Molecular Immwwingy, Vol. 17. pp. I 105-l I 15. iDPergamon Press Ltd. 1980. Prkted in Great Britain.

0161~5890/80/0901-I 10s $02.00/O

SECRETORY ANTIBODY RESPONSE TO LOCAL INJECTION OF SOLUBLE OR PARTICULATE ANTIGENS IN RATS

D. S. COX, M. A. TAUBMAN,* J. L, EBERSOLE and D. J. SMITH

Department of Immunology, Forsyth Dent& Center, and Harvard School of Dental Medicine, Boston, MA 02115, U.S.A.

(Received 13 September 1979)

Abstract-The locat antibody response to soluble (dinitrophenylated bovine gamma globulin: DNP-BGG) or particulate (DNP-~~re~~ffcoce~ mutans) antigens was examined in the rat. IgA and IgG antibody characteristics (concentration, activity and avidity) were determined in milk after injection of DNP-BGG or DNP-S. mutans into the vicinity of the mammary gland during gestation. More IgG antibody activity was detected in milk collected from rats injected with the soluble antigen relative to those injected with the particulate antigen using haptenated bacteriophage neutralization analysis. Analyses of milk antibody concentrations by combined immunoadsorption, elution and radial immunodiffusion confirmed the relative difference in response between the two antigens. IgA antibody levels in milk were similar in rats injected with soluble or particulate antigen (26 + 1 vs 27 + 7 pg/ml). However, the IgG antibody levels were higher in rats injected with the soluble antigen (24 f 2 vs 1 1 + 3 pg/ml). IgA antibody concentrations in saliva, determined by an enzyme linked immunosorbant assay, were also approximately the same after injection of soluble or particulate antigen (3 16 & 33 ng/ml vs 310 + 85 ng/ml) in the vicinity of the salivary glands. IgG antibody was only detected in the saliva of rats injected with soluble antigen (128 f 26 ng/ml). Therefore, differences in the local responses to the two antigens seemed to be confined to IgG antibody. Inhibition of bacteriophage neutralization by DNP-lysine and an analysis of neutralization kinetics revealed that IgA antibody may have a greater avidity for a multideterminant antigen than does IgG antibody in the same secretion.

Initiation of a humoral immune response by the injection of antigen results in the production of antibodies of several different isotypes. The distribution of isotypes of the antibody that results from immunization of an animal can vary depending upon the nature of the antigen (Larralde & Janof, 1972; MIkell et al., 1971; Naor et al., 1974), the route of administration of antigen (Mestecky et al., 1978; Monigomery et al., 1974), the dose of antigen (Svehag& Mandel, 1964a; Uhr, 1964) and schedule of antigen administration (Sahiar & Schwartz, 1965; Svehag & Mandel, 19643). The ability to influence the distribution of antibody isotypes following the administration of antigen could be advantageous because of different functional characteristics of the various isotypes.

Previous investigations have indicated that local injection of antigen at a secretory site can lead to the production of IgA antibody in the secretion (Genco & Taubmari, 1969; Taubman & Genco, 1971; Eddie et at., 1971; Montgomery et al., 1974). In general it appears that particulate antigens induce a substantially better local

*To whom requests for reprints should be sent.

immune response than do soluble preparations (Strannegard & Yurchison, 1969; Waldman et al., 197.0; Ebersole & Molinari, 1978). Preliminary studies of Montgomery et al. (1974) using dinitrophenylated antigen also indicated that the physical character of the antigen that is used to stimulate a local response can have an effect on the nature of the IgA antibody. In that case, a particulate DNP antigen seemed to produce a greater IgA antibody response than a soluble DNP antigen administered to rabbits. We have investigated the role of soluble and particulate antigens in the selective induction of local IgA antibody in the rat. The relative activity, concentration and avidity of IgA and IgG antibody from milk was examined. We found that the IgA antibody concentration in an individual secretory fluid (milk, saliva) was similar after local immunization with either soluble or particulate antigen. However, IgG antibody concentration in these secretions was elevated when soluble antigen was used.

MA~RrA~ AND METHODS

Antigen ~r~pur~t~~~

Dinitrophen~lated bovine gamma globulin 1105

1106 D. S. COX cr irl.

(DNP-BGG), the soluble antigen, was prepared and characterized according to the method of Eisen el ul. (1953). Bovine gamma globulin (Miles Laboratories, Inc., Elkhart, IN) was reacted with

3 x recrystallized dinitrobenzenesulfonic acid (Eastman Kodak Co., Rochester, NY). The preparation was characterized as having 55.4

DNP groups per molecule of protein by dry weight and by optical density analysis at 360 nm

(Z$$” for DNP-L-lysine = 17,530). Dinitrophenylated Streptococcus mutans 6715

was prepared accorG;ng to the method of Montgomery & Pincus (1973). Briefly, S. mutuns

(4.5 x 1 Oy bacteria per ml in 240 ml distilled H,O)

was adjusted to pH 11.0 with 5N NaOH.

