Vol. 15, No. 2

INMCTION AND IMMUNITY, Feb. 1977, P. 360-369

Printed in U.S.A.

Copyright © 1977 American Society for Microbiology

Immunoglobulin and Specific-Antibody Synthesis In Vitro by Enteral and Nonenteral Lymphoid Tissues After Subcutaneous Cholera Immunization ANN-MARI SVENNERHOLM AND JAN HOLMGREN* Institute of Medical Microbiology, University of Goteborg, Goteborg, Sweden

Received for publication 20 July 1976

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

Cholera, the prototype for the "enterotoxic enteropathies" (7), is a disease in which local immunity is important. Neither the infectious agent, Vibrio cholerae, nor its secreted diarrheagenic enterotoxin appears to penetrate the intestinal epithelium, and consequently antibodies directed against either the vibrio or the enterotoxin must be present in the gut lumen or at the mucosal surface in order to be effective (15). The circumstance that intestinal antibodies may originate not only from local sites of synthesis but also from the circulation has complicated the evaluation of gastrointestinal immunity in infections. Furthermore, it meets with considerable difficulties in quantitating immunoglobulin and specific antibodies accurately in intestinal secretions due to the heterogeneity in size of particularly immunoglobulin A (IgA) (26) and to proteolytic degradation of immunoglobulin and specific antibodies (10, 26). To overcome the previously encountered problems in measuring local cholera antibodies and in evaluating their intestinal or systemic origin, we have attempted to quantify directly

the systhesis of immunoglobulins and specific antibodies by intestinal and extraintestinal lymphoid tissues of rabbits. The approach has been to let the tissue produce 14C-labeled protein during an in vitro incubation period and then to isolate the newly formed IgG, IgA, and IgM as well as the specific antibodies against V. cholerae lipopolysaccharide (LPS) and enterotoxin by affinity chromatography. It is demonstrated that immunization by the subcutaneous (s.c.) route induces a local immune response in the small intestine against both of these protective antigens of V. cholerae, and that this response comprises IgA and IgG.

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MATERIALS AND METHODS Animals. New Zealand white rabbits, weighing 1.0 to 1.3 kg at the onset of immunization, were used. Antigens. Crude cholera toxin was prepared by filtering culture filtrate of V. cholerae strain 569B Inaba (lot 4493G, supplied by the National Institute of Allergy and Infectious Diseases, Bethesda, Md.) through a pellicon membrane with a cutoff of 104 daltons and lyophilizing the retentate (13). This material contained approximately 10% LPS and 0.1%

VOL. 15, 1977

IMMUNOGLOBULIN SYNTHESIS BY LYMPHOID TISSUES

exoenterotoxin on a dry-weight basis (16). Highly purified cholera toxin (CT) was prepared by R. A. Finkelstein, Dallas, Tex. (8), and supplied by C. Miller at the National Institute of Allergy and Infectious Diseases. Purified LPS of V. chokerae strain 35A3 Inaba was prepared by hot phenol-water extraction followed by repeated ultracentrifugation (21). Immunizations. Sixteen rabbits were divided into two equal groups, one immunized with crude cholera toxin and the other given only phosphatebuffered saline (PBS; 145 mmol/mol of sodium chloride, 50 mmol/mol of phosphate, pH 7.2). The antigen was dissolved in PBS, and 12.5 mg was injected s.c. in two 0.5-ml portions above each of the posterior legs; this procedure was repeated 2 weeks later. All animals were bled before immunization and immediately before sacrifice, 5 days after the second injection. Other rabbits were similarly immunized with crude toxin and used for procedural control experiments.

