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The Journal of Immunology, 1999, 163: 898-905.
Copyright © 1999 by The American Association of Immunologists

Neonatal Exposure to Idiotype Induces Schistosoma mansoni Egg Antigen-Specific Cellular and Humoral Immune Responses

M. Angela Montesano*,{dagger}, Daniel G. Colley{dagger}, George L. Freeman, Jr.{dagger} and W. Evan Secor1,{dagger}

* Departamento de Microbiologia, Immunologia e Parasitologia, Universidade Federal de Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil; and {dagger} Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA 30341


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Exposure of neonatal mice to appropriate, cross-reactive Id (CRI) preparations alters immune responsiveness, ameliorates pathology, and prolongs survival of animals upon subsequent Schistosoma mansoni infection. However, because schistosome infections profoundly affect host immunobiology, which responses are effected by neonatal Id exposure alone and which responses are influenced by infection is unclear. To directly examine the schistosome soluble egg Ag (SEA)-specific immune responses altered by CRI exposure, neonatal mice were injected with CRI-expressing (CRI+) SEA-specific Ab preparations, SEA-specific Abs that did not express CRI (CRI-), or normal mouse Ig. At 9 wk of age, only mice that were neonatally exposed to CRI+ anti-SEA Abs displayed significant SEA-specific IgG serum levels and spleen cell proliferative responses. SEA-stimulated spleen cells from these CRI+-exposed mice also produced IFN-{gamma}, although not at significantly higher levels than mice receiving CRI- Id or normal mouse Ig. If CRI+-exposed mice were also injected with SEA at 8 wk of age, the 9-wk IFN-{gamma} responses were significantly higher than those of the other neonatal injection groups. The presence of both CRI and anti-CRI in the sera of animals neonatally injected with CRI, but receiving no exposure to S. mansoni Ags or infection, suggested a functional idiotypic network led to these responses. These data demonstrate that appropriate idiotypic exposure induces B and T cell responsiveness to the Ag recognized by the Id and support the hypothesis that neonatal idiotypic exposure can be an important immunoregulatory factor in schistosomiasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic infectious diseases present unique opportunities for the investigation of immunoregulation. In infections where the host is not immediately able to clear the infectious agent nor is rapidly killed by the disease, a fine immunologic balance often ensues. Some degree of host immune response is necessary to prevent damage of host tissues by the infecting agent; however, too much of an unregulated immune response results in pathologic consequences that are similarly detrimental. Understanding the components important for striking this balance can provide valuable insights into regulation of immune responses in such infections, autoimmunity, and neoplasms.

In experimental Schistosoma mansoni infections, failure to mount a granulomatous immune response to parasite eggs lodged in the liver results in hepatic necrosis caused by egg enzymes (1, 2). Conversely, an unregulated immune response to egg Ags leads to increased liver fibrosis and is associated with increased morbidity and mortality in the chronic phase of infection (3, 4, 5, 6, 7). A balanced response to egg Ags is also necessary for minimum pathophysiology in human schistosomiasis. Patients who progress to the relatively asymptomatic, intestinal chronic form of disease demonstrate reduced schistosome soluble egg Ag (SEA)2-specific PBMC responses following the acute stage of infection, while patients who are in the early stages of hepatosplenic disease have continued strong PBMC responses to SEA (8, 9, 10). Thus, a failure to regulate the acute-stage responses to egg Ags correlates with the development of severe schistosomiasis, while immunoregulation of SEA-specific responses is associated with less severe and even asymptomatic disease.

Immunoregulatory, cross-reactive Ids (CRI) expressed on anti-SEA Abs are strongly associated with less severe chronic schistosomiasis mansoni in both humans and mice (4, 11, 12, 13, 14). Patients with the chronic intestinal form of schistosomiasis and 20-wk infected mice with the analogous moderate splenomegaly syndrome (MSS) express these CRI on their circulating anti-SEA Abs. Patients with the intestinal form of disease and MSS mice share Ids as shown by their comparable recognition by the same rabbit anti-Id Ab preparations. Conversely, patients with severe hepatosplenic disease and mice with the analogous hypersplenomegaly syndrome (HSS) fail to express these immunoregulatory CRI, despite having high titers of anti-SEA Ab in their sera (4, 11, 12, 13, 14). Ids prepared from sera of MSS mice (MSS Id) and 8-wk-infected mice (8WkId) both express CRI (are CRI+). MSS Id and 8WkId stimulate autologous cells to produce cytokines associated with immunoregulation in schistosomiasis (IFN-{gamma} and IL-10, respectively) (14). In contrast, Id prepared from HSS mouse sera (HSS Id) does not express CRI (is CRI-) and is not stimulatory for cells from any source. Immunoregulatory Id has also been demonstrated in the sera of mice and humans with Schistosoma japonicum infections (15, 16, 17).

