Characterization of contrasensitizing antibodies

CELLULAR

IMMUNOLOGY

11,

272-285 (1974)

Characterization ALFRED Webb-Waring

J. CROWLE, Lung

of Contrasensitizing KUNIO

Institute, School

C. C. Hu, AND YUTAKA

YONEMASU,

Department

of Medicine, Received

Antibodieslf*

of Dewer, June

Microbiology, Colorado

University 80220

FUJITA

of Colorado

29, 1973

This paper describes experiments on the nature of humoral antibodies which suppress induction of delayed hypersensitivity in mice to purified proteins, i.e., contrasensitizing antibodies. These antibodies may be analogous to those responsible for afferent or early central immunosuppression in immunological enhancement phenomena and for similar forms of immunosuppression sometimes referred to as immune deviation, split tolerance, or preimmunization tolerance. CF-1 mice were sensitized with either human serum albumin or ovalbumin in incomplete Freuncl adjuvant, were injected with contrasensitizer-rich mouse antisera or fractions thereof, and ,subsequently were skintested to measure the relative capacities of antisera or their components to interfere with sensitization. Agar gel zone electrophoresis located contrasensitizing activity among the 71 globulins, and this identification was confirmed by DEAE cellulose chromatography. Fractionation on Sephadex G-200 dextran columns showed the inhibitory activity to be among molecules of approximately 170,000 molecular weight, and a combination of chromatographic procedures further indicated that this activity is due to specific antibodies closely associated with mouse IgF or 7s 71 globulin. Contrasensitizing antibodies also were found to be heat-stable, and although precipitin and passive hemagglutination antiserum titers often correlated well with immunosuppressive activity, neither was equivalent to it.

INTRODUCTION Immunological enhancement (facilitation) originally was defined as an antibodymediated specific suppression of tumor immunity (1). But more recently its scope has broadened, and it has been considered a centrally important form of homeostatic immunoregulation particularly affecting induction of delayed hypersensitivities (2). As studied by its interference with tumor immunity or allograft rejection, immunological enhancement is complicated by the many ways in which various humoral anltibodies can affect different tumors and grafts and their rejection, and by the fact that these foreign tissues which are to be rejected or enhanced are both inducers and targets of cellular immunity (i.e., delayed hypersensitivity). Thus, the relative effects of afferent, central, and efferent forms of immunosuppression tend to be too closely interwoven for experimentation to make ready distinctions among them. The intent of our investigations has been to make such distinctions beforehand by experimenting with a small and proportionally simplified facet of immunologic enhancement in which certain humoral antibodies can 1 Presented in par,t at the 53rd Annual s Supported by USPHS AI09867-03.

Meeting

272 Copyri ht All ng.i!its

1974 by Academic Press, reproduction in any form

of ‘FASEB,

April,

1969.

CONTRASENSITIZIh-C

ASTIIlODIES

‘773

suppress early events in lthe induction of delayed hypersensitivity to purified protein antigens in the mouse. Previously, we have shown that this immunosuppression is antigen-specific and is due to humoral antibodies (3). We have presented evidence that it acts in afferent or early central inhibiltion, and we have suggested that the immunosuppressive or contrasensitizing antibodies belong to a certain immunoglobulin class in the mouse, perhaps a 7s yl globulin (4). The results which we present here confirm and extend these findings, indicating that these antibodies are heat-stable 7S globulins of yl electrophoretic mobility most closely associated, among the known classes of mouse immunoglobulin, with 7S yl (5) or TgF (6, 7). MATERIALS

