INFECTION AND IMMUNrrY, Sept. 1974, p. 458-463Copyright 0 1974 American Society for Microbiology

Vol. 10, No. 3Printed in U.S.A.

Local Antibody Production Against the Murine Toxin ofYersinia pestis in a Golf Ball-Induced Granuloma

ROBYN P. HILLAM, ROBERT P. TENGERDY, AND GEORGE L. BROWN

Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, and ImmunologySection, Clinical Investigation Service, Fitzsimmons Army Medical Center, Denver, Colorado 80240

Received for publication 8 April 1974

The method of Garra and Baygorria, for localized antibody production, hasbeen adapted for obtaining high-titered and monospecific antibodies against themurine toxin of Yersinia pestis. A hollow, perforated plastic golf ball surgicallyimplanted under the skin of rabbits induced the formation of a granuloma. Whenthe murine toxin of Y. pestis was injected directly into the granulomatous cavity,an increased amount of antibody was found in the granuloma fluid as comparedwith serum or with serum antibody obtained by conventional immunization. Thegranuloma antibody consisted mainly of immunoglobulin G, probably producedlocally by lymphoid cells of the granuloma. The immune granuloma fluid and thegranuloma tissue were rich in plasma cells and lymphocytes. The chemicalcomposition of the granuloma fluid indicated that it was a transudate.

The purpose of this investigation was theadaptation of a novel, but little used, methQodfor the improved production of high-titeredantibody against the murine toxin of Yersiniapestis. The murine toxin of Y. pestis is one ofthe main antigenic components of this bacte-rium and may serve for an immunologicaldifferentiation of various Y. pestis strains (8).Garra and Baygorria reported the localized

production of antibody in granulomatous cavi-ties that was induced by subcutaneous surgicalimplantation of perforated plastic table tennisballs into rabbits (3, 4). The antigens, eithererythrocytes or human growth hormone, wereinjected into the interior of the ball. The fluiddraining into this granulomatous cavity con-tained antibodies with higher titer and in-creased specificity than could be obtained fromsera by conventional immunization.Using the implantation technique, Paseyro et

al. obtained antithyroid antibodies with titers16 times higher than those in the serum of thesame rabbits (6). Similarly, Somma Moreiraand Tosi obtained antibody against Coxsackievirus (group A, type 1) with titers 10 tmeshigher in the granuloma fluid than in the serumof conventionally immunized rabbits (7).We demonstrate in this report that this new

method of immunization yields more and easilyobtainable antibody for the identification of Y.pestis strains and characterize some immuno-logical and chemical properties of the granu-

loma fluid and granuloma tissue that shouldfacilitate the general application of the method.

MATERIALS AND METHODSPreparation of murine toxin from Y. pestis. Y.

pestis strain JAVA 1241 (Communicable DiseaseCenter, Fort Collins, Colo.) was the source of thetoxin. The bacteria were grown in casein hydrolysatebroth at 28 C for 7 days. The autolyzed cells wereharvested, and the toxin was precipitated from thesupernatant of the autolysate with (NH4)2SO4. Thiscrude murine toxin was purified further by differen-tial (NH4)2SO4 precipitation to separate the Fl frac-tion, following the procedures of Baker et al. (2) andAjl et al. (1). Finally, the toxin was purified by gelfiltration, first on a Sephadex G-200 and then on asuperfine G-150 column. The immunochemical char-acterization of this antigen was described previously(9).

Induction of granuloma. Hollow, perforated plas-tic practice golf balls (Target brand) were implantedsurgically on both sides, under the dorsal skin, of3-month-old New Zealand white male rabbits. Theballs settled firmly in one place in about 3 monthswith a granulomatous tissue covering them and withfluid draining into the lumen of the balls. The golfball with its surrounding tissue at the end of theexperiment is shown in Fig. 1.

Immunization procedures. One hundred and fortydays after implantation, the four rabbits were immu-nized by injection of toxin (1.25 mg in 1.5 ml of saline)into the lumen of each golf ball (2.5 mg total perrabbit). At day 70 after immunization, the rabbitswere rechallenged with 0.25 mg of toxin in 1.5 ml ofsaline injected into each granuloma.

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FIG. 1. Granuloma tissue surrounding the im-planted golf ball. The picture was taken 240 days afterimplantation (100 days after immunization). Histo-logic tests described in text were made from thistissue.

