Clin. exp. Immunol. (1978) 33, 30-37.

Analysis of immunosuppression during early acute infection of mice with Ascaris sMum R. B. CRANDALL, CATHERINE A. CRANDALL &JOYE F. JONES* Department of Immunology and Medical Microbiology, and Department of Pathology, University ofFlorida College ofMedicine, USA

(Received 30 January 1978)

SUMMARY

Immunosuppression was examined at 10 to 12 days following oral inoculation of 10,000 to 12,000 embryonated Ascaris suum eggs. Reduced antibody responses to sheep red cells (SRC) following systemic immunization were confirmed in CD-1 and C57BI/6 mice. Infection alone induced antibody reactive with DNP equivalent to that observed after immunization with DNPFicoll. There was a decrease in thymus and spleen size by day 8 of infection, followed by a splenic proliferative response during the second week. In the second week, serum antibodies reactive with SRC, chicken erythrocytes, DNP and bacterial lipopolysaccharide were demonstrated, suggesting polyclonal B-cell stimulation. The cellular basis of immunosuppression was investigated by in vitro culture of splenocytes from C57B1/6 mice. Differential leucocyte counts of splenocytes before culture demonstrated a relative increase in plasma cells, blastoid cells, complement receptor-bearing lymphocytes and eosinophils, with a relative decrease in small lymphocytes. The splenocytes had reduced responses to T-cell mitogens, as measured by thymidine incorporation in vitro, and reduced antibody responses to SRC and DNP-Ficoll. In vitro, cell mixing experiments did not demonstrate suppressor cells in the spleens of infected mice.

INTRODUCTION Experimental nematode infections have been shown to induce various alterations in immune responsiveness, including immunosuppression (Faubert & Tanner, 1971; Shimp, Crandall & Crandall, 1975; Potaro, Britton & Ash, 1976). Such alterations could be of significance in both the pathogenesis of infection and development of acquired resistance. The causes of these alterations and the conditions of infection which induce them are not well defined, and have been examined in only a few experimental models. A previous study demonstrated immunosuppression during acute, primary Ascaris suum infection in mice (Crandall & Crandall, 1976). This experimental infection has been well characterized and serves as a model for a common type oflarval nematode infection of man and certain animals. The purpose of this study was to investigate the mechanisms of immunosuppression produced by Ascaris infection. Infection with Ascaris is self-limiting in the mouse and induces a complex, rapidly changing host response; therefore, mediation of suppression may vary during the course of infection. For this reason a single time period, 10 to 12 days after infection, was selected for study. At this time a high IgM response to the parasite has been induced, and antibody responses and delayed hypersensitivity to systemic antigenic stimulation by sheep erythrocytes (SRC) are depressed (Crandall & Crandall, 1976). *Present address: National Cancer Institute, NIH, Bethesda, Maryland 20014, USA. Correspondence: Dr R. B. Crandall, Department of Immunology and Medical Microbiology, Box J-266 JHMHC, University of Florida College of Medicine, Gainesville, Florida 32610, USA. 0099-9104/78/0700-0030$02.00 ©) 1978 Blackwell Scientific Publications

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Ascaris suum infection in mice

31

MATERIALS AND METHODS Animals and experimental infections. The mice used in these experiments were 3-4 month old female CD-1 (Charles River, Wilmington, Massachusetts), and C57BI/J6 (The Jackson Laboratories, Bar Harbour, Maine). The mice were maintained in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Methods used for obtaining and embryonating A. suum eggs, and the infection of the mice, have been described (Arean & Crandall, 1963). In this study mice were infected by oral inoculation of 10,000-12,000 embryonated A. suum eggs. Antigens and immunization. Sheep erythrocytes (SRC) were obtained from Baltimore Biological Laboratories, Cockeysville, Maryland or Colorado Serum Co., Denver, Colorado). DNP-Ficoll was prepared as described by Sharon et al. (1975). The preparation used in these experiments contained 52 mol of DNP-lysine per 4x 104 daltons of carbohydrate. E. coli lipopolysaccharide (LPS) 0127 :B8 was obtained from Difco Laboratories, Detroit, Michigan.) Procedures for immunization with SRC were described pre iously (Crandall & Crandall, 1976). Immunization with DNP-Ficoll was done by i.p. injection; serum antibody and splenic antibody-forming cells (AFC) were measured 7 days after injection. Immunization with LPS was done by i.v. injection; serum antibody was measured 8 to 9 days later. For in vitro immunization, the Click, Benck & Alter (1972) technique for the culture of splenocytes, as described prexiously (Jones, Crandall & Crandall, 1976), was used. Briefly, 107 cells, to which SRC or DNP-Ficoll were added, were cultured at 370C in gas-tight boxes and were gassed daily with a mixture of 5% CO,, 12% °2 and 83% N2. Cultures were assayed for the number of direct AFC on day 4 with DNP-Ficoll immunization, or day 5 with SRC immunization. Three to fiv e replicates were cultured for each experimental group. Measurement of antibody. Direct (IgM) AFC and haemagglutinin (HA) titres were assayed as described previously (Shimp et al., 1975). For anti-DNP assays, hapten-conjugated anti-SRC Fab fragments coupled to SRC were used (Strausbach, Sulica & Gixol, 1970). Serum antibodies to LPS were measured as described by Anderson & Blomgren (1971). All sera were absorbed with SRC prior to measurements for LPS and DNP antibodies. A1itogen stimulation and determination of cell populations. Micro-methods described by Jones et al. (1976) were used to measure the proliferation of splenic lymphocytes stimulated by mitogens. Several concentrations of each mitogen were used to determine optimal responses. Differential cell counts were made on slides prepared with a cytocentrifuge (Shandon Scientific Co. Ltd, London) and stained with May-Grunwald-Giemsa stain. Complement receptor-bearing lymphocytes were enumerated by the bacterial antibody-complement (BAC) rosette assay described by Gormus, Crandall & Shands