AXD

METHODS

An&n&. CF-1 female mice, 8-12 weeks of age, were used in groups of 10. These were purchased from Carwortb Farms (Portage, Michigan) and maintained on commercial mouse food and water. Albino female rabbits purchased locally were used for making various antisera. Antigens. The antigens used for studying contrasensitizer were crystalline chicken ovalbumin (OVX) and crystalline human serum albumin (HSA) , the first purchased from Nutritional Biochemicals Corporation (Cleveland, Ohio) and the second from Miles Laboratories (Kankakee, Illinois). E.t-perimcntnl ~zodel. The capacity of an antiserum or its antibodies to suppress induction of delayed hypersensitivity was measured in mice as described previously (4, 8). Briefly, mice nere sensitized on days 0 and 7 (9). Mice in groups receiving an antiserum were injected intraperitoneally with 0.1 ml volumes of 10C;b antiserum, diluted with physiologic phosphate buffer, on days O-4 and 7-11. All mice in an experiment were skin-tsted at 3, 5. and 7 weeks on opposite flanks with 0.02 ml volumes of 0.1% antigen in physiologic saline, and reactions were rpad at 3 and 24 hr indicating, respectively, Arthus and delayed hypersensitivities (4, 8). Prodzection of co?ztrasensiti,ner-rich n&seru~~, A procedure, described pseviously (4), has been standardized to high reproducibility. Sensitized mice are injected intraperitoneally on 3 succeedin g days with 0.1, 1.0, and either 1.0 mg or IO mg quantities of the antigen in 0.2 ml volumes of physiologic phosphate buffer. and 6 days after the third injection they are bled out. Their sera are harvested, pooled, and stored at -20°C until needed. An antiserum was considered acceptable when it could reduce induction of delayed hypersensitivity in mice trea’ted with it as described in the “experimental model,” above, bv_ SOS or more as comp;lred with control mice sensitized but not treated with it. /f vtibody fifratiow. Antibodies detectable by passive hemagglutination wcrc quantitated with tanned, formalinized sheep erythrocytes sensitized with antigen (10). Their titers are expressed as the reciprocal of the highest in a series of doubling dilutions ,to produce hemagglutination. Precipitins were titered by two different techniques. In most of the experiments, a series of doubling dilutions of an antiserum mere tested by a template double immunodiffusion technique (11 j. The reciprocal of the greatest dilution forming ;I visible band of precipitate with antigen is the titer recorded. In one experiment, testing contrasensitizer for heat stability, a new ( I 3) reversed one-dimensional single electroimmunodiffusion ‘test was used. This test is superior to the previously

274

CROWLE

ET

AL.

used doubIe immunodiffusion test in several respects, notably in measuring precipitin titers in continuous scale and in readily detecting differences of as little as 2 percent in these ti,ters (12). The test is performed on a double-width microscope slide in 1.5% agar in pH 7.4 phosphate buffer of ionic strength 0.05. Each of a dozen wells near the anodic end of the slide is filled with a constant volume, estimated to be 0.4 ~1, of 10% antiserum being testted, and 0.003% antigen is allowed to diffuse for 7.5 min at 24°C into the cathodic two-thirds of the slide by a method originally devised for two-dimensional electroimmunodiffusion (13). Then the excess antigen is rinsed off, electrophoresis is performed for 2 hr at 5 V/cm, the plate is washed and stained for proteins, and the height of resulting chevrons of precipitate is measured by projection-magnification. In the present paper titers are expressed in ratio to a coatrol antiserum. Each test antiserum was titered in triplicate, and the reported values are means of the three individual measurements. Scheidegger’s (14) micromethod of qualitative immunoelectrophoresis was employed, and a microtemplate method ( 15) was used for qualitative double immunodiffusion analyses. Antisera for analyzing mouse sera and immunoglobulins by these tests were prepared in our laboratory, usually by routine procedures. Some special methods are described in the Results section. Mouse homocytotropic anltibodies were quantitated by passive cutaneous anaphylaxis tests, heat-stable being distinguished from heat-labile antibodies by the empirical test of challenging mice with antigen in 2.5-3.5 hr for the former and 24 hr for the latter (16). Results are reported as reciprocals of greatest antiserum dilutions causing specific blueing. Other procedures, Preparatory zone electrophoresis was performed in 2% agar dissolved in pH 8.6 barbital-acetate buffer of ionic strength 0.025 and cast 1 cm thick on a 15.5 x 10 cm silicone grease-polished glass plate. Electrode vessels were filled with the same buffer bult at double ionic strength. An origin 0.5 cm wide and 13 cm long was cut in this gel and was filled with 6.7 ml of 1 :l mixture of an antiserum, previously dialyzed against the electrophoresis buffer, and 2% agar, both mixed at 52°C immediately before casting. The site of the origin is indicated in Fig. 2. Eleotrophoresis was performed at 7.5 V/cm for 4 hr and 40 min at 4°C and during this time the surface of the agar gel was covered with Saran Wrap. Antiserum constituents were eluted from sections, cut from the resulting electropherogram, by freezing and thawing the agar gel and expressing fluid from iit through a fine mesh wire screen disc in the bottom of a large syringe. Conditions for chromatographic fractionations are described in appropriate figure legends below. Protein concentrations were measured with a Coleman-Hitachi double beam ultraviolet spectrophotometer, either in relationship to a standard prepared with bovine serum albumin or directly by optical density at 280 nm. RESULTS Electrophoretic location of contrasensitizer. Immunoelectrophoretic comparison of contrasensitizer-rich antiserum with normal mouse serum revealed the development pattern of several “new” antigens, but no change in the immunoelectrophoretic was so prominent as intensification of the IgF arc of precipitate. This is illustrated in Fig. 1. Previous experiments have indicated that contrasensitizer is an antibody (3, 17)) and this immunoelectrophoretic change suggested that the antibody might be a yl globulin. Therefore, mouse antisera rich in this immunosuppressive sub-