Previous studies have shown that the antigen dose used for immunization gives optimal serum antibody titers against CT as well as LPS (16), and that protection against experimental cholera is at its peak 4 to 5 days after the booster (17). Affinity chromatography. Gels for isolation of rabbit IgG, IgA, or IgM were prepared by coupling the respective anti-immunoglobulin to cyanogen bromide (CNBr)-activated Sepharose. Activated Sepharose 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 of the antisera was ascertained 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% saturated ammonium sulfate, was directly coupled to 1.5 ml of the swollen activated gel (1). Columns for purification of specific cholera antibodies were prepared by covalent coupling of CT or LPS to Sepharose. CT was coupled directly to activated Sepharose by reacting 0.25 mg of CT and 0.5 mg of bovine serum albumin with 0.75 ml of swollen commercial CNBr-Sepharose (1). The LPS, on the other hand, was coupled to laboratory-activated Sepharose over a spacer, diaminohexane, as described previously (24). After CNBr treatment of the LPS, 0.25 mg of this material was reacted with 1 ml of swollen diaminohexane-substituted Sepharose. Before use, all gels were incubated with 1 M ethanolamine for 2 to 3 h at room temperature to block any remaining reactive sites. Sepharose 4B derivatized with protein A of Staphyiococcus aureus (Protein A-Sepharose) was purchased from Pharmacia, Uppsala, Sweden. The derivatized gels were packed on top of a bottom layer of Sepharose 4B in Pasteur pipettes. The columns were equilibrated with starting buffer (50 mmol/mol of phosphate, 500 mmol/mol of glycine, 500 mmol/mol of sodium chloride, pH 7), and the samples were run through the gels. Nonspecifically attached material was washed out with starting buffer. Elution of specifically bound material was performed with an acid buffer (1 mol/mol of acetic

361

acid, 500 mmol/mol of glycine, 500 mmol/mol of sodium chloride, pH 3). Protein synthesis. The method of Smiley et al. (23) with incorporation of 14C-labeled amino acids into the synthesized proteins from in vitro-incubated tissues was used with slight modifications. The procedure is outlined in Fig. 1. After sacrifice of the animal for study, the spleen, mesenteric lymph nodes, Peyer's patches and small intestinal segments distant from Peyer's patches were quickly excised. Ten to twenty mesenteric lymph nodes were dissected free from surrounding tissue, as were all the Peyer's patches along the small bowel. Five to six intestinal specimens were taken in the middle between Peyer's patches, and the mucus was removed by rubbing against paper. The tissues, 200 to 300 mg, were thoroughly washed in incubation medium, minced into 1- to 2-mm pieces, and placed into conical tubes containing 8 ml of freshly prepared modified Eagle medium, pH 7.2. This medium lacked the amino acids contained in the 14C label and was supplemented with 5% heat-inactivated normal rabbit serum and 100 U of penicillin-streptomycin per ml. The incubation tube had a glass capillary sealed to its bottom through which a 95% 02-5% C02 mixture was slowly introduced to keep the pH at 7.2 to 7.4. After incubation at 370C for 15 min, a mixture of 14C-labeled L-arginine, L-leucine, L-lysine, and Lvaline (Schwarz-Mann, Orangeburg, N.Y.) was added to the medium (4 .Ci/g of tissue), and the incubation continued for 6 h. After incubation was completed the medium was centrifuged at 3,000 x g for 10 min to remove particulate material. Each supernate was added with 100 mg of Casamino Acids and dialyzed for 3 days against 1,000 volumes of PBS to eliminate nonincorporated 14C-labeled amino acids. The amount of protein synthesized was determined on an aliquot of the dialyzed medium by precipitation with 10% trichloroacetic acid followed by liquid scintillation in Instagel (Packard Instrument Co., Downers Grove, Ill.). A Packard series 3000 liquid scintillator with 81% efficiency for 14C was used, and the radioactivity was expressed as counts per minute per gram (wet weight) of tissue. Determination of de novo-synthesized immunoglobulin. IgG, IgA, and IgM were isolated on the columns with class-specific anti-immunoglobulin coupled to Sepharose. Samples, 0.5 to 1.0 ml, of dialyzed medium were run through the columns. After washing with at least 5 ml of starting buffer, the specifically bound immunoglobulin was eluted with 5 to 10 ml of acid buffer. By liquid scintillation on a portion ofthe neutralized acid eluate, the newly synthesized immunoglobulin was determined as counts per minute per gram of tissue and as percentage of the total protein synthesized. Determination of newly produced specific antibodies. Specific cholera antibodies were similarly measured by liquid scintillation after isolation by affinity chromatography on the CT- or LPS-coupled gels. Enzyme-linked immunosorbent assay (ELISA). Titrations of specific IgG, IgA, and IgM antibodies were performed as earlier described, using CT and LPS as solid-phase antigens (14).