(1974). Statistical analysis. Data were analysed using Student's t-test with log transformations of the AFC data.

RESULTS

Antibody responses Initial experiments confirmed previous results (Crandall & Crandall, 1976) that antibody responses to SRC were depressed in CD-i mice following intravenous or intraperitoneal immunization on day 11 after oral infection with 10,000-12,000 A. swinm eggs. Immunization at the same period of A. slium infection with a T-independent immunogen, LPS, did not demonstrate significant differences in antibody responses between infected and control mice (Table 1). To determine if a similar degree of immunosuppression was induced by A. situm infection in an inbred mouse strain, which could be used in cell culture studies, C57B1/6 mice were infected and subsequently immunized on day 11 with SRC or DNP-Ficoll, a T-independent immunogen. The results were similar to those observed with CD-i mice (Table 2). Infected mice had a reduced antibody response to SRC, but their responses to DNPFicoll were similar to those of the controls. However, mice infected with A. swtum which had received no antigen had antibody titres to DNP which were almost as high as those of mice immunized with DNP-Ficoll. The antibody reactive to DNP induced by infection in C57B1/6 mice and the high serum IgM levels previously observed suggested that infection might induce non-specific B-cell activation. To determine if infection induced antibodies with specificities consistent with a polyclonal B-cell activation (PBA), CD-1 mice were infected with 10,000 A. snum eggs, bled at intervals and antibodies reactive with SRC, chicken erythrocytes, LPS and DNP were titred. Antibodies reactive with these antigens were demonstrated after day 8 of infection; the initiation and time-course of these antibody responses appeared similar, although the data are not extensive enough to make detailed comparisons. Antibody titres to SRC were essentially at the background level by 21 days, while titres to DNP and LPS remained elevated (Fig. 1). The antibody titres to LPS induced by A. snum infection were similar to those observed in infected mice immunized with LPS. The antibody reactive with DNP was exclusively IgM, as judged

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R. B. Crandall, Catherine A. Crandall 5 Joye F. Jones TABLE 1. Serum haemagglutinin titres after immunization with sheep erythrocytes or lipopolysaccharide in CD-1 mice infected with Ascaris suum

Antigen*

Dosage (route)

SRC

2 x 106 (i.v.) 2 x 108 (i.v.) 2 x 108 (i.p.)

LPS

20-0 ig (i.v.) 80 0 [tg (i.v.)

Days after antigen injection 4 6 11 4 6 11 4 6 11 8 11 8 11

HA titre

+s.e. (log2)t

Infected

Control

2-2 + 0-6t

5-2 ± 0 4

4-4±07t 4-4 ± 07t

6*4±0*3

6-5 ± 0 3t 5-5± 05t 5-5 + 0-9t 6-6 ± 04t

5-8±0-2t 7-2 ± 0-4t 95 ±03 90 ±04 10-5 0-6

90±0-5

7-2 ± 8-2 ± 7-8± 8-4 ± 9-6 ±

0*4 0-6 0-8 0-6 07 80± 0-5 9-6 ± 0-7 100 ± 04 10-0 ± 05 9-6 ± 0-2 9-6±0-4

* Mice were injected with SRC 10 days and LPS 11 days after Ascaris infection.

t Titres are means + s.e. of five or six mice in each group. t Significantly less than the control value (P< 0.05). TABLE 2. Antibody response to immunization with sheep erythrocytes and DNP-Ficoll in C57BI mice infected with Ascaris suum

Treatment Infected Non-infected Infected Infected Non-infected Non-infected

AFC per Number of spleen + s.e. mice Immunization* (x 10-3)t 6 6 6 5 6 2

SRC SRC DNP-Ficoll None DNP-Ficoll None

HA titre ± s.e.