FIG. 1. Iln17lul70electrop~~oretic contrasellsitizcr rich mouse Cathotlc to the left.

serum

comparison (lo\vrr

of normal mouse serum (upper pattern) showing intellrification of

pattern 1 n-itI1 IgF (arrow ),

stance were fractionated by agar zone elecStrophoresis, and eluates from different parts of the resulting electropherograms were tested for immunosuppressiveness. For the first of two such experiments reported here, the electropherogram ux divided into large sections correspondin g to the major serum components. The results and some essential experimental details are recorded in Table 1. These results indicate that contrasensitizing activity centers in the /3 zone; there was Itone ill the y region of classic zone electrophoresis. A second fractionation was effcctc~l similarly but improved by using narrower cuts for better resolution, and by supplcmenting i~z V~ZYI assay for immunosuppressiveness with immunoelectro~)hor~tic analyses of each eluate for mouse serum constituents. A barbital-acetic acid buffer MYISused for this fracltionation to improve resolution of the ,13-y region, The results,

Group

Treatment 3 12’tYks I_A

fI~l,cr.iensitivit.y 5 \Veeks ___-

II 0

0

0

IO0 0

100 111

0

100 100

80 GO

78 20 100

0 10 x0

at : t \\(Ykh

.A

1)

.\

I)

LO II)0 0 0 100 100 78 40 100

0 90 0 (I 00 70 I1 II 00

ho IO0 50 XII 100 IO0 100 X0

I) 100 0 0 101) 50 .so ‘I,

IO0

01)

‘I Group 1 mice rereived only skin tests at 3, 5, and 7 weeks; positive control Group 2 mi(,c ~verc sensitized on days 0 and 7 and skin-tested. Succeeding groups were sensitized and in addition \VE~C injected intraperitoneally with 0.1 ml volumes of antiserum or elcctrophorctic fraction\ thcrcllt’ ;LS indicated on days 0 through 4 and 7 through 11. Each fraction MXS adjllstcd to c.ontain aplx-oximately the same amounts of its constituents as 1.55, whole antiserum, and whole anti+rlrlll itself was used at 1.5:,. “A” and “11” indicate percentages of nlicc dcv~lnpi~~g d\rthrl> c\~l~l d~~l.~ycd hypersensitivity skin reactions.

