e.g. Rabellino et aL, 1971). However, some doubt about the significance of this finding in Xenopus arises because of the large number of Ig-bearing cells in the thymus. Another finding at least consistent with an antibody-secreting line in the tadpoles is the appearance of specific rosette-forming cells following immunization with sheep erythrocytes (Kidder et al., 1973). Mammalian lymphocytes which rosette thus can apparently be either B or T (Schlesinger, 1970; Ashman & Raft, 1973). The following studies were designed to demonstrate whether or not a humoral antibody can be 8

MATERIALS AND METHODS Animals Adult and tapole Xenopus laevis were reared and maintained in our own colony as previously described (Maclean & Jurd, 1971). Preparation o f antigens Light chains were prepared from human IgG as described by Stevenson & Dorrington (1970).A solution containing 1 mg/rnl light chain in 0.2 M Tris-HCl, was adsorbed on to aluminium hydroxide ("alum-precipitated") after the method of Proom (1943). The precipitate was immediately spun down at 5000 g and air-dried. "Alum-precipitated" Limulus haemocyanin (Calbiochem Ltd., Los Angeles, U.S.A.) was similarly prepared from a solution containing 1 mg/ml of the protein in 0.2 M Tris-HCl. Immunization and bleeding of tadpoles Stage 48 (Nieuwkoop & Faber, 1967) Xenopus tadpoles were anaesthetized in a 0.02% aqueous solution of tricaine methane sulphonate (MS 222---Sandoz Products Ltd., London). The tadpoles were removed from the anaesthetic and placed on moist falter paper in an open Petri dish. Working with the aid of a binocular dissecting microscope, a small incision, approximately 0.5 mm long, was made with a scalpel blade in the dorsal skin of the tadpole in the proximity of the left thymus. Using a fine tungsten electrolysis needle, a fragment of dried, alumprecipitated antigen, approximately 20 t~g in weight, was inserted under the skin. During the operative procedure the tadpole was kept irrigated with tap water. After implantation of the antigen the animal was returned to 65

66

RICHARD D. JtlRD, SHEILAM. LUTHER-DAVIES AND GEORGE T. STEVENSON

aerated, aged tap water, and was allowed to recover from anaesthesia. It was found that approximately 75 per cent of the tadpoles survived the operation, and that the antigenic implant, visible beneath the tadpoles' clear skins, was retained by almost all the survivors. Tadpoles were immunized on days 1 and 36, varying the sites of implantation. On day 43 (by which time the animals had reached stages 53-56) the anaesthetized tadpoles were placed, ventral side up, on moist filter paper, and were blotted dry with tissue. Two methods of bleeding were used. Either an incision was made into the ventricle with the tip of a scalpel blade and the exuding blood was drawn by capillarity or cardiac pumping into the mouth of a Pasteur pipette placed over the whole heart or, alternatively, a Pasteur pipette was drawn out to terminate in a fine capillary which was used as a hypodermic needle to penetrate the ventricle directly--blood rose into the pipette by capillarity. The blood was blown from the pipette or the capillary into a small ignition tube where it was pooled with the blood from other tadpoles and allowed to clot by standing for 6 hr at room temperature. The serum was spun at 2500 g for 30 rain in microhaematocrit tubes.

Immunization and bleeding of adult Xenopus Human Ig (2.0 mg) light chain, or 2-0 mg of Limulus haemocyanin, in 0"5 ml of amphibian saline solution was emulsified in Complete Freund's Adjuvant (Difco Laboratories, Detroit, U.S.A.) and injected intraperitoneally on day 1. Booster doses of 1 mg of antigen in Complete Freund's Adjuvant were given intraperitoneally on days 36, 64 and 85. One week after each booster injection up to 2 ml of blood with withdrawn from the ventricle of the toad (which had previously been anaesthetized by immersion in 0"5~ aqueous MS 222). The blood was allowed to clot by standing for 6 hr at room temperature; the serum was spun for 20rain at 10,000 g to remove platelets.

Purification of tadpole and adult anti-light chain antibody An immunoadsorbent comprising 2 mg of h u m a n Ig light chain coupled to 2 ml of Sepharose 4B (Pharmacia Ltd., Uppsala, Sweden) was prepared following the cyanogen bromide coupling method of Axen et aL (1967), as modified by Cuatrecasas (1970). Tadpole antiserum (1 ml) was incubated with 1 ml of the packed antigen-coupled Sepharose 4B for 24 hr at room temperature. The incubation was carried out in double-ended tubes having a sintered-glass filter at one end, constructed as described by Stevenson & Eady (1973): the tubes were rotated on a blood-cell suspension mixer ( M a t b u m Surgical Instrument Co., Portsmouth, U.K.). After incubation, the supernatant was spun out of the Sepharose which was then washed three times in 0'2 M Tris-HCl. The coupled Sepharose was treated with its own bed volume of molar NH4OH for 5 rain at room temperature to break the antibody-antigen bond. The supernatant, containing the antibody, was spun out of the Sepharose which was then twice washed with 2 ml 0"2 M Tris-HCl. The washings were pooled with the supernatant which was then dialysed into 0.2 M Tris-HCl. The dialysate was adsorbed overnight at room temperature with a few beads of Sephadex G-25 (fine) coupled to