Another unique aspect of chronic infectious diseases such as schistosomiasis is the effect maternal infection may have on the developing immune response of their offspring. Typically, people become infected with schistosomiasis during childhood and, barring chemotherapeutic intervention, usually maintain their infections into middle age; a span that includes the child-bearing years of adult women. Thus, most children born in areas endemic for schistosomiasis may well be born of infected mothers. Studies of children of infected mothers have demonstrated the presence of anti-SEA Abs as well as Id-responsive lymphoid cells in umbilical cord blood (18, 19, 20). Such in utero exposure to Id-bearing, Ag-specific Abs, and possibly parasite Ags, is likely to influence the developing immune environment of children born to infected mothers (18, 21, 22) and may predispose them to respond with an altered, perhaps more immunoregulated form of schistosomiasis. Anecdotally, persons born in endemic areas who become infected with schistosomiasis present with less severe symptoms than travelers or immigrants into an endemic area who become infected and whose mothers would not have been infected (23, 24, 25). Recently, we used an experimental model to test the effects of neonatal Id exposure on mice that were subsequently infected with schistosomiasis (26). Animals that received CRI+ anti-SEA Abs at birth (8WkId or MSS Id) had altered profiles of cytokine and Ab isotypes at both 8 and 20 wk after subsequent infection compared with animals that had received CRI- Ab preparations (HSS Id or normal mouse Ig; NoMoIgG). Also, recipients of CRI+ Abs demonstrated decreased granuloma size and liver fibrosis at 8 wk after infection as well as dramatically increased survival at 20 wk after infection, indicating that neonatal exposure to CRI is distinctly beneficial during subsequent experimental schistosomiasis (26). The florid granulomatous response at 8 wk after infection is associated with a Th2-type cytokine response (27, 28, 29) and can be down-regulated by IFN-{gamma} (30, 31, 32). Animals that received CRI+ Abs as neonates and had ameliorated pathology at 8 and 20 wk after infection demonstrated increased IFN-{gamma} responses to SEA (Ref. 26 and M. A. Montesano et al., manuscript in preparation). The current study is designed to more directly investigate the immunologic effects of neonatal Id exposure by evaluating the Ag-specific responses of mice that were injected with different Id preparations at birth but were not subsequently infected with S. mansoni.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
SEA and Id preparation

The methods used to prepare SEA have been presented elsewhere (33). Briefly, S. mansoni eggs were isolated by differential centrifugation from homogenized liver tissue of CF-1 mice (Charles Rivers Laboratories, Wilmington, MA) infected with 300 cercariae for 7–8 wk. Soluble material from purified eggs was obtained by homogenization in Dulbecco’s PBS and subsequent ultracentrifugation. For Id preparations (14, 34), sera from mice at 8 wk of infection or 20 wk of infection (segregated into MSS and HSS by spleen weight and pathologic characteristics) were collected by cardiac puncture and stored at -20°C. For anti-SEA Ab preparations, SEA was coupled to cyanogen bromide-activated Sepharose 4B (Sigma, St. Louis, MO). Pooled sera from MSS or HSS mice were passed over this SEA-Sepharose column. Anti-SEA Abs (Id) were eluted using 0.1 M glycine-HCl, pH 2.8, and collected into 0.025 M borax for neutralization. The eluates were concentrated, dialyzed against saline, and their protein concentrations were determined. All Id preparations (i.e., those from different serum sources) used in a given experiment were prepared over the same SEA affinity column. To verify that they were not contaminated with SEA leached from the immunoaffinity column, Id preparations were subjected to SDS-PAGE under reducing conditions and silver stained using the Pierce (Rockford, IL) Gelcode-SilverSNAP stain kit according to the manufacturer’s instructions (data not shown).