362

SVENNERHOLM AND HOLMGREN

INFECT. IMMUN. 95% 02 - 5% C02

Incubate with

I

Mince tissue in small pieces

14C-amino acids

t g

Centrifuge

3rC, 6h

3000 x g ,10 min.

Pialyze supernate against PBS Precipitate with 107. TCA

Affinity chromatography 1.Application of sample CT2.Wash with buffer pH7 aEluate with buffer pH 3

Total protein

VP

P

P

P

IgG

IgA

IgM

anti-

CT

^

P Discard Neutralizewashes acid eluates anti-

LPS

Liquid scintillation vL r!_of TCA precipitate

and acid eluate fractions

FIG. 1. Outline of procedure for protein synthesis in vitro by various tissues and for isolation by affinity chromatography of newly synthesized immunoglobulins and specify' antibodies to CT and LPS. TCA, Trichloroacetic acid. Immunodiffusion-in-gel. The Ouchterlony double-diffusion (27) and the Mancini single-radial-immunodiffusion methods (20) were used. Protection tests. The protection studies were performed as earlier described, using the small-bowel loop technique (25). Immunized and control rabbits were concurrently challenged in ligated intestinal loops with graded doses of live vibrios (strain 569B, Inaba) or toxin (National Institutes of Health V. cholerae culture filtrate 4493G). Five doses of bacteria (105 to 10k) or toxin (0.1 to 10 mg) were tested in multiple positions along the gut, and the dose giving rise to half-maximal fluid accumulation in loops (50% effective dose) was determined. Procedural controls: (i) protein synthesis. (a) Dialysis of the medium in which the various tissues

had been incubated for more than the usual 72 h did not further decrease the radioactivity. With all samples, 85 to 95% of the radioactivity of the dialyzed medium was precipitable with trichloroacetic acid. The reproducibility of the acid precipitations was within ±3%. (b) To ensure that the radioactive protein represented de novo synthesis, the 14C label was added to the medium at the end of the incubation. After dialysis of the medium, the radioactivity of the acid precipitate was only 1 to 3% of that obtained when the various tissues had been incubated with the label for 6 h. When the '4C label was present from the beginning but incubation was performed at 0 to SoC (ice bath), the radioactivities were 5 to 10% of those obtained with the standard procedure.

IMMUNOGLOBULIN SYNTHESIS BY LYMPHOID TISSUES

VOL. 15, 1977

(c) The possibility of proteolytic degradation of immunoglobulin during incubation was studied. Rabbit immune sera were added to the medium and incubated with intestinal tissue according to the standard procedure. As measured with the ELISA, the antibody titers of the various immunoglobulin classes in serum were unchanged by the tissue incubation. (ii) Isolation of immunoglobulins. (a) The specificity, capacity, and yield of the class-specific antiimmunoglobulin columns were studied using the ELISA and the single-radial-immunodiffusion technique. It was found that >90% of the IgA and IgM but 90% of rabbit IgG binds to protein A in this manner (19). We made use of this reaction to further ascertain the specificity of our anti-IgG Sepharose columns. Thus, we showed that 290% of the anti-IgG column binding radiolabeled material in incubation media of spleen and intestine was removed on passage through a column with Protein A-Sepharose. (iii) Isolation of specific cholera antibodies. (a) The gels coupled with V. cholerae antigens. were

