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Macroglobulin antibody response to Ascaris suum infection. A comparison of precipitating antibody in rats and mice
EXI'ERIXUINTAL
21,
PARASITOLOGY
Macroglobulin
391-402
(1967)
Antibody
A Comparison
of Precipitating
Catherine Departments
Response
A.
of
Crandall
Pathology
U&e&y (Submitted
of
for
Antibody and
and Florida,
to Ascaris
publication,
infection.
in Rats and
Richard
Microbiology, Gainesville,
suum
Mice’
B. Crandall
College of Medicine, Florida 32601 25 April
1967)
CRANDALL, CATHERINE A., AND CRANDALL, &CHARD B. 1967. Macroglobulin antibody response to Ascaris suum infection. A comparison of precipitating antibody in rats and mice. Experimental Parasitology 21, 391402. Precipitating antibody was detected by micro-Ouchterlony slides in the sera of mice and rats infected with one per OS dose of embryonated Asca& suum eggs. Characterization of the antibody by gel filtration through Sephadex G-200, sensitivity to 2-mercaptoethanol, ultracentrifugation in a sucrose gradient, and immunoelectrophoresis revealed that the principal precipitating antibody in mice was a macroglobulin and in rats was a 7S globulin. The antigen( s ) used to detect the precipitating antibodies was an extract of lyophilized adult ascaris. The extract had a high carbohydrate to protein ratio and the antigen activity was heat stable. Activity was not destroyed by pronase, lysozyme, mild acid, or alkaline hydrolysis, but periodic acid oxidation decreased activity. The antigenic activity was not separated by 5% trichloroacetic acid precipitation. The precipitating antibody in the mouse induced by A. suum infection cross reacted with extracts of several other helminth species. This precipitin could not be related to the human isohemagglutinins or heterophil antibody by the tests employed.
Larval ascarid infections in man (visceral larva migrans) induce extensive host reaction including a hypergammaglobulinemia with an increased level of IgM (Huntley et al., 1965). Study of the immunoglobulin classes in the antibody response to larval ascarid infections of experimental animals would be of interest in determining whether a marked IgM response is characteristic of this type of infection. The purpose of the present investigation was to study the precipitin response in larval Ascaris suum infections in two rodent species with particular reference to production of macroglobulin antibody.
MATERIALS
Preparation
AND
METHODS
of Antigens
Adult A. suum worms were obtained from a local abattoir. They were homogenized in a Waring Blendor containing demineralized water, then lyophilized, and stored at -20°C. The lyophilized material was extracted with ether at 4°C and the residue dried on filter paper in a Biichner funnel with vacuum. This delipidized material was extracted in 0.01 M sodium phosphate buffered water pH 7.6, using a mortar and pestle for grinding 2-3 times and centrifuged at 10,000 rpm; the residue was discarded and the supernate fluid was stored at -20°C. Five ml samples were gel filtrated on a column (2.5 X 60 cm) containing Sephadex G-100 (Pharmacia
‘This investigation was supported in part by Public Health Service grants AI-03212 and AI05345 from the National Institute of Allergy and Infectious Diseases. 391
392
CRANDALL
AND
Fine Chemicals, Inc., Piscataway, N.J.) equilibrated in the phosphate buffer described above. Five-ml portions were collected at a flow rate of 10 ml/hour. Two protein fractions were obtained and the first protein peak contained the antigenic activity. It was pooled and pressure dialyzed against the phosphate buffer (see above). This crude antigenic extract was stored at -20°C or autoclaved (20 psi for 15 minutes) and stored at 4°C. The autoclaved extract was the material used for all antigenic characterization studies. An extract of hatched second-stage A. suum larvae was prepared as described above. Cuticular antigen was obtained through the courtesy of Dr. A. L. L. Moore, Department of Microbiology, University of Florida. An additional somatic antigen was prepared by homogenizing ascarid tissues in a Waring blender after the cuticle was removed. After centrifugation at 10,000 rpm, the supernate fluid was used as an antigen. Dirofkiu immitis, adult and larval Nippostrongylus bra&en&s and larval Trichinella spiralis extracts were made by grinding the lyophilized helminths in a mortar with a small amount of saline or phosphate buffered water (pH 7.6). In some cases, sand was added to the morter to aid in the grinding and extraction process. After centrifugation, the supernates of the extracts were stored at -20°C. Characterization
of the Ascaris Antigen
Precipitation with trichloroacetic acid. One volume of 10%trichloroacetic acid was added to one volume of antigenic extract. After centrifugation, the supernate fluid was removed, and the precipitate was washed twice with demineralized water, redissolved in 0.1 N NaOH, and both the precipitate and supernate were dialyzed against phosphate buffer. Acid and alkaline hydrolysis. One ml of the antigenic extract was adjusted to ap-
CRANDALL
proximately pH 1 with 0.1 N HCl and placed in a boiling water bath for 20 minutes. It was neutralized with 0.1 N NaOH and dialyzed against phosphate buffer. For mild alkaline hydrolysis, 0.5 ml of the extract was adjusted to pH 9 by the addition of 0.5 N NaOH, placed in a boiling water bath for 15 minutes, then neutralized with 1 N HCl. Severe alkaline hydrolysis was done by boiling 2 ml of the extract in 30%KOH for 11 minutes, then neutralizing with concentrated HCI before testing for activity. Periodic acid oxidation. In one experiment, 1 ml of the extract was adjusted to pH 3 with 0.1 N HCI, 50 mg of sodium metaperiodate were added for 24 hours at room temperature in the dark. In another experiment, 50 mg of periodic acid were added to 1 ml of the antigenic extract for 24 hours at room temperature. The pH was adjusted to neutral with 0.5 N NaOH in both experiments before testing for activity. Lysozyme. The ascaris extract was dialyzed against 0.07 M sodium phosphate buffer containing 0.0625 BI NaCl pH 5.85. One mg of lysozyme (Worthington Biochemical Corp., Freehold, N.J.) was added to 1 ml of the extract containing 1: 10,000 merthiolate and incubated for 20 hours at 37°C. A control, 1 ml of extract with merthiolate without lysozyme, was also incubated. Pronase. The method of Nomoto et al. (1960) was used. One PU of pronase (Calbiochem, Los Angeles, California) in 0.1 ml of phosphate buffer (sterilized by filtration through a millipore filter) was added to 2 ml of autoclaved extract and incubated at 40°C for 20 hours. The material was dialyzed against phosphate buffer and tested for antigenic activity. Protein and carbohydrate determinations. Total proteins were determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard, and total hexoses were measured by the an-
MACBOGLOBULIN
ANTIBODY
RESPONSE
throne method described by Stewart-Tull et al. ( 1965), using dextrose as the standard.
TO
ASCARIS
INFECTION
393
Gel filtration of serum. Two to 5 ml of sera were filtr’ated on a Sephadex laboratory column (2.5 x 100 cm) containing Sephadex G-200 equilibrated in 0.1 M phosphate infection of Animals buffer, pH 6.8. Fractions corresponding to Embryonated A. suum eggs were ob- 19s or 7S (Flodin and Killander, 1962) tained as described previously (Arean and were pooled and concentrated by pressure Crandall, 1963). Mixed sexesof white Swiss dialysis against 0.15 M phosphate buffered mice of the ICR strain and rats of the saline, pH 7.5, to approximately the same Wistar stiain were purchased from the protein concentrations. In some casesit was Dublin Laboratories, Dublin, Virginia. The necessary to concentrate fractions further in mice weighed between 25-30 gm and the dialysis bags surrounded by Ficoll (Pharrats from 250-300 gm at the beginning of macia Fine Chemicals). the experiments. Female C&BIJs mice Reduction with 2-mercaptoethunol. Sera were purchased from Jackson Memorial in 0.5- or l-ml amounts, undiluted or diLaboratory, Bar Harbor, Maine. luted, were dialyzed against 0.1 M 2-merThe animals were inoculated with em- captoethanol (2-ME ) for 16-18 hours at bryonated eggs by stomach intubation and room temperature, then against 0.15 M bled by decapitation or severing the brach- phosphate buffered saline, pH 7.2 for 48 ial artery. Some groups of mice were hyperhours at 4%. Controls were treated identiinfected as described previously (Crandall cally except buffered saline was substituted and Arean, 1964). for the 2-mercaptoethanol. Sucrose-gradient determinations. UltraCharacterization of the Antibody centrifugations in a sucrose gradient were Gel diffusion and immunoelectrophoresis. carried out as described by Lindenmann Detection of the precipitating antibody was (l!=). done in micro-Ouchterlony slides containIsohemagglutinin and sheep red cell heing 0.8% Ionagar (Consolidated Labora- molysin determinations. Isohemagglutinins tories, Inc., Chicago Hts., Ill.) in 0.15 M in mouse sera were titrated with human A, phosphate buffered saline pH 7.5 ( PBS ) B, and 0 cells and inhibition of the human containing 1: 10,000 merthiolate. For im- isohemagglutinins was carried out with dimunoelectrophoresis, slides were prepared lutions of the ascaris extract described in using 0.05 M sodium barbital buffer, pH the first paragraph. The Jeme technique 8.6. (Jeme et al., 1963) was used to measure A rabbit antimouse immunoglobulin sheep red cell hemolysins from spleen cells serum was prepared by injection of an of mice infected with ascaris. Titers of antigen-antibody precipitate prepared at sheep cell agglutinins in mouse serum were equivalence in 0.01 M EDTA (ethylenedetermined with a 2.5% suspension of diamine tetraacetate ). The antigen-antiwashed sheep erythrocytes in a 1:250 dilubody precipitate was prepared with ascaris- tion of absorbed rabbit seturn in PBS. infected mouse serum and ascaris extract. The precipitate was washed three times RESULTS with PBS and suspended in Freund’s complete adjuvant. Four mg of protein per Antibody Response in Mice rabbit were injected subcutaneously in In an initial experiment, mice were inocmultiple sites. The rabbits were bled 1 ulated with lO,OOO-12,000 embryonated ascarid eggs and bled thereafter at intervals month after the injection.