Mice, neonatal exposure, and SEA injections

CBA/J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in the American Association for Accreditation of Laboratory Animal Care-approved animal care facilities of the Centers for Disease Control and Prevention. Newborn (<24 h old) mice from uninfected parents received transthoracic, intraperitoneal injections of 50 µg immunoaffinity-purified anti-SEA Abs or commercial NoMoIgG (Sigma) in PBS. The male offspring were used for infection studies (26), and the female mice were used in these experiments. At 8 wk of age, some of the mice were injected i.p. with 100 µg SEA. At 9 wk of age, all mice were sacrificed, blood was collected by cardiac puncture for sera analyses, and spleens were harvested for cellular assays.

Determination of anti-SEA Ab levels

Ab level analyses were performed by specific ELISAs to detect anti-SEA activity. SEA (0.25 µg/well in 0.1 M NaHC03, pH 9.6) was adsorbed onto flat-bottom Immunolon II microtiter plates (Dynatech, Chantilly, VA) overnight at 4°C. Following a blocking step of PBS with 0.3% Tween 20 (Sigma), plates were incubated with 1:40 dilutions of mouse sera in PBS plus 0.05% Tween 20. Anti-SEA Abs bound to the SEA-coated plate were detected using peroxidase-conjugated, affinity-purified IgG fractions of isotype-specific goat anti-mouse IgM or anti-mouse IgG (Boehringer Mannheim, Indianapolis, IN), diluted 1:1000 in PBS plus 0.3% Tween 20. Assays were developed with 3,3',5,5'-tetramethylbenzidine peroxidase substrate (TMB; Kirkegaard & Perry Laboratories, Gaithersburg, MD), stopped with 1 M H2SO4, and read at a wavelength of 450 nm on a microplate reader (Molecular Devices, Sunnyvale, CA).

Lymphocyte proliferation assays

Single-cell suspensions of spleen cells were prepared and cultured at 5 x 105 cells/well for 3 days in flat-bottom 96-well microtiter plates (Costar, Cambridge, MA) in 200 µl RPMI 1640 (Life Technologies, Grand Island, NY) containing 5% heat-inactivated FBS (Life Technologies), 2% penicillin-streptomycin (10,000 U/ml and 10,000 µg/ml stock; Life Technologies), and 2 mM L-glutamine (Life Technologies). Cells were stimulated with media, 1 µg/ml Con A (Sigma), or 4 µg/ml SEA and were maintained in a humidified atmosphere containing 5% CO2 at 37°C. The cultures were pulsed for the final 8 h with 0.5 µCi [3H]TdR (sp. act., 17 Ci/mM; New England Nuclear, Boston, MA), harvested onto glass fiber filters, and incorporated radioactivity counted in a Betaplate (Wallac, Turku, Finland) liquid scintillation counter.

Cytokine production and measurement

Spleen cells were cultured in 48-well plates (Costar) at 2.5 x 106 cells/well in 1 ml RPMI 1640 supplemented with 2% FBS, 2% penicillin/streptomycin, and 2 mM L-glutamine. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 and stimulated with medium alone, hamster anti-mouse CD3-{epsilon} at 1 µg/ml (PharMingen, San Diego, CA), or SEA at 4 µg/ml. Supernatant fluids were harvested after 24 h (IL-4) or 48 h (IL-10 and IFN-{gamma}) and stored at -70°C. Cytokine levels were measured by capture ELISA. Plates were coated with anti-mouse cytokine mAb (PharMingen) at 2 (IL-10) or 2.5 (IL-4 and IFN-{gamma}) µg/ml in 0.1 M NaHCO3, pH 9.6, overnight. Following a blocking step of PBS with 0.3% Tween 20 and 5% nonfat dry milk, plates were incubated with 100 µl of culture supernatant for 2 h at room temperature. Bound cytokines were detected by sequential incubation with 1 µg/ml biotinylated specific anti-cytokine mAbs (PharMingen), HRP-conjugated streptavidin (Sigma), and TMB peroxidase substrate solution. Plates were washed three times with PBS and 0.05% Tween 20 between each step. The rate change in OD of each well was measured at 650 nm with a Vmax kinetic microplate reader (Molecular Devices). Levels of supernatant cytokines were calculated from log2 dilution curves of homologous recombinant cytokine standards (BioSource International, La Jolla, CA) that were included in each assay.