rabbit anti-(human Ig light chain) to remove any light chain broken off the Sepharose-light chain immunoadsorbent by the action of NH4OH. Agar-gel double diffusion on Ouchterlony plates showed a precipitin line when the adsorbed dialysate was reacted against h u m a n Ig light chain, showing that it had retained its antibody activity. Antibody was stored frozen at - 10°C.

lodination of antibody Tadpole antibody (100 t~g) to h u m a n Ig light chain was iodinated with 1 mCi of x~5I (NaX~SI, specific activity on day of iodination: > 14 mCi/t~g, conc. 100 m C i / m l - Radiochemical Centre, Amersham, U.K.) following the Chloramine T method of Hunter & Greenwood (1962). Adult antibody (25/zg) was similarly iodinated with 0.25 mCi JzsI. The iodinated antibodies were passed down a 2 0 0 × 2 2 m m Sephadex G-25 (coarse) column, presaturated with Xenopus whole serum, to remove noncoupled 125I. Radioactivity was measured in a Wallac GTL G a m m a Sample Counter (Wallac OY, Finland). Approximately 50 per cent of the iodine was bound to the proteins.

Characterization of antibody by gel-filtration chromatography A 1-ml sample of iodinated tadpole or adult antibody containing approximately 50/~Ci x25I per ml was diluted with 3 ml adult Xenopus anti-(human Ig light chain) serum (to act as a carrier) and 2 ml 0.2 M Tris-HCl. The sample was passed, in 0.2 M Tris-HC1, through an upward-flowing Sephadex G-150 column, 9 0 0 × 2 5 m m . Eluent fractions were assayed for total protein content by optical absorption at ~ = 280 nm, and for radioactivity.

Raising of antisera in rabbits' Antisera to whole adult Xenopus serum, to Xenopus IgM and to Xenopus IgG were raised in New Zealand White rabbits, using the schedules described by Jurd & Stevenson (1973).

Cellulose acetate electrophoresis Cellulose acetate membrane electrophoresis of serum proteins was performed in a Beckman Model R100 Microzone Electrophoresis apparatus as described by K o h n (1968).

Double diffusion in agar gels and immunoeleetrophoresis Double diffusion Ouchterlony plates were set up using a modification of Ouchterlony's (1949) technique. Ionagar 1 ~ (Oxoid Division, Oxo Ltd., London) gels were used, made up in 0'02 M Tris-HCl/saline with 0.02~ N a N 3 added. The gels were prepared on washed glass slides; 1 m m dia. wells were cut using the tip of a Pasteur pipette. Immunoelectrophoresis was carried out as described by Feinstein (1968). Ouchterlony and immunoelectrophoresis plates were washed first in 0"85% NaCI solution and then in distilled

IZI

(b)

!

(c) Fig. 5(a). lmmunoelectrophoresis/autoradiograph plate showing patterns obtained when tadpole anti(human lg light chain), diluted with adult serum carrier (above), and adult serum (below) were electrophoresed and reacted with rabbit anti(Xenopus whole serum). Anode to left. (b). Part of albumin line (white arrow in a), showing absence of silver autoradiograph grains. (c). Part of Ig G line (black arrow in a), showing presence of silver atttoradiograph grains.

Larval antibodies in water t o remove unprecipitated proteins before drying and staining with amido black after the methods of Uriel (1964).

