Heligmosomoides polygyrus (=Nematospiroides dubius): Suppression of antibody response to orally administered sheep erythrocytes in infected mice

Heligmosomoides polygyrus (=Nematospiroides dubius): Suppression of antibody response to orally administered sheep erythrocytes in infected mice

EXPERIMENTAL PARASITOLOGY 38, 257-269 ( 19%) Heligmosomoides polygyrus (= Nematospiroides dubius) : Suppression of Antibody Response to Orally Ad...

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EXPERIMENTAL

PARASITOLOGY

38,

257-269

( 19%)

Heligmosomoides polygyrus (= Nematospiroides dubius) : Suppression of Antibody Response to Orally Administered Sheep Erythrocytes in Infected Mice ROBERT G. SHIMP,~ RICHARD B. CRANDALL, Department Immunology College

AND CATHERINE

A. CRANDALL

Medicine, Division of Comparatiue Medicine, Department and Medical Microbiology and Department of Pathology, of Medicine, University of Florida, Gainesoille, Florida 32610 of

Accepted

March

25,

of

1975

SHIMP, R. G., CRANDALL, R. B., AND CRANDALL, C. A. 1975. Heligmosomoides polygyrvs ( = Nematospiroides dubius) : Suppression of antibody response to orally administered sheep erythrocytes in infected mice. Experimental Parasitology 38, 257-269. Mice infected with 200 to 300 Heligmosomoides polygyrus had reduced serum hemagglutinin titers following a series of oral inoculations of sheep erythrocytes (SRBC) when compared to similarly inoculated uninfected mice. Study of antibody-producing cells by the indirect hemolytic plaque technique demonstrated a low splenic response to oral immunization in which IgA predominated. No alteration was evident in the proportions of antibody-containing cells in the different Ig classes with infection. Comparison of the immune response to oral and intraperitoneal routes of SRBC inoculation in infected and uninfected mice demonstrated a similar reduction in antibody titer with both routes of inoculation, although immunosuppression following intraperitoneal inoculations was not consistantly observed. The data are discussed in relation to the influence of the helminth infection on intestinal immune response and systemic immune response. INDEX DESCRIPTORS: Heligmosomoides polygyrus ( = Nematospiroides dubius); Mouse; Immunosuppression; Oral immunization; Sheep red blood cells; Hemagglutinin; Antibody forming cells; Immunity.

The histopathologic changes induced by infection with the common species of gastrointestinal nematodes of medical and veterinary importance have been well described, but the concomitant functional changes, which may contribute to pathogenesis, often have not been examined critically ( Symons 1969). Alteration in the immunologic function of the intestinal tract could be a significant feature of this type of parasitism. The intestinal lymphoid cells constitute a major lymphoid tissue, func1 Present address: Division of Laboratory Animal Resources, Medical College of Georgia, Augusta, Georgia 30902. 257 Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

tionally related with the peripheral lymphoid tissues and under continuous antigen stimulation from materials entering the mucosal surfaces. The intestinal lymphoid tissue, as part of the secretory immunologic system, has a protective role in preventing intestinal infection (Tomasi 1972) and, it has been suggested, a more general regulatory or protective function in controlling antigen absorption from the intestinal tract (Walker, Isselbacher, and Block 1972). Changes in digestive function, intestinal motility, and mucosal permeability produced by helminth infection could influence the amount and quality of antigenic

SHIMP,

258

CRANDALL AND CRANDALL

exposure from the intestinal lumen; changes in intestinal lymphoid cell populations, with inflammation and local immune responses to the nematode, could also alter the intestinal response to local antigenic stimulation and the subsequent systemic response. The present study was undertaken to determine if an infection with H. polygyrus, a trichostrongylid nematode parasite of the rodent small intestine, altered the humoral immune response to an orally administered antigen, sheep erythrocytes (SRBC). This helminth infection is well characterized in the mouse (Baker 1954) ; it produces an acute and chronic inflammatory response of the upper intestinal tract ( Liu 1965a, b ) and changes in the intestinal lymphoid cell populations (Crandall, Crandall, and Franc0 1974). The infection has been demonstrated to slow intestinal passage of ingested materials (Bawden 1969) and to increase experimental colonization of E. coli in the lumen and intestinal wall (Cypcss et ~1. 1974). With the available information on the pathologic and immune responses induced by the parasite, this infection in the mouse offers a convenient model for study of immunologic alterations in this common type of gastrointestinal parasitism. MATERIALS