Granuloma fluid was withdrawn with a sterilesyringe from each rabbit prior to immunization andbiweekly thereafter. The rabbits were not anesthe-tized for this procedure. An average of 9 ml (range: 4to 10 ml) fluid was collected each time from each golfball.Two control rabbits were immunized with 2.5 mg of

toxin emulsified in Freund complete adjuvant by amodification of a proven immunization procedure forthe murine toxin (10). The injections were distributedequally at five different sites: two intramuscularly ineach hind flank, two subcutaneously in the axillaryregion, and one subcutaneously between the shoul-ders. At day 28, a booster shot of 0.5 mg of toxin was

given intravenously.All rabbits were bled from the marginal ear vein for

tests after 12 h of starvation.Chemical and immunological characterization

of granuloma fluids and serum. Cellulose acetateelectrophoresis, acrylamide disk electrophoresis, im-munodiffusion, immunoelectrophoresis, quantitativeprecipitin test, and passive hemagglutination were

performed by sta,ndard techniques (11-13). The bloodchemistry profile of whole blood versus granulomafluid was obtained by Autotechnicon SMA 6/60 andSMA 12/60 analysis.

Electroimmunodiffusion was performed, as de-scribed previously (9), by using a modification of thetechnique of Merrill et al. (5).The antibody-containing fraction of the granuloma

fluid was isolated by Sephadex G-200 gel filtration ona Pharmacia K25/45 column equilibrated with 0.1 Mborate buffer (pH 8.3) containing 0.2% sodium azide.The flow rate was maintained at 15 ml/h. Theapproximate molecular weights of the fractions weredetermined from the elution volume by comparisonwith standard purified proteins, bovine gamma globu-lin (BGG), ovalbumin, bovine serum albumin (BSA),and trypsin.

RESULTS

The local, "in granuloma" immunization of

rabbits resulted in substantial production andaccumulation of antibodies in the granulomafluid draining into the lumen of the perforatedgolf ball (Table 1). During the course of immu-nization the hemagglutination antibody titer ofthe granuloma fluid was consistently 3 to 5 log2units higher than the serum titer in the samerabbits. The titer was also about 3 log2 unitshigher than the serum titer of conventionallyimmunized rabbits (Table 2). Furthermore, thetiter remained high for more than 60 days in thein granuloma-immunized rabbits; after thebooster immunization given on day 70, the titerincreased and remained high for at least 4 moreweeks.Table 1 indicates that two of the eight golf

balls implanted became infected either duringimplantation or fluid withdrawal. With propersurgical and sampling techniques such infec-tions can be eliminated.The antibody content of the granuloma fluid

42 days postimmunization, as determined bythe quantitative precipitin test, was 2.84 mg/mlcompared with 1.02 mg/ml found in the serumof conventionally immunized rabbits (Table 3).The granuloma fluid contained only about halfas much protein as the serum, yet it contained ahigher percentage of gamma globulin and morespecific antibody than the serum. In the preim-munization stage, the granuloma fluid con-tained mostly albumin, some beta globulins,and little gamma globulin, but after immuniza-tion the gamma globulin increased from 1 to26%, with a corresponding 10-fold immunoglob-ulin G (IgG) increase. Much of this IgG (53.9%)was specific antibody. In the immune serumobtained by conventional immunization, theincrease in IgG was much smaller, and thespecific antibody constituted only 10.3% of thetotal IgG. All antibody activity of the granu-loma fluid was contained in one SephadexG-200 elution peak. This peak had an approxi-mate molecular weight of 160,000 based oncomparison with the elution volume of bovineserum albumin, bovine gamma globulin, oval-bumin, and trypsin. This fraction also gave aline of identity with purified rabbit 7S IgG in animmunodiffusion test against goat anti-rabbitgamma globulin and anti-normal rabbit serum,thus indicating that this antibody was the IgGtype.Acrylamide gel-disk electrophoretic patterns

also revealed differences between pooled im-mune granuloma fluid and pooled immuneserum. The gamma globulin band was morepronounced in the granuloma fluid than in

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TABLE 1. Passive hemagglutination titers of the granuloma fluid and serumgranuloma" with the murine toxin of Y. pestisa

of rabbits immunized "in

Hemagglutination log2 titera

Day after Golf Granuloma fluid Serumimmunization ballb

jc 2 3 4 1 2 3 4

0 A 0 0 -d 0 0 0 0 0B 0 0 0 0

14 A 6 6 - 4 3 4 2 3B 5 6 4 2

28 A 9 10 - 8 6 6 5 6B 9 9 8 7

42 A 12 13 - 10 8 9 7 8B 13 14 9 -

56 A 10 11 - 9 8 8 6 8B 11 11 8 -

70 A 9 9 - 8 7 7 5 79 10 6 -

84 A 9 9 - 7 7 7 5 6B 9 9 6 -

98 A 12 12 - 9 8 8 5 8B 12 13 8 -

a The samples were analyzed in triplicate sets. The error of detection in this test is + 1 log2 unit, thus 2-unitdifferences are regarded significant."The arrows indicate the booster immunization.c Rabbit number.d, No sample obtained due to infection in the ball.