Saturated CNBr (180 ml) (J. T. Baker, Philipsburg, NJ) was added to the bacterial suspension and the pH maintained at 11.0. s-DNP-L-lysine (Sigma, St. Louis, MO) in H,O (1.3 mg/mI; 240 ml) was added to the ‘activated cells and stirred overnight at 4°C. The washed RNP-S. rn~tu~~ cells were resuspended in

phosphate buffered saline (PBS; 0.02M PO,, pH 7.5). This particulate antigen vaccine was characterized by spectrophotometric analysis at

360 nm following hydrolysis of the cells with 1.0 N NaOH at 100°C. There were 1.7 x 10s DNP groups per bacterium in the vaccine.

Mammary injections

Pregnant Sprague-Dawley rats (n = 15; Charles River Breeding Laboratories, Wilmin-

gton, MA) were injected in multiple sites in the vicinity of the mammary glands (MGV) with a

dinitrophenylated (DNP) soluble of particulate antigen on day 1 and on day 11 of pregnancy (Gene0 & Taubman, 1969). One group of rats (n = 6) received, as soluble antigen, 200 pg of DNP-BGG in complete Freund’s adjuvant

(CFA). Another group of rats (n = 7) received DNP-S. mutans, a particulate antigen,

containing an equimolar amount of DNP per

injection (7.4 x IO-* moles) in CFA. Sham- immunized rats received injections of PBS in CFA.

Salivary gland injections

Adult female Sprague-Dawley rats (n = 19) were injected once in the vicinity of each of the 4 major salivary glands (SGV) (Taubman~ 1973; Taubman & Smith. 1974) with either DNP-BGG (n = 9), DNP-S. mutm.s (n = 7), or PBS (n = 3) all in CFA. Antigen concentrations were as described for mammary gland immunization.

Collection qf milk

After parturition, milk was collected at daily

intervals for 21 days. Oxytocin (Pitocin, Parke, Davis & Co., Detroit, MI) was injected

intraperitoneally (2.0 units/rat) to stimulate the milk supply. Milk was expressed by digital

manipulation and collected into a Pasteur pipette. The samples were immediateIy frozen at -2OY and were thawed and clarified by

centrifugation (27,000 R for 40 min) only at the time of analysis.

Collection of saliva and serum

Saliva was collected 11 days after SGV

injection. Pilocarpine (1 .O mg/lOO g body wt) was used to stimulate saliva from ether-anesthetized rats. Serums were collected 11 days after SGV injection or 16 days after the last MGV injection

and treated as described previously (Taubman & Smith, 1974).

Antisera

Monospecific rabbit anti-rat IgM serum was

prepared as previously described (Ebersole et al.

1979a). Rabbit anti-rat IgG antiserum was prepared

from rabbits immunized with Fc rich fractions of

rat serum IgG prepared by DEAE-cellulose chromatography, (NH,),SO, precipitation and papain digestion. Light chain activity was removed by extensive adsorption with insolubilized rat IgM and IgA (Axen rf al.. 1967). Ill~lnunoelectrophoretic and gel diffusion analyses of the adsorbed material demonstrated reactivity with both rat IgG, and IgG.,

myeloma proteins (Dr. H. Bazin, Brussels, Belgium) and no reactivity with other serum proteins.

Monospecific rabbit anti-rat secretory IgA

was prepared as previously described (Taubman & Smith, 1977).

Removal of IgM ji-om milk

Our radial immunodiffusion analyses have

indicated that IgM is present in rat milk at concentrations in the order of 90-100 pg/ml. To ensure that antibody activity was only IgA or IgG, IgM was removed before analysis. Rabbit anti-rat p chain serum coupled to CNBr “activated’ Sepharose 4B (Wilchek et al., 1971) was used for this purpose. IgM was not detectable in any sample treated in this manner (sensitivity less than 1.6 pg/ml).

Response to Soluble or Particulate Antigens 1107

~epuration of milk IgA from IgG

Milk samples (1.1-2.5 ml) were applied to tandem pr~alibrated Sephadex G-200 columns (1.6 x 90 cm; 4°C) to separate the secretory IgA from IgG. Single radial immunodiffusion (Mancini et al., 1965) with monospecific rabbit anti-rat c1 or y chain reagent was used to identify IgA-containing and IgG-containing fractions (4 ml). No IgG was detectable in the IgA fractions to a sensitivity of less than 0.8 pg/ml and no IgA was detectable in the IgG fractions to a sensitivity of less than 2.5 fig/ml (Fig. 1). Peak milk fractions (72-158 iu.g/rnl IgA; 35-53 pg/ml IgG) were then used for studies of bacteriophage neutralization, inhibition of bacteriophage neutralization and the determination of neutraiization kinetics.