394
CRANDALL
AND
from 8 to 27 days. The sera from 15 to 25 mice were pooled at each time interval and tested for precipitating antibody on microOuchterlony slides with ascaris extract. Antibody was detected in most of these serum pools. A single major precipitin band was seen and with many sera an additional minor band was present. Those sera producing a strong precipitin band were subjected to gel filtration on Sephadex G-200 and treated with 0.1 M 2-mercaptoethanol The major precipitin was detected only in the fraction corresponding to the macroglobulin peak eluted from the Sephadex column and the precipitating activity of the serum was sensitive to reduction with 2-mercaptoethanol. The minor precipitins detected by gel diffusion were not observed consistently after any treatment or dilution of the sera and were not further investigated. In sera from two groups of mice hyperinfected with ascarid eggs, the major precipitating antibody was also a macroglobulin as indicated by gel filtration and 2-mercaptoethanol reduction (Table I, Fig. 1) . These experiments were repeated with
CRANDALL TABLE I Characterization by Gel Filtration and bMerco.ptoethanol Treatment 05 Antibody in the Sera of Mice and Rats Infected with .Yscaris suunl No. of emhryonated eggs per animal Mice lo-12,000 N-12,000 lo-12,000 lo-12,000 Hyperinfecteh Hyperinfectedc Rata 17,000 Hyperinfcctedd
1 1 1 1
dose dose dose dose
1 dose
Bled days post-
Sephadex G-200 gel
inoculation
filt,rat,ion
i4
W
1.5 21 8 15
IgM IqM kM kM IgM
1:: 1 .:
IgG IgG
‘27
‘2-m+
IX Ti
a S = sensitive to 2-mercaptoethanol; R= resistant,. b Inoculated with 5,000 embryonated eggs given twice with a 2-week interval and challenged 4 months after the last infection with 10,000 embryonated eggs. c Inoculated with 9-10,000 embryonated eggs followed by ll-12,000 l-month later. d Inoculated with 12,000 embryonated eggs followed by 17,000 2-months later.
FIG. 1. Localization by gel filtration and sensitivity to 2-mercaptoethanol of mouse precipitating antibody to A. suum. Center wells A-C contain ascaris extract. A. Gel filtration of pooled mouse sera obtained 8 days after inoculation. S = whole sera; G = 7s peak from G-200 column; M = macroglobulin peak from G-200 column. B. Two-mercaptoethanol treatment of pooled sera: 1 is sera from 8-day infection; 2 is from 21-day infection; 3 is from 27-day infection. C. Identical sera to B not treated with %mercaptoethanol.
MACBOGLOBULIN
ANTIBODY
RESPONSE
another strain of mice, C5,B1JF, with essentially the same results. Further confirmation of the macroglobulin response in mice to ascarid infections was obtained by gel-filtration profiles, immunoelectrophoresis, and sucrose-gradient ultracentrifugation. A comparison of elution profiles from a Sephadex G-200 column of 2.5 ml of pooled normal mouse sera with pooled sera from mice 12 days after inoculation with 10,000 ascarid eggs revealed a marked increase in the initial (macroglobu-
TO
ASCARIS
INFECTION
395
from infected mice and comparison of the precipitin bands developed with ascarid extract and a rabbit antiserum to mouse immunoglobulins demonstrated that the precipitating antibody migrated as the macroglobulin, IgM (Fig. 3). Sucrose-
1.2
I.1
1.0
0.9
FIG. 3. Immunoelectrophoretic sera demonstrating antibody troughs contain rabbit anti-mouse serum or ascaris extract. Well 1 mouse sera. Wells 2 and 3 are mice infected with A. suum.
0.9
EE‘
07
b. c
06
I 8
05
i c
04
analysis of mouse in IgM. Serum immunoglobulin is pooled normal pooled sera from
gradient ultracentrifugation of pooled sera from an 8-day infection also revealed that the major precipitin was a macroglobulin. Detectable precipitin activity with ascarid extract was confined to the fastest moving protein peak in the gradient.
8 03
02
Antibody Response in Ruts 01
00
30
40
50 FRACTION
60
70
a0
so
NUMBER
FIG. 2. Comparison of mouse sera by gel filtration on Sephadex G-200. A-A pooled normal mouse sera. 0 - - - - Q pooled sera from mice infected with A. suum.
lin) protein peak with the sera from infected mice (Fig. 2)) but what part of this increase was due to specific antibody was not determined. Electrophoresis of serum
Similar studies to those conducted with mice were carried out with rats. Infection with lO,OOO-12,000 ascarid eggs did not consistently produce a detectable precipitating antibody and never as early as the S-days postinoculation, when antibody was consistently detected in mice. Subsequently, some rats were given a higher dosage of eggs and others were hyperinfected. Two rats were given 17,000 eggs and bled at 13 days; in addition, four rats, previously inoculated with 12,000 eggs, were given I7,OOO eggs 2 months after the initial ex-
396
CRANDALL
AND
posure and also bled at 13 days. The sera from these animals gave a single prominent precipitin band in Ouchterlony slides with ascarid extract, but not of the intensity shown by mouse sera. In contrast to the mouse precipitin the detectable antibody was 2-ME resistant, and eluted from the G-200 Sephadex column in the fraction corresponding to a “7s” immunoglobulin (Table I, Fig. 4-4).
CFUNDALL
Time and Dose Dependency body Response
of the Anti-
A more extensive experiment was conducted to compare the time relationship and dose dependency of the antibody response to ascarid infection in rats and mice. One hundred and fifty mice were divided equally into three groups and given 10,000, 7,500, and 3,000 ascarid eggs. Twenty-six
FIG. 4. Localization by gel filtration and sensitivity to 2mercaptoethanol of rat precipitating antibody to A. suum. Center wells A-D contain ascarid extract. A. Gel filtration of a rat serum obtained 13 days after inoculation (left) and 13 days after hyperinfection (right) : yG = 7S peak from G-200 column; yM = macroglobulin peak from column; S = whole sernm. B-D: 2mercaptoethanol-treated sera in wells on right (ME); untreated sera on left, B. Sera from B-day infection. Upper wells contain mouse sera (M); lower wells, rat sera after inoculation with 50,000 embryonated eggs (EE). C. Rat sera from 15day infection with 50,000 EE. D. Rat sera from 21-day infection with 50,000 EE.
To determine whether a detectable macroglobulin antibody could be induced in rats by a larger dosage of eggs, three groups of rats containing three rats each were given 70,000, 50,000, and 25,000 eggs. One rat from each dosage group was bled on day 8, 15, and 21. All of the rats given the two larger doses produced antibody at all the times checked, but a precipitin response in rats given 25,000 eggs was found only on day 15 with no response on day 8, and only a barely detectable response on day 21. AI1 the sera that had detectable antibody were treated with 2-ME and the precipitin activity of all sera was 2-ME resistant (Figs. 4B, C, and D).