Preparation of rabbit anti-CRI

Rabbit anti-CRI was prepared by immunizing a rabbit (Myrtle’s Rabbitry, Thompson Station, TN) s.c. with 200 µg MSS Id preparation mixed 1:1 in RIBI adjuvant (RIBI ImmunoChem Research, Hamilton, MT). Three injections were given at 15-day intervals. Fifteen days after the final injection, the rabbit was bled. Ig was purified from the rabbit serum using a T-Gel purification kit (Pierce). Id-specific antiserum was prepared by exhaustively absorbing the immunized rabbit serum with NoMoIgG (Sigma) coupled to cyanogen bromide-activated Sepharose 4B (10 mg Ig/g Sepharose). Repeated absorptions (15, 16, 17, 18, 19, 20) were required to yield reagent, which did not react with NoMoIgG by ELISA, while reactivity with the original immunizing MSS Id preparation was maintained.

Competitive ELISAs for Id and for anti-Id

The competitive ELISA used for measurement of serum Id levels has been described previously (11). Briefly, MSS Id preparation (0.25 µg/well in 0.1 M NaHCO3, pH 9.6) was adsorbed to the wells of Immunlon II plates as described above. Following a blocking step, plates were incubated with anti-MSS Id rabbit sera that had been preincubated with different dilutions of mouse sera in a microcentrifuge tube for 30 min. If Id is present in the mouse sera, it binds to the rabbit anti-MSS Id Abs and prevents them from binding the MSS Id on the plate. The level of rabbit anti-MSS Id bound to MSS Id on the plate was determined by the addition of biotinylated donkey anti-rabbit (Amersham Life Sciences, Arlington Heights, IL), followed by streptavidin-peroxidase (Sigma) and TMB substrate (Kirkegaard & Perry Laboratories). Reactions were stopped with 1 M H2SO4 and read at a wavelength of 450 nm. A standard inhibition curve was prepared for each plate by preincubating known concentrations of MSS Id with the rabbit anti-MSS Id.

To detect anti-Id, the blocked MSS Id-coated plates were incubated with different dilutions of mouse sera before the addition of the rabbit anti-Id. Anti-Id in the mouse sera binds to the MSS Id on the plate, and unrelated Abs, including Id, are then washed off. The bound anti-Id that remains competes with the subsequent binding of added rabbit anti-MSS Id Abs. Assays were developed using biotinylated anti-rabbit Ig, streptavidin-peroxidase, and TMB substrate as above.

Statistical analyses

Statistical analyses were performed using GraphPad Instat (GraphPad Software, San Diego, CA). Group means were compared by two-tailed t test for comparisons of two groups; two-tailed ANOVA and multiple comparison tests were employed for comparison of three or more groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ab responses of mice neonatally exposed to CRI+ anti-SEA Abs

Neonatal mice were injected with 50 µg of NoMoIgG or Id preparations within 24 h of birth. At 8 wk of age, half of the mice were injected i.p. with 100 µg of SEA. One week later, all animals were sacrificed, blood was taken for serum, and levels of SEA-specific IgM and IgG were determined (Fig. 1Go). Mice that were exposed to Id or NoMoIgG as neonates displayed negligible levels of anti-SEA IgM in their sera at 9 wk of age (Fig. 1Go, left). Injection of SEA 1 wk earlier induced higher anti-SEA IgM levels in all groups of animals. Those animals that had received CRI+ preparations (8WkId, top, or MSS Id, bottom) did have a significantly higher serum level of SEA-specific IgM than animals neonatally injected with NoMoIgG or HSS Id at birth. In contrast to the IgM responses, animals neonatally injected with CRI+ preparations had strong SEA-specific IgG responses at 9 wk of age, while animals that received NoMoIgG or HSS Id had little to no SEA-specific IgG (Fig. 1Go, right). Animals exposed to CRI+ Ab at birth, injected with SEA at 8 wk of age, and sacrificed at 9 wk had an increased SEA-specific IgG response compared with animals that had received only CRI+ Abs at birth. However, SEA-specific sera IgG levels of animals injected with NoMoIgG or HSS Id at birth and SEA at 8 wk of age remained low.