TABLE 2. Passive hemagglutination titers of rabbitsimmunized with the murine toxin of Y. pestis in

Freund complete adjuvanta

Day after Hemagglutination log2 titerimmunization Rabbit 5 Rabbit 6

0 0 014 4 4,28" 7 842 9 1156 9 1070 9 1084 8 998 7 9

aThe samples were analyzed in triplicate sets. Theerror of detection in this test is 1 log2 unit, thus2-unit differences are regarded significant.

b The arrow indicates the booster immunization.

serum, but three bands corresponding to alphaand beta globulins were missing in the granu-loma fluid (Fig. 2). The immunoelectrophoreticpatterns against goat anti-rabbit serum cor-roborated these differences. When the anti-serum was adsorbed with the granuloma fluid,the remaining lines with rabbit serum werethose in the alpha and beta macroglobulinregion, demonstrating the absence of theseproteins in the granuloma fluid (Fig. 3).

The blood chemistry profile of immune andnormal granuloma fluid shown in Table 4 sug-gested that it was a transudate. The granulomafluid was different from the blood serum in itschemical composition; notably, it was higher inCO2 but much lower in inorganic phosphate,cholesterol, bilirubin, and alkaline phospha-tase.These data indicated clearly that the granu-

loma fluid was different from blood serum,suggesting that the secretion of this transudatewas most likely under the control of the sur-rounding granuloma tissue.The differential leukocyte count in the granu-

loma fluid of immunized rabbits also indicatedlocal antigenic stimulation (Table 5). Notablewas the high initial neutrophil count, alsoobserved by others around a site of infection (8).The neutrophil/lymphocyte ratio progressivelyreversed as immunization proceeded. The lym-phocyte count and plasma cell count becameunusually high by day 42 after infection andresembled cell reaction associated with lymph-oid tissue.

Histological tests confirmed the above obser-vations. The granuloma tissue covering theholes of the golf ball consisted primarily offibrovascular connective tissue with resolvinggranulation tissue interspersed with focal areasrich in plasma cells. Small numbers of macro-

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TABLE 3. Protein fractions and antibody content of immune granuloma fluid versus immune seruma

Protein distribution (%)Total IgG Antibody

Sample protein Globulins (mg/ml) (mg/ml)(mg/ml) Albumin

a, a2 1 Vy

Pooled immune granuloma fluid from7 granulomas .................... 25.6 67 _b _ 7 26 5.27 2.84

Pooled immune serum from 4 granu-loma rabbits ..................... 53.2 62 7 8 8 15 6.5 0.16

Pooled immune serum from 2 normalrabbits ......................... 59.2 57 7 6 7 23 9.9 1.02

Pooled preimmunization granulomafluid from 7 granulomas .......... 18.0 94 - - 5 1 0.5

Pooled preimmunization normalserum from 6 rabbits ............. 52.4 65 7 6 12 10 4.2

aGranuloma fluid and serum were taken before and 42 days after immunization. The percent proteindistribution was calculated from cellulose acetate electrophoregrams, total protein was assayed by the FolinLowry test, IgG was assayed by electroimmunodiffusion, and antibody was assayed by the quantitativeprecipitin test.b_,Below limit of determination.

phages, neutrophils, and lymphocytes were also... p ,.:'observed.

Iin_b DISCUSSIONLocal antibody production in a golf ball-

induced granuloma is a novel and convenientway for obtaining specific, high-titered anti-bodies.Examination of the granuloma tissue sur-

rounding the golf ball and the leukocyte countsin the granuloma fluid suggest that this granu-*X i-2 loma tissue, in attempting to ward off theimplanted foreign body, mounts a local immuneresponse. The localized antigen within the ballpresents a constant and lasting antigenic stimu-lus to the surrounding granuloma tissue rich inlymphocytes and plasma cells, resulting in asustained production of high-titered antibody.The high neutrophil counts in the granuloma

fluid at the early stage of antigenic stimulationand the progressive reversal of the neutrophil/lymphocyte ratio are consistent with the pictureof a localized antigenic stimulation. The differ-ent composition of the granuloma fluid from

U that of serum and the blood chemistry profilestrongly suggest that this transudate is excreteddirectly by the granuloma tissue.