276

CROWLE

Electrophoretic

ET

AL.

froctlons

2. Immunoelectrophoretic nature and agar zone electrophoretic location of contrasensitizing activity for delayed and Arthus hypersensitivities. Results shown are from 7 and 5 week skin tests, for delayed and Arthus reactions, respectively, because these showed the best resolution of immunosuppressive activities in the eluates. Numbers along the abscissa indicate successive sections of the agar electropherogram from which eluates were tested, and the immunoelectrophoretic diagram is a composite prepared from immunoelectrophoretic analyses of each eluate individually. The large black arrow and “0” indicate the location of the origin of the antiserum electrophoresed. FIG.

diagrammed in Fig. 2, indicate that greatest contrasensitizing activity is in the anodic portion of the yl globulin region. Whether the mobility of immunosuppressive substance affecting Arthus sensitization differs from that suppressing induction of delayed hypersensitivity, as Fig. 2 modestly suggests,remains to be determined by future experimentation ; it was not pursued further in these experiments. Reversed absorption of contrasensitizer. To better identify contrasensitizing antibodies with some class of immunoglobulin by preparing precipitins against its class and using these for reversed absorption and specific depression of contrasensitizing activity in a whole antiserum, three groups of rabbits were immunized with different doses of the substance semipurified by agar zone electrophoresis. The first group received 0.8 mg protein equivalent in 1 ml of water-in-oil emulsion on day 0, was boosted on days 27 and 28 with 9 mg quantities in 1.5 ml volumes of alum precipitate (18), and was bled out on day 34. Animals in the second and third groups received the same regimen of injections, but only 0.01 and 0.0001 of the quantities of eluted protein equivalent used in the first group. Immunodiffusion and immunoelectrophoretic analyses of the resulting antisera indicated that those from the first group detected up to 6 different antigens in the eluate but those from the second detected only one, as illustrated in Fig. 3. Reactions of sera from the third group were marginal. Reversed absorption Itherefore was attempted with a pool of two sera from the second group of rabbits.

CONTRASEN~ITIZIXG

ANTIHODIES

277

These antisera had been prepared by immunizing the rabbits with a concentrate of contrasensitizer specific for HSA. Since this class of antibodies probably would be the same regardless of which antigen ilt was manufactured to recognize, reversed absorbtion was performed upon a mouse antiserum with contrasensitizer to OVX instead of HSA. Six ml volumes of 4Oc/r, 491, and 0.4%, concentrations of this antiserum were mixed with equal volumes of undiluted rabbit antiserum to contrasensitizer. The mixtures were allowed to stand for 1 hr at 24”C, during which time no precipitate formed. After additional standing for 72 hr at 4”C, moderate, heav!-, and ver!’ heavy precipitation had developed in these three concentrations of mouse antiserum, respectivelv. The mixtures lvere centrifuged at 39,OOOc/ for 1 llr at 4”C, the upper four-fifths of the resulting clear supernatant fluids were harvested, and these were compared with equivalent concentrations (20, 2.0, and OL.?~L) (Jf unabsorbed mouse an’tiserum of the same batch for capacity to inhibit sensitization to OVA. The results, summarized in Fig. 4, indicate that although absorption was too inefficient to diminish the strong immunosuppressiveness of 207, mouse antiserum, it did appear to suppress that of the weaker 2Fj antiserum: the activity against sensitization of 0.2% antiserum was too weak to provide a definite indic:ition of reversed absorbtion, but a suggestion of this effect is given hy the 3 we& skin test results. The results of this experiment. then, tend to associate rnntr:lsensitizing activity with mouse IgF. M~lccul~r size of coutrascnsitizing anfibodics. This was determined by chromatographing mouse antiserum on Sephadex (i-200 destran and testing resulting cluates for ill ZGO activity. Results, shown in Fig. 5, indicate that contrasensitizer-rich antiserum contains considerably more middle-molecular weight (i.e., “7s” I com-

‘FIG. 3. Imtnunoelectrophoretic comparisons of rahhit antiserum try whole mwse serum ( 1 ), antiserum from a rahbit receiving the largest amount of antigen ( 2; see text ). and atltiserunl from a rahhit receiving 0.01 that quantity of antigen (3), when tested against agar z~,nc electroIhoresis-purified mouse serum components including contrasensitizer.