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  		FIGURE 1. Neonatal injection of CRI induces SEA-specific IgG. Mice were injected with 50 µg Id or NoMoIgG in the first 24 h of life. At 8 wk of age, some mice were injected with 100 µg SEA i.p. At 9 wk of age, mice were sacrificed and the sera were tested for SEA-specific IgM (left) or IgG (right). The neonatal injection is indicated in the key and the total n values are shown in parentheses as: (number of mice in group with no injection; number of mice in group injected with SEA at 8 wk). *, p < 0.001 compared with animals neonatally injected with NoMoIgG or HSS Id.

 
SEA-specific spleen cell proliferative responses of mice neonatally exposed to CRI

As with the anti-SEA serum IgG responses, spleen cells from animals that were injected with 8WkId or MSS Id at birth and that received no further exposure to Id or SEA had significant in vitro proliferative responses to SEA (Fig. 2Go). Injection of animals with SEA at 8 wk of age increased the SEA-specific proliferative response for all groups but animals that had received CRI+ preparations at birth maintained a significantly higher proliferative response than animals receiving NoMoIgG or HSS Id at birth. Proliferative responses to mitogen (Con A) were similar among all groups, regardless of neonatal exposure or injection with SEA (data not shown).



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  		FIGURE 2. Neonatal injection of CRI induces SEA-specific proliferative responses. Mice were injected with 50 µg Id or NoMoIgG in the first 24 h of life. At 8 wk of age, some mice were injected with 100 µg SEA i.p. At 9 wk of age, mice were sacrificed and the spleen cells were stimulated with SEA. Data in upper panel represent the results from three experiments, and data in lower panel represent the results from two experiments. The neonatal injection is indicated in the key and the total n values are shown in parentheses as: (number of mice in group with no injection; number of mice in group injected with SEA at 8 wk). Data are reported as mean experimental c.p.m. divided by control c.p.m. ± SEM. The ranges of the mean control c.p.m. values for the various groups were 547–701 (upper panel) and 658–783 (lower panel). *, p < 0.001 compared with animals neonatally injected with NoMoIgG or HSS Id.

 
Spleen cell cytokine responses to anti-CD3 and SEA

Some of the spleen cells prepared for the proliferation experiments were stimulated with anti-CD3-{epsilon} or SEA. The cell supernatants from these cultures were harvested and tested for IFN-{gamma} (Fig. 3Go), IL-4 (Fig. 4Go), and IL-10 (Fig. 5Go) by cytokine-specific ELISA. Anti-CD3-{epsilon} stimulation of spleen cells induced similar cytokine levels for all neonatal exposure groups in a given experiment ( Figs. 3–5GoGoGo). Spleen cells from animals that had received CRI+ Abs at birth produced more IFN-{gamma} when stimulated in vitro with SEA than did spleen cells from animals that had received NoMoIgG or HSS Id at birth (Fig. 3Go). These increased levels were measurable but not significant for mice that were not also injected with SEA at 8 wk of age. However, Id-mediated priming for IFN-{gamma} production upon exposure to SEA was apparent, as the SEA-stimulated IFN-{gamma} levels were significantly elevated (p < 0.001) for CRI+-exposed animals that had received an SEA injection 1 wk before sacrifice and in vitro stimulation (Fig. 3Go). SEA-specific IL-4 and IL-10 responses were generally not higher than the lower detection limit of the assays (Figs. 4Go and 5Go).



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  		FIGURE 3. Neonatal injection of CRI primes for SEA-specific IFN-{gamma} production. Mice were injected with 50 µg Id or NoMoIgG in the first 24 h of life. At 8 wk of age, some mice were injected with 100 µg SEA i.p. At 9 wk of age, mice were sacrificed and the spleen cells were stimulated with anti-CD3-{epsilon} (left) or SEA (right). Supernatants were harvested at 48 h and tested in a sandwich ELISA for IFN-{gamma}. Data represent the results from two experiments. The neonatal injection is indicated in the key, and the total n values are shown in parentheses as: (number of mice in group with no injection; number of mice in group injected with SEA at 8 wk). Data are reported as mean experimental U/ml minus control U/ml ± SEM. *, p < 0.01 compared with animals neonatally injected with NoMoIgG or HSS Id.