AND METHODS

Animals, Experimental Infections, and Immunization CD-l female mice (Charles River, Wilmington, MA), 8 to 10 weeks old, were used in all experiments. They were maintained in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Methods of infecting mice with H. polygyrus and determining worm burdens have been described (Crandall, Crandall, and Franc0 1974). Larvae for infection were stored in water at 4°C for periods up to 2 months before use. The inoculating dos-

age, 200 larvae, was determined by counting only larvae motile at room temperature; this procedure generally underestimated the numbers of viable larvae inoculated. Sheep red blood cells in Alsever’s solution (purchased from Becton, Dickinson and Co., Cockeysville, MD) were washed three times with phosphate buffered saline (PBS, 0.15 M NaCl, pH 7.5) and resuspended to a concentration of 2Oc/(,in PBS. For oral immunization, mice were given 0.1 ml of a 20%, suspension of SRBC by placing the suspensiondropwise in the oral cavity or by stomach intubation using a blunted 17-gauge needle and l-ml syringe. Gastric intubation was employed in all studies of splenic response; other studies used oral inoculation. For parenteral immunization, mice were injected intraperitoneally ( IP ) with 0.1 ml of a 20% suspension. Mice were bled individually from the orbital sinus with lOO-~1 pipettes (Levy Lang). The blood was diluted immediately into 0.10 ml of PBS. After removal of the clot, the supernate was considered a 1:2 dilution for antibody titer determination. Undiluted serum was collected from the mice sacrificed at the time hemolytic plaque assayswere done. The diluted and undiluted sera were stored at -20°C. Immunologic Assays Hemagglutination (HA) titers were determined using microtiter plates and a multimicrodiluter (Cooke Engineering Co., Alexandria, VA.). Normal rabbit serum (1:lOO in PBS) was used as a diluent. Mercaptoethanol ( 2-ME ) resistant antibody was determined as described by Scott and Gershon (1970). The hemolytic plaque assay of Jerne, Nordin, and Henry ( 1963), modified by Plotz, Talal and Asofsky ( 1968), M&hell and Dutton (1967), and Pierce (1969), was used to determine antibody-forming cells (AFC) in mouse spleens, The augmenting sera, anti-IgG and anti-IgA, were

SUPPRESSION OF HETEROLOGOUS

used at a dilution of 1:50, demonstrated to be optimum for plaque development; lyophilized guinea pig serum (Grand Island Biologicals, Grand Island, NY) at a 1: 10 dilution was used as the complement source for hemolytic plaque production. Plaque assays were done on duplicate slides for each Ig. For analysis of spleen AFC following oral inoculation, approximately 2 X 10” cells per animal were assayed for IgG and IgA. The mean number of direct plaques ( IgM) was subtracted from the number of indirect plaques in IgA and IgG AFC determinations. The results were calculated for AFC per total number of nucleated cells in the spleen because an estimate of total splenic response was desired, and the uniformly larger cell population of infected animals could bias results expressed per 10” cells or per other unit of cell population. The rabbit antisera used in the indirect plaque technique were prepared as described previously (Crandall and Crandall 1971). The anti-IgG was multivalent; on immunoelectrophoresis (IE ) with mouse sera, it reacted with IgG, and IgGs. These antisera gave precipitin bands on ouchterlony anaIysis with myeloma proteins MOPC 141 ( IgGlb) and MOPC 21 (IgG, ), but not MOPC 104E ( IgM ) or MOPC 167 ( IgA). Anti-IgA on IE, gave a precipitin band with IgA and an additional arc corresponding to an alpha globulin. These antisera gave precipitin bands with IgA myeloma protein but not with IgM or IgG myeloma proteins. To confirm immunoglobulin (Ig) specificity of these IgG and IgA reagents under the experimental conditions employed, it was demonstrated that each reagent facilitated a different population of plaque-forming cells (PFC); the numbers of indirect AFC detected with each reagent were additive when these reagents were used together in a single hemolytic plaque test. Absorption of reagents with mouse

