INMCTION AND IMMUNITY, Feb. 1977, P. 360-369Copyright © 1977 American Society for Microbiology

Vol. 15, No. 2Printed in U.S.A.

Immunoglobulin and Specific-Antibody Synthesis In Vitro byEnteral and Nonenteral Lymphoid Tissues After

Subcutaneous Cholera ImmunizationANN-MARI SVENNERHOLM AND JAN HOLMGREN*

Institute ofMedical Microbiology, University of Goteborg, Goteborg, Sweden

Received for publication 20 July 1976

An in vitro culture technique with synthesis of "4C-labeled protein has beenused to study immunoglobulin and specific-antibody formation by spleen, mes-enteric lymph nodes, Peyer's patches, and small intestine of rabbits, which wereimmunized twice subcutaneously with Vibrio cholerae lipopolysaccharide (LPS)and enterotoxin; saline-injected rabbits served as controls. Newly synthesizedimmunoglobulin G (IgG), IgA, and IgM were quantitated by liquid scintillationafter their isolation by means of affinity chromatography from columns withimmunoglobulin class-specific antibodies coupled to Sepharose. Specific antibod-ies were similarly measured after purification from gels derivatized with LPS orcholera toxin. The isolated antibodies had full biological activity as studied inprotection tests. The immunization increased the overall IgM synthesis in thespleen. It also enhanced the production of IgA and IgG in Peyer's patches and ofIgA in intestine. Significant synthesis ofradiolabeled antibodies against both V.cholerae LPS and enterotoxin was found in spleen as well as in Peyer's patches ofimmunized animals. Titrations with an enzyme-linked immunosorbent assay(ELISA) showed significant levels of IgG as well as IgA antibodies in incubationmedium from all the studied tissues, whereas specific IgM was only found forspleen and mesenteric lymph nodes. Simultaneous tissue incubations at 370Cand in an ice bath indicated that the major part of the antibodies registered withthe ELISA represented de novo synthesis.

Cholera, the prototype for the "enterotoxicenteropathies" (7), is a disease in which localimmunity is important. Neither the infectiousagent, Vibrio cholerae, nor its secreted diar-rheagenic enterotoxin appears to penetrate theintestinal epithelium, and consequently anti-bodies directed against either the vibrio or theenterotoxin must be present in the gut lumenor at the mucosal surface in order to be effective(15). The circumstance that intestinal antibod-ies may originate not only from local sites ofsynthesis but also from the circulation has com-plicated the evaluation of gastrointestinal im-munity in infections. Furthermore, it meetswith considerable difficulties in quantitatingimmunoglobulin and specific antibodies accu-rately in intestinal secretions due to the hetero-geneity in size of particularly immunoglobulinA (IgA) (26) and to proteolytic degradation ofimmunoglobulin and specific antibodies (10,26).To overcome the previously encountered

problems in measuring local cholera antibodiesand in evaluating their intestinal or systemicorigin, we have attempted to quantify directly

the systhesis of immunoglobulins and specificantibodies by intestinal and extraintestinallymphoid tissues of rabbits. The approach hasbeen to let the tissue produce 14C-labeled pro-tein during an in vitro incubation period andthen to isolate the newly formed IgG, IgA, andIgM as well as the specific antibodies againstV. cholerae lipopolysaccharide (LPS) and enter-otoxin by affinity chromatography. It is demon-strated that immunization by the subcutaneous(s.c.) route induces a local immune response inthe small intestine against both of these protec-tive antigens of V. cholerae, and that this re-sponse comprises IgA and IgG.

MATERIALS AND METHODSAnimals. New Zealand white rabbits, weighing

1.0 to 1.3 kg at the onset of immunization, wereused.

Antigens. Crude cholera toxin was prepared byfiltering culture filtrate of V. cholerae strain 569BInaba (lot 4493G, supplied by the National Instituteof Allergy and Infectious Diseases, Bethesda, Md.)through a pellicon membrane with a cutoff of 104daltons and lyophilizing the retentate (13). This ma-terial contained approximately 10% LPS and 0.1%

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IMMUNOGLOBULIN SYNTHESIS BY LYMPHOID TISSUES 361

exoenterotoxin on a dry-weight basis (16). Highlypurified cholera toxin (CT) was prepared by R. A.Finkelstein, Dallas, Tex. (8), and supplied by C.Miller at the National Institute ofAllergy and Infec-tious Diseases. Purified LPS of V. chokerae strain35A3 Inaba was prepared by hot phenol-water ex-

traction followed by repeated ultracentrifugation(21).

Immunizations. Sixteen rabbits were divided intotwo equal groups, one immunized with crude chol-era toxin and the other given only phosphate-buffered saline (PBS; 145 mmol/mol of sodium chlo-ride, 50 mmol/mol ofphosphate, pH 7.2). The antigenwas dissolved in PBS, and 12.5 mg was injected s.c.

in two 0.5-ml portions above each of the posteriorlegs; this procedure was repeated 2 weeks later. Allanimals were bled before immunization and imme-diately before sacrifice, 5 days after the second injec-tion. Other rabbits were similarly immunized withcrude toxin and used for procedural control experi-ments.