M. The minimum DNP-r_-lysine concentration that would produce a 50% reduction in bacteriophage neutralization activity was determined. The reciprocal of this molar concentration of the monovalent inhibitor can be used to represent the average relative affinity constant of an antibody preparation (Make15 et al., 1967; Sarvas & Make& 1970).

neutralization kinetics

Haptenated bacteriophage neutralization

The neutralization of haptenated 4X174 by either IgA or IgG antibody fractions from the same milk sample was compared after incubation for varying periods of time (up to 60 min) before addition of E. co&. The fractions were assayed as described previously (Hornick & Karush, 1969). Neutralization was expressed as the fraction of surviving bacteriophage.

Bacteriophage 4X174 (Miles Laboratories, Elkhart, IN) was substituted with DNP by incubation with 2,4-dinitro-benzenesulfonate (0.005% w/v) in 0.2 M Na,CO, (pH 11.0) for 24 hr at 4°C. Individual fractions of the gel filtered (G-200) milk samples were examined for relative antibody activity to DNP by means of a haptenated bacteriophage neutralization pro- cedure (Hornick & Karush, 1969). Briefly, a portion (IO-100 ~1) of each fraction was incubated for 1 hr at 37°C with a known number of DNP-#Xl74 bacteriophage in a total volume of 0.5 ml diluent (0.004 MPO,, 0.03 MNaCl, pH 7.5, with 1 mg/ml human serum albumin). Then 0.2 ml of a 3.5 hr culture of Escherichia coli C406 (originally provided by Dr. R. L. Sinsheimer) grown in tryptose phosphate broth (Difco Laboratories, Detroit, MI) was added to each reaction tube and incubated for an additional IO min at 37°C. After incubation, each reaction tube was mixed with 2.5 ml of nutrient agar at 52°C and plated onto a petri dish containing a base of solidified nutrient agar. The relative antibody activity was determined by the reduction in the number of lytic plaques seen on the petri dish after incubation at 37°C for 3 hr. The result was expressed in terms of percentage reduction in the number of plaques produced compared to controls which contained diluent instead of a milk fraction,

Radial immunodiffusion (RID)

Inhibition of haptenated bacteriophage neutrali- zation

Concentrations of IgA and IgG antibody in the unfractionated milk samples were determined in eluates following adsorption of a sample (400 ~1) with an excess of DNP-HSA- Sepharose 4B immunoadsorbent for 2 hr at room temperature. The amount of immunoadsorbent needed to remove all anti-DNP antibody was determined by bacteriophage neutralization analysis of the supernatant after adsorption. The amount selected for use was found to be in excess of that necessary to remove all the neutralizing activity from any milk sample examined. The adsorption procedure was followed by a mock elution with 0.1 M carbobenzoxy (CBZ) glycine (Sigma Chemical Co., St. Louis, MO) for a period of 30 min. DNP-r_-lysine, at a concentration of 2 x 10W3 M, was then used to elute the antibody (Eisen, 1964). These eluates were applied to radial immunodiffusion plates containing monospecific rabbit anti-rat IY or y chain reagents and were compared to appropriate dilutions of a standard milk sample (IgA, 1.78 mg/ml; IgG, 1.08 mg/ml). Subsequent elutions with 5 x 10V3 iM DNP-L-lysine and 3.0 M NaSCN were carried out to ensure that no residual antibody remained on the adsorbent. Normal rat milk or milk from sham-immunized rats was treated in the same fashion to determine if non-specific binding occurred. Only the fluids from immune animals demonstrated the presence of bound immunoglobulin.

DNP-r-lysine was added to the bacteriophage Enzyme linked immunosorbent assay (ELISA)

reaction mixture described previously. Con- A modified ELISA (Engvall & Perlmann, centrations ranged from 1 x 10m3 M to 1 x 10W9 1972) technique was utilized to confirm the

1108 D. S. COX rt cd

results obtained by the adsorption and elution procedure with milk samples and to further

determine the antibody concentrations in saliva and serum. We have previously used this technique for the quantitation of anti-DNP

antibody of the IgA and IgG isotypes in

secretions and serum (Ebersole et al., 1979b). DNP was conjugated to human serum albumin