rats were divided into four groups: two were given 200,000 eggs; eight were given 100,000, 75,000, and 30,000 eggs. Seven mice given the higher dosages of eggs died between days 7 and 14 apparently from Ascaris-induced lung damage; three rats in the groups given 75,000 and 100,000 died between days 8-14. The two rats given 200,006 eggs were bled 6 days after inoculation at which time they were moribund and their serum was negative for precipitating antibody. Between eight and 16 mice from each group were bled on days 8, 15, 21, 23, and 33; sera from two mice were pooled for testing. Similarly, one or two rats were bled
MACROGLOBULIN
ANTIBODY
RESPONSE
from each of the dosage groups at these time intervals. Sera were tested in microOuchterlony slides with the ascarid extract and the precipitin response roughly graded at 24 hours as just detectable ( + ), medium intensity ( + ) and strong ( ++ ). The precipitin response with sera obtained on day 15 of infection is illustrated in part in Figs. 5A, B, and C. The results of this experiment summarized in Table II indicate that the precipi-
TO
ASCARIS
INFECTION
397
precipitin band formed in agar gel by mouse serum and ascarid extract typically formed nearer the serum well and curved toward the well, in contrast to the precipitin band with rat serum which usually formed a straight line more distant from the serum well. In five serum pools from the above experiment the precipitin band formed by mouse serum did not appear completely typical and resembled that produced by rat serum. These five serum pools
FIG. 5. Gel diffusion analysis of antibody to A. suum in mouse and rat sera. Center wells A-F contain ascarid extract. A. Precipitin reaction with rat sera from 1.5day infections with different doses of embryonated ascarid eggs (EE ). 1 = 100,000 EE; 2 = 75,000 EE; 3 and 4 = 30,000 EE. B. Precipitin reaction with mouse sera from 15-day infections with 10,000 EE. C. Precipitin reaction with mouse sera from 15day infection with 7,500 EE. D-F. Comparison of antibody specificity in rat and mouse antiascarid sera by Ouchterlony tests. D-E. Sera from 8-day infections: 1 = mouse sera after inoculation with 10,000-12,000 EE; 2 = rat sera after inoculation with 70,000 EE; 3 = rat sera after 30,000 EE. F. Sera from 15-day infections: 1 = pooled mouse sera after lO,OOO-12,000 EE; 2 = rat sera after 70,000 EE; 3 = rat sera after 30,000 EE.
tating antibody in mice appeared somewhat earlier and more animals gave a strong response with the higher dosage of eggs than with the lower. The peak antibody response appeared to be between weeks 2 and 3 of infection and declined by day 33. The comparative study with rats employed too few animals to show any definite pattern of response; however, with the higher dosages antibody could be detected by day 8 of infection. In the above experiment, as well as those reported previously in this study, the major
were treated with 2-mercaptoethanol and in one pool (two mice infected for 21 days with 10,000 eggs), the precipitating ability was not completely destroyed. To confirm the presence of a 7s precipitin, pooled sera from mice collected 2-4 weeks after inoculation of 10,000 eggs and after hyperinfection were fractionated on a Sephadex G-200 column and the 7s peak highly concentrated by pressure dialysis. This peak gave no IgM line on immunoelectrophoresis with anti-mouse immunoglobulin serum but a light precipitin band with ascarid extract
398
CRANDALL
Precipitin
Response
qf Mice
Bled days postinoculation
8 15 21 23 33
a Number the indicated
+(3): +(3) f(l)
~‘IJ~S
eggs per animal 3,000
neg@!
w(8) + + (7) ; neg(1)
++@I; +W
neg(l!
f (3); w(2)
75,000
neg(1)
+Oi; +(I) *(I) 5 (1) k-(l)
in parenthesis refers to the number of serum pools response. For grading precipitin response see text.
on Ouchterlony slides. Reexamination of 7S peaks from mouse serum pools in the initial experiment did not reveal a precipitin with ascarid extract and if precipitating antibody were present, it was at a concentration below the sensitivity of the test employed. Relationship (Identity or Nonidentity) Functional Antigen(s)
+ (4) ;
++(I);
+ + (4) ; nedI) +(I)
100,000 +!l); +(l) f(l) +(I) * (2)
suum
7,500
f(2); nenW ++@I ++(4); ++(a; neg(1);
Doses qf Ascaris
of embryonated
10,000
Rats 8 1.5 21 23 33
CRANDALL
TABLE II and Rats to Graded
Number
Mice
AND
of
The preceeding experiments demonstrated that mice uniformly produced precipitating antibody of the mactioglobulin class in response to Ascaris infection, but the detectable precipitins produced by the rat were not macroglobulins. It was con.sidered that this apparent difference in the immunoglobulin class of the antibody response to ascarid infection might reflect a response to a different antigen(s) by the two rodent species. Ouchterlony tests to detect identity or nonidentity of antigens to which the two species responded gave somewhat equivocal results. A reaction of partial identity or a failure of precipitin bands to fuse was observed (Figs. 5D and E). Although various arrangements of anti-
neg(4!
+@I; ned5) neg(2)
30,000 neg(2)
nedl)
+(l); f(l) + (2) neg(l); + (1)
(mice)
or number
of animals
(rat,s)
giving
gen- and antisera-containing reservoirs in the Ouchterlony slides were tried, as well as several dilutions of antisera, no definite crossing of precipitin bands indicating complete nonidentity was found; but in some instances it appeared that the precipitin band formed by the rat serum with ascarid extract was identical with a minor precipitin band of the mouse (Fig. 5F). In two cases sera from infected rats gave a precipitin band in Ouchterlony slides with mouse sera; these rat sera also reacted with sera from uninfected mice. Relationship to Heterophil Antibody Zsohemagglutinins
and
The possible heterophilic nature of the antigen inducing the macroglobulin response in the mouse was investigated by testing sera for human isohemagglutinins and heterophil antibody. Tests for human isohemagglutinins were conducted on the same serum pool. The titer with type-A erythrocytes was 1: 128, type-B erythrocytes 1: 16, and type-0 erythrocytes 1: 32. Pooled normal sera was weakly positive with A and
MACROGLOBULIN
ANTIBODY
RESPONSE
B erythrocytes at a 1:2 dilution and negative with 0 erythrocytes. Absorption of the mouse serum with A, B, and 0 cells until the isohemagglutinin titer was removed did not remove the precipitating antibody to ascarid extract; but absorption of human serum with the extract removed the isohemagglutinins. The titer of heterophil antibody as measured by sheep cell agglutination was not increased in mouse serum after infection nor were the number of spleen cells producing antibody capable of sensitizing sheep cells for hemolysis in the presence of complement (Jeme technique) increased. Species Specificity Antigen
and Localization
of
Pooled mouse sera taken 8 days after inoculation of 10,000 ascaris eggs produced precipitin bands with saline extracts of adult and larval N. braxiliensis, adult D. immitis, and T. spiralis muscle larvae. Absorption of antisera was attempted only with D. immitk extract, which completely absorbed out the precipitin to ascarid extract. The localization in Ascaris of the antigen inducing the major precipitin in mice was studied by testing extracts of cuticle, somatic tissues, and extracts of second-stage larvae by gel diffusion. All these tissues contained the antigen. Characterization of the Antigen Preliminary studies were conducted to determine the chemical properties of the antigen in the ascarid extract. The extract used throughout most of this study contained 3.7 mg/ml of protein and 5 mg/ml of carbohydrate by the tests employed. The antigen in the extract was heat stable: autoclaving for 15 minutes at 20 Ibs pressure did not destroy the precipitating ability. Activity was not destroyed by the proteolytic enzyme pronase or by lysozyme. The activity was resistant to mild acid and
TO
ASCABIS
INFECTION
399
alkaline hydrolysis but destroyed by hydrolysis with 30%KOH. Periodate oxidation markedly decreased the precipitability of the antigen. Precipitation of the protein in the extract with 5%TCA failed to segregate the antigen although a stronger precipitin band was produced with the supemate fluid which contained 0.1 mg/ml protein as compared with the redissolved precipitate which contained 1.7 mg/ml of protein. DISCUSSION
In this study the differentiation of the macroglobulin, IgM, and 7s antibody response has been based primarily on the sensitivity of antibody activity to reduction by 0.1 M 2-mercaptoethanol and fractionation by gel filtration on Sephadex G-200. The sensitivity of macroglobulin to 2-mercaptoethanol and the relative stability of IgG has been widely employed as a method of differentiating these immunoglobulin classes since the study of Deutsch and Morton ( 1957). Reliance on this criterion alone has been questioned (Schrohenloher et al., 1964) and mouse 7S antibody produced soon after immunization with protein antigens has been shown to lose significant precipitating ability after 2-mercaptoethano1 treatment and alkalation (Adler, 1965 b ). However, the correlation between 2mercaptoethanol sensitivity with data from other procedures (gel filtration, sucrosegradient ultracentrifugation, and immunoelectrophoresis) confirms that the major precipitin induced by ascaris infection in the mouse is a macroglobulin. By the same criteria, 2-mercaptoethanol sensitivity and gel filtration, the precipitins in rat serum were not macroglobulins, but the immunoglobulin classes represented were not further defined. It should be emphasized that this study of the precipitin response in the rat and mouse does not reveal the total range of antibody response to A. s2cuminfection because of the type and comparative insensi-