Previous studies have shown that the antigendose used for immunization gives optimal serumantibody titers against CT as well as LPS (16), andthat protection against experimental cholera is atits peak 4 to 5 days after the booster (17).

Affinity chromatography. Gels for isolation ofrabbit IgG, IgA, or IgM were prepared by couplingthe respective anti-immunoglobulin to cyanogenbromide (CNBr)-activated Sepharose. ActivatedSepharose 4B was purchased from Pharmacia,Uppsala, Sweden, and goat antisera to rabbit IgG,IgA, or IgM were purchased from Nordic, Tilburg,The Netherlands. The specificity ofthe antisera wasascertained by double-diffusion-in-gel analyses (27).The gamma globulin fraction of 0.6 to 1 ml of serum,prepared by repeated precipitation with 50% satu-rated ammonium sulfate, was directly coupled to 1.5ml of the swollen activated gel (1). Columns forpurification of specific cholera antibodies were pre-pared by covalent coupling of CT or LPS to Sepha-rose. CT was coupled directly to activated Sepharoseby reacting 0.25 mg of CT and 0.5 mg of bovineserum albumin with 0.75 ml of swollen commercialCNBr-Sepharose (1). The LPS, on the other hand,was coupled to laboratory-activated Sepharose overa spacer, diaminohexane, as described previously(24). After CNBr treatment of the LPS, 0.25 mg ofthis material was reacted with 1 ml of swollen dia-minohexane-substituted Sepharose.

Before use, all gels were incubated with 1 Methanolamine for 2 to 3 h at room temperature toblock any remaining reactive sites.

Sepharose 4B derivatized with protein A ofStaph-yiococcus aureus (Protein A-Sepharose) was pur-chased from Pharmacia, Uppsala, Sweden.The derivatized gels were packed on top of a bot-

tom layer of Sepharose 4B in Pasteur pipettes. Thecolumns were equilibrated with starting buffer (50mmol/mol of phosphate, 500 mmol/mol of glycine,500 mmol/mol of sodium chloride, pH 7), and thesamples were run through the gels. Nonspecificallyattached material was washed out with startingbuffer. Elution of specifically bound material was

performed with an acid buffer (1 mol/mol of acetic

acid, 500 mmol/mol of glycine, 500 mmol/mol of so-dium chloride, pH 3).

Protein synthesis. The method of Smiley et al.(23) with incorporation of 14C-labeled amino acidsinto the synthesized proteins from in vitro-incu-bated tissues was used with slight modifications.The procedure is outlined in Fig. 1. After sacrifice ofthe animal for study, the spleen, mesenteric lymphnodes, Peyer's patches and small intestinal seg-ments distant from Peyer's patches were quicklyexcised. Ten to twenty mesenteric lymph nodes weredissected free from surrounding tissue, as were allthe Peyer's patches along the small bowel. Five tosix intestinal specimens were taken in the middlebetween Peyer's patches, and the mucus was re-moved by rubbing against paper. The tissues, 200 to300 mg, were thoroughly washed in incubation me-dium, minced into 1- to 2-mm pieces, and placed intoconical tubes containing 8 ml of freshly preparedmodified Eagle medium, pH 7.2. This mediumlacked the amino acids contained in the 14C label andwas supplemented with 5% heat-inactivated normalrabbit serum and 100 U of penicillin-streptomycinper ml. The incubation tube had a glass capillarysealed to its bottom through which a 95% 02-5% C02mixture was slowly introduced to keep the pH at 7.2to 7.4. After incubation at 370C for 15 min, a mixtureof 14C-labeled L-arginine, L-leucine, L-lysine, and L-valine (Schwarz-Mann, Orangeburg, N.Y.) wasadded to the medium (4 .Ci/g of tissue), and theincubation continued for 6 h. After incubation wascompleted the medium was centrifuged at 3,000 x gfor 10 min to remove particulate material. Eachsupernate was added with 100 mg ofCasamino Acidsand dialyzed for 3 days against 1,000 volumes ofPBSto eliminate nonincorporated 14C-labeled aminoacids. The amount of protein synthesized was deter-mined on an aliquot of the dialyzed medium byprecipitation with 10% trichloroacetic acid followedby liquid scintillation in Instagel (Packard Instru-ment Co., Downers Grove, Ill.). A Packard series3000 liquid scintillator with 81% efficiency for 14Cwas used, and the radioactivity was expressed ascounts per minute per gram (wet weight) oftissue.

Determination of de novo-synthesized immuno-globulin. IgG, IgA, and IgM were isolated on thecolumns with class-specific anti-immunoglobulincoupled to Sepharose. Samples, 0.5 to 1.0 ml, ofdialyzed medium were run through the columns.After washing with at least 5 ml of starting buffer,the specifically bound immunoglobulin was elutedwith 5 to 10 ml of acid buffer. By liquid scintillationon a portion ofthe neutralized acid eluate, the newlysynthesized immunoglobulin was determined ascounts per minute per gram of tissue and as percent-age of the total protein synthesized.