(HSA) by incubation for 40 hr and the molar ratio determined spectrophotometrically (Eisen et al., 1953). The DNP-HSA conjugate (26.9

moles DNP/mole HSA) was bound at 0.67 pg/ml in 200 ~1 to the wells of microtiter plates (Flow

Laboratories, Hamden, CT) by incubating at 37’C for 3-5 hr. After washing, 200 ~1 of a dilution (at

least l/4) of the unknown sample was applied to the well and incubated (2 hr). After washing,

monospecific rabbit anti-rat x or 7 chain reagents were added and the plate was incubated at room

temperature ( 16 hr). After further washing, goat anti-rabbit IgG (Miles Laboratories Inc., Elkhart, IN) conjugated with alkaline phosphatase (Sigma

Type VII) was added to the wells, incubated (2 hr) and washed out. The substrate, p-nitro- phenylphosphate (200 pl), was then added and after 30 min the reaction was stopped by the

addition of 100 ~1 of 1 .O N NaOH. The reaction product, p-nitrophenolate, was then quantitated spectrophotometrically at 400 nm. The amount

of antibody in the sample could then be determined by comparison with various dilutions of a known standard run in the same assay. The standard was a milk sample from an immune animal characterized for antibody concentration by adsorption, elution and RID as

described above (22 pg/ml IgA, 25 pg/ml IgG).

RESULTS

Relative IgA and IgG antibody activity

Milk samples from rats that were immunized with soluble DNP-BGG antigen, particulate DNP-S. mutans antigen, or milk samples from sham-immunized rats were gel filtered.

Haptenated bacteriophage neutralization analyses were performed on the milk fractions after separation. Four representative elution profiles are shown in Fig. 1. Two major peaks of neutralizing activity which approximate the areas of elution of exocrine IgA and of IgG were detected. Anti-DNP antibody activity appears to be present in both the IgA and IgG populations of milk antibody. However, differences in the relative neutralizing activity of IgA and IgG

0.5 _

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I

Ia 1.0 -

I, I\

Milk IO

I I

Milk II

O-51 ,J$JJ 30 50 70 90

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100

50

100

50 5

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100 z. 2

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100

50

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Fig. 1. Gel filtration of clarified rat milk on columns of Sephadex G-200. Rats 1 and 3 were injected in the MGV with DNP-BGG and rats 10 and 11 were injected with DNP-S. mutans. Optical density at 280 nm and haptenated bacteriophage neutralization activity are represented. The IgA and IgG regions were defined by radial immunodiffusion

analyses (see text).

antibody can be seen in the representative profiles. In the milk from rats injected with

soluble antigen (samples 1 and 3), the peak neutralizing activity of IgG fractions was greater than the peak neutralizing activity of fractions in the IgA region of the elution profile. However, in animals injected with the particulate antigen (samples 10 and 1 l), peak neutralizing activity of IgA fractions was greater than that of fractions

in the IgG region. Milk samples from sham- immunized animals, gel filtered in a like manner, never demonstrated neutralizing activity above maximum plating error (10)1/,).

The difference between the milks of animals injected with soluble and particulate antigens was consistent in all 19 samples that have been examined and is emphasized by a comparison of

Response to Soluble or Particulate Antigens 1109

Table 1. Neutralizing activity of antibody from milk of rats immunized with soluble or particulate DNP-substituted

antigen

Neutralization Immunizing Animal Day of ratio*

antigen number collection” (IgA/IgG)

1 13 0.8 1 15 0.7 3 8 0.7

Soluble 3 15 0.5 DNP-BGG 3 16 0.5

3 20 0.9 6 8 0.7 P” 1-21 0.8

10 15 1.9 10 19 3.4 11 13 1.3

Particulate 11 14 1.3 DNP-S. mutans 12 8 1.4

12 13 1.4 13 19 2.4 14 8 1.2 14 13 1.2 16 8 1.7 16 13 1.3

“Days post parturition. bRatio of % neutralization of the tube of greatest

neutralizing activity in the IgA region and in the tube of greatest neutralizing activity in the IgG region of the elution profile.

‘Pool of milk collected from 9 rats.

the IgA/IgG ratios of the peak percentage neutralization (Table 1). Eight milk samples collected at various times post-parturition from 4 randomly chosen animals immunized with

soluble antigen were anaiysed after gel ~ltration. IgG antibody consistently demonstrated greater activity than IgA in every sample from these animals (IgA/IgG neutralization ratio 10.9). When samples collected from a single animal (number 3) were examined at various times during lactation, no appreciable change in the IgA/IgG ratio was observed, with the ratio remaining 50.9 in all samples.