In the particular case reported here, theantigen stimulated the increased production of

AB7.5% polyacrylamide gel. The gels were electro-phoresed, by the standard Davis and Ornstein proce-dure, for approximately 2 h at a constant current of2.5 mA per tube. The gels were stained with amido

FIG. 2. Protein patterns of pooled immune rabbit black and scanned in a Canalco model G microdensi-serum (A) and Dooled immune granuloma fluid (B) in tometer.

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IgG-type antibody. Since only the IgG fractionof the fluid contained antibody, it is concludedthat most or all antibodies produced by this

I A

1 ~~~~B

t C~~~

2::: :: ': ':: ;

FIG. 3. Immunoelectrophoretic patterns of pooled,immune granuloma fluid (1) and pooled immunerabbit serum (2). The antisera in the troughs are

nonadsorbed antiserum against normal rabbit serum

(A), anti-normal rabbit serum absorbed with the fluidfrom the golf ball granuloma (B), and anti-normalrabbit serum adsorbed with normal rabbit serum (C).

granuloma were of the IgG class. The amount ofantibody represented more than 50% of the totalIgG content of the granuloma fluid, a very

respectable yield compared with conventionalimmunization where 10.3% of the total IgG was

specific antibody.This method of immunization presents some

real and potential advantages over conventionalimmunization, as summarized below.

(i) The antibody is conveniently produced ina clear fluid, virtually free of erythrocytes andconcomitant hemolysis problems. In this experi-ment the biweekly withdrawal of 9 ml of granu-loma fluid per ball, or 18 ml per rabbit, repeatedfour times in 8 weeks yielded 144 ml of fluidwith an average antibody content of 2.84 mg/ml, i.e., about 205 mg of IgG-type antibody.Conventional immunization, assuming the bi-weekly withdrawal of a maximum of 40 ml ofblood (about 20 ml of serum) repeated fourtimes, would give 80 ml of serum with a

maximum of 1.02 mg of antibody per ml, i.e.,81.5 mg of antibody total. Isolation and purifi-cation of the antibody are easier because thefluid contains more antibody but less and fewerkinds of other proteins.

(ii) The antigen is present in a well-localizedgranuloma and is probably retained there for a

long time, thus providing a constant antigenicstimulation judged from the sustained high

TABLE 4. Blood chemistry profile of granuloma fluid versus seruma

Immune ImmuneImmune serum serum Preimmune Preimmune

Constituent granuloma from from granuloma serumfluid granuloma normal fluid

rabbit rabbit

Cl (mg/liter). 117 116 110 115 109CO2 (mg/liter).18 13.5 12 21 14K+ (mg/liter).4.05 5.1 3.6 3.9 5Na+ (mg/liter).152 150 143 146 145Blood urea.N2 (mg/100 ml).21 21 21 21 22Glucose (mg/100 ml).42 45 120 90 90Ca2+ (mg/100 ml) ................. 10.1 14 12.1 10.2 13.1Inorganic P (mg/100 ml).4.1 5 6.4 3.8 5.3Cholesterol (mg/100 ml).20 40 40 12.5 40Uric acid (mg/100 ml).0.3 0.3 0.4 0.25 0.35Creatine (mg/100 ml).1.35 1.6 1.5 1.15 1.35Total bilirubin (mg/100 ml).0.1 0.25 0.3 0.1 0.2Alkaline phosphatase (mU/ml).5 60 65 0 35Creatine phosphokinase (mU/ml) 0 0 0 0 0Lactic dehydrogenase (mU/ml).170 270 150 50 375Serum glutamic oxalic transaminase(mU/ml) ...................... 25 30 27.5 30 35

a Data were obtained by Autotechnicon SMA 6/60 and 12/60 analysis. The values for normal rabbit serum arewithin accepted values reported in the literature. The samples used for this analysis are the same as those inTable 3.