363

analyzed in a similar manner as the anti-immunoglobulin columns. Application of an anti-culture filtrate serum, containing antibodies to both CT and LPS, to the CT- or the LPS-Sepharose columns resulted in passage of >99% of the antibodies of the heterologous specificity but of 90% of the specifically bound antibodies from both types of columns as tested with immune sera. Moreover, for all of the tissue incubation samples, >80% of the specifically bound radioactivity was recovered on acid elution. (b) The biological activity of antibodies obtained by acid elution was examined after pH neutralization. The anti-LPS antibodies had a protective titer (24) against intestinal challenge with live vibrios which accounted for more than 90% of that observed for the antiserum from which the antibodies had been isolated. Similarly, purified anti-CT antibodies had almost the same toxin-neutralizing capacity in the intradermal neutralization test (3) as did the unfractionated anti-CT serum. RESULTS

Protective capacity of immunization. The immunization used induced a .160-fold increase in resistance against intestinal challenge with live vibrios as based on comparison of 50% effective dose values for immunized and concurrently tested control rabbits. The protection against toxin challenge was fivefold. Protein synthesis by different tissues. The amounts of protein synthesized and secreted from various organs of immunized and control rabbits during in vitro culture for 6 h are shown in Table 1. In the nonimmunized animals, the magnitude of protein synthesis was rather similar from spleen, mesenteric lymph nodes, and Peyer's patches, whereas the intestinal tissue distant from Peyer's patches produced 20 to 30% less protein. After immunization, increased protein synthesis was only registered from spleen and mesenteric lymph nodes (Table 1). Immunoglobulin synthesis. For all the tissues examined, a substantial fraction of the newly synthesized protein was immunoglobulin. As a mean, 40% of the total radioactive protein secreted by the spleen and 32% of that s.c.

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

Immunized (n = 8) Controls (n = 8)

Spleen 62,000 + 8,100a

nodes 57,200 + 3,600

42,400 ± 4,800

Intestine 31,300 ± 2,500

47,800 ± 6,800

43,200 ± 6,000

43,200 ± 5,600

33,800 ± 6,200

P > 0.20

P > 0.20

P 0.20b Mean value + standard error of mean. Student's t test.

P

a b

Protein synthesis (cpm/g of tissue) Mesenteric lymph

>


0.20

P < 0.20

P < 0.20

P > 0.20

P > 0.20b P > 0.20 Mean value + standard error of mean. b Student's t test. a

formed by the mesenteric lymph nodes of control animals was immunoglobulin, for both organs distributed on IgG, IgA, and IgM as 6:1:2 (Tables 2 and 3). Immunization increased the production of IgG and IgM from the spleen, whereas no such increase was noted for the mesenteric lymph nodes. The immunoglobulin formation in Peyer's patches and intestinal tissue of control animals was lower than in the nonenteral tissues (Tables 4 and 5). Only 20% of the protein synthesized in Peyer's patches or in intestinal tissue was immunoglobulin. The proportion of IgA in the enterally formed immunoglobulin was considerably higher than for the other tissues. Thus, for nonimmunized animals 25% of the immunoglobulin synthesized by Peyer's patches and 20% of that produced by intestinal tissue was IgA, whereas this class made up only 10% ofthe immunoglobulin formed by the spleen and mesenteric lymph nodes. Cholera immunization increased the synthesis of particularly IgA but also of IgG in Peyer's patches (Table 4). Relative to the total protein synthesis, increased IgA formation was also observed for intestinal tissue (Table 5). The finding of significant immunoglobulin synthesis in Peyer's patches was surprising in view of the scarcity of antibody-producing cells in this tissue as studied by immunofluorescence (4, 5). A preponderance of immunoglobulin-containing cells in the intestine adjacent to the Peyer's patches has, however, been described (2). To examine the possibility that contamina-