Eleven milk samples from 6 randomly chosen animals immunized with particulate antigen were examined in a similar fashion (Table 1). In this case, IgA antibody consistently de- monstrated greater peak neutralizing activity than IgG antibody (IgA/IgG neutralization ratio 2 1.2). Again samples selected from the same rat (numbers 10, 11, 12, 14 or 16) demonstrated similar ratios and always remained 2 1.2. One possible explanation for the differences found between the soluble and particulate antigens might lie in differences in the absolute concentrations of IgA and IgG antibody in the samples examined. Therefore, the concen- trations of anti-DNP IgA and IgG antibody in milk samples collected from rats injected with soluble or particulate DNP antigen were examined.

Quantitation of ZgA and ZgG in milk

Individual milk samples were adsorbed with an excess of DNP-HSA Sepharose 4B

Table 2. ~on~ntrations of anti-DNP antibody in the milk of rats injected with DNP-BGG or DNP-S. gleans

Antigen Animal Day of

collection”

Ab concentration (Pg/mllb

IgA IgG IgA/IgG ratio

DNP-BGG 1 19 22 23 3 19 31 33 3 22 22 25 6 13 29 22 8 13 38 31 8 14 11 15

Mean + SE. 26+1 2422 1.1+0.1

DNP-S. mutans 11 8 11 8 13 10 22 5 13 23 16 8 14 9 40 20 15 8 37 10

Mean f SE.’ 27+7 lli3 2.4+0.5

n Days post parturition. b Mean of 2-6 replicate determinations. ‘The mean of the IgA or IgG antibody concentrations of both days of collection for

animals 3,8 and 13 was used to compute the overall means of antibody concentrations for the group of animals.

1110 D. S. COX et al.

adsorbent. After elution with 2 x 10m3 M

DNP-L-lysine, the IgA and IgG antibody

concentrations were determined by radial immunodiffusion (Table 2). The IgA antibody concentration in samples collected from animals injected with soluble DNP antigen was about the

same as the IgA antibody concentrations in samples collected from animals injected with the particulate DNP antigen (26 + 1 vs 27 f 7 pg/ml).

However, the IgG antibody concentration is much lower in animals injected with a particulate

antigen than in animals injected with a soluble antigen (11 f 3 vs 24 k 2 pg/ml). The differences

in antibody concentration that are seen among the rats injected with soluble antigen and those injected with particulate antigen are differences in the IgG antibody response. The modified

ELISA was used to verify these results. The same milk samples reported in Table 2 were assayed by ELISA to determine the relative concentrations

of IgA and IgG antibody. The ELISA antibody determinations were related to the respective RID concentrations. Linear regression analyses comparing these two methods gave correlation coefficients of 0.83 and 0.84 for IgA and IgG

antibody determinations, respectively. Both coefficients were statistically significant at the

0.01 level. Taken together, these results indicate that the IgG antibody response to particulate antigen, DNP-S. mutans, is approximately half of the IgA response, while responses of both isotypes are similar after injection of the soluble DNP-BGG antigen. It would appear that the

differences in IgG concentration observed could account for the relative differences in haptenated bacteriophage neutralization in the IgA and IgG regions of the elution profiles.

Relative avidity of milk antibody

Another explanation for the difference in relative peak neutralizing activity between IgA and IgG antibody produced by either soluble or particulate antigen could involve the affinity of the antibody. The relative affinity of an antibody can be determined by inhibition of the haptenated bacteriophage neutralization re- action with a monovalent inhibitor (Sarvas & Makela, 1970). DNP-L-lysine was added to the reaction mixture at concentrations varying from 1 x 10e9 to 1 x 10m3 M and the concentration that produced a 50”/, inhibition of bacteriophage neutralization is shown in Table 3. All IgG samples were inhibitable to 50% at concentrations which ranged from 1 x lo- 5 to 1 x lop9 A4 DNP-L-lysine. On the other hand,

Table 3. Minimum DNP-L-lysine concentration needed to inhibit neutralization by milk fractions to SO<‘/,

Antigen Animal Day” IgA (M) IgG (M)

DNP-BGG 1 13 1 15 3 8 3 16 3 20

DNPS. mutans 10 19 11 13 13 19 14 8

> 10-3 >10-3 > 10-3 >10-3 > IO_ 3

> 10-s > 10-3 > 10-s >lO_”

“Day of milk collection after parturition. b ND = not done.

no IgA samples could be inhibited to 50% by DNP-L-lysine at a concentration of 1 x 10 - 3 M.