IMMUNITY

BY ~digT?lOXl~Oid~~

259

sera blocked al1 facihtation; absorption with mouse myeloma proteins, IgG1, IgG2,, IgG,,, IgM, and IgA, at a single concentration (10 pug/ml) resulted in a 100% plaque reduction in anti-IgA absorbed with IgA and from 8 to 18% reduction in anti-IgA absorbed with the other myeloma proteins, In the absorption with anti-IgG, plaque reductions were 54% with IgG1, 56% with W-Lx, 46% with IgGz,,, 12% with IgM, and 33% with IgA. Serum Fractionation Serum pools were made by combining equal serum aliquots from individual mice in an experimental group. Equal volumes of serum were fractionated by stepwise elution with 0.04 M, 0.09 M, 0.19 M NaCl in 0.01 M phosphate buffer from a DEAEcellulose column equilibrated in 0.01 M buffer, pH 7.5. The 0.01 M, 0.05 M, 0.10 M, and 0.20 M eluates were concentrated by negative pressure dialysis against PBS to equal volumes and HA titers and Ig ( IgA and IgGl ) levels measured. Ig levels were measured by radial immunodiffusion as described previously (Crandall and Crandall I971 >. Statistical Analysis For comparison of antibody titers and AFC, the t test was used; numbers were converted to logarithms for testing. A probability of 0.05 or less was considered significant. EXPERIMENTAL

DESIGN AND RESULTS

influence on HA Response to Oral Inoculation In an initial experiment 40 mice were given oral inoculations of SRBC for 11 consecutive days. Twenty of these mice were inoculated with 200 infective larvae on the first day of immunization. Mice were bled before immunization and at 12, 16, 26, and 30 days following initiation of immuni-

260

SHIMP,

CRANDALL

AND

CRANDALL

days; there was no significant decrease in these titers with 2-ME treatment. The infected mice had consistently lower mean titers than uninfected mice; however, following initial exposure to antigen, only at Day 12 was this reduction statistically significant (P = 0.01). Following reexposure to antigen, the infected animals had significantly (I’ = 0.025 to 0.01) lower titers at the two time periods tested after completion of immunization. The mean number of worms per mouse in the infected group was 153.8 * 7.8. As a control study, HA titers to SRBC were measured in mice never inoculated with SRBC and in mice only infected with H. polygyrus. Normal CD-l mice and mice infected with 200 larvae were bled at intervals, five mice per time period, for 3 months. In addition, serum HA titers were measured in groups of mice of various ages and in H. polygyrus infected mice used as stock animals. The mean HA titer of normal and helminth infected mice not immunized

8’0 DAYS

Fig. 1. Mean hemagglutinin titers in Heligmosomoides polygyrw infected and uninfected mice following primary and secondary oral inoculations of sheep erythrocytes (SRBC). Oral inoculation initiated at time of Heligmosomoides polygyrus infection. Vertical line = 2 SE of mean.

zation. On Day 44, a second series of eight oral immunizations was started, and the mice were bled on Days 48, 51, and 62. HA titers were measured and the results shown in Fig. 1. Both groups of orally immunized mice developed titers within 12

1

14 UNINFECTED UNINFECTED, ANTIHELMINTHIC TREATED INFECTED, ANTIHELMINTHIC TREATED

IO

20

30

40

50

60

70

110

I20

130 140

150

160

DAYS

Fig. 2. Mean hemagglutinin titers in Heligmosomoides polygyrus infected and uninfected mice following primary, secondary, and tertiary oral inoculations with sheep erythrocytes (SRBC). Oral inoculations initiated on Day 15 of Heligmosomoides polygyws infection. Antihelminthic (levamisole) given to half of the mice in infected and control groups prior to final inoculations of SRBC. Vertical line = 2 SE of mean.