(log2)t

9 9 + 0 54 7-2 + 0.5t 140-6 15-0 10-3 0-8 18-5 ± 8-1 15-0 ± 04 19-4 ± 4.9 16-8 ± 1-6 26-7 i 4-9 11-5 0-3 0 5-5 ± 05

* Mice were injected i.p. with 2 x 108 SRC or 80-0 tig DNP-Ficoll 11 days after an oral inoculation of 10,000-12,000 A. suum eggs. t Direct (IgM) splenic AFC and serum HA titres were measured 5 days after immunization with SRC and 7 days after immunization with DNP-Ficoll. t Significantly less than the control value (P < 0 05).

by exclusion from Sephadex G-200. Adsorption of sera with A. suum extract failed to reduce significantly the antibody titre to DNP (data not shown). In a similar experiment, splenic antibody responses (direct haemolytic plaques) to DNP were measured. The mean numbers of antibody-forming cells (AFC) per spleen were 81-3±24, 1338±269, 869±185 at 8, 10 and 13 days, respectively, after infection. The numbers ofAFC in uninfected, control mice ranged from fifty to 160 per spleen.

Changes in splenocyte populations Quantitative and functional changes in splenocyte populations during early A.

suum

infection were

33

Ascaris suum infection in mice

2

a

0

0

4

4

8

11

16

21 0

4

8

11

16

21

Days post-infection FIG. 1. Haemagglutinin titres to (a) sheep erythrocytes (SRC) and chicken erythrocytes (CRC), and (b) to, lipopolysaccharide (LPS) and dinitrophenyl (DNP) in mice infected with Ascaris suum. For (a): (a-) CRC control; (a) CRS, A. suum; ( 0) SRC control; (a) SRC, A. suum. For (b): (0) LPS control; (a) LPS, A. suum; ( 0) DNP control; (-) DNP, A. suum.

investigated. In conjunction with the experiment measuring splenic antibody to DNP, the total splenocyte numbers were counted. Mean numbers were 192±18, 93±5, 180±22 and 330±74 (x 106) at 6, 8, 10 and 13 days, respectively, after infection. The mean of the uninfected was 160±9. This decrease in cell numbers by day 8, followed by an increase to above-normal numbers by the end of the second week of A. suum infection, was consistently observed by splenic size changes in both C57BI/6 and CD-1 mice. Visual examination of the thymus indicated a similar change: thymus size was greatly reduced by day 8; it subsequently enlarged, but was still obviously less than control size at day 13 of infection. Detailed examination of changes in splenocyte populations in C57BI/6 mice on day I11 of infection was made on cell suspensions used for in vitro cultures. Differential leucocyte counts indicated a relative increase in blastoid cells and large lymphocytes, plasma cells and eosinophils, with a relative decrease in small lymphocytes; these cell populations had approximately 10% more complement receptor-bearing cells (primarily B lymphocytes) than control populations (Table 3). The total number of nucleated cells per spleen was somewhat variable, but generally about 20% higher than in the infected mice. A comparison of responses to mitogen stimulation in culture demonstrated in the splenocytes of infected mice an increased background incorporation of [3H] thymidine, a reduced response to the T-lymphocyte TABLE 3. Splenic cell population of C57BI mice infected with Ascaris suum

Mean percentage cell type ± s.e.* Cell type Small lymphocytes Large lymphocytes and blastoid Plasmacells Macrophages Neutrophils Eosinophils Complement receptor

Infectedt

Non-infected

55 0 ± 50

76-0 i 30

27-0 ±30

19 0 i40 07 05 05 0-2 2-7 ± 10 0-8 03 38-2 1-5

6-0±2-0 17 i 05 4-2 ± 07 5-7 0-8 53 0 3-6

Counts were based on five spleen pools; each pool contained spleens from five to ten mice. t Spleens were removed 11 days after an oral inoculation of A. suum. *

C

R. B. Crandall, Catherine A. Crandell & Joye F. Jones

34

mitogens concanavalin A (Con A) and PHA, and an increased response to the B-cell mitogen LPS (Fig. 2). No difference in the mitogen concentrations inducing the maximum lymphocyte responses was detected for infected and control mice. Additional experiments on mitogen responsiveness using a different culture medium, containing 2-ME, always confirmed the reduced response of splenocytes from infected mice to T-cell mitogens, but the response to LPS was variable, often with no significant differences from that of control cultures (data not shown). Antibody response in vitro To investigate the cellular basis of immunosuppression, in vitro studies of the primary antibody response were conducted with splenocytes of C57B1/6 mice. Cultures from infected mice consistently gave reduced antibody responses to SRC (Table 4a); often these responses were not above the back10.f0 _

.0

1.0

E

0

~~~~~A

A

0.1~~~~

091_

50 /Lg Con A LPS (P