400
CRANDALL
AND
CRANDALL
tivity of the test for antibody employed, experimental Toxocara infection would be and the use of only one kind of antigen of value in determining whether this repreparation. The differences demonstrated sponse is a general phenomenon with asin antibody response of the two species may carid infections. IgM is detected ineffibe only quantitative, but at least on a ciently by precipitin tests (Pike et al., quantitative basis the mouse responds with 1966); thus the strong precipitin reaction, a macroglobulin precipitin to ascaris while in addition to the gel-filtration profile with the rat does not. Further, the major macromouse serum, suggest a high IgM level. A globulin antibody produced by the mouse concentration of IgM antibody detectable in early infection is not directed toward by precipitin test is not usually induced by antigenic determinants identical to those an infectious agent and the response in the recognized by the detectable precipitins of rat is probably more typical than the mouse the rat. in that the precipitins observed were preA number of comparative studies of the sumably IgG. IgM and IgG antibody response has been Generally IgM is considered to be promade with a variety of species after antigen duced early in a primary antibody response injection, and in experimental and natural followed by a greater and more persistent infections; but the factors controlling the 7s response, although recently this serelative amounts of each immunoglobulin quence in immunoglobulin production has class produced and the significance of been questioned ( Robbins et al., 1965)) and species differences in the response are only with certain antigens and immunization partially known (Stavitsky, 1966). The imschedules only a prolonged IgM response munoglobulin classes of both rodent species has been observed (Sandberg and Stollar, have been studied, and both possess the 1966). Mice have been reported to exhibit major classes recognized in other mammals the macroglobulin to 7s transition in anti(Fahey et al., 1964; Arnason et al., 1964; body activity after experimental Salmonella Nussenzweig and Binaghi, 1965). No obinfection (Turner et al., 1964) and antigen vious basis for the observed species differinjection (Adler, 1965a). In the present ence in immunoglobulin response to ascaris study the IgM-precipitin response appeared infection can be stated. Whether it repreduring the second week of infection and sents an inherent species difference in reapparently declined after the fourth week; action to a particular antigen or a difference and this response could be restimulated by in the nature of the antigenic stimulus proreinfection. There was no evidence that the duced by ascaris infection in the two spe- IgM precipitin was followed by a major cies will require further study. During IgG precipitin, and only occasionally a 7s ascaris infection the availability and conantibody was detected in the serum of mice centration of antigens, physical state of infected with ascaris. The IgM response in the mouse was deantigens, duration and site of antigen release and other factors known to influence tected during the acute phase of the inantibody response and immunoglobulin fection and appeared to be dosage dependclass may differ significantly in the two ent with the maximum response produced species. by levels of infection which induced extenThe macroglobulin precipitin induced in sive tissue damage (Crandall et al., 1966). the mouse is of interest in view of the Varying the infective dose, however, did increased IgM concentration reported in not appear to qualitatively alter the prehuman larval ascarid infections, and studies cipitin response in either rats or mice. of the IgM response in the mouse during The antibody specificity of the macro-
MACBOGLOBULIN
ANTIBODY
RESPONSE
globulin in mice is not unique to ascaris as indicated by the precipitin reaction with extracts of other helminth species. The preliminary tests on the nature of the antigen were consistent with a polysaccharide or polysaccharide-protein complex structure. Antigens of this type have been described in ascaris (Kagan et al., 1958). These antigens show little species specificity (Kagan et al., 1959) and the antigenic determinants are probably found in many parasitic helminths (Oliver-Gonzalez, 1946). Heterophi1 antibody titers and isohemagglutinins have been demonstrated to increase in larval ascarid infections (So&by, 1958; Silver et nl., 1952; Heiner and Kevy, 1956) but no evidence was obtained in this study that the mouse macroglobulin response was related to either of these antibodies. REFERENCES ADLER, F. L. 1965a. Studies on mouse antibodies. I. The response to sheep red cells. JoumaZ of Immunology 95, 26-38. ADLER, F. L. 196513. Studies on mouse antibodies. II. Mercaptoethanol-sensitive 7S antibodies in mouse antisera to protein antigens. Journal of Immunology 95, 3947. AREAN, V. M., AND CRANDALL, C. A. 1963. Experimental ascaridic endophthalmitis. Archives of Ophthalmology 69, 585-594. ARNASON, B. G., DE VAUX ST-CRY, C., AND RELYVELD, E. H. 1964. Role of the thymus in immune reactions in rats. IV. Immunoglobulins and antibody formation. International ATchives of Allergy and Applied Immunology 25, 206-224. CRANDALL, C. A., AND AREAN, V. M. 1964. In vivo studies of Ascaris suum larvae planted in diffusion chambers in immune and nonimmune mice. Journal of Parasitology 50, 685688. CRANDALL, R. B., CRANDALL, C. A., HUNTER, G. W., III, AND AREAN, V. M. 1966. Studies on cross-resistance in schistosome and Ascaris suum infections of mice. Annals of Tropical Medicine and Parasitology 60, 70-77. DEUTSCH, H. F., AND MORTON, J. 1. 1957. Dissociation of human serum macroglobulins. Science 125, 600-601. FAHEY, J. L., WITNDERLICN, J., AND MISHELL, R. 1964. The immunoglobulins of mice. I. Four
TO
ASCARIS
INFECTION
401
major classes of immunoglobulins: 7s yr, and 18s ylM-globulins. 7S yr, YIA (BRA)-, Journal of Experimental Medicine 120, 223242. FLODIN, P., AND K&LANDER, J. 1962. Fractionation of human-serum proteins by gel filtration. Biochimica et Biophysics Acta 63, 403-410. HEINER, D. C., AND KEVY, S. V. 1956. Visceral larva migrans. Report of the syndrome in three siblings. New England Journal of Medicine 254, 629-636. HUNTLEY, C. C., COSTAS, M. C., AND LYERLY, A. 1965. Visceral larva migrans syndrome: Clinical characteristics and immunologic studies in 51 patients. Pediatrics 36, 523536. JERNE, N. K., NOI~KIN, A. A., AND HENRY, C. The agar plague technique for recognizing antibody producing cells. In “Cell-Bound Antibodies” (B. Amos and H. Koprowski, eds.), pp. 109-125. Wistar Institute Press, Philadelphia, 1963. KAGAN, I. G., JESKA, E. L., AND GENTZKOW, C. J. 1958. Serum-agar double diffusion studies with ascaris antigens. II. Assay of whole worm and tissue antigen complexes. ~ournaZ of Immunology 80, 400406. KAGAN, I. G., NORMAN, L., AND ALLAIN, D. S. 1959. Studies on the serology of visceral larva migrans. I. Hemagglutination and flocculation tests with purified ascaris antigens. Journal of Immunology 83, 297301. LINDENMANN, J. 1964. Immunity to transplantable tumors following viral oncolysis. I. Mechanism of immunity to Ehrlich ascites tumor. Journal of Immunology 92, 912-919. LOWRY, 0. H., ROSERROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurements with the Folin phenol reagent. lournal of Biological Chemistry 193, 265-275. NOMOTO, M., NARAHASHI, Y., AND MURAKAMI, M. 1960. A proteolytic enzyme of Streptomyces griseus protease. Journal of Biochemistry 48, 593-602. NUSSENZUTEIG, V., AND BINAGHI, R. A. 1965. Heterogeneity of rat immunoglobulins. International Archives of Allergy and Applied Immunology 27, 355-360. OLIVER-G• NZ~~LEZ, J. 1946. Immunological relationships among polysaccharides from various infectious organisms. Journal of Infectious Diseases 79, 221-225. PIKE, R. M., SCHULZE, M. L., AND CHANDLER, C. H. 1966. Agglutinating and precipitating capacity of rabbit anti-Salmonella typhosa yG and yM antibodies during prolonged immunization. .lonrnol of Bacteriology 92, 880-886.
402
CRANDALL
AND
ROBBINS, J. B., KENNY, K., AND SUTER, E. 1965. The isolation and biologic activities of rabbit yM and yG anti-Salmonella typhimurium antibodies. ]ournal of Experimental Medicine 122, 385-402. SANDBERG, A. L., AND STOLLAR, B.D. 1966.A 19s anamnestic response to DNA-methylated bovine serum albumin complexes. Immunology 11, 547-556. SCHROHENLOHER, R. E., KUNKEL, H. G., AND TOMASI, T. B. 1964. Activity of dissociated and reassociated 19s anti-y-globulins. Journal of Experimental Medicine 120, 1215-1229. SILVER, H. K., HENDERSON, P., AND CONTOPOULOS, A. 1952. Extreme eosinophilia, increased blood heterophile-agglutination titer and hyperglobulinemia. American Journal of Diseases of Children 83, 649-653.
CRANDALL
SOULSBY, E. J. L. 1958.
Studies on the heterophile antibodies associated with hehninth infections. I. Heterophile antibody in Ascaris Zumbricoio!es infection in rabbits. Journal of Comparative Pathology 68, 71-81. STAVITSKY, A. B. 1966. Comments on malarial antibodies. Military Medicine 131 (Supplement). 1167-1170. STEWART-TULL, D. E. S., WILKINSON, P. C., AND WHITE, R. G. 1965. The affinity of a mycobacterial glycopeptide for guinea-pig gammaglobulin. immunology 9, 151-160. TURNER, K. J., JENKIN, C. R., AND ROWLEY, D. 1964. The basis for immunity to mouse typhoid. II. Antibody formation during the carrier state. Aus&aliun Journal of Experimental Biology and Medical Science 42, 215228.