SUPPRESSION

OF

HETEROLOGOUS

with SRBC remained less than 1:2 over a 3-month period. Other “normal” mice tested did not develop significant HA titers ( > 1:4) by 6 months of age, but in a group of mice infected with 200 H. polygyrus for 1 year, the mean HA titer was 5.4 (log 2)) and in pooled sera from uninfected mice of the same age, the titer was 9 (log 2). These results indicate that unimmunized CD-l female mice and mice infected only with H. polygyrus do not develop detectable titers to antigens of SRBC over the time periods covered by most of the experiments reported in this study, but some mice do develop titers on aging. A second experiment was carried out to confirm that an established H. polygymcs infection suppressed humoral, immune response to oral immunization. This experiment examined HA responses to primary, secondary, and tertiary immunization and attempted to determine if antihehninthic treatment modified the immunosuppression induced by the helminth infection, A group of 19 mice infected with 200 H. polygyrus Iarvae I5 days previously and a similar uninfected control group were given an 11-day oral immunization with SRBC, followed by another S-day oral immunization from Days 44 to 51 after initiation of the first immunization period and then a final oral immunization on Days 143 to 150. On Day 136, 1 week before the final oral immunization, one-half of the mice in each group was given a single oral dose (50 mg/kilo body weight) of the antihelminthic Tramisol (levamisole hydrochloride, American Cyanamid Co., Princeton, NJ). Mice were bled before oral immunization and at intervals thereafter, and HA titers were measured. The experimental results are shown in Fig. 2. The mean HA titers of infected mice were significantly (P = 0.01 to 0.005) lower than the titers of uninfected mice at all periods tested following primary and secondary immunization, with the exception of the first time period ( 12 days). HA titers were

IMMUNITY

BY

fk~ig7?lO~O~OiGh

261

not significantly reduced by 2-Me treatment. Following tertiary immunization, a reduced antibody titer in the infected mice as compared with uninfected mice was demonstrated in the mice not treated with the antihelminthic. Levamisole apparently was immunosuppressive at the dosage used, and it was subsequently found that immunosuppression by this drug, under the conditions employed, had been reported (Renoux and Renoux 1974). The drugtreated, uninfected mice had a lower (P = 0.05) mean titer than the untreated, uninfected mice; the antihelminthic-treated and untreated, infected animals had similar HA responses to immunization. The antihelminthic treatment was effective in eliminating the helminths. Untreated animals had a mean worm count of 248.2 * 38.7, and the treated animals had a mean count of < 1. Influence on Splenic AFC Response Primary Oral and IP Zmmunization

to

To compare splenic responses to oral immunization, 58 mice were divided into two groups; one group was inoculated with 200 H. polygyrus larvae 15 days prior to the initiation of an D-day series of oral inoculations with SRBC. IgA, IgG and IgM antibody-producing cells from four to five mice in each group were counted on Days 4, 6, 9, 12, 18, and 32 after initiation of oral immunization. Two experiments of similar design were carried out to compare responses following IP inoculation of SRBC. Helminth-infected and control mice were injected with 0.1 ml of a 20% suspension of SRBC. In the first experiment, splenic plaques were assayed in four to five mice at 4, 7, 12, and 15 days after erythrocyte inoculation; in the second experiment, AFC were assayed at 2, 4, and 6 days. HA titers were measured in sera, and worm burdens were determined. The experimental results are summarized in Table I. During oral immunization a small, but definite, splenic response was detected by

262

SHIMP,

CRANDALL

AND

TABLE Inlmune

Group

Rrsponse oj Mire Infrcted with to Oral and Zntrapcritoncal &lean

Dayb

plaqnes/spleen -__

I

Heligmosornoidrs Inoculation

x

1OF

CRANDALL

polygyrus and Uninjcctrtl of Sheep ErythrocyteP Nean

(SE)

______

Uninfected

Infected

Mice

hemagglutinin log 2, (8E)c

[Jninfected

Infected IgG

IgM

W

IgG

IgXl

Id

PBS -___ Oral

Od

6

0.12 (0.03)

0.18 (0.05)

0.89 (0.75)

0.41 (0.15)

0.33 (0.11)

0.60 (0.19)

1.10 (0.56)

1.53 (0.61)

0.20 (0.20)

0.83 (0.83)

12 18 32

2-111X

PBS

2-&lE

__-

4

9

titer,

0

0

0

0

0

0

0.18 (0.05)

0.09 (0.06)

0.83 (0.62)

1.19 (0.91)

0.78 (0.34)

2.70 (0.10)

4.26 (1.56)

0

0.13 (0.07)

0.54 (0.06)

1.38 (0.33)

0.11 (0.06)

0.20 (0.10)

0.80 (0.65)

0.65 (0.30)

0

0.36 (0.06) 0

-


0

0

-.