278

CROWLE

0

ET

--

A ,e+-

AL.

--

3

--

5 Weeks

after

--;“z

a----

-L

7

sensitization

FIG. 4. Reversed absorption of contrasensitizing activity from O.Z%, 2.0%, and 20% mouse antisera by rabbit antiserum to mouse contrasensitizer. The ordinate indicates the percentages of mice developing delayed hypersensitivity. The solid line indicates the course of sensitization in positive control mice not treated with any antiserum, and the broken lines indicate courses of sensitization for the various test groups, ‘solid data points representing unabsorbed antisera an.d hollow data points indicating absorbed antisera.

than normal mouse serum but that it is not different in content of the other two major varieties of molecules (i.e., “19.5” and “3.5S”). They also show that all contrasensitizing activity is confined to the 7s peak and, more specifically, to the descending limb of this peak. Thus, in size the immunosuppressive antibodies are a homogeneous population of approximately 170,000 molecular weight. Similar Sephadex G-200 chromatography of mouse antiserum rich in contrasensitizer to the antigen HSA instead of OVA confirmed these results. Distinctiom between yl and yZ globulins. Agar zone electrophoresis already had suggested that contrasensitizer is not a yz globulin, but since separation of yr from yZ globulins by this technique is poor, better data were sought by using DEAE cellulose column chromatography. Mouse antiserum with contrasensitizer to OVA was fractionated and pools of eluates were tested in viva for immunosuppressiveness and in z&o for passive hemagglutinins and for precipitins. The results of this experiment, summarized in Fig. 6, confirm that sensitization-inhibiting activity being studied in these experiments is concentrated in the yl rather than the ys globulin fraction. They also indicate that this activity did not correspond well for the antiserum that was fractionated with passive hemagglutinin titers although it did correlate well with precipitin tilters. Abselzce of correlation with precipitins or passive hewzagglutimins.To examine possibilities of using either precipitin or passive hemagglutination tests as indicaitors of potential contrasensitizing activity in mouse antisera, we reviewed data from a number of our experiments in which individual mouse antisera had been compared for titers of these three different antibody activities. The most significant ponents

CONTRASENSITIZING

27!)

ANTIBODIE.’

data are graphed in Fig. 7. There are examples, among these, of low contrasensitization glutinin

and high precipitin titers, of both being high, of both being high but hemagtiters being low, of the precipitin and hemagglutinin titers being high but and of hemagglutinin titer being high and both contrasensitizer titer being low, precipitin and contrasensitizing titers being low. Thus, neither of these ilz zitro tests can reliably measure the quantities in an antiserum of the imnlunosul,prcssi~~, antibodies that we have been studying. Further evidence that neither precipitin nor hemagglutinating activities themselves are responsible for contrasensitization, and also that there may be some species specificity to this latter effect, is presented in Table 2, showing results from a comparison of four rabbit antisera with a mouse antiserum i/z Z&W and ilz vitro. Although t\vo of the rabbit antisera were equivalent in prrcipitin and passive hemagglutin;~tio~~ titers to the mouse antiserum, neither these nor the other t\vo rabbit antisera exhibited immunosuppressiveness against induction of delayed hypersensitivit\comparable to that exerted by the mouse antiserum. Nea.G sk!Dilitg of cou2~ascnsiti~i~~g nrltibodics. Recently, there has been some intlication that antibndies detectable in mice by passive cutaneous anaphylaxis may be of enhancing (i.e.. immunosuppressive) type (I?,). Since one of the two l
/10

20

30

40 EllJtion

IO tube

20

30

40

number

ITIC:. 5. Sephadcx G-ZOO chromatographic fractionation of contrascnsitizcr rich antiserum in comparison bvith similar fractionation of normal mouse serunl, sho\viug relative conrelltratious of 19S, 7S, and 3.5s serum components and indicating where in the chromatogram of the antiserum rontrasensitizing activity is found by in vko tests. A 2 X 36 cm column \vas used \\-itI1 0.1 M NaCl mixed 9:l with 0.1 M pH 8.0 borate buffer as solvent. .A 1.7 ml volume oi st’rum was chromatographed, and 3 ml volumes of eluate were collected. Pools of tubes 8-11, 12 -15, 1619, 20-23, 24-27, 28-31, and 32-40 were tested in zGz*o. As intlicatetl, immunosul)l)res~i~~t~ activity was found only in the pool of tubes 20-23.