tion with intestinal tissue was responsible for the antibody production from the routinely prepared Peyer's patch specimens, the immunoglobulin synthesis by the very center of Peyer's patches from which the mucosa had been carefully removed was compared with that of the surrounding intestinal tissue, taken 1 to 2 mm immediately around the patch (19,200 cpm/g of tissue), and also with that of intestine taken in the middle between patches (27,100 cpm/g of tissue). The proportion of immunoglobulin and its distribution on various classes in the newly synthesized protein was similar for these various tissues. To investigate whether the incubation time had a differential influence on the synthesis of the various types of immunoglobulins, spleen and Peyer's patch specimens from four animals were incubated for 2, 6, and 24 h. The amount of protein produced from both organs increased almost proportionally with time for at least 24 h, indicating a rather constant rate of synthesis (Fig. 2). The immunoglobulin formation of the spleen also increased through the entire incubation period, although to a less extent than the total protein synthesis. In Peyer's patches, on the other hand, the immunoglobulin synthesis increased only little between 6 and 24 h. The class distribution of the newly synthesized immunoglobulin was rather similar after the various incubation times (Fig. 2). In relation to the total protein synthesis, the amount of the different immunoglobulins formed decreased slightly with time, being 1.5- to 2-fold lower

IMMUNOGLOBULIN SYNTHESIS BY LYMPHOID TISSUES

VOL. 15, 1977

after 24 than after 2 h of incubation (Fig. 2, inserts). Synthesis of specific cholera antibodies. The amounts of newly synthesized specific cholera antibodies were determined after isolation on the CT- and LPS-coupled gels. Significant formation of antibodies to CT as well as LPS was shown both in spleen and in Peyer's patches after immunization (Fig. 3). Local as well as systemic antibody formation was also indicated by antibody titrations with the ELISA on dialyzed incubation medium. For all the tissues studied, the immunized rabbits showed higher levels ofboth anti-LPS and antiCT antibodies than did the control rabbits (Fig. 4). The increased anti-LPS formation comprised IgG as well as IgM in spleen and mesenteric lymph nodes,,whereas the enhanced titers in the enteral tissues were due to IgG and IgA. The increase in anti-CT titers comprised IgG as 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 37 and at 0 to 5OC (ice bath). Since incubation in the cold decreased protein synthesis by 90 to 95%, titers observed in the 0 to 5C incubation medium should mainly represent preformed antibodies released into medium, and increases in titer on incubation at 370C should represent the de novo synthesis. For spleen, an approximately 5-fold increase in IgG titer was seen at 370C to both LPS and CT; a 2- to about 10-fold increase in IgA and a 2- to 4-fold increase in

IgM were seen. For intestine, the increase in IgG was 2- to 5-fold, and the IgA titers rose 4to 20-fold. DISCUSSION An in vitro culture technique with synthesis of radiolabeled protein followed by affinity chromatography isolations of immunoglobulins and specific antibodies has been developed for the study of systemic and intestinal cholera immunity. By measuring de novo synthesis in the incubated tissue, the method permits direct determination of the enteral antibody formation without interference of contaminating antibodies derived from serun. The control experiments indicate that proteolytic degradation, often encountered with intestinal immunoglobulins in vivo, is also avoided by the in vitro culture. The results show that protective immunization with a mixture of V. cholerae LPS and enterotoxin by the s.c. route induces specific-antibody formation both in spleen and intestine. The in vitro protein synthesis method has previously been used in studies of immune responses in rheumatoid arthritis (23) and experimental urinary tract infection (18). In these studies, immunoglobulins synthesized by synovial membrane or infected kidney were isolated by diethylaminoethyl chromatography followed by immunoprecipitation, and specific antibodies were determined after immunopreor adsorption to bacteria. The use of cipit#tion affinity chromatography for isolation of immu-

TABLE 4. In vitro immunoglobulin synthesis in Peyer's patches Percent of total protein Synthesis (cpm/g of tissue) Animals

Immunized (n = 8) Controls (n = 8)

6,800 ± 900a

4,700 - 600

IgM 2,600 + 400

IgG 16.1 ± 1.6a

11.3 ± 1.8

IgM 6.3 ± 1.1

4,700 ± 700

2,200 + 400

2,000 ± 300

12.3 ± 1.7

5.6 ± 0.8

5.0 ± 0.6

P > 0.20

P < 0.10

IgG

IgA

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

IgA

P < 0.01

P > 0.20

a

Animals

Immunized

(n

=

7)