Determination of newly produced specific anti-bodies. Specific cholera antibodies were similarlymeasured by liquid scintillation after isolation byaffinity chromatography on the CT- or LPS-coupledgels.

Enzyme-linked immunosorbent assay (ELISA).Titrations of specific IgG, IgA, and IgM antibodieswere performed as earlier described, using CT andLPS as solid-phase antigens (14).

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362 SVENNERHOLM AND HOLMGREN

95%02 - 5% C02

I Incubate withMince tissue 14C-amino acidsin small pieces t g 3rC, 6h

Centrifuge 3000 x g ,10 min.

Pialyze supernateagainst PBS

Precipitatewith 107. TCA

Affinity chromatography

1.Application of sample

CT2.Wash with buffer pH7

aEluate with buffer pH 3

VP P P P P Discard washesNeutralize acid eluatesTotalprotein IgG IgA IgM anti- anti-

CT LPS

^ vL Liquid scintillationr!_of TCA precipitate

and acid eluate fractions

FIG. 1. Outline ofprocedure for protein synthesis in vitro by various tissues and for isolation by affinitychromatography of newly synthesized immunoglobulins and specify' antibodies to CT and LPS. TCA,Trichloroacetic acid.

Immunodiffusion-in-gel. The Ouchterlony dou-ble-diffusion (27) and the Mancini single-radial-im-munodiffusion methods (20) were used.

Protection tests. The protection studies were per-formed as earlier described, using the small-bowelloop technique (25). Immunized and control rabbitswere concurrently challenged in ligated intestinalloops with graded doses of live vibrios (strain 569B,Inaba) or toxin (National Institutes of Health V.cholerae culture filtrate 4493G). Five doses of bacte-ria (105 to 10k) or toxin (0.1 to 10 mg) were tested inmultiple positions along the gut, and the dose givingrise to half-maximal fluid accumulation in loops(50% effective dose) was determined.Procedural controls: (i) protein synthesis. (a) Di-

alysis of the medium in which the various tissues

had been incubated for more than the usual 72 h didnot further decrease the radioactivity. With all sam-ples, 85 to 95% of the radioactivity of the dialyzedmedium was precipitable with trichloroacetic acid.The reproducibility of the acid precipitations waswithin ±3%.

(b) To ensure that the radioactive protein repre-sented de novo synthesis, the 14C label was added tothe medium at the end of the incubation. Afterdialysis of the medium, the radioactivity of the acidprecipitate was only 1 to 3% of that obtained whenthe various tissues had been incubated with thelabel for 6 h. When the '4C label was present fromthe beginning but incubation was performed at 0 toSoC (ice bath), the radioactivities were 5 to 10% ofthose obtained with the standard procedure.

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IMMUNOGLOBULIN SYNTHESIS BY LYMPHOID TISSUES 363

(c) The possibility of proteolytic degradation ofimmunoglobulin during incubation was studied.Rabbit immune sera were added to the medium andincubated with intestinal tissue according to thestandard procedure. As measured with the ELISA,the antibody titers of the various immunoglobulinclasses in serum were unchanged by the tissue incu-bation.

(ii) Isolation of immunoglobulins. (a) The speci-ficity, capacity, and yield of the class-specific anti-immunoglobulin columns were studied using theELISA and the single-radial-immunodiffusion tech-nique. It was found that >90% of the IgA and IgMbut <1% of the IgG of a serum sample, diluted 1:20,that was applied in a 0.5-ml volume to the anti-IgGcolumn passed directly through the gel. Subsequentacid elution released 80 to 100% of the specificallybound IgG as determined with the ELISA. With thesingle radial immunodiffusion, an apparently loweryield, 40 to 80%, was registered, the discrepancyprobably being due to aggregation or partial dena-turation of the immunoglobulin after treatmentwith the acid buffer. Less than 1% of the total IgAand IgM of the applied serum sample was recoveredin the acid eluate from the anti-IgG column. Similartesting of the anti-IgA and anti-IgM columns gaveresults in close agreement with those for the anti-IgG column.

(b) The de novo synthesis of immunoglobulin wasdetermined as the radioactivity isolated from theimmunoglobulin class-specific columns. No signifi-cant radioactivity bound to underivatized Sepharose4B. More than 95% of the total radioactivity appliedto the derivatized columns was recovered in thewash and the acid eluate. The reproducibility of theimmunoglobulin determinations was within ±14%when tested on different occasions with various gelbatches.

(c) Staphylococcal protein A selectively binds IgGthrough a reaction with the Fc portion (9); it hasbeen reported that >90% of rabbit IgG binds toprotein A in this manner (19). We made use of thisreaction to further ascertain the specificity of ouranti-IgG Sepharose columns. Thus, we showed that290% of the anti-IgG column binding radiolabeledmaterial in incubation media ofspleen and intestinewas removed on passage through a column withProtein A-Sepharose.