 


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  		FIGURE 4. IL-4 production by spleen cells of mice neonatally injected with Id or NoMoIgG. Experimental set-up was as described in Fig. 3Go but supernatants were harvested at 24 h after stimulation with anti-CD3-{epsilon} (left) or SEA (right). Data represent the results from two experiments. The neonatal injection is indicated in the key, and the total n values are shown in parentheses as: (number of mice in group with no injection; number of mice in group injected with SEA at 8 wk). Data are reported as mean experimental ng/ml minus control ng/ml. ± SEM. There were no statistical differences between neonatal injection groups.

 


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  		FIGURE 5. IL-10 production by spleen cells of mice neonatally injected with Id or NoMoIgG. Experimental set-up was as describe in Fig. 3Go with supernatants harvested at 48 h after stimulation with anti-CD3-{epsilon} (left) or SEA (right). Data represent the results from two experiments. The neonatal injection is indicated in the key, and the total n values are shown in parentheses as: (number of mice in group with no injection; number of mice in group injected with SEA at 8 wk). Data are reported as mean experimental U/ml minus control U/ml ± SEM. There were no statistical differences between neonatal injection groups.

 
Id and anti-Id in sera of mice neonatally exposed to CRI

How neonatal exposure to anti-SEA CRI+ Abs induces B and T cell reactivities to SEA is not known. One possible mechanism would include an idiotypic network, resulting from CRI injection, that would be perturbed by exogenous Ag. Thus, the sera of neonatally injected animals that had or had not received further Ag exposure were tested for Id and anti-Id by competitive ELISAs. Fig. 6Go demonstrates the standard competitive CRI curve (right) and competition curves over a range of sera dilutions (left) for Id measurement in the competitive ELISA. Table IGo presents the data derived from this assay as the concentration of Id in the sera of mice from the various neonatal Id exposure groups that did or did not receive subsequent SEA injection. Only mice that received CRI+ anti-SEA Abs at birth (8WkId, MSS Id) demonstrated measurable levels of CRI in their sera at 9 wk of age. Injection of 100 µg SEA at 8 wk of age enhanced the level of Id expression in the sera of these mice by 9 wk. In contrast, sera of animals that received NoMoIgG or CRI- HSS Id had no detectable Id in their sera by 9 wk of age, even with injection of SEA at 8 wk of age (Fig. 6Go and Table IGo). Competitive sera preincubated with SEA-coated beads before their inclusion in this competitive ELISA no longer inhibited binding of rabbit anti-Id Abs, indicating that SEA-reactive Id was responsible for the competitive effects of these sera (data not shown).



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  		FIGURE 6. CRI levels in sera as detected by competitive ELISA. Assay details are presented in Materials and Methods. The left panel demonstrates representative competition by various dilutions of sera from 9-wk-old animals that were neonatally injected with Id or NoMoIgG. At 8 wk of age, some animals had also received an injection of 100 µg SEA i.p. Data represent results from three to five mice per group. The right panel depicts the standard curve that was used for calculation of Id levels in sera of mice. This information was used to choose the appropriate dilution for testing of all the mice and determination of serum Id concentration. Results are shown in Table IGo.

 

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  		Table I. Id levels in sera of mice neonatally injected with Id or NoMoIgG1

 
Parallel results were obtained when the competitive ELISA was reconfigured to detect anti-Id in the sera of mice exposed to Id or NoMoIgG as neonates (Fig. 7Go and Table IIGo). Mice that had been injected with 8WkId or MSS Id at birth had anti-CRI activity in their sera at 9 wk of age, while animals that were neonatally injected with NoMoIgG or HSS Id did not. Similarly, the levels of anti-CRI in sera of 9-wk-old animals that had been injected with 8WkId or MSS Id at birth were elevated in response to SEA injection at 8 wk of age, while those of animals receiving NoMoIgG or HSS Id were not.



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  		FIGURE 7. Anti-Id levels in sera as detected by competitive ELISA. Assay details are presented in Materials and Methods. Representative competition by various dilutions of sera from 9-wk-old animals that were neonatally injected with Id or NoMoIgG. At 8 wk of age, some animals had also received an injection of 100 µg SEA i.p. Data represent results from three to five mice per group. This information was used to choose the appropriate dilution for testing of all the mice and determination of percent competition by serum anti-Id. Results are shown in Table IIGo.