However, in some cases, IgA neutralization could be inhibited by 5 x lo- 3 M DNP-L-lysine. This would indicate that either the affinity of the antibody in the sample is very low, or that the binding of the antibody to the DNP+X174 is more avid than the binding of the antibody to the

B

s f 04-

:. I3 Ol.,,... ,

0 5 10 20 30 40 50 60

Fig. 2. Neutralization of DNP+X174 by rat IgA (-A-) and IgG (--t) from the same milk sample. The ordinate is the fraction of surviving bacteriophage (P/PO) and 2 standard errors of the mean are enclosed in the brackets. (A). DNP-$X174 was incubated with rat IgA or IgG (Rat 3; DNP-BGG immunized) in 12 reaction tubes for each isotype. Three reaction mixtures were sampled in triplicate at the series of time intervals shown. (B). Milk IgA or IgG (Rat 7; DNP-BGG immunized) was incubated in 4 reaction tubes for each isotype. Each reaction tube was sampled at each time

interval shown.

Response tg Soluble or Particulate Antigens 1111

DNP-L-lysine. Kinetic studies of haptenat~ bacteriophage neutralization comparing milk IgA with milk IgG from DNP-BGG immunized rats further supported the latter suggestion (Fig. 2A). Although the IgA preparation contained less neutralizing activity at 60 min (53.6% + 3.5%) than the IgG (84.4% f 3. lx), the initial rate of neutralization after 10 min was greater for IgA (39.1%$-2.0x) than for IgG (28.6% + 5.4%). A second experiment (Fig. ZB), using a different milk sample, also showed a greater initial neutralization rate with IgA than IgG, although the eventual IgG neutralizing activity was slightly greater. These kinetic experiments have been repeated three times using 2 different sets of samples with comparable results.

Antimony in saliva czzd serum

The antibody response at a second secretory site (salivary glands), produced after local injection of soluble or particulate DNP antigen, was examined by the ELISA technique (Table 4). The results indicate that a single SGV injection of

Table 4. IgA and IgG antibody concentrations in rat saliva” produced following injection of soluble or particulate antigen

in salivary gland vicinity

Antibody concentration

Antigen Animal IgA(ng’ml)h IgG

DNP-BGG A 384 240 B 296 108 C 136 44 D 288 108 E 192 20 F 448 220 G 368 60 H 356 172 I 372 180

Mean + S.E. 316&33 128526

DNP-S. mutans J 208 8 K 572 80 L 572 44 M 128 4 N 160 52 0 48 16 P 484 9

Sham

Mean 4 SE.

17 18 19

MeanIt:S.E.

31Ok8.5 30+11

55 42 20 30 10 7

28+14 26klO

“Saliva samples are collected 11 days post injection. b ELISA determinations of antibody concentration were

performed using an antibody standard (milk sample 15) which was quantitated by RID.

0) ANTIGEN

ROUTE OF ANTIGEN ADMlNlS~rlON

Fig. 3. Serum IgG antibody concentration as determined by ELISA after either 2-MGV injections or a single SGV injection of DNP-BGG in CFA or DNP-S. rnz&mS in CFA. Also shown for the MGV route is a sham-immunized group (PBS in CFA). A group of non-immune normal rats is designated by ‘None’ as the route of administration. Brackets enclose 2 standard errors of the mean and the number of

animals in each group is shown above the brackets.

the soluble DNP-BGG antigen produced significant IgA (316 rt 33 ng/ml) and IgG (128 &- 26 ng/ml) antibody responses in the saliva. Similarly, injection of particulate DNP-S. mutans gave rise to significant IgA antibody responses (3 10 & 85 ng/ml) in saliva. As observed with milk, the IgA levels were virtually identical after administration of either antigen. However, IgG antibody in saliva was only seen in samples from animals immunized with the soluble antigen. After immunization with particulate antigen, IgG antibody levels were equal to IgG levels in sham-immunized animals, These data confirm the observations of milk antibody that the differences in the local antibody response elicited by soluble or particulate antigen are restricted to the IgG isotype.

IgG antibody was quantitated in serum samples collected from rats locally injected in the MGV or SGV with the soluble or particulate DNP antigens (Fig. 3). A significant amount of IgG antibody was found in the serum of rats injected with soluble antigen (MGV: 622+ 50 pg/ml; SGV: 337f 39 p&/ml). However, there was considerably less IgG antibody in the serum of rats injected with the particulate antigen (MGV: 193154 pg/ml; SGV: 155k46 pug/ml). The difference in response may be related to the localization of the particulate antigen at the site of inoculation.