Autoradiography Dried, stained Ouchterlony and immunc~lectrophoresis plates were coated with Ilford K5 Nuclear Emulsion (Ilford Ltd., Ilford, U.K.) diluted 1 : 1 with water at 50°C. The coated slides were dried and left at 4°C in light-tight boxes for 10 days prior to photographic development and fixation. Autoradiographs were examined under an oil-film using a light microscope with a x 10 eyepiece and x 10 or x 40 objectives. RESULTS Serum samples from unimmtmized tadpoles at various developmental stages were submitted to immunoelectrophoresis against rabbit anti-(Xenopus whole serum) antiserum. A protein migrating in the y-globulin region of the gel was first detectable in stage 47 tadpoles. At stage 53, a second y-protein appeared. The proportion of these proteins relative to the total serum proteins increased until, by stage 59, the immunoelectrophoresis pattern of the serum proteins was indistinguishable from that of adult Xenopus serum. Serum from tadpoles immunized with human Ig light chain, or haemocyanin, respectively, together with normal Xenopus tadpole serum and serum from adults immunized with light chain or haemocyanin were reacted with antigens on agar-gel Ouchterlony plates. A preo'pitin line, indicative of antibody, formed between the antigen wells and the wells containing sera from the appropriately immunized adults and immunized tadpoles (Fig. 1). Tadpole anti-light chain did not cross-react with haemocyanin, or vice versa. Immunoelectrophoresis and cellulose acetate electrophoresis revealed somewhat enhanced levels of proteins migrating in the y-globulin region in sera from immunized tadpoles compared with the levels in unimmunized animals (Fig. 2). In an attempt to identify and quantitate the classes of protein constituting the tadpole antibody, the antibody was purified by immunoadsorption, radioiodinated and mixed with antiserum raised in adults to the same antigen as a carrier/marker, and subjected to gel chromatography. Figure 3 illustrates the elution profile obtained when iodinated anti-(human Ig light chain) from the pooled sera of ten immunized tadpoles, bled 6 weeks after the primary and 1 week after the booster injection, was chromatographed, with adult serum carrier, on Sephadex G-150. It will be seen that the overwhelming majority of the antibody elutes from the column in the first peak, identifiable from previous work (Jurd & Stevenson, 1973) as the 19 S, IgM peak. A small, though significant amount of iodinated tadpole antibody elutes in the 7 S protein peak.

Xenopus

67

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Fig. 3. Elution profile obtained when radio-iodinated anti-(human Ig light chain) from pooled sera of ten immtmized tadpoles, diluted with adult serum carrier, was chromatographed on Sephadex G-150. Figure 4 shows, for comparison, the elution profile obtained when iodinated anti-light chain antibody from adult Xenopus, bled 6 weeks after the primary and 1 week after the booster immunization, was chromatographed. Most of the antibody is ehited from the column in the 7 S protein peak, only a small amount eluting in the 19 S peak. I 3o 19S

Adult

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Fig. 4. Elution profile obtained when radio-iodinated anti-(human Ig light chain)from an adult toad, diluted with adult serum carder, was chromatographed on Sephadex G-150. Dipping-film autoradiographs were prepared from Ouchterlony and immunoelectrophoresis plates on which the iodinated adult or tadpole antibodies had been analysed. In each case, the iodinated antibody had been diluted with whole serum from an adult Xenopus, immunized against human Ig light chain, as a carrier/marker. Reaction of both tadpole and adult iodinated antibody with light chain on an Ouchterlony plate caused a deposition of silver grains on the autoradiograph co-incident with the precipitin line caused by the carrier antibody, confirming that it was antibody which was iodinated. Reaction on an Ouchterlony plate of adult iodinated antibody with rabbit antiserum to whole

68

RICHARDD. JURD,SHEILAM. LUTHER-DAvIESAND GEORGET. STEVENSON

Xenopus serum revealed, by autoradiography, the presence of at least two precipitin lines. Iodinated adult antibody was also precipitated on an Ouchterlony plate by both rabbit anti-Xenopus-IgM and rabbit anti-Xenopus-IgG antisera. Similar results were obtained with iodinated tadpole antibody. When the iodinated adult antibody was subjected to immunoelectrophoresis with reaction against rabbit anti-Xenopus whole serum, and made into an autoradiograph, most of the deposition of silver grains occurred over the line, identifiable from previous work (Jurd & Stevenson, 1973), corresponding to IgG. Some antibody was precipitated in the IgM line. Tadpole antibody was also precipitated in both IgG and IgM lines, with the grain count in the latter proportionately higher than in the case of the adult antibody (Fig. 5). Surprisingly, however, even the tadpole antibody gave most of its grain counts over the IgG line. DISCUSSION The larvae of X. laevis have been shown to synthesize humoral antibody in response to immunization by "alum-precipitated" soluble antigens. Six weeks after the primary, and 1 week after the booster immunization, most of the antibody in the tadpole is confined to the 19 S protein fraction, although a small, but significant amount is found in the 7 S fraction: in adult toads immunized and bled at the same times, the overwhelming majority of the antibody is in the 7 S protein fraction. From these studies it is not possible to say definitely if the tadpole antibody comprises both IgM and IgG, or solely of polymeric (19 S) and monomeric (7 S) IgM. The antibody reacts with specific antibodies to adult Xenopus IgG and IgM, respectively, on Ouchterlony plates: however, this is not in itself sufficient evidence to identify the presence of IgG, since light chain determinants on an IgM molecule may precipitate anti-IgG antibody. Stronger evidence for the presence of IgG in the tadpole is provided by immunoelectrophoresis combined with autoradiography. By these techniques, tadpole antibody is precipitated in the electrophoresis gel in lines corresponding to those of both IgM and IgG from the adult. Unfortunately, the relative proportions of IgM and IgG demonstrated by this method conflict with the proportions of 19 S and 7 S antibody shown by gel chromatography. Two possible explanations for this phenomenon can be proposed. (i) The tadpole IgG is partially polymerized so that some of it migrates in the 19S Sephadex peak: occasional human IgG molecules are polymerized thus (Kochwa et aL, 1966; Capra & Kunkel, 1970). (ii) Some of the carrier Xenopus anti-(human Ig light chain) serum has antibody activity to allotypic Xenopus Ig characters (such antibody activity is well known in t uman serum), which could cause tadpole IgG to