 

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  		Table II. Percent competition by anti-Id in sera of mice neonatally injected with Id or NoMoIgG1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Immunologic manipulations of animals as neonates using either Ag or Id-expressing Abs clearly affects their subsequent B and T cell responses to antigenically related material (35, 36, 37, 38, 39, 40). Studies that have investigated immunization with Id or anti-Id provide useful information on the ontology of the immune response and the generation of immunologic repertoire diversity (41, 42, 43, 44, 45, 46, 47). By transplacental passage of Id-expressing Abs and/or Ag, chronic infectious diseases that coincide with the childbearing years provide evidence that naturally occurring neonatal immune exposures are normal (18, 19, 20, 21, 22, 48, 49) and influence responses to infectious agent Ags encountered later in life (50, 51).

We have previously demonstrated that neonatal exposure to certain Id-expressing anti-SEA Abs alters cytokine responses, pathologic presentations, and survival rates in mice infected with S. mansoni infections as adults (26). The present studies were designed to determine the immunologic changes wrought by these neonatal Id-related manipulations in the absence of subsequent infection. Neonatal exposure to appropriate anti-SEA Id preparations induced both B and T cell responsiveness to SEA with patterns characteristic of primed, secondary immune responses. For example, anti-SEA Abs present in sera of animals neonatally exposed to CRI but no other schistosome-related Ag or Ab are of IgG isotypes rather than IgM. The typical primary Ab response to SEA is, as is true for most Ags, an IgM isotype (Fig. 1Go). The presence of anti-SEA-specific IgG found in the sera of CRI-injected mice is not merely residual Ab activity from the neonatal injection, as the half life of IgG injected into a mouse is only 4 to 8 days (52) and these studies were done 63 days after injection. Furthermore, the boost effect observed following SEA injection could only be the result of B cell expansion and Ab production, not from residual anti-SEA activity. The idiotypic specificity of this response is demonstrated by injection of anti-SEA Abs that contain no CRI (HSS Id). HSS Id preparations contain similar overall anti-SEA Ab reactivity (14) and were injected in the same amounts as the MSS Id and 8WkId, but there was no anti-SEA IgG found in the sera of mice neonatally injected with HSS Id.

As observed for the B cell responses, spleen cells from mice neonatally injected with CRI+ anti-SEA Abs alone demonstrated proliferative and cytokine responses to SEA that were not observed with spleen cells from mice injected with either NoMoIgG or HSS Id. These results demonstrate that Id exposure in the absence of subsequent infection or Ag injection is sufficient to initiate SEA-specific responses. Similarly, the elevated IFN-{gamma} production by spleen cells from CRI-injected mice suggest that neonatally influenced animals subsequently infected with schistosomes would have a different "initial" immune response to SEA than animals with no neonatal exposure to CRI+ anti-SEA Abs.

The mechanism of how neonatal exposure to CRI-expressing Abs reactive with SEA can induce Ab and cellular responsiveness to SEA is still under investigation. However, we have demonstrated the presence of both Id and anti-Id in the sera of mice neonatally injected with CRI whether they received no further exposure to Ag or infection (Figs. 6Go and 7Go and Tables I and II), were injected with SEA at 8 wk of age and were sacrificed at 9 wk (Figs. 6Go and 7Go and Tables I and II), or were infected with S. mansoni at 8 wk of age and sacrificed at 8 or 20 wk after infection (M. A. Montesano et al., manuscript in preparation). It is likely that some of the anti-Id Abs could contain an internal image of critical SEA epitopes and thereby initiate expansion of SEA-specific B and T cells. Although it may seem unusual that the conformation on Id or anti-Id could activate T cells, it should be remembered that the TCR recognizes a conformational shape made up of MHC and Ag. It is conceivable that the conformation of an Id or anti-Id could also represent such a shape. In fact, SEA-specific Id has been shown to directly stimulate T cell proliferation from schistosomiasis patients in the absence of APCs as long as it is cross-linked and a source of IL-1 is provided (53). Nevertheless, we still need to define whether Id stimulation of T cells occurs through the TCR or another receptor on T cells. Similar results suggesting that exposure to certain Ids induces an idiotypic network including Abs and T