21,
PARASITOLOGY
Macroglobulin
391-402
(1967)
Antibody
A Comparison
of Precipitating
Catherine Departments
Response
A.
of
Crandall
Pathology
U&e&y (Submitted
of
for
Antibody and
and Florida,
to Ascaris
publication,
infection.
in Rats and
Richard
Microbiology, Gainesville,
suum
Mice’
B. Crandall
College of Medicine, Florida 32601 25 April
1967)
CRANDALL, CATHERINE A., AND CRANDALL, &CHARD B. 1967. Macroglobulin antibody response to Ascaris suum infection. A comparison of precipitating antibody in rats and mice. Experimental Parasitology 21, 391402. Precipitating antibody was detected by micro-Ouchterlony slides in the sera of mice and rats infected with one per OS dose of embryonated Asca& suum eggs. Characterization of the antibody by gel filtration through Sephadex G-200, sensitivity to 2-mercaptoethanol, ultracentrifugation in a sucrose gradient, and immunoelectrophoresis revealed that the principal precipitating antibody in mice was a macroglobulin and in rats was a 7S globulin. The antigen( s ) used to detect the precipitating antibodies was an extract of lyophilized adult ascaris. The extract had a high carbohydrate to protein ratio and the antigen activity was heat stable. Activity was not destroyed by pronase, lysozyme, mild acid, or alkaline hydrolysis, but periodic acid oxidation decreased activity. The antigenic activity was not separated by 5% trichloroacetic acid precipitation. The precipitating antibody in the mouse induced by A. suum infection cross reacted with extracts of several other helminth species. This precipitin could not be related to the human isohemagglutinins or heterophil antibody by the tests employed.
Larval ascarid infections in man (visceral larva migrans) induce extensive host reaction including a hypergammaglobulinemia with an increased level of IgM (Huntley et al., 1965). Study of the immunoglobulin classes in the antibody response to larval ascarid infections of experimental animals would be of interest in determining whether a marked IgM response is characteristic of this type of infection. The purpose of the present investigation was to study the precipitin response in larval Ascaris suum infections in two rodent species with particular reference to production of macroglobulin antibody.
MATERIALS
Preparation
AND
METHODS
of Antigens
Adult A. suum worms were obtained from a local abattoir. They were homogenized in a Waring Blendor containing demineralized water, then lyophilized, and stored at -20°C. The lyophilized material was extracted with ether at 4°C and the residue dried on filter paper in a Biichner funnel with vacuum. This delipidized material was extracted in 0.01 M sodium phosphate buffered water pH 7.6, using a mortar and pestle for grinding 2-3 times and centrifuged at 10,000 rpm; the residue was discarded and the supernate fluid was stored at -20°C. Five ml samples were gel filtrated on a column (2.5 X 60 cm) containing Sephadex G-100 (Pharmacia
‘This investigation was supported in part by Public Health Service grants AI-03212 and AI05345 from the National Institute of Allergy and Infectious Diseases. 391
392
CRANDALL
AND
Fine Chemicals, Inc., Piscataway, N.J.) equilibrated in the phosphate buffer described above. Five-ml portions were collected at a flow rate of 10 ml/hour. Two protein fractions were obtained and the first protein peak contained the antigenic activity. It was pooled and pressure dialyzed against the phosphate buffer (see above). This crude antigenic extract was stored at -20°C or autoclaved (20 psi for 15 minutes) and stored at 4°C. The autoclaved extract was the material used for all antigenic characterization studies. An extract of hatched second-stage A. suum larvae was prepared as described above. Cuticular antigen was obtained through the courtesy of Dr. A. L. L. Moore, Department of Microbiology, University of Florida. An additional somatic antigen was prepared by homogenizing ascarid tissues in a Waring blender after the cuticle was removed. After centrifugation at 10,000 rpm, the supernate fluid was used as an antigen. Dirofkiu immitis, adult and larval Nippostrongylus bra&en&s and larval Trichinella spiralis extracts were made by grinding the lyophilized helminths in a mortar with a small amount of saline or phosphate buffered water (pH 7.6). In some cases, sand was added to the morter to aid in the grinding and extraction process. After centrifugation, the supernates of the extracts were stored at -20°C. Characterization
of the Ascaris Antigen
Precipitation with trichloroacetic acid. One volume of 10%trichloroacetic acid was added to one volume of antigenic extract. After centrifugation, the supernate fluid was removed, and the precipitate was washed twice with demineralized water, redissolved in 0.1 N NaOH, and both the precipitate and supernate were dialyzed against phosphate buffer. Acid and alkaline hydrolysis. One ml of the antigenic extract was adjusted to ap-
CRANDALL
proximately pH 1 with 0.1 N HCl and placed in a boiling water bath for 20 minutes. It was neutralized with 0.1 N NaOH and dialyzed against phosphate buffer. For mild alkaline hydrolysis, 0.5 ml of the extract was adjusted to pH 9 by the addition of 0.5 N NaOH, placed in a boiling water bath for 15 minutes, then neutralized with 1 N HCl. Severe alkaline hydrolysis was done by boiling 2 ml of the extract in 30%KOH for 11 minutes, then neutralizing with concentrated HCI before testing for activity. Periodic acid oxidation. In one experiment, 1 ml of the extract was adjusted to pH 3 with 0.1 N HCI, 50 mg of sodium metaperiodate were added for 24 hours at room temperature in the dark. In another experiment, 50 mg of periodic acid were added to 1 ml of the antigenic extract for 24 hours at room temperature. The pH was adjusted to neutral with 0.5 N NaOH in both experiments before testing for activity. Lysozyme. The ascaris extract was dialyzed against 0.07 M sodium phosphate buffer containing 0.0625 BI NaCl pH 5.85. One mg of lysozyme (Worthington Biochemical Corp., Freehold, N.J.) was added to 1 ml of the extract containing 1: 10,000 merthiolate and incubated for 20 hours at 37°C. A control, 1 ml of extract with merthiolate without lysozyme, was also incubated. Pronase. The method of Nomoto et al. (1960) was used. One PU of pronase (Calbiochem, Los Angeles, California) in 0.1 ml of phosphate buffer (sterilized by filtration through a millipore filter) was added to 2 ml of autoclaved extract and incubated at 40°C for 20 hours. The material was dialyzed against phosphate buffer and tested for antigenic activity. Protein and carbohydrate determinations. Total proteins were determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard, and total hexoses were measured by the an-
MACBOGLOBULIN
ANTIBODY
RESPONSE
throne method described by Stewart-Tull et al. ( 1965), using dextrose as the standard.
TO
ASCARIS
INFECTION
393
Gel filtration of serum. Two to 5 ml of sera were filtr’ated on a Sephadex laboratory column (2.5 x 100 cm) containing Sephadex G-200 equilibrated in 0.1 M phosphate infection of Animals buffer, pH 6.8. Fractions corresponding to Embryonated A. suum eggs were ob- 19s or 7S (Flodin and Killander, 1962) tained as described previously (Arean and were pooled and concentrated by pressure Crandall, 1963). Mixed sexesof white Swiss dialysis against 0.15 M phosphate buffered mice of the ICR strain and rats of the saline, pH 7.5, to approximately the same Wistar stiain were purchased from the protein concentrations. In some casesit was Dublin Laboratories, Dublin, Virginia. The necessary to concentrate fractions further in mice weighed between 25-30 gm and the dialysis bags surrounded by Ficoll (Pharrats from 250-300 gm at the beginning of macia Fine Chemicals). the experiments. Female C&BIJs mice Reduction with 2-mercaptoethunol. Sera were purchased from Jackson Memorial in 0.5- or l-ml amounts, undiluted or diLaboratory, Bar Harbor, Maine. luted, were dialyzed against 0.1 M 2-merThe animals were inoculated with em- captoethanol (2-ME ) for 16-18 hours at bryonated eggs by stomach intubation and room temperature, then against 0.15 M bled by decapitation or severing the brach- phosphate buffered saline, pH 7.2 for 48 ial artery. Some groups of mice were hyperhours at 4%. Controls were treated identiinfected as described previously (Crandall cally except buffered saline was substituted and Arean, 1964). for the 2-mercaptoethanol. Sucrose-gradient determinations. UltraCharacterization of the Antibody centrifugations in a sucrose gradient were Gel diffusion and immunoelectrophoresis. carried out as described by Lindenmann Detection of the precipitating antibody was (l!=). done in micro-Ouchterlony slides containIsohemagglutinin and sheep red cell heing 0.8% Ionagar (Consolidated Labora- molysin determinations. Isohemagglutinins tories, Inc., Chicago Hts., Ill.) in 0.15 M in mouse sera were titrated with human A, phosphate buffered saline pH 7.5 ( PBS ) B, and 0 cells and inhibition of the human containing 1: 10,000 merthiolate. For im- isohemagglutinins was carried out with dimunoelectrophoresis, slides were prepared lutions of the ascaris extract described in using 0.05 M sodium barbital buffer, pH the first paragraph. The Jeme technique 8.6. (Jeme et al., 1963) was used to measure A rabbit antimouse immunoglobulin sheep red cell hemolysins from spleen cells serum was prepared by injection of an of mice infected with ascaris. Titers of antigen-antibody precipitate prepared at sheep cell agglutinins in mouse serum were equivalence in 0.01 M EDTA (ethylenedetermined with a 2.5% suspension of diamine tetraacetate ). The antigen-antiwashed sheep erythrocytes in a 1:250 dilubody precipitate was prepared with ascaris- tion of absorbed rabbit seturn in PBS. infected mouse serum and ascaris extract. The precipitate was washed three times RESULTS with PBS and suspended in Freund’s complete adjuvant. Four mg of protein per Antibody Response in Mice rabbit were injected subcutaneously in In an initial experiment, mice were inocmultiple sites. The rabbits were bled 1 ulated with lO,OOO-12,000 embryonated ascarid eggs and bled thereafter at intervals month after the injection.