Controls (n = 7)

TABLE 5. In vitro immunoglobulin synthesis in intestinal tissue Percent of total protein Synthesis (cpm/g of tissue) IgA IgM IgM IgA IgG IgG 8.1 ± 1.3 8.0 ± 1.2 11.7 ± 1.6a 2,300 ± 400 2,300 ± 400 2,900 ± 600a 4,000 - 1,200

1,700 ± 500

P > 0.20 a Mean value ± standard error of mean. b Student's t test. P > 0.20

365

2,300 ± 500

12.2 ± 2.4

5.1 ± 0.7

7.3 ± 1.6

P > 0.20

P > 0.20

P < 0.10

P > 0.20

366

SVENNERHOLM AND HOLMGREN a

INFECT. IMMUN.

SPLEEN

b

100 -

100

-

10

-

I0

I0

D

z e)

PEYER'S PATCHES

10

-

6 E

1-~

1-

, I

2

6

24

2

h

24

6

h

h

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 of immunoglobulins ofvarious classes in relation to total protein formation. Mean values and standard error of mean for two to four animals are given.

10

10

a)

a)

20

0

QL

C 0

5 5

5

a)

c

a)

Anti-LPS

Anti-CT

Anti-LPS

Anti-CT

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

noglobulins and specific antibodies has allowed convenient and more effective purifications with improved yields. All the derivatized gels used were highly specific, since they retained less than 1% of heterologous immunoglobulins or antibodies. The desorption conditions were based on previous experiments with pH gradient elutions, which indicated that the chosen procedure would elute at least 80% of the bound

antibodies, including those with high affinity (24). In the present study, yields between 80 to 100% were achieved in the isolations of immunoglobulins as well as of specific antibodies, when tested both with serum samples and with the actual radioactive tissue culture specimens. Although immunodiffusion tests suggested that the isolated immunoglobulins were aggregated to a certain extent, examinations with the

IMMUNOGLOBULIN SYNTHESIS BY LYMPHOID TISSUES

VOL. 15, 1977

367

10 000

1000 100

10 000

1000

0)

100

.)

-

w

10000

1000 100

10000 1000 100

bgG

IgA Anti- LPS

IgM

IgG

IgA

Anti- CT

FIG. 4. Titers of antibodies of various immunoglobulin classes against LPS and CT in incubation medium from various tissues of immunized (0) and control (0) rabbits as determined with the ELISA. Mean values and standard error of mean are indicated (eight animals in each group).

ELISA demonstrated that they retained full antigen-binding capacity as well as reactivity with heavy-chain specific antibodies. Furthermore, the antibodies eluted from the LPS- and CT-coupled Sepharose had intact biological activity as estimated in protection experiments.

The relative amounts of IgG, IgA, and IgM synthesized by spleen and mesenteric lymph nodes of the control rabbits are in close agreement with those reported by Smiley et al. (23) for normal human spleen and lymph node. In view ofthe predominance of IgA plasma cells in