(iii) Isolation of specific cholera antibodies. (a)The gels coupled with V. cholerae antigens. were

analyzed in a similar manner as the anti-immuno-globulin columns. Application of an anti-culture fil-trate serum, containing antibodies to both CT andLPS, to the CT- or the LPS-Sepharose columns re-

sulted in passage of >99% of the antibodies of theheterologous specificity but of <5% of the homolo-gous antibodies. Elution with acid buffer released>90% of the specifically bound antibodies fromboth types of columns as tested with immune sera.Moreover, for all of the tissue incubation samples,>80% of the specifically bound radioactivity was re-covered on acid elution.

(b) The biological activity of antibodies obtainedby acid elution was examined after pH neutraliza-tion. The anti-LPS antibodies had a protective titer(24) against intestinal challenge with live vibrioswhich accounted for more than 90% of that observedfor the antiserum from which the antibodies hadbeen isolated. Similarly, purified anti-CT antibodieshad almost the same toxin-neutralizing capacity inthe intradermal neutralization test (3) as did theunfractionated anti-CT serum.

RESULTSProtective capacity of immunization. The

s.c. immunization used induced a .160-fold in-crease in resistance against intestinal chal-lenge with live vibrios as based on comparisonof 50% effective dose values for immunized andconcurrently tested control rabbits. The protec-tion against toxin challenge was fivefold.

Protein synthesis by different tissues. Theamounts of protein synthesized and secretedfrom various organs of immunized and controlrabbits during in vitro culture for 6 h are shownin Table 1. In the nonimmunized animals, themagnitude of protein synthesis was rather sim-ilar from spleen, mesenteric lymph nodes, andPeyer's patches, whereas the intestinal tissuedistant from Peyer's patches produced 20 to 30%less protein. After immunization, increasedprotein synthesis was only registered fromspleen and mesenteric lymph nodes (Table 1).Immunoglobulin synthesis. For all the tis-

sues examined, a substantial fraction of thenewly synthesized protein was immunoglobu-lin. As a mean, 40% of the total radioactiveprotein secreted by the spleen and 32% of that

TABLz 1. Protein synthesis in vitro by different tissues from cholera-immunized and control rabbits

Protein synthesis (cpm/g of tissue)Animals Mesenteric lymph

Spleen nodes Peyer's patches Intestine

Immunized 62,000 + 8,100a 57,200 + 3,600 42,400 ± 4,800 31,300 ± 2,500(n = 8)

Controls 47,800 ± 6,800 43,200 ± 6,000 43,200 ± 5,600 33,800 ± 6,200(n = 8)

P > 0.20b P < 0.10 P > 0.20 P > 0.20

a Mean value + standard error of mean.b Student's t test.

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364 SVENNERHOLM AND HOLMGREN

TABLE 2. In vitro immunoglobulin synthesis in spleen

Synthesis (cpm/g of tissue) Percent of total proteinAnimals

IgG IgA IgM IgG IgA IgMImmunized 16,700 + 3,100a 2,900 ± 900 8,300 ± 3,600 26.2 ± 2.5a 5.0 ± 1.4 14.4 ± 3.0

(n = 8)Controls 12,400 ± 3,000 2,000 + 300 4,700 ± 1,200 20.3 ± 3.6 4.7 ± 0.7 8.8 ± 1.9

(n = 8)P > 0.20t P > 0.20 P >0.20 P > 0.20 P > 0.20 P < 0.10

a Mean value ± standard error of mean.b Student's t test.

TABLE 3. In vitro immunoglobulin synthesis in mesenteric lymph nodes

Synthesis (cpm/g of tissue) Percent of total proteinAnimals

IgG IgA IgM IgG IgA IgMImmunized 8,500 ± 1,900a 2,000 ± 300 4,400 ± 800 14.7 ± 3.9a 3.3 ± 0.4 7.1 ± 1.3

(n = 5)Controls 8,600 ± 1,400 1,300 ± 500 3,800 ± 600 17.3 ± 2.0 2.8 ± 0.9 7.3 ± 0.4

(n = 4)P > 0.20b P > 0.20 P > 0.20 P < 0.20 P < 0.20 P > 0.20

a Mean value + standard error of mean.b Student's t test.

formed by the mesenteric lymph nodes of con-trol animals was immunoglobulin, for both or-gans distributed on IgG, IgA, and IgM as 6:1:2(Tables 2 and 3). Immunization increased theproduction of IgG and IgM from the spleen,whereas no such increase was noted for themesenteric lymph nodes. The immunoglobulinformation in Peyer's patches and intestinal tis-sue of control animals was lower than in thenonenteral tissues (Tables 4 and 5). Only 20% ofthe protein synthesized in Peyer's patches or inintestinal tissue was immunoglobulin.The proportion ofIgA in the enterally formed

immunoglobulin was considerably higher thanfor the other tissues. Thus, for nonimmunizedanimals 25% of the immunoglobulin synthe-sized by Peyer's patches and 20% of that pro-duced by intestinal tissue was IgA, whereasthis class made up only 10% ofthe immunoglob-ulin formed by the spleen and mesentericlymph nodes. Cholera immunization increasedthe synthesis ofparticularly IgA but also ofIgGin Peyer's patches (Table 4). Relative to thetotal protein synthesis, increased IgA forma-tion was also observed for intestinal tissue (Ta-ble 5).The finding of significant immunoglobulin