280

CROWLE

40 Contrasensitizer

Precipitation

20 -

-

Hemagglutination-

ET

AL.

30

40

r

-I-

+-

-

2+

-t

-

2+

-

-

3t

50

FIG. 6. DEAE chromatographic fractionation of contrasensitizer-rich mouse antiserum using 1 X 22 cm column, 2 ml of antiserum, pH 8.3 potassium phosphate-borate buffer, gradient elution with NaCl ranging from 0.015 M through 0.3 M, and collection of 3 ml samples. The smaller numbers along the abscissa indicate sample tube numbers, and the larger numbers the five successive poolings of these samples tested in &IO and in vitro for contrasensitizer, for passive hemagglutinins, and for precipitins. For simplicity in comparisons, the relative activity of fractions by each of these three different measures is indicated by an arbitrary - to 4+ scale. The relative locations of ?a and yI globulins were determined by immunoelectrophoretic analyses of the pooled eluates.

tested the susceptibility of ithis substance to heating and obtained the results shown in Fig. 8. Since these indicate that it is heat-stable, its immunosuppressive activity would not seemto be equivalent in mouse antiserum to heat-labile passive cutaneous anaphylaxis activity. As might have been expected, precipitins and the other variety of mouse passive cutaneous anaphylaxis antibody were both heat stable. Indeed, their titers may have been increased somewhat by heating. IndkiduuJ C&WSof the w&body. The data above pointed collectively to IgF (i.e., yl 7s globulin) as being closely associated with contrasensitizer. This evidence was reexamined in an additional experiment using chromatographic purifications. First, contrasensitizer-rich antiserum (for HSA) was absorbed with glutaraldehyde-insolubilized HSA to separate its nonantibody constituents from its antibodies which, in turn, then were acid-eluted from the insolubilized antigen (21). Most of the pool of eluted antibodies next was chromatographed on Sephadex G-200 dextran and then rechromatographed either on G-200 (to purify IgM globulin) or DEAE cellulose. In this manner the antibodies eluted from glutaraldehyde-insolubilized HSA were separated into fractions containing predominantly IgM, IgG, IgA, or IgF, according rto immunoelectrophoretic analyses. These separate fractions and a reconstituted pool of them were compared with the original unfractionated pool of antibodies and with the whole antiserum as well as with the nonantibody

COKTRASENSITIZING Anttbody

Correlations ___-

titer

Hemoggiulln!n

I

281

ANTIIK~DIES

I Precipitln

tfter

M449H

M436H Experiment

FIG. 7. Data from separate experiments in mice comparing titers of precipitins and passive hemagglutinins Gth contrasensitizing activity for different pools of mouse antiserum. Hemagglutinin and precipitin titers (double diffusion test) are indicated as reciprocals of highest antiserum dilutions reacting, and immunosuppressive activity is indicated by the percent of inhibition of the induction of delayed hypersensitivity as measured relative to positive control mice at the 5 R.eck skin testing. Numbers along the abcissa refer to individual experiments. portion of the anmtiserum for contrasensitizing activity in vk~. The various fractions were adjusted to protein content equivalent to that of each estimated presvl:t in the original antiserum. Results from this experiment are graphed in Fig. 9. They indicate that mo>t

contrasensitizing activity is associated with the IgF antibodies ; but they also suggest that purifying contrasensitizer may labilize it, for neither the IgF fraction n(or

Precipitill titer 3 \\‘c&s 1

2 .i 4 5 6

Rabbit Rabbit Rabbit Rabbit Mouse None

no. no. no. no.