DISCUSSION

We examined the antibody response to local

1112 D. S. COX et ut

injection of a hapten (DNP) which was presented on either a soluble (BGG) or a particulate (5‘. ~~~~~~) carrier molecule. The relative IgA,IgG antibody activity was always greater in the rats that were locally injected with the particulate DNP antigen. IgA antibody

concentration in either the milk or in the saliva was found to be the same whether the animal had

been injected with soluble or particulate antigen. However, a significantly higher IgG antibody concentration was induced by injection of

soluble antigen than by particulate antigen injection. In addition, examination of inhibition of bacteriophage neutralization and the kinetics of neutralization suggested that secretory IgA

antibody may bind more avidly to a multivalent antigen than IgG antibody.

In our experiments we have found that the relative IgA/IgG antibody activity was always greater after injection of DNP-S. mutans, the

particulate antigen. Differences between the two

antigens, other than the physical structure of the carrier, could potentially explain the results which were obtained. Differences that exist between the soluble and particulate antigens may involve the microenvironment present at the

point of DNP recognition by the lymphocyte. Although the vast majority of the DNP groups are bound to the s-amino group of lysine, DNP

can also conjugate to OH groups of tyrosine and SH groups of cysteine (Little & Eisen, 1967). The microenvironment of the DNP group, partic-

ularly the adjacent amino acid, can also determine the character of the immune response elicited (Campos-Neto et al., 1978). However, these constraints do not appear to influence this

system since we have obtained identical results using soluble and particulate antigens with the DNP-hapten contained in the same microen- vironment, i.e. DNP-BGG and DNP-BGG-S.

mutans (Cox & Taubman, 1980). Although the immunizations in our

experiments were controlled in order to ensure that the same number of DNP groups would be administered with either antigen, the density of the DNP haptenic groups on the surface of the carrier may have been different on the particulate than on the soluble antigen. Various investigators have suggested that increasing the density of haptenic determinants on the same carrier seems to result in an increase in the IgM/IgG antibody ratio (Make& et al., 1969; Make& et al., 1971; Kontiainen, 1971; Naor et al., 1974). It has been suggested that the relative elevation of the IgM/IgG antibody ratio may lie

in the degree of thymus independency of the antigen (And, 1971) and therefore in its ability to stimulate the generally thymus independent IgM response.

Other investigators (Furuichi rt al., 1976) have

shown that the thymus independent character rather than the particulate nature of the antigen

(DNP-lipopolysaccharide from E. r.nfi vs DNP-E. co/i) can account for the stimulation of a relatively T lymphocyte independent antibody response. On the other hand, the secretory IgA antibody response, with which we are dealing,

has been shown to be quite T cell dependent (Ebersole et al., 1979~). Therefore, the presumptive T independent nature of DNP-

S. rn~t~~~ antigen, based on possible differences in density of DNP haptenic groups, cannot

account for the elevated IgA/IgG ratios seen in our experiments.

l‘he difference in the physical structure of the carriers may account for the differences observed

in antibody response. The most obvious difference in the nature of the carriers lies in their soluble (DNP-BGG) vs particulate (DNP- S. mz&m.sf character. Investigations have indicated that, in general, particulate antigens induce a greater local immune response than do

soluble antigens (Strannegard 81 Yurchison, 1969; Waldman CI: al., 1970: Ebersole & Molinari, 1978). The difference in response

observed between soluble and particulate antigens may be related to a greater localization of the particulate antigen at the site of

inoculation, as has been suggested by others (Waldman et at., 1970; Ebersole & Molinari, 1978). If the soluble DNP-BGG is able to freely

pass into the circulation while the particulate antigen remains localized, one would expect to see a serum antibody response to the soluble antigen caused by stimulation of more distant lymphoid tissue. The particulate antigen, however, would not gain such access to the peripheral circulation and might remain in the vicinity of the site of injection. As a result, the particulate antigen would produce less serum antibody. This would seem to be the case in our studies where serum IgG antibody was always higher after local immunization with soluble antigen. Furthermore, examination of salivary antibody after the 2 MGV injections with DNP-BGG (Cox et al., 1979) demonstrated the presence of IgA and IgG antibody. No antibody of either isotype was detected in saliva after the MGV regimen with DNP-S. mutans. Thus. only the soluble antigen may gain access to

Response to Soluble or Particulate Antigens 1113

immunocompetent lymphoid tissue in the gut (Craig & Cebra, 1971), in other mucosal associated lymphoid tissues (Bienenstock et al., 1978) or in the salivary glands (Gray, 1973).

The differences observed between the soluble and particulate antigens may also be explained on the basis that IgA in milk and rat saliva is largely locally synthesized (Hochwald et al., 1964; Hurlimann & Darling, 1971), while the IgG may be serum derived, as has been shown in rabbits injected with antigen in CFA (Hurlimann & Lichaa, 1976). Since the IgA antibody is locally synthesized, one might expect similar IgA antibody responses to be produced in the respective secretion due to a similar deposition of soluble or particulate antigen at the injection site. However, since the soluble antigen may be better able to stimulate serum antibody and since the IgG in the external secretion can reflect the serum antibody level, one might expect an increased level of IgG antibody in the secretions of animals injected with the soluble antigens as we found.