synthesis in Peyer's patches was surprising inview of the scarcity of antibody-producing cellsin this tissue as studied by immunofluorescence(4, 5). A preponderance ofimmunoglobulin-con-taining cells in the intestine adjacent to thePeyer's patches has, however, been described(2). To examine the possibility that contamina-

tion with intestinal tissue was responsible forthe antibody production from the routinely pre-pared Peyer's patch specimens, the immuno-globulin synthesis by the very center of Peyer'spatches from which the mucosa had been care-fully removed was compared with that of thesurrounding intestinal tissue, taken 1 to 2 mmimmediately around the patch (19,200 cpm/g oftissue), and also with that of intestine taken inthe middle between patches (27,100 cpm/g oftissue). The proportion of immunoglobulin andits distribution on various classes in the newlysynthesized protein was similar for these var-ious tissues.To investigate whether the incubation time

had a differential influence on the synthesis ofthe various types of immunoglobulins, spleenand Peyer's patch specimens from four animalswere incubated for 2, 6, and 24 h. The amount ofprotein produced from both organs increasedalmost proportionally with time for at least 24h, indicating a rather constant rate of synthesis(Fig. 2). The immunoglobulin formation of thespleen also increased through the entire incu-bation period, although to a less extent thanthe total protein synthesis. In Peyer's patches,on the other hand, the immunoglobulin synthe-sis increased only little between 6 and 24 h. Theclass distribution of the newly synthesized im-munoglobulin was rather similar after the var-ious incubation times (Fig. 2). In relation to thetotal protein synthesis, the amount of the dif-ferent immunoglobulins formed decreasedslightly with time, being 1.5- to 2-fold lower

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IMMUNOGLOBULIN SYNTHESIS BY LYMPHOID TISSUES 365

after 24 than after 2 h of incubation (Fig. 2,inserts).Synthesis of specific cholera antibodies.

The amounts ofnewly synthesized specific chol-era antibodies were determined after isolationon the CT- and LPS-coupled gels. Significantformation of antibodies to CT as well as LPSwas shown both in spleen and in Peyer'spatches after immunization (Fig. 3).

Local as well as systemic antibody formationwas also indicated by antibody titrations withthe ELISA on dialyzed incubation medium. Forall the tissues studied, the immunized rabbitsshowed higher levels ofboth anti-LPS and anti-CT antibodies than did the control rabbits (Fig.4). The increased anti-LPS formation comprisedIgG as well as IgM in spleen and mesentericlymph nodes,,whereas the enhanced titers inthe enteral tissues were due to IgG and IgA.The increase in anti-CT titers comprised IgGas well as IgA antibodies (Fig. 4).To examine to what extent the ELISA titers

represented de novo synthesis of antibodies,tissues were simultaneously incubated at 37and at 0 to 5OC (ice bath). Since incubation inthe cold decreased protein synthesis by 90 to95%, titers observed in the 0 to 5C incubationmedium should mainly represent preformedantibodies released into medium, and increasesin titer on incubation at 370C should representthe de novo synthesis. For spleen, an approxi-mately 5-fold increase in IgG titer was seen at370C to both LPS and CT; a 2- to about 10-foldincrease in IgA and a 2- to 4-fold increase in

IgM were seen. For intestine, the increase inIgG was 2- to 5-fold, and the IgA titers rose 4-to 20-fold.

DISCUSSIONAn in vitro culture technique with synthesis

of radiolabeled protein followed by affinitychromatography isolations of immunoglobulinsand specific antibodies has been developed forthe study of systemic and intestinal choleraimmunity. By measuring de novo synthesis inthe incubated tissue, the method permits directdetermination of the enteral antibody forma-tion without interference of contaminating an-

tibodies derived from serun. The control exper-iments indicate that proteolytic degradation,often encountered with intestinal immunoglob-ulins in vivo, is also avoided by the in vitroculture. The results show that protective im-munization with a mixture of V. cholerae LPSand enterotoxin by the s.c. route induces spe-cific-antibody formation both in spleen and in-testine.The in vitro protein synthesis method has

previously been used in studies of immune re-

sponses in rheumatoid arthritis (23) and experi-mental urinary tract infection (18). In thesestudies, immunoglobulins synthesized by syn-ovial membrane or infected kidney were iso-lated by diethylaminoethyl chromatographyfollowed by immunoprecipitation, and specificantibodies were determined after immunopre-cipit#tion or adsorption to bacteria. The use ofaffinity chromatography for isolation of immu-

TABLE 4. In vitro immunoglobulin synthesis in Peyer's patches

Synthesis (cpm/g of tissue) Percent of total proteinAnimals

IgG IgA IgM IgG IgA IgM

Immunized 6,800 ± 900a 4,700 - 600 2,600 + 400 16.1 ± 1.6a 11.3 ± 1.8 6.3 ± 1.1(n = 8)

Controls 4,700 ± 700 2,200 + 400 2,000 ± 300 12.3 ± 1.7 5.6 ± 0.8 5.0 ± 0.6(n = 8)

p < 0. lob P < 0.01 P > 0.20 P < 0.10 P < 0.01 P > 0.20a Mean value ± standard error of mean.b Student's t test.