1 2 3 4

128 32 128 32 128

40,000 5,120 80,000 5,120 80,000

60 00 00 60 10 100

5 l\‘Wkh 60 00 80 80 20 100

7 \\‘CC!Q~ 100 IOl) 100 100 70 100

lL Mouse groups indicated were used to detect contrasensitizing activity. In thczc, each antiserum was used at 10’;; concentration as described in Table 1. LJevelopment of delayed hypersensitivity is expressed as percent positive at 3, 5, and 7 weeks after sensitization. Precipitins MYTC’ titered by double diffusion tests as described in Materials and Method>.

282

CROWLE

ET

AL.

either pool of purified antibodies were as effective as whole, unfractionated antiserum. The nonantibody portion of the fractionated mouse antiserum had no inhibitory effect. DISCUSSION We found contrasensitizer-rich mouse antisera to exhibit abnormally intense IgF precipitin arcs in immunoelectrophoresis (Fig. 1). But this effect seems to be characteristic of hyperimmunized mouse serum (22), and since several antibody activities are found in IgF (2, 20), this observation alone could only suggest that one such activity mighlt be specific immunosuppression. Preparatory agar zone electrophoresis confirmed this suggestion by locating the peak of immunosuppressive activity in the anodic region of the yl globulins (Fig. 2)) and it provided evidence against association of this activity wi’th either of the two known mouse yZ globulins. Additional support for this identification was obtained by reversed absorption of contrasensitizer-rich antiserum with rabbit antiserum selectively reactive with mouse IgF, for this procedure diminished the antiserum’s immunosuppressivness as tested in z&o (Fig. 4). IgM could be absolved of contrasensitizing activity because among fractions of antiserum obtained by Sephadex G-200 dextran chromatography only the descending limb of the 7s peak was immunosuppressive (Fig. 5). Improved distinction between yl and y2 immunoglobulins was achieved by DEAE cellulose column chromatography (Fig. 6). With results closely resembling ithose of Chard (23) and of Kinsky et al. (19), this DEAE fractionation of mouse antiserum associated the inhibitory activity with yl rather than y2 globulins. Precipitins were also found in this region

PCP

Group

5 Minutes

I

I

50

100 Percent

FIG. 8. Comparative effects of heating at on titers of heat stable (S) and heat labile

Precipitin

1f.,

-L

1280

160

1.0

2560


1.1

5120

< IO

1.1

5120


1.3

5120


1 .o

positive

56°C for different periods of time, as indicated, (L) passive cutaneous anaphylaxis antibodies, of precipitins as determined by reversed electroimmunodiffusion, and of contrasensitizing activity. The latter is indicated in terms of inhibition of the development of delayed hypersensitivity retained by an antiserum as measured 5 weeks after sensitization in comparison with semitization in untreated control mice or mice treated with unheated antiserum. Heating quickly inactivated PCAI, antibodies but not PCAs, precipitins, or contrasensitizer.

CONTRASENSITIZING

ANTIBODIES

Recombnned

Nonant,body

Eluotc

5

3 Weeks

After

7

Sensltlzation

FIG 9. Inhibitory effects of various purified immunoglohulin and nonimmuuoglclbulin fractions of a mouse antiserum on induction of delayed hylwsensitivity as measured 3, j, a11d 7 weeks after sensitization. Strongest prolonged inhibition was effected by unfractionated antiserum, but purified antibodies and their IgF fraction also \2-ere inhibitory. Protein concrntrations of the various preparations were adjusted to equivalence by conlpari.m~ n-it11 their rchtivc estimated concentrations in the original unfractionatetl antiwrum.