The relative avidity of the antibody population for DNP-#Xl74 was determined by inhibition of haptenated bacteriophage neutralization. Theoretically, the average relative affinity constant for an antibody population can be determined using this technique (Makehi ef al., 1967; Sarvas & Make%, 1970) and is expressed as the reciprocal of the molar concentration of inhibitor needed to produce a 50% reduction in antibody activity. However, the concentrations of DNP-t-lysine required to inhibit the samples of IgA antibody to 50% were in excess of 1 x 10e3 M (Table 3). Therefore, the IgA antibody was either of very low affinity for the antigen (less than 1 x 10” l/IM or the avidity of the IgA antibody for the haptenated bacteriophage was greater than the avidity for the monovalent DNP-L-lysine. Since a relative average affinity of IgA antibody of less than I x IO” l/M is inconsistent with immunoglo- bulin being antibody (Pauhng, 1940), and since we have shown that this IgA is antibody (Fig. 1, Table 2), we believe that inhibition was impeded by a more avid binding of the IgA antibody for the haptenated bacteriophage than for the inhibitor. We have, in fact, found that the avidity of secretory IgA antibody is probably greater for the haptenated bacteriophage than IgG antibody by examination of the binding kinetics (Fig. 2). The initial rate of binding for IgA antibody was greater than for IgG antibody from the same secretion, although the total neutralization capacity of the IgG antibody was greater than

that of IgA. Furthermore, previous studies (Taubman & Genco, 1971; Montgomery, 1974) have indicated that the affinjty of rabbit IgA antibody is the same or even greater than IgG antibody from the same animal. It is therefore unlikely that the IgA antibody affinity in the rat would be as much as 106-fold less than the IgG antibody affinity from the same secretion. Although the IgG antibody was always inhibitable to the 50% point in our studies, it is still possible that the IgG antibody may have a greater avidity for the DNP-+X 174 than for the DNP-lysine, but that the difference is simply not as great as with the IgA antibody.

It has been suggested that most of the combining sites of rabbit colostral IgA antibody bind to a single molecule possessing multiple identical antigenic determinants as opposed to cross-linking two or more such molecules. This property, termed monogamous polyvalency, was proposed to account for the lack of precipitation of polyvalent group A streptococcal polysac- charide by IgA antibodies with a valence of 4 (Taubman & Genco, 1971). Newcomb & Sutoris (1974) showed that IgA bound to an immunoadsorbent would not cross-link with the same polyvalent antigen presented in soluble form, again suggesting the possibility of monogamous binding. We believe the expe~ments reported in this paper further support the concept that milk IgA can exhibit monogamous polyvalency. If such were the case, given a multivalently substituted antigen (DNP+X174) and sufficient flexibility in the antibody molecule, it is possible that two or more Fab regions could bind to the same multivalent antigen molecule or particle. Such an association between the antibody and antigen results in an association constant that is far in excess of the association constant that exists between a single Fab region and a single determinant (Hornick & Karush, 1972). This explanation would be entirely consistent with the inability to inhibit IgA antibody mediated DNP-#X174 neutrali- zation by monovalent DNP-lysine.

Although IgA is the most prominent Ig in secretions, total antibody concentrations are low (in the order of 10-7-10-1’ M) as compared to approximately 10 -6 M in serum. Enhanced avidity due to monogamous polyvalency may be important in protective interactions with bacteria and viruses where multiple identical epitopes are present. Indeed, SIgA antibody from cervicovaginal mucus has been found to immobilize ~~~r~o~t~~ without clumping and to

Ill4 D. S. COX et ui.

immobilize more efficiently than IgG antibody Research (Edited by Eisen H.) Vol. 10, pp. 94-102. Year

(Corbeil et al., 1974). Furthermore, these high Book Medical Publishers, Chicago.

avidity SIgA bacterial interactions may be quite E&en H. N., Belman S. & Carsten M. E. (1953) The reaction

of 2, 4-dinitrobenzelle-suffoni~ acid with free amino important in inhibiting aspects of bacterial

infection associated with dental and other diseases.

Acknowk~d~en2e,zts--The research upon which this publication was based was supported by a National Research Service Award DE-05050 (tdfi.S.C.), grant DE-04733 from the National Institute of Dental Research and bv Public Health Service Career Development Awards DE&024 (to D.J.S.) and DE-00075 (to J.L.E.). The authors would like to express appreciation for the help of Dr. V. J. Iacono.

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