TABLE 5. In vitro immunoglobulin synthesis in intestinal tissue

Synthesis (cpm/g of tissue) Percent of total proteinAnimals

IgG IgA IgM IgG IgA IgMImmunized 2,900 ± 600a 2,300 ± 400 2,300 ± 400 11.7 ± 1.6a 8.0 ± 1.2 8.1 ± 1.3

(n = 7)Controls 4,000 - 1,200 1,700 ± 500 2,300 ± 500 12.2 ± 2.4 5.1 ± 0.7 7.3 ± 1.6

(n = 7)P > 0.20 P > 0.20 P > 0.20 P > 0.20 P < 0.10 P > 0.20

a Mean value ± standard error of mean.b Student's t test.

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100 -

I0

z 10 -e)

6

1-

a SPLEEN

100 -

I0D 10 -

E

1-~

I

2 6 24 h

b PEYER'S PATCHES

6

FIG. 2. Influence of incubation time on de novo synthesis of total protein (a), IgG (I), IgA (a), and IgM(A) in (a) spleen and (b) Peyer's patches. Inserts show the synthesis ofimmunoglobulins ofvarious classes inrelation to total protein formation. Mean values and standard error ofmean for two to four animals are given.

10

a)

20

C

5 5a)

10

a)0

QL

0

5c

a)

Anti-LPS Anti-CT Anti-LPS Anti-CT

FIG. 3. Synthesis ofantibodies to LPS and CT in (a) spleen and (b) Peyer's patches ofimmunized (a) andcontrol (E) rabbits. Specific antibody formation is expressed as percentage ofthe total protein synthesis ofthetissue sample. Mean values and standard errors of mean are shown (eight animals in each group).

noglobulins and specific antibodies has allowedconvenient and more effective purificationswith improved yields. All the derivatized gelsused were highly specific, since they retainedless than 1% of heterologous immunoglobulinsor antibodies. The desorption conditions werebased on previous experiments with pH gra-dient elutions, which indicated that the chosenprocedure would elute at least 80% ofthe bound

antibodies, including those with high affinity(24). In the present study, yields between 80 to100% were achieved in the isolations of immu-noglobulins as well as of specific antibodies,when tested both with serum samples and withthe actual radioactive tissue culture specimens.Although immunodiffusion tests suggested thatthe isolated immunoglobulins were aggregatedto a certain extent, examinations with the

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10 000

1000

100

10 000

1000

0).)

-

w

100

10000

1000

100

10000

1000

100

bgG IgAAnti- LPS

IgM IgG IgAAnti- CT

FIG. 4. Titers ofantibodies ofvarious immunoglobulin classes againstLPS and CT in incubation mediumfrom various tissues ofimmunized (0) and control (0) rabbits as determined with the ELISA. Mean valuesand standard error of mean are indicated (eight animals in each group).

ELISA demonstrated that they retained fullantigen-binding capacity as well as reactivitywith heavy-chain specific antibodies. Further-more, the antibodies eluted from the LPS- andCT-coupled Sepharose had intact biological ac-

tivity as estimated in protection experiments.

The relative amounts of IgG, IgA, and IgMsynthesized by spleen and mesenteric lymphnodes of the control rabbits are in close agree-ment with those reported by Smiley et al. (23)for normal human spleen and lymph node. Inview ofthe predominance ofIgA plasma cells in

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368 SVENNERHOLM AND HOLMGREN

the intestinal lamina propria, as observed withimmunofluorescence, the high proportion ofIgG synthesized by the rabbit intestine in vitrowas unexpected (4, 5). Based on control experi-ments, using columns with protein A coupled toSepharose, the possibility that radioactive non-immunoglobulin material was bound to theanti-IgG column, giving rise to falsely high IgGvalues, could be eliminated. Protein A, whichselectively binds IgG (9), removed > 90% of theanti-IgG column binding material from tissueculture medium of both spleen and intestine. Itis possible, however, that the expression ofIgGprecursor cells is favored by the in vitro incuba-tion. We tested whether incubation periodsother than 6 h for the in vitro protein synthesiswould result in different proportions of IgG,IgA, and IgM. No such change was seen byvarying the time between 2 and 24 h usingspleen and Peyer's patches. It is noteworthythat a substantial in vitro synthesis ofIgG andIgA, as well as IgM, occurred in the Peyer'spatches, since only few plasma cells or immu-noglobulin-containing cells have been observedwithin the lymphoid follicles of the patches (4-6, 28). However, IgA-, IgG- and IgM-containingcells are abundant on the edge of the folliclesand in the adjacent intestinal mucosa (2, 11). Apolarity, with the IgA cells to the mucosal sideand the IgG and IgM cells to the serosal edge ofthe follicles, has been described (2). Althoughin control experiments the immunoglobulinsynthesis per gram of tissue in the rabbitPeyer's patch preparations was unchanged bycareful removal of the mucosa, interfollicularcells might still have contributed to the immu-noglobulin synthesis registered. Using an in vi-tro radiolabeling procedure, Vitetta et al. re-cently showed synthesis and secretion of IgG,IgA, and IgM by mouse Peyer's patch cells (30),and Henry et al. (12) found that rabbit Peyer'spatch cells responded as well with antibodyformation as did spleen cells when stimulatedwith antigen in in vitro culture (600 plaque-forming cells [PFC]/107 cells). Veldkamp et al.demonstrated a significant PFC response inmouse Peyer's patches (400 to 500 PFC/107 cells)after repeated intraduodenal or intraperitonealimmunization with V. cholerae (29).The fact that the synthesis of immunoglobu-