migrate, as part of an antibody-antigen complex, in the 19 S zone. Because of the quantitative discrepancy between the gel chromatography and immunoelectrophoresis results all that can be said is that the tadpole is capable of synthesizing both IgM and IgG antibodies, and in the case examined the proportion of the former in the antibody population was greater than in an antibody population raised in adult Xenopus. Although considered phylogenetically primitive, Xenopus tadpoles have already been shown to possess lymphomyeloid elements from a relatively early stage in development (Manning & Horton. 1969) and to exhibit cell-mediated immunity (Horton, 1969; Horton & Manning, 1972). The present demonstration of antibody production in response to challenge by soluble antigens reinforces a view that Xenopus larvae have a relatively sophisticated immune system, possibly developed in response to adaptive pressure from an aquatic environment containing numerous naturally occurring antigens. The presence of immune responses in other anuran tadpoles, including Rana eatesbeiana (Maniatis et aL, 1969; Baculi & Cooper, 1972; Moticka et al., 1973; Haimovich & du Pasquier, 1973), Rana esculenta (du Pasquier, 1969) and Alytes obstetricans (du Pasquier, 1969, 1970), subject to similar pressures, have been described. The existence of both 19 S and 7 S antibody is of great interest. Moticka et al. (1973) were only able to detect an lgM response to sheep erythrocytes in R. catesbeiana tadpoles, and were unable to elicit a true anamnestic response in such animals. Similarly, in Necturtts masculosus, a neotenous urodele, Marchalonis & Cohen (1973) found only an IgM immunoglobulin. In Rana pipiens, Marchalonis (1971) reported that the tadpoles of the species possessed IgM immunoglobulin similar to the 7M macroglobulins of adult frogs, first detectable at stage 25 of ontogenesis. Marchalonis was unable to detect lgG molecules in tadpoles, although a major serum component, intermediate in size between lgM and IgG, and with an electrophoretic mobility and a molecular weight similar to IgA, was found in late metamorphic animals. However, the presence of 7 S Ig in larval X. laevis suggests that the absence of such proteins in other larval amphibians may be either the result of a loss of 7 S Ig at some stage during evolut i o n - t h e r e is evidence for multiple classes of Ig among certain galeoid sharks (Gitlin et al., 1973)--or result from the occurrence of metamorphosis in these amphibians at a relatively earlier stage in the ontogenetic scale of serum protein appearance, before synthesis of IgG commences. SUMMARY Precipitating humoral antibodies to human Ig light chain and to Limulus haemocyanin were found in the larvae of Xenopus laevis following immunization with the relevant antigens.

Larval antibodies in Xenopus Gel chromatography of the purified, radioiodinated antibody, diluted with adult Xenopus serum as a marker/carrier, showed that most of the antibody was in the 19 S serum protein fraction, but that a small, though significant amount of antibody was in the 7 S fraction. Further analysis of the radio-iodinated antibody by Ouchterlony plate immunodiffusion and by immunoelectrophoresis showed that Xenopus tadpoles synthesized both IgM and IgG. The possession of IgG by Xenopus tadpoles appears to be unique among amphibian larvae so far investigated.

Acknowledgements--This work was supported by Tenovus of Cardiff and the Wessex Regional Hospital Board. We are grateful to Dr. Roy Eady for help in the preparation of the immuno-adsorbents.

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RICHARD D. JURD, SHEILAM. LUTHER-DAvIESAND GEORGE T. STEVENSON

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Key Word Index--Amphibian; amphibian immune response; amphibian larvae; amphibian larval immune response; autoradiograph immunoelectrophoresis; immunoadsorbent; immunoglobulin; immunoglobulin classes; immunophylogeny; phylogeny of immune response; Xenopus laevis.