(ii)

(ii)

(ki)

4.0 (1.4)

6.8 (0.6)

(0.2)

(V)

9.0 (0.8)

9.4

6.0

8.6

(0.6)

(2.8)

IP 1

4

0

0

7

79.3 (15.0)

23.4 (15.6)

64.4 (20.7) 38.1

12 15 2

0

0

(1.7)

55.9 (5.8)

12.t; (3.3)

2.3 (1.5)

(7.8)

20.7 (4.8)

0

0

2 4 6

150.8 (70.3)

<4 83.9 (23.6)

<4 25.0

(8.6)

210.7 (15.7)

7.6 (2.4)

15.6 (4.5)

9.1 (3.1)

13.3

40.2 (8.1)

15.6 (2.5)

0.8 (0.8)

14.4

0

47.3 (6.6)

19.4 (6.4)

0

22.2 (1.3)

26.0 (2.4)

1.7 (0.6)

0

0

0.8 (0.1)

0

0

233.9 (21.2)

0

0

178.7 (25.4)

13 (2:l)4

(E)

10.5 0.1)

5.9

9.4 (3.9)

a See text for details. * Days after beginning oral inocrilat,ions of 20% SI1BC. c Titers measured in PBS, resuspended, if significantly different from saline titer. d Plaque numbers not above background

156.1 (30.2)

wit,h

52.7 (9.5)

0.1 ml

and measllred levels,


Day 9 in both groups. This response was characterized by a high IgA content with comparatively little IgM response. In the uninfected mice, the IgA and IgG plaque counts and HA titers appeared maximal at

of 20!1{; SI:BC with

0.0.5 M

x 103/spleen

4.3 (2.0)

7.5.0 (7.3) 14.5 (0.4)

(0.8)

16.2 (2.5)

(2.W

or single 2-.\IE;

t.it.ers

7.3 (0.9)

10.2 (5.8)

(T::‘)

4.4 (0.2)

12.7 Q-w -_____ IP inoculation wit.h

2-1IE

of 0.1 ml shown

only

for Ighl.

Day 12, whereas a lower maximum response in infected mice was observed at Day 18. HA titers were not altered by 2ME treatment. Statistical analysis demonstrated significantly (P = 0.05) fewer IgA

suwwss10N

0~ 25

1

HETEROLOGOUS

BY Heligmosomoides

IMMUNITY

263

a UNlNFECTED , IPSRBC

IO

I

I

20

30

I

40

f

I

50

60

DAYS

Fig. 3. Comparison of hemagglutinin titers in Heligmosomoides polygyrus infected and uninfected mice following primary and secondary oral and intraperitoneal (IP) immunization with sheep erythrocytes (SRBC). Initial immunization on Day 15 of Heligmosomoides polygyrus infection. Vertical line = 2 SE of mean.

and IgG antibody-containing cells and lower HA titers in infected mice only at Day 12. The mean worm burden of the infected mice was 211.1 * 1.8. The splenic response to IP immunization was typical of that usually reported for this route of immunization; there was an early IgM response followed by a predominantly IgG response. There were no consistent, significant differences in plaque or HA responses observed between infected and uninfected mice. The mean worm counts were 237.0 -+ 4.8 and 237.2 * 7.7 in the first and second experiments. Influence Seconday

on Splenic ACF Response Oral and IP Immunization

to

A comparison of the effect of helminth infection on immune responses to secondary IP and oral inoculation with SRBC was made in a single experiment. One-half of a group of 78 mice was inoculated with 200 H. polygyrus larvae. On Day 15 after inoculation, four groups of 19 to 20 mice each were established: two groups, one hehninth-infected and one uninfected, were injected IP with 0.10 ml of a 20% suspension of SRBC; two identical groups

were given an 11-day course of oral SRBC inoculations. Mice in all groups were bled on Days 12, 17, 30, and 36 following initiation of SRBC inoculation. On Day 44 the two groups that received oral inoculations were rechallenged by an %-day course of oral inoculations, and mice in the IPinoculated groups were given a single IP injection of SRBC. The IgA, IgG, and IgM AFC in the spleens of six mice in each of the orally inoculated groups were counted at 6, 9, and I2 days after initiation of secondary SRBC inoculations. In the IPinoculated groups, plaque counts were made at 2, 6, and 9 days after SRBC inoculation. HA titers were measured in sera from all groups, and worm burdens were determined at the time of plaque analysis. Results are shown in Fig. 3 and Table II. Mean HA titers following primary exposure to both oral and IP immunization were significantly (P = 0.005) lower in the infected groups at all the times tested (Fig. 3). Following a second antigen exposure, orally immunized, infected mice had lower (P = 0.05) titers than uninfected mice at 6 and 9 days but similar titers at Day 12. Following IP challenge, mean