lin and its distribution on various classes mightbe influenced by the conditions of the in vitroincubation does not detract from the usefulnessof the method in registering changes in forma-tion ofimmunoglobulins and specific antibodiesinduced by specific immunization. It is evidentthat s.c. immunization, being highly protectiveagainst experimental cholera (.160-fold in thisstudy against intestinal challenge with live

cholera vibrios), gives rise to substantial syn-thesis of radiolabeled antibodies to both V.cholerae LPS and enterotoxin in spleen. It alsoincreases the overall synthesis of IgM in thistissue. Furthermore, the immunization resultsin increased specific ELISA antibody titers inincubation medium of spleen and mesentericlymph nodes. The immunoglobulin class distri-bution ofthese titers closely corresponds to thatpreviously found for antibodies to LPS and CTin serum (17).

In rats, Pierce and Gowans found that paren-teral (intraperitoneal) immunization with chol-era toxoid only gave rise to very few antitoxin-containing cells in the gut, but primed the in-testine for a subsequent secondary IgA cell an-titoxic immune response to an enteral boost(22). In rabbits, low levels of both anti-CT andanti-LPS antibodies, IgG as well as IgA, havepreviously been found in small intestinal wash-ings after s.c. vaccination (17, 25). However, ithas not been possible until now to determinewhether these antibodies are produced by theintestine itself or are derived from the circula-tion. In the present investigation, a local syn-thesis of specific, radiolabeled antibodies to V.cholerae LPS and enterotoxin after s.c. immu-nization is established for Peyer's patches. Wedid not test for formation of radiolabeled spe-cific antibodies from the intestinal tissue apartfrom Peyer's patches. However, the titrationson tissue incubation medium with the ELISAclearly indicate specific-antibody productionalso by the intestine. Thus, in addition to ob-serving higher antibody titers in incubationmedium from the immunized rabbits than fromthe control ones, we show that the titers ofimmunized animals increase during proteinsynthesis, as tested by simultaneous incubationof tissue at 37 and at 0 to 5°C. For intestinaltissue, a 4- to 20-fold increase was seen for theIgA antibodies, whereas the rise in IgG wassmaller (2- to 5-fold). For spleen, the increase inIgA antibodies was about 2- to 10-fold, and inspecific IgG it was about 5-fold.

ACKNOWLEDGMENTSJames W. Smith, Dallas, Tex., is gratefully acknowl-

edged for advice on the in vitro protein synthesis procedure.The skillful technical assistance of Ulla Westerberg-Berndtsson is highly appreciated.

This work was supported by grants from the SwedishMedical Research Council (project 16X-3382), FOA, Sweden(5061H563), and the World Health Organization.

LITERATURE CITED1. Axin, R., V. Porath, and S. Ernback. 1967. Chemical

coupling of peptides and proteins by means of cyano-gen halides. Nature (London) 214:1302-1304.

2. Crabbe, P. A., D. R. Nash, H. Bazin, H. Eyssen, and J.F. Heremans. 1970. Immunohistochemical observa-

INFECT. IMMUN.

on Septem

ber 1, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

IMMUNOGLOBULIN SYNTHESIS BY LYMPHOID TISSUES 369

tions on lymphoid tissues from conventional andgerm-free mice. Lab. Invest. 22:448-457.

3. Craig, J. P. 1965. A permeability factor (toxin) found incholera stools and culture filtrates and its neutraliza-tion by convalescent cholera sera. Nature (London)207:614-616.

4. Craig, S. W., and J. J. Cebra. 1971. Peyer's patches; anenriched source of precursors for IgA immunocytes inthe rabbit. J. Exp. Med. 134:188-200.

5. Craig, S. W., and J. J. Cerbra. 1975. Rabbit Peyer'spatches, appendix, and popliteal lymph node B lym-phocytes: a comparative analysis of their membraneimmunoglobulin components and plasma cell precur-sors. J. Immunol. 114:492-502.

6. Faulk, W. P., J. N. McCormick, J. R. Goodman, J. M.Yoffey, and H. H. Fudenberg. 1970. Peyer's patches:morphologic studies. Cell. Immunol. 1:500-520.