394
CRANDALL
AND
from 8 to 27 days. The sera from 15 to 25 mice were pooled at each time interval and tested for precipitating antibody on microOuchterlony slides with ascaris extract. Antibody was detected in most of these serum pools. A single major precipitin band was seen and with many sera an additional minor band was present. Those sera producing a strong precipitin band were subjected to gel filtration on Sephadex G-200 and treated with 0.1 M 2-mercaptoethanol The major precipitin was detected only in the fraction corresponding to the macroglobulin peak eluted from the Sephadex column and the precipitating activity of the serum was sensitive to reduction with 2-mercaptoethanol. The minor precipitins detected by gel diffusion were not observed consistently after any treatment or dilution of the sera and were not further investigated. In sera from two groups of mice hyperinfected with ascarid eggs, the major precipitating antibody was also a macroglobulin as indicated by gel filtration and 2-mercaptoethanol reduction (Table I, Fig. 1) . These experiments were repeated with
CRANDALL TABLE I Characterization by Gel Filtration and bMerco.ptoethanol Treatment 05 Antibody in the Sera of Mice and Rats Infected with .Yscaris suunl No. of emhryonated eggs per animal Mice lo-12,000 N-12,000 lo-12,000 lo-12,000 Hyperinfecteh Hyperinfectedc Rata 17,000 Hyperinfcctedd
1 1 1 1
dose dose dose dose
1 dose
Bled days post-
Sephadex G-200 gel
inoculation
filt,rat,ion
i4
W
1.5 21 8 15
IgM IqM kM kM IgM
1:: 1 .:
IgG IgG
‘27
‘2-m+
IX Ti
a S = sensitive to 2-mercaptoethanol; R= resistant,. b Inoculated with 5,000 embryonated eggs given twice with a 2-week interval and challenged 4 months after the last infection with 10,000 embryonated eggs. c Inoculated with 9-10,000 embryonated eggs followed by ll-12,000 l-month later. d Inoculated with 12,000 embryonated eggs followed by 17,000 2-months later.
FIG. 1. Localization by gel filtration and sensitivity to 2-mercaptoethanol of mouse precipitating antibody to A. suum. Center wells A-C contain ascaris extract. A. Gel filtration of pooled mouse sera obtained 8 days after inoculation. S = whole sera; G = 7s peak from G-200 column; M = macroglobulin peak from G-200 column. B. Two-mercaptoethanol treatment of pooled sera: 1 is sera from 8-day infection; 2 is from 21-day infection; 3 is from 27-day infection. C. Identical sera to B not treated with %mercaptoethanol.
MACBOGLOBULIN
ANTIBODY
RESPONSE
another strain of mice, C5,B1JF, with essentially the same results. Further confirmation of the macroglobulin response in mice to ascarid infections was obtained by gel-filtration profiles, immunoelectrophoresis, and sucrose-gradient ultracentrifugation. A comparison of elution profiles from a Sephadex G-200 column of 2.5 ml of pooled normal mouse sera with pooled sera from mice 12 days after inoculation with 10,000 ascarid eggs revealed a marked increase in the initial (macroglobu-
TO
ASCARIS
INFECTION
395
from infected mice and comparison of the precipitin bands developed with ascarid extract and a rabbit antiserum to mouse immunoglobulins demonstrated that the precipitating antibody migrated as the macroglobulin, IgM (Fig. 3). Sucrose-
1.2
I.1
1.0
0.9
FIG. 3. Immunoelectrophoretic sera demonstrating antibody troughs contain rabbit anti-mouse serum or ascaris extract. Well 1 mouse sera. Wells 2 and 3 are mice infected with A. suum.
0.9
EE‘
07
b. c
06
I 8
05
i c
04
analysis of mouse in IgM. Serum immunoglobulin is pooled normal pooled sera from
gradient ultracentrifugation of pooled sera from an 8-day infection also revealed that the major precipitin was a macroglobulin. Detectable precipitin activity with ascarid extract was confined to the fastest moving protein peak in the gradient.
8 03
02
Antibody Response in Ruts 01
00
30
40
50 FRACTION
60
70
a0
so
NUMBER
FIG. 2. Comparison of mouse sera by gel filtration on Sephadex G-200. A-A pooled normal mouse sera. 0 - - - - Q pooled sera from mice infected with A. suum.
lin) protein peak with the sera from infected mice (Fig. 2)) but what part of this increase was due to specific antibody was not determined. Electrophoresis of serum
Similar studies to those conducted with mice were carried out with rats. Infection with lO,OOO-12,000 ascarid eggs did not consistently produce a detectable precipitating antibody and never as early as the S-days postinoculation, when antibody was consistently detected in mice. Subsequently, some rats were given a higher dosage of eggs and others were hyperinfected. Two rats were given 17,000 eggs and bled at 13 days; in addition, four rats, previously inoculated with 12,000 eggs, were given I7,OOO eggs 2 months after the initial ex-
396
CRANDALL
AND
posure and also bled at 13 days. The sera from these animals gave a single prominent precipitin band in Ouchterlony slides with ascarid extract, but not of the intensity shown by mouse sera. In contrast to the mouse precipitin the detectable antibody was 2-ME resistant, and eluted from the G-200 Sephadex column in the fraction corresponding to a “7s” immunoglobulin (Table I, Fig. 4-4).
CFUNDALL
Time and Dose Dependency body Response
of the Anti-
A more extensive experiment was conducted to compare the time relationship and dose dependency of the antibody response to ascarid infection in rats and mice. One hundred and fifty mice were divided equally into three groups and given 10,000, 7,500, and 3,000 ascarid eggs. Twenty-six
FIG. 4. Localization by gel filtration and sensitivity to 2mercaptoethanol of rat precipitating antibody to A. suum. Center wells A-D contain ascarid extract. A. Gel filtration of a rat serum obtained 13 days after inoculation (left) and 13 days after hyperinfection (right) : yG = 7S peak from G-200 column; yM = macroglobulin peak from column; S = whole sernm. B-D: 2mercaptoethanol-treated sera in wells on right (ME); untreated sera on left, B. Sera from B-day infection. Upper wells contain mouse sera (M); lower wells, rat sera after inoculation with 50,000 embryonated eggs (EE). C. Rat sera from 15day infection with 50,000 EE. D. Rat sera from 21-day infection with 50,000 EE.
To determine whether a detectable macroglobulin antibody could be induced in rats by a larger dosage of eggs, three groups of rats containing three rats each were given 70,000, 50,000, and 25,000 eggs. One rat from each dosage group was bled on day 8, 15, and 21. All of the rats given the two larger doses produced antibody at all the times checked, but a precipitin response in rats given 25,000 eggs was found only on day 15 with no response on day 8, and only a barely detectable response on day 21. AI1 the sera that had detectable antibody were treated with 2-ME and the precipitin activity of all sera was 2-ME resistant (Figs. 4B, C, and D).