of the chromatogram, whereas antibodies detectable by passive 17ell7agglutiliatior1 eluted slightly later than the precipitins and contrasensitizing antibodies. The identity of contrasensitizing antibodies was further elucidated by repeating chromatographic fractionations of antibodies eluted from insolubilized antigen and obtaining from G-ZOO Sephadex and DEAE columns antibody fractions predominating individually in IgM, IgG, IgA, and IgF. Among these only the IgF fraction exhibited contrasensitizing activity comparable with that of the unfractionated antibodies (Fig. 9). In several years of working with this simplified model of the afferent branch of immunological enhancement, we have used both precipitin and passive hemagglutination tests as convenient its vitro indicators of the potency of a mouse antiserunl to suppress induction of delayed hypersensitivity. and recently we have also ens.ployed passive cutaneous anaphylaxis tests for the same purpose because of evidence (unpublished work) that contrasensitizing antibodies may be cytotropic. However, w-e have shown here that neither precipitin nor passive hemagglutination tests can be relied on to detect these antibodies (Fig. 7 j. This was confirmecl as well by our inability to detect contrasensitizing activity in mice for rabbit antisera of good precipitin and passive hemagglutination titers (Table 2). One of two

284

CROWLE

ET

AL.

known varieties of mouse passive cutaneous anaphylaxis antibody is heat labile ( 16) ; since we have demonstrated here that contrasensitizer is heat stable (Fig. 8)) it probably is not equivalent to the immunobiologic effects of heat-labile anaphylactic antibody. Several attempts to identify classes of humoral antibodies which have enhancing or contrasensitizing activities in more complex experimental systems than ours (i.e., tumor and allograft enhancement) have been reported. Most reports agree, as we find, that some classes of immunoglobulin are more effective than others (2, 24-28), that therefore something more than just complexing with antigen is required for enhancing antibody to interfere with sensitization (2), and ithat its Fc fragment probably plays an important role in its biologic activity (2, 24). There also seems to be general agreement with our finding here that the immunosuppressive antibodies are not IgM (2, 25, 29, 30), and there is some evidence against their being IgA (30). Our results confirm reports that although precipitin and, especially, passive hemagglutination titers frequently may parallel immunosuppressive activity, they are not equivalent to it (2, 26, 30). Although most laboratories agree that immunosuppressive antibodies are 7S, they do not agree on whether these are yl or y2 globulins (cf. 2, 26). This uncertainty probably reflects the complexities of immunologic enhancement itself and resultant differences in the fine aspects of the various experimental models being employed. Our results may be useful in helping decrease this uncertainty by providing definitive information relating selectively to initial development of delayed hypersensitivity. Thus, they suggest that reports implicating 7s yl globulin antibodies in immunologic enhancement (e.g., 31) describe results of experiments focusing on the early stages and characteristics of enhancement analogous to what we have examined in our experimental systems, i.e., afferent or early central inhibition. In addition to classic immunological enhancement, our data probably also apply to related forms of immunological unresponsiveness selectively interfering with delayed hypersensitivity to nonliving antigens, like proteins and contactants, and frequently referred to as immune deviation, split tolerance, and preimmunization tolerance (cf. 2, 3, 32, 33). The results reported here should not be construed as having identified contrasensitizing antibodies with mouse IgF. They only indicate a close association between the two. Thus, contrasensitizer could be a subclass of IgF, an IgF recognizing antigen in a certain way (34) or acting in special fashion biologically (16, ZO), or a different class of immunoglobulin with physicochemical characteristics closely resembling IgF but not readily distinguishable from it by the analytic techniques so far employed. Additional refined experimentation therefore is required to identify #this class of immunosuppressive antibody definitively. REFERENCES 1. 2. 3. 4. 5. 6.

Kaliss, N., Ann. NY Acad. Sci. 129, 155, 1966. Voisin, G. A., Progr. Allergy 15, 328, 1971. Yonemasu, K., and Crowle, A. J., Immunology, 25, 541, 1973. Crowle, A. J., and Hu, C. C., J. Allergy 43, 209, 1969. Fahey, J. L., Wunderlich, J., and! Mishell, R., J. Exp. Med. 120, 223, 1964. Herzenberg, L. A., and Warner, N. L., In “Regulation of the Antibody Response” Cinader, Ed.), pp. 322-348. Chas. Thomas, Springfield, Ill., 1968.

(B.

7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 2.7. 24.

25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

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