7. Finkelstein, R. A. 1973. Cholera. Crit. Rev. Microbiol.2:553-623.

8. Finkelstein, R. A., and J. J. LoSpalluto. 1969. Patho-genesis of experimental cholera: preparation and iso-lation of choleragen and choleragenoid. J. Exp. Med.130:185-202.

9. Forsgren, A., and J. Sjoquist. 1966. "Protein A" fromS.areus. I. Pseudo-immune reaction with human y-globulin. J. Immunol. 97:822-827.

10. Fubara, E. S., and R. Freter. 1972. Source and protec-tive function of coproantibodies in intestinal disease.Am. J. Clin. Nutr. 25:1367-1363.

11. Guy-Grand, D., C. Griscelli, and P. Vassal. 1974. Thegut-associated lympoid system: nature and propertiesof the large dividing cells. Eur. J. Immunol. 4:435-443.

12. Henry, C., W. P. Faulk, L. Kuhn, J. M. Yoffey, and H.H. Fudenberg. 1970. Peyer's patches: immunologicstudies. J. Exp. Med. 131:1200-1210.

13. Holmgren, J., I. LUnnroth, and 0. Ouchterlony. 1971.Immunochemical studies of two cholera toxin-con-taining standard culture filtrate preparations of Vi-brio cholerae. Infect. Immun. 3:747-755.

14. Holmgren, J., and A.-M. Svennerholm. 1973. Enzyme-linked immunosorbent assays for cholera serology.Infect. Immun. 7:759-763.

15. Holmgren, J., and A.-M. Sennerholm. 1976. Mecha-nisms of disease and immunity in cholera. J. Infect.Dis. Suppl., in press.

16. Holmgren, J., A.-M. Svennerholm, and 0. Ouchter-lony. 1972. Experimental studies on cholera immuni-zation. I. The response ofneutralizing and vibriocidalantibodies in rabbits after immunization with culturefiltrate material from V. cholerae. Acta Pathol. Mi-crobiol. Scand. Sect. B 80:489-496.

17. Holmgren, J., A.-M. Svennerholm, 0. Ouchterlony, A.

Andersson, G. Wallerstrom, and U. Westerberg-Berndtsson. 1975. Antitoxic immunity in experimen-tal cholera: protection and serum and local antibodyresponses in rabbits after enteral and parenteral im-munization. Infect. Immun. 12:1331-1340.

18. Lehmann, J. D., J. W. Smith, T. E. Miller, J. A. Bar-nett, and J. P. Sanford. 1968. Local immune responsein experimental pyelonephritis. J. Clin. Invest.47:2541-2550.

19. Lind, I., and B. Mansa. 1968. Further investigation ofspecific and non-specific adsorption of serum globu-lins to Staphylococcus aureus. Acta Pathol. Micro-biol. Scand. 73:637-645.

20. Mancini, G., A. 0. Carbonara, and J. F. Heremana.1965. Immunochemical quantitation of antigens bysingle radial immunodiffusion. Immunochemistry2:235-254.

21. Orskov, F., I. Orskov, K. Jann, E. Mfiller-Seitz, and 0.Westphal. 1967. Immunochemistry ofEscherichia coliO antigens. Acta Pathol. Microbiol. Scand. 71:339-358.

22. Pierce, N. F., and J. L. Gowans. 1975. Cellular kineticsofthe intestinal immune response to cholera toxoid inrats. J. Exp. Med. 142:1550-1563.

23. Smiley, J. D., C. Sachs and M. Ziff. 1968. In vitrosynthesis of immunoglobulin by rheumatoid synovialmembrane. J. Clin. Invest. 47:624-632.

24. Svennerholm, A.-M. 1975. Experimental studies oncholera immunization. IV. The antibody response toformalinized Vibrio chokrae and purified endotoxinwith special reference to protective capacity. Int.Arch. Allergy Appl. Immunol. 49:434452.

25. Svennerholm, A.-M., and J. Holmgren. 1975. Synergis-tic protective effect in rabbits of immunization withVibrio cholerae lipopolysaccharide and toxin/toxoid.Infect. Immun. 13:735-740.

26. Tomasi, B., and J. Bienenstock. 1968. Secretory immu-noglobulins. Adv. Immunol. 9:1-96.

27. Wadsworth, C. 1957. A slide microtechnique for theanalysis of immune precipitates in gel. Int. Arch.Allergy Appl. Immunol. 10:355-360.

28. Waksman, B. H., H. Ozer, and H. E. Blythman. 1973.Appendix and M-antibody formation. VI. The func-tional anatomy of the rabbit appendix. Lab. Invest.28:614-626.

29. Veldkamp, J., R. van der Gaag, and J. M. N. Willers.1973. The role of Peyer's patch cells in antibody for-mation. Immunology 25:761-771.

30. Vitetta, E. S., M. McWilliams, J. M. Phillips-Quag-liata, M. E. Lamm, and J. Uhr. 1975. Cell surfaceimmunoglobulin. XIV. Synthesis, surface expression,and secretion of immunoglobulin by Peyer's patchcells in the mouse. J. Immunol. 115:603-05.

VOL. 15, 1977

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ber 1, 2020 by guesthttp://iai.asm

.org/D

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