264

SHIMP,

CRANDALL

AND

TABLE Immune

Group

Response of Mice Secondary Oral

Dayb

Mean

CRANDALL

II

Infected with Heligmosomoides and Intraperitoneal Inoculation plaques/spleen

X lo+

Infected

polygyrus and Uninfected of Sheep Erythrocytes”

(SE)

Mice

Mean hemagglutinin titer, log2 (SE)

Uninfected Infected

kG

Oral

to

Ig4

IgM

[email protected]

W

IgM

Uninfected

6

1.05 (0.75)

0.81 (0.49)

1.39 (1.27)

4.68 (1.59)

5.49 (1.16)

2.39 (1.59)

6.7 (1.4)

12.8

9

3.50 (0.95)

4.30 (1.12)

1.25 (0.66)

9.74 (3.33)

11.8 (3.59)

1.25 (0.44)

12.2 (1 ..i)

16.8 (1.4)

12

8.44 (4.32)

8.27 (3.66)

1.11 (0.47)

5.41 (2.38)

8.73 (3.11) ______.__~

2.65 (1.67)

13.3 (1.8)

14.2 (0.9)

19.4

(ii)

(E)

18.5 (2.5)

(k4,

7.5

(Y:“,)

13.3 (0.4)

(2.2)

IP 2

(0.8) 6 9

62.9 (16.7)

28.8

WI

80.1 (11.3)

39.6 (5.0)

3.7 (0.9)

17.5) (0.4)

20.7 (1.3)

39.7 (8.0)

21.8 (3.7)

32.2 (3.7)

10.9 (3.1)

I.1 (0.5)

18.,? (0.5)

18.5 (1.1)

a See text for details. b Days after beginning ml of 20y0 SRBC.

oral inoculations

(E)

(2.0)

with

0.1 ml of 20y0 SRBC

titers were lower at borderline significance (P = 0.05 to 0.10) in the infected groups at 2 and 6 days and were nearly identical at Day 9. The mean worm burdens of the two helminth-infected groups were not significantly different; the combined mean was 291.7 f 36.5. Following secondary oral immunization, the splenic response was characterized by a relatively high content of IgA antibodyproducing cells, as was observed following initial immunization. Paralleling the HA titers, mean numbers of IgA- and IgG-containing cells were marginally lower (P = 0.05 to 0.10) in the infected animals at 6 and 9 days but equal to the uninfected group at 12 days. In the splenic response to IP immunization, IgG was the predominant class of antibody-producing cells. The infected group appeared to have a lower maximum response, but differences between infected and uninfected groups at

or days aft.er

single

II’

injection

of 0.1

the three time periods tested were not significant. To determine if infection altered the Ig class composition of the splenic AFC response, ratios of 1gA:IgG AFC were compared from all experiments at each time period. No significant differences were observed in these ratios between infected and uninfected groups or in ratios following primary and secondary immunization. The means of ratios for all time periods following primary oral immunization were 2.0 and 1.8 for infected and uninfected groups, respectively; following secondary oral immunization, the means were 1.76 and 1.84. Following primary IP inoculation, these ratios were 0.42 and 0.41 for infected and normal mice in one experiment and 0.37 and 0.32 in the second experiment; following secondary inoculation, the ratios were 0.77 and 0.42; the high ratio, 0.77, was due to the 2-day response

SUPPRESSION

OF

HETEROLOGOUS TABLE

Distribution

Inoculation groups”

IMMUNITY

BY

Heligmosomoides

III

of Antibody to Sheep Erythocytes and Immunoglobulins Fractions from Orally and Parenterally Immunized Mice Serum fractionsb