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TABLE 5. Differential leukocyte counts in granuloma fluid during the course of immunizationa

Days after Plasma Segmented Macrophage Monocyte Lymphocyteimmunization cell neutrophil

14 7 (3-21) 60 (51-88) 1 (0-3) 0 32 (16-43)28 6 (4-8) 29 (3-68) 2 (0-5) 0 (0-1) 62 (22-88)42 24 (21-26) 16 (1-42) 0 (0-1) 0 60 (33-76)56 7 (3-16) 6 (0-7) 1 (0-3) 0 84 (42-94),70 2 (1-5) 6 (3-34) 1 (0-3) 0 91 (92-99)84 15 (5-23) 36 (25-63) 1 (0-1) 0 43 (19-76)98 9 (3-20) 49 (16-90) 0 (0-1) 0 42 (16-86)

a Numbers are the average percentages of total leukocyte count with the range given in parentheses. Sevengranuloma fluids were averaged. Preimmunization values are not available due to loss of samples. The averagetotal leukocyte count of the fluids was 1,450/mm3 with a range of 1,200 to 3,450/mm8. The arrow indicates theday of the booster immunization. Comparative literature values for normal rabbit blood are: total leukocytes,9,000 (6,000 to 13,000) per mm3; neutrophils, 46% (36 to 52%); lymphocytes, 39% (30 to 52%); monocytes, 8% (4to 12%); basophil, 5% (2 to 7%); eosinophils, 2% (0.5 to 3.5%) (In E. C. Albritton, [ed.], Standard values inblood, W. B. Saunders, Philadelphia, 1951, p. 53).

antibody titer in the granuloma fluid. Thistechnique assures a very economic production ofantibody with a small expenditure of antigen.

(iii) The localization of the antigen inside thegranuloma may be a particular advantage inworking with toxic antigens. The presumablyminor and slow escape of small quantities ofsuch substances from the granuloma, judgedfrom the low antibody response in the serum,may prevent it from reaching toxic concentra-tions in vital areas of the body.

ACKNOWLEDGMENTSWe thank Bruce Hudson at the Communicable Disease

Center, Ecologic Investigations Unit, Fort Collins, Colo., forintroducing us to the concept of golf ball antibody production,for providing the Y. pestis strain, and for many helpful ideasand practical help during the course of this work.

This investigation was supported by Public Health re-search grant CC 00546 from the National CommunicableDisease Center.

LITERATURE CITED1. Ajl, S. J., J. S. Reedal, E. L. Durrum, and J. Warren.

1955. Studies on plague. I. Purification and propertiesof the toxin of Pasteurella pestis. J. Bacteriol.70:158-169.

2. Baker, E. E., H. Sommer, L. E. Foster, E. Meyer, and K.F. Meyer. 1952. Studies on immunization againstplague. I. The isolation and characterization of thesoluble antigen of Pasteurella pestis. J. Immunol.68:131-145.

3. Garra, A., and G. Cendon de Baygorria. 1959. Producci6nlocal de anticuerpos. Ann. Fac. Med. Montevideo44:544-547.

4. Garra, A., G. Cendon de Baygorria, J. Morato Manaro,and P. Paseyro. 1960. El granuloma immunitario comometodo para la producci6n de anticuerpos de la hor-mona de crecimiento humana. Ann. Fac. Med. Monte-video 45:339-344.

5. Merrill, D., T. F. Hartley, and H. N. Claman, 1967.Electroimmunodiffusion: a simple report method forquantitation of immunoglobulins in dilute biologicalfluids. J. Lab. Clin. Med. 69:151-159.

6. Paseyro, P., E. Negrin, J. Morato Manaro, and 0. F.Grosso. 1961. El granuloma immunitario de Garra yBaygorria como metodo par obtener anticuerpos an-titiroideos en el conejo. Ann. Fac. Med. Montevideo46:297-303.

7. Somma Moreira, R. E., and H. C. Tosi. 1962. Elgranuloma immunitario de Garra y Baygorria y su em-pleo en virologia. Ann. Fac. Fac. Med. Montevideo 47:152-156.

8. Speirs, R. S., and E. E. Speirs. 1963. Cellular localizationof radioactive antigen in immunized and non-immu-nized mice. J. Immunol. 90:561-575.

9. Tengerdy, R. P., and R. P. Hillam, 1973. Quantitativedifferentiation of Yersinia pestis strains by their mu-rine toxin and fraction I contents. Bull. W.H.O.48:279-287.

10. Warren, J., W. Ursula, J. S. Reedal, and S. J. Ajl. 1955.Studies on plague. II. Immunological properties ofpurified Pasteurella pestis toxin. J. Bacteriol.70:170-176.

11. Williams, C. A., and M. W. Chase (ed.). 1967. Methods inimmunology and immunochemistry, vol. 1. AcademicPress Inc., New York.

12. Williams, C. A., and M. W. Chase (ed.). 1968. Methods inimmunology and immunochemistry, vol. 2. AcademicPress Inc., New York.

13. Williams, C. A., and M. W. Chase (ed.). 1971. Methods inimmunology and immunochemistry, vol. 3. AcademicPress Inc., New York.

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