rats were divided into four groups: two were given 200,000 eggs; eight were given 100,000, 75,000, and 30,000 eggs. Seven mice given the higher dosages of eggs died between days 7 and 14 apparently from Ascaris-induced lung damage; three rats in the groups given 75,000 and 100,000 died between days 8-14. The two rats given 200,006 eggs were bled 6 days after inoculation at which time they were moribund and their serum was negative for precipitating antibody. Between eight and 16 mice from each group were bled on days 8, 15, 21, 23, and 33; sera from two mice were pooled for testing. Similarly, one or two rats were bled
MACROGLOBULIN
ANTIBODY
RESPONSE
from each of the dosage groups at these time intervals. Sera were tested in microOuchterlony slides with the ascarid extract and the precipitin response roughly graded at 24 hours as just detectable ( + ), medium intensity ( + ) and strong ( ++ ). The precipitin response with sera obtained on day 15 of infection is illustrated in part in Figs. 5A, B, and C. The results of this experiment summarized in Table II indicate that the precipi-
TO
ASCARIS
INFECTION
397
precipitin band formed in agar gel by mouse serum and ascarid extract typically formed nearer the serum well and curved toward the well, in contrast to the precipitin band with rat serum which usually formed a straight line more distant from the serum well. In five serum pools from the above experiment the precipitin band formed by mouse serum did not appear completely typical and resembled that produced by rat serum. These five serum pools
FIG. 5. Gel diffusion analysis of antibody to A. suum in mouse and rat sera. Center wells A-F contain ascarid extract. A. Precipitin reaction with rat sera from 1.5day infections with different doses of embryonated ascarid eggs (EE ). 1 = 100,000 EE; 2 = 75,000 EE; 3 and 4 = 30,000 EE. B. Precipitin reaction with mouse sera from 15-day infections with 10,000 EE. C. Precipitin reaction with mouse sera from 15day infection with 7,500 EE. D-F. Comparison of antibody specificity in rat and mouse antiascarid sera by Ouchterlony tests. D-E. Sera from 8-day infections: 1 = mouse sera after inoculation with 10,000-12,000 EE; 2 = rat sera after inoculation with 70,000 EE; 3 = rat sera after 30,000 EE. F. Sera from 15-day infections: 1 = pooled mouse sera after lO,OOO-12,000 EE; 2 = rat sera after 70,000 EE; 3 = rat sera after 30,000 EE.
tating antibody in mice appeared somewhat earlier and more animals gave a strong response with the higher dosage of eggs than with the lower. The peak antibody response appeared to be between weeks 2 and 3 of infection and declined by day 33. The comparative study with rats employed too few animals to show any definite pattern of response; however, with the higher dosages antibody could be detected by day 8 of infection. In the above experiment, as well as those reported previously in this study, the major
were treated with 2-mercaptoethanol and in one pool (two mice infected for 21 days with 10,000 eggs), the precipitating ability was not completely destroyed. To confirm the presence of a 7s precipitin, pooled sera from mice collected 2-4 weeks after inoculation of 10,000 eggs and after hyperinfection were fractionated on a Sephadex G-200 column and the 7s peak highly concentrated by pressure dialysis. This peak gave no IgM line on immunoelectrophoresis with anti-mouse immunoglobulin serum but a light precipitin band with ascarid extract
398
CRANDALL
Precipitin
Response
qf Mice
Bled days postinoculation
8 15 21 23 33
a Number the indicated
+(3): +(3) f(l)
~‘IJ~S
eggs per animal 3,000
neg@!
w(8) + + (7) ; neg(1)
++@I; +W
neg(l!
f (3); w(2)
75,000
neg(1)
+Oi; +(I) *(I) 5 (1) k-(l)
in parenthesis refers to the number of serum pools response. For grading precipitin response see text.
on Ouchterlony slides. Reexamination of 7S peaks from mouse serum pools in the initial experiment did not reveal a precipitin with ascarid extract and if precipitating antibody were present, it was at a concentration below the sensitivity of the test employed. Relationship (Identity or Nonidentity) Functional Antigen(s)
+ (4) ;
++(I);
+ + (4) ; nedI) +(I)
100,000 +!l); +(l) f(l) +(I) * (2)
suum
7,500
f(2); nenW ++@I ++(4); ++(a; neg(1);
Doses qf Ascaris
of embryonated
10,000
Rats 8 1.5 21 23 33
CRANDALL
TABLE II and Rats to Graded
Number
Mice
AND
of
The preceeding experiments demonstrated that mice uniformly produced precipitating antibody of the mactioglobulin class in response to Ascaris infection, but the detectable precipitins produced by the rat were not macroglobulins. It was con.sidered that this apparent difference in the immunoglobulin class of the antibody response to ascarid infection might reflect a response to a different antigen(s) by the two rodent species. Ouchterlony tests to detect identity or nonidentity of antigens to which the two species responded gave somewhat equivocal results. A reaction of partial identity or a failure of precipitin bands to fuse was observed (Figs. 5D and E). Although various arrangements of anti-
neg(4!
+@I; ned5) neg(2)
30,000 neg(2)
nedl)
+(l); f(l) + (2) neg(l); + (1)
(mice)
or number
of animals
(rat,s)
giving
gen- and antisera-containing reservoirs in the Ouchterlony slides were tried, as well as several dilutions of antisera, no definite crossing of precipitin bands indicating complete nonidentity was found; but in some instances it appeared that the precipitin band formed by the rat serum with ascarid extract was identical with a minor precipitin band of the mouse (Fig. 5F). In two cases sera from infected rats gave a precipitin band in Ouchterlony slides with mouse sera; these rat sera also reacted with sera from uninfected mice. Relationship to Heterophil Antibody Zsohemagglutinins
and
The possible heterophilic nature of the antigen inducing the macroglobulin response in the mouse was investigated by testing sera for human isohemagglutinins and heterophil antibody. Tests for human isohemagglutinins were conducted on the same serum pool. The titer with type-A erythrocytes was 1: 128, type-B erythrocytes 1: 16, and type-0 erythrocytes 1: 32. Pooled normal sera was weakly positive with A and
MACROGLOBULIN
ANTIBODY
RESPONSE
B erythrocytes at a 1:2 dilution and negative with 0 erythrocytes. Absorption of the mouse serum with A, B, and 0 cells until the isohemagglutinin titer was removed did not remove the precipitating antibody to ascarid extract; but absorption of human serum with the extract removed the isohemagglutinins. The titer of heterophil antibody as measured by sheep cell agglutination was not increased in mouse serum after infection nor were the number of spleen cells producing antibody capable of sensitizing sheep cells for hemolysis in the presence of complement (Jeme technique) increased. Species Specificity Antigen
and Localization
of
Pooled mouse sera taken 8 days after inoculation of 10,000 ascaris eggs produced precipitin bands with saline extracts of adult and larval N. braxiliensis, adult D. immitis, and T. spiralis muscle larvae. Absorption of antisera was attempted only with D. immitk extract, which completely absorbed out the precipitin to ascarid extract. The localization in Ascaris of the antigen inducing the major precipitin in mice was studied by testing extracts of cuticle, somatic tissues, and extracts of second-stage larvae by gel diffusion. All these tissues contained the antigen. Characterization of the Antigen Preliminary studies were conducted to determine the chemical properties of the antigen in the ascarid extract. The extract used throughout most of this study contained 3.7 mg/ml of protein and 5 mg/ml of carbohydrate by the tests employed. The antigen in the extract was heat stable: autoclaving for 15 minutes at 20 Ibs pressure did not destroy the precipitating ability. Activity was not destroyed by the proteolytic enzyme pronase or by lysozyme. The activity was resistant to mild acid and
TO
ASCABIS
INFECTION
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alkaline hydrolysis but destroyed by hydrolysis with 30%KOH. Periodate oxidation markedly decreased the precipitability of the antigen. Precipitation of the protein in the extract with 5%TCA failed to segregate the antigen although a stronger precipitin band was produced with the supemate fluid which contained 0.1 mg/ml protein as compared with the redissolved precipitate which contained 1.7 mg/ml of protein. DISCUSSION
In this study the differentiation of the macroglobulin, IgM, and 7s antibody response has been based primarily on the sensitivity of antibody activity to reduction by 0.1 M 2-mercaptoethanol and fractionation by gel filtration on Sephadex G-200. The sensitivity of macroglobulin to 2-mercaptoethanol and the relative stability of IgG has been widely employed as a method of differentiating these immunoglobulin classes since the study of Deutsch and Morton ( 1957). Reliance on this criterion alone has been questioned (Schrohenloher et al., 1964) and mouse 7S antibody produced soon after immunization with protein antigens has been shown to lose significant precipitating ability after 2-mercaptoethano1 treatment and alkalation (Adler, 1965 b ). However, the correlation between 2mercaptoethanol sensitivity with data from other procedures (gel filtration, sucrosegradient ultracentrifugation, and immunoelectrophoresis) confirms that the major precipitin induced by ascaris infection in the mouse is a macroglobulin. By the same criteria, 2-mercaptoethanol sensitivity and gel filtration, the precipitins in rat serum were not macroglobulins, but the immunoglobulin classes represented were not further defined. It should be emphasized that this study of the precipitin response in the rat and mouse does not reveal the total range of antibody response to A. s2cuminfection because of the type and comparative insensi-