265

Hemagglutinin titer (log 2)c PBS

2-ME

in St-rum

Immunoglobulin concentration (mg/ml) IgG,

Id

1. Oral, primary 12-day

Whole serum 0.01 M 0.20 M

9 8 3

ND 8 3

1.1 0.5 0

0.21 0 0.12

2. IP, primary B-day

Whole serum 0.01 M 0.20 M

17 17 10

ND 17 4

5.9 1.2 <0.4d

0.34 0 0.16

3. Oral, g-day

Whole serum 0.01 M 0.20 M

>24 17 6

ND 17 6

2.2 0.8 < 0.4d

0.46 0 0.19

Whole serum 0.01 M 0.20 M

>24 >24 14

ND >24 8

4.8 1.4 < 0.4d

0.50 0 0.20

secondary

4. IP, secondary 6-day

a See text for details. b Serum fractionated on DEAE cellulose. Fractions shown eluted with 0.01 M and 0.20 M NaCl column equilibrated in 0.01 M PO4 buffer, pH 7.5. c Titers measured in PBS, resuspended, and treated with 2-ME. ND = not done. d IgG, detectable, but concentrations (<0.4) could not be estimated accurately with standards Approximate values are 0.2 mg/mi for oral group and 0.3 mg/ml for IP groups.

in infected animals in which the mean ratio was 1.2 with a 0.8 standard error. Although no differences were detected in Ig distribution of splenic AFC with infection, there was an obvious difference in 1gG:IgA ratio following oral and IP inoculation. To determine if the serum HA antibodies following oral and IP inoculation reflected this Ig class difference in splenic response, sera were fractionated by ionexchange chromatography (DEAE-cellulose) to obtain fractions containing predominantly IgG or IgA-IgM. HA titers and Ig levels were compared on these fractions. Four serum pools were examined; these were from mice of uninfected groups in which splenic AFC assays had been done. These groups were ( 1) primary oral inoculation of SRBC at 12 days, (2) primary IP inoculation of SRBC at 6 days, (3) secondary oral inoculation at 9 days, and (4) secondary IP inoculation at 6 days (see Tables I and II). Under the

from

used.

elution conditions employed, previous study had shown that the 0.01-M and 0.05-M elution fractions contained predominantly IgG ( IgG, and IgG2); and the 0.20-M, IgA and IgM. Partial results, including only 0.01-M and 0.20-M serum fractions, are shown in Table III. The results demonstrate that HA antibody was primarily in IgG following both oral and IP immunization, with higher IgGi levels and antibody responses in IgG on IP inoculation. IgM antibody, as measured by 2-ME sensitivity, was observed only in IP immunization. IgA serum concentrations and antibody titers following 2-ME reduction in fractions containing IgA were similar following both routes of immunization. The actual titers of IgA antibody cannot be determined from these results because low levels of IgG also were present in the fractions containing IgA. An additional fractionation was carried out on pooled sera from uninfected mice bled at four

266

SHIMP,

CRANDALL

intervals following primary oral inoculation (Fig. 2) with essentially identical results to those obtained in the other orally inoculated groups examined. DISCUSSION

This study was undertaken to evaluate the influence of an intestinal nematode infection on the antibody response to antigenie stimulation from the intestinal tract, Antigenic exposure from the intestinal tract may result in both local and general antibody responses (Heremans 1971; Robertson and Cooper 1972). Typically IgA predominates in the local intestinal response in many species including the mouse ( Crabb& et nE. 1969). The relationship between local intestinal response and circulating antibody detected following oral immunization is variable. Peroral immunization may lead to circulating antibody in which Ig classes are similar to those following a parenteral immunization, with IgG as the major Ig class, or circulating antibody may have IgA as the predominant Ig class paralleling the intestinal response (Heremans 1971). The determining factors for the “enteric” or “parenteral” types of response following oral immunization are not completely clear, although immunogenic levels of antigen reaching the circulatory system from the intestinal tract would be anticipated to induce a parenteral type of response. In the enteric type of response, there is good evidence that the gut-associated lymphoid tissues (GALT) are the principal source of serum antibody, and systemic sensitization to antigen is produced primarily by migration of lymphoid cells from GALT to the spleen and peripheral lymphoid tissues rather than direct antigenic stimulation (Dolezel and Bienenstock 1971; Rothberg, Kraft, and Michalek 1973). Therefore, in this study changes with helminth infection in serum antibody titers and splenic responses following oral immunization could reflect alterations in either intestinal re-