368

SVENNERHOLM AND HOLMGREN

the intestinal lamina propria, as observed with immunofluorescence, the high proportion of IgG synthesized by the rabbit intestine in vitro was unexpected (4, 5). Based on control experiments, using columns with protein A coupled to Sepharose, the possibility that radioactive nonimmunoglobulin material was bound to the anti-IgG column, giving rise to falsely high IgG values, could be eliminated. Protein A, which selectively binds IgG (9), removed > 90% of the anti-IgG column binding material from tissue culture medium of both spleen and intestine. It is possible, however, that the expression of IgG precursor cells is favored by the in vitro incubation. We tested whether incubation periods other than 6 h for the in vitro protein synthesis would result in different proportions of IgG, IgA, and IgM. No such change was seen by varying the time between 2 and 24 h using spleen and Peyer's patches. It is noteworthy that a substantial in vitro synthesis of IgG and IgA, as well as IgM, occurred in the Peyer's patches, since only few plasma cells or immunoglobulin-containing cells have been observed within the lymphoid follicles of the patches (46, 28). However, IgA-, IgG- and IgM-containing cells are abundant on the edge of the follicles and in the adjacent intestinal mucosa (2, 11). A polarity, with the IgA cells to the mucosal side and the IgG and IgM cells to the serosal edge of the follicles, has been described (2). Although in control experiments the immunoglobulin synthesis per gram of tissue in the rabbit Peyer's patch preparations was unchanged by careful removal of the mucosa, interfollicular cells might still have contributed to the immunoglobulin synthesis registered. Using an in vitro radiolabeling procedure, Vitetta et al. recently 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's patch cells responded as well with antibody formation as did spleen cells when stimulated with antigen in in vitro culture (600 plaqueforming cells [PFC]/107 cells). Veldkamp et al. demonstrated a significant PFC response in mouse Peyer's patches (400 to 500 PFC/107 cells) after repeated intraduodenal or intraperitoneal immunization with V. cholerae (29). The fact that the synthesis of immunoglobulin and its distribution on various classes might be influenced by the conditions of the in vitro incubation does not detract from the usefulness of the method in registering changes in formation ofimmunoglobulins and specific antibodies induced by specific immunization. It is evident that s.c. immunization, being highly protective against experimental cholera (.160-fold in this study against intestinal challenge with live

INFECT. IMMUN.

cholera vibrios), gives rise to substantial synthesis of radiolabeled antibodies to both V. cholerae LPS and enterotoxin in spleen. It also increases the overall synthesis of IgM in this tissue. Furthermore, the immunization results in increased specific ELISA antibody titers in incubation medium of spleen and mesenteric lymph nodes. The immunoglobulin class distribution of these titers closely corresponds to that previously found for antibodies to LPS and CT in serum (17). In rats, Pierce and Gowans found that parenteral (intraperitoneal) immunization with cholera toxoid only gave rise to very few antitoxincontaining cells in the gut, but primed the intestine for a subsequent secondary IgA cell antitoxic immune response to an enteral boost (22). In rabbits, low levels of both anti-CT and anti-LPS antibodies, IgG as well as IgA, have previously been found in small intestinal washings after s.c. vaccination (17, 25). However, it has not been possible until now to determine whether these antibodies are produced by the intestine itself or are derived from the circulation. In the present investigation, a local synthesis of specific, radiolabeled antibodies to V. cholerae LPS and enterotoxin after s.c. immunization is established for Peyer's patches. We did not test for formation of radiolabeled specific antibodies from the intestinal tissue apart from Peyer's patches. However, the titrations on tissue incubation medium with the ELISA clearly indicate specific-antibody production also by the intestine. Thus, in addition to observing higher antibody titers in incubation medium from the immunized rabbits than from the control ones, we show that the titers of immunized animals increase during protein synthesis, as tested by simultaneous incubation of tissue at 37 and at 0 to 5°C. For intestinal tissue, a 4- to 20-fold increase was seen for the IgA antibodies, whereas the rise in IgG was smaller (2- to 5-fold). For spleen, the increase in IgA antibodies was about 2- to 10-fold, and in specific IgG it was about 5-fold. ACKNOWLEDGMENTS James W. Smith, Dallas, Tex., is gratefully acknowledged for advice on the in vitro protein synthesis procedure. The skillful technical assistance of Ulla WesterbergBerndtsson is highly appreciated. This work was supported by grants from the Swedish Medical Research Council (project 16X-3382), FOA, Sweden (5061H563), and the World Health Organization. LITERATURE CITED 1. Axin, R., V. Porath, and S. Ernback. 1967. Chemical coupling of peptides and proteins by means of cyanogen halides. Nature (London) 214:1302-1304. 2. Crabbe, P. A., D. R. Nash, H. Bazin, H. Eyssen, and J. F. Heremans. 1970. Immunohistochemical observa-

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