400
CRANDALL
AND
CRANDALL
tivity of the test for antibody employed, experimental Toxocara infection would be and the use of only one kind of antigen of value in determining whether this repreparation. The differences demonstrated sponse is a general phenomenon with asin antibody response of the two species may carid infections. IgM is detected ineffibe only quantitative, but at least on a ciently by precipitin tests (Pike et al., quantitative basis the mouse responds with 1966); thus the strong precipitin reaction, a macroglobulin precipitin to ascaris while in addition to the gel-filtration profile with the rat does not. Further, the major macromouse serum, suggest a high IgM level. A globulin antibody produced by the mouse concentration of IgM antibody detectable in early infection is not directed toward by precipitin test is not usually induced by antigenic determinants identical to those an infectious agent and the response in the recognized by the detectable precipitins of rat is probably more typical than the mouse the rat. in that the precipitins observed were preA number of comparative studies of the sumably IgG. IgM and IgG antibody response has been Generally IgM is considered to be promade with a variety of species after antigen duced early in a primary antibody response injection, and in experimental and natural followed by a greater and more persistent infections; but the factors controlling the 7s response, although recently this serelative amounts of each immunoglobulin quence in immunoglobulin production has class produced and the significance of been questioned ( Robbins et al., 1965)) and species differences in the response are only with certain antigens and immunization partially known (Stavitsky, 1966). The imschedules only a prolonged IgM response munoglobulin classes of both rodent species has been observed (Sandberg and Stollar, have been studied, and both possess the 1966). Mice have been reported to exhibit major classes recognized in other mammals the macroglobulin to 7s transition in anti(Fahey et al., 1964; Arnason et al., 1964; body activity after experimental Salmonella Nussenzweig and Binaghi, 1965). No obinfection (Turner et al., 1964) and antigen vious basis for the observed species differinjection (Adler, 1965a). In the present ence in immunoglobulin response to ascaris study the IgM-precipitin response appeared infection can be stated. Whether it repreduring the second week of infection and sents an inherent species difference in reapparently declined after the fourth week; action to a particular antigen or a difference and this response could be restimulated by in the nature of the antigenic stimulus proreinfection. There was no evidence that the duced by ascaris infection in the two spe- IgM precipitin was followed by a major cies will require further study. During IgG precipitin, and only occasionally a 7s ascaris infection the availability and conantibody was detected in the serum of mice centration of antigens, physical state of infected with ascaris. The IgM response in the mouse was deantigens, duration and site of antigen release and other factors known to influence tected during the acute phase of the inantibody response and immunoglobulin fection and appeared to be dosage dependclass may differ significantly in the two ent with the maximum response produced species. by levels of infection which induced extenThe macroglobulin precipitin induced in sive tissue damage (Crandall et al., 1966). the mouse is of interest in view of the Varying the infective dose, however, did increased IgM concentration reported in not appear to qualitatively alter the prehuman larval ascarid infections, and studies cipitin response in either rats or mice. of the IgM response in the mouse during The antibody specificity of the macro-
MACBOGLOBULIN
ANTIBODY
RESPONSE
globulin in mice is not unique to ascaris as indicated by the precipitin reaction with extracts of other helminth species. The preliminary tests on the nature of the antigen were consistent with a polysaccharide or polysaccharide-protein complex structure. Antigens of this type have been described in ascaris (Kagan et al., 1958). These antigens show little species specificity (Kagan et al., 1959) and the antigenic determinants are probably found in many parasitic helminths (Oliver-Gonzalez, 1946). Heterophi1 antibody titers and isohemagglutinins have been demonstrated to increase in larval ascarid infections (So&by, 1958; Silver et nl., 1952; Heiner and Kevy, 1956) but no evidence was obtained in this study that the mouse macroglobulin response was related to either of these antibodies. REFERENCES ADLER, F. L. 1965a. Studies on mouse antibodies. I. The response to sheep red cells. JoumaZ of Immunology 95, 26-38. ADLER, F. L. 196513. Studies on mouse antibodies. II. Mercaptoethanol-sensitive 7S antibodies in mouse antisera to protein antigens. Journal of Immunology 95, 3947. AREAN, V. M., AND CRANDALL, C. A. 1963. Experimental ascaridic endophthalmitis. Archives of Ophthalmology 69, 585-594. ARNASON, B. G., DE VAUX ST-CRY, C., AND RELYVELD, E. H. 1964. Role of the thymus in immune reactions in rats. IV. Immunoglobulins and antibody formation. International ATchives of Allergy and Applied Immunology 25, 206-224. CRANDALL, C. A., AND AREAN, V. M. 1964. In vivo studies of Ascaris suum larvae planted in diffusion chambers in immune and nonimmune mice. Journal of Parasitology 50, 685688. CRANDALL, R. B., CRANDALL, C. A., HUNTER, G. W., III, AND AREAN, V. M. 1966. Studies on cross-resistance in schistosome and Ascaris suum infections of mice. Annals of Tropical Medicine and Parasitology 60, 70-77. DEUTSCH, H. F., AND MORTON, J. 1. 1957. Dissociation of human serum macroglobulins. Science 125, 600-601. FAHEY, J. L., WITNDERLICN, J., AND MISHELL, R. 1964. The immunoglobulins of mice. I. Four
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major classes of immunoglobulins: 7s yr, and 18s ylM-globulins. 7S yr, YIA (BRA)-, Journal of Experimental Medicine 120, 223242. FLODIN, P., AND K&LANDER, J. 1962. Fractionation of human-serum proteins by gel filtration. Biochimica et Biophysics Acta 63, 403-410. HEINER, D. C., AND KEVY, S. V. 1956. Visceral larva migrans. Report of the syndrome in three siblings. New England Journal of Medicine 254, 629-636. HUNTLEY, C. C., COSTAS, M. C., AND LYERLY, A. 1965. Visceral larva migrans syndrome: Clinical characteristics and immunologic studies in 51 patients. Pediatrics 36, 523536. JERNE, N. K., NOI~KIN, A. A., AND HENRY, C. The agar plague technique for recognizing antibody producing cells. In “Cell-Bound Antibodies” (B. Amos and H. Koprowski, eds.), pp. 109-125. Wistar Institute Press, Philadelphia, 1963. KAGAN, I. G., JESKA, E. L., AND GENTZKOW, C. J. 1958. Serum-agar double diffusion studies with ascaris antigens. II. Assay of whole worm and tissue antigen complexes. ~ournaZ of Immunology 80, 400406. KAGAN, I. G., NORMAN, L., AND ALLAIN, D. S. 1959. Studies on the serology of visceral larva migrans. I. Hemagglutination and flocculation tests with purified ascaris antigens. Journal of Immunology 83, 297301. LINDENMANN, J. 1964. Immunity to transplantable tumors following viral oncolysis. I. Mechanism of immunity to Ehrlich ascites tumor. Journal of Immunology 92, 912-919. LOWRY, 0. H., ROSERROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurements with the Folin phenol reagent. lournal of Biological Chemistry 193, 265-275. NOMOTO, M., NARAHASHI, Y., AND MURAKAMI, M. 1960. A proteolytic enzyme of Streptomyces griseus protease. Journal of Biochemistry 48, 593-602. NUSSENZUTEIG, V., AND BINAGHI, R. A. 1965. Heterogeneity of rat immunoglobulins. International Archives of Allergy and Applied Immunology 27, 355-360. OLIVER-G• NZ~~LEZ, J. 1946. Immunological relationships among polysaccharides from various infectious organisms. Journal of Infectious Diseases 79, 221-225. PIKE, R. M., SCHULZE, M. L., AND CHANDLER, C. H. 1966. Agglutinating and precipitating capacity of rabbit anti-Salmonella typhosa yG and yM antibodies during prolonged immunization. .lonrnol of Bacteriology 92, 880-886.
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ROBBINS, J. B., KENNY, K., AND SUTER, E. 1965. The isolation and biologic activities of rabbit yM and yG anti-Salmonella typhimurium antibodies. ]ournal of Experimental Medicine 122, 385-402. SANDBERG, A. L., AND STOLLAR, B.D. 1966.A 19s anamnestic response to DNA-methylated bovine serum albumin complexes. Immunology 11, 547-556. SCHROHENLOHER, R. E., KUNKEL, H. G., AND TOMASI, T. B. 1964. Activity of dissociated and reassociated 19s anti-y-globulins. Journal of Experimental Medicine 120, 1215-1229. SILVER, H. K., HENDERSON, P., AND CONTOPOULOS, A. 1952. Extreme eosinophilia, increased blood heterophile-agglutination titer and hyperglobulinemia. American Journal of Diseases of Children 83, 649-653.
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SOULSBY, E. J. L. 1958.
Studies on the heterophile antibodies associated with hehninth infections. I. Heterophile antibody in Ascaris Zumbricoio!es infection in rabbits. Journal of Comparative Pathology 68, 71-81. STAVITSKY, A. B. 1966. Comments on malarial antibodies. Military Medicine 131 (Supplement). 1167-1170. STEWART-TULL, D. E. S., WILKINSON, P. C., AND WHITE, R. G. 1965. The affinity of a mycobacterial glycopeptide for guinea-pig gammaglobulin. immunology 9, 151-160. TURNER, K. J., JENKIN, C. R., AND ROWLEY, D. 1964. The basis for immunity to mouse typhoid. II. Antibody formation during the carrier state. Aus&aliun Journal of Experimental Biology and Medical Science 42, 215228.