AND

CRANDALL

sponses, or in systemic immunization and response, or both. This study has demonstrated a reduced HA response to oral immunization in E-I. polygyrz~ infected mice. The reduction in mean antibody titers of infected animals was usually less than three (log 2), but this reduction was highly significant statistically in experiments that compared large ( >15) groups of mice. The reduced titers persisted for at least several weeks following immunization and were equally evident after secondary and tertiary immunization. These results indicate a sustained suppression of circulating antibody levels by the helminth infection. The nature of this titer reduction was not examined in the sera, but IgM was apparently not involved, and HA antibodies were principally in IgG following oral immunization. The experimental data are too limited to allow a detailed comparison of splenic AFC responses in infected and uninfected mice to primary and secondary oral immunization, but the results suggest a retardation and reduction in the maximum splenic IgA and IgG responses of infected mice. The IgM responses were low and variable in both infected and uninfected groups, correlating with the resistance of HA titers to 2-ME reduction. Comparison of AFC responses by Ig class gave no evidence of differences with helminth infection. Differences would be expected if infection resulted in altered exposure of the spleen to direct antigenic stimulation via the circulatory system or in changes of Ig classes in the GALT response. The comparison of HA titers in infected and noninfected mice following IP and oral immunization, in which large experimental groups were used, demonstrated similar titer reductions in infected animals with both inoculation routes. Experiments examining the splcnic responses and HA titers to primary IP immunization did not confirm significant differences

SVPPRESSION OF HETEROLOGOUS

between infected and uninfected mice, but these results may be due to the small size of the experimental groups. In total, the results support the conclusions that suppression of circulating antibody observed in this study was not unique to the oral route of immunization and a general immunosuppressive effect was induced by infection. Studies with other immunogens also have demonstrated immunosuppression in mice infected with this helminth (Cypess, Lubiniecki, and Hammon 1973; Chowaniec, Wescott, and Congdon 1972). It should be noted, however, that this study does not exclude the possibility of immunologic alterations due to intestinal damage by the helminth. The intestinal, secretory immune response was not examined, nor was the immune response investigated in detail during acute intestinal inflammatory stages of infection or following repeated infections. Although this study did not show qualitative changes with infection in the immune response to either oral or IP immunization, there was a difference in splenic and humoral responseswith the two routes of immunization. Following oral immunization, the relatively low splenic response in relation to HA titers, in comparison with IP immunization, and the relatively high IgA content of the splenic response are generally consistent with an enteric type of response. Other investigators have reported a similar Ig distribution of splenic AFC following oral SRBC immunization, although fewer IgG-producing cells and an Ig distribution more closely paralleling that of the intestinal lymphoid tissues were observed (Andre, Bazin, and Heremans 1973). The predominance of IgG HA antibody following oral immunization in the present study does not suggest a GALT source but does not exclude it. The possible influence on the nature of the immune response of the two different methods of oral immunization used, gastric intubation and oral inoculation, requires

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further investigation and may explain differences in this study and that of Andre, Bazin, and Heremans (1973). Gastric intubation was used in studies of splenic AFC, and HA titers in these studies generally were higher than those in which oral inoculation was employed. Minor trauma to mucosal surfaces can lead to increased systemic immunization by orally administered antigens (Thomas et nl. 1973), and the possibility of some immunization through damaged mucosal surfaces due to gastric intubation cannot be excluded. The basis of the reduced immune responsein H. polygyms infected mice is unknown. Reduced antibody responses to unrelated antigens have been reported in several nematode infections; antigenic competition, immunosuppressive substances from the helminth, and loss of antibody from the intestinal mucosa have been suggested as causes of suppression, but no conclusive proof of mechanism has been presented (Faubert and Tanner 1971; Cypess, Lubiniecki, and Hammon 1973; Chowaniec, Wescott, and Congdon 1972). Undernutrition has been demonstrated in H. polygyrus infections (Symons and Jones 1971) and could be a contributing factor. The high, persistant IgG levels in H. polygyrus infections (Crandall, Crandall, and Franc0 1974) indicate sustained antigenie stimulation and suggest antigenic competition as a reasonable possibility. ACKNOWLEDGMENTS This work was supported in part by Public Health Service Grants AI-03212 and AI-05345 from ,the NIAID. Mr. Robert Shimp w’as supported by NIH Training Grants RR 050-03 and 5TI AI 0128. We thank Ms. Charlene Francis for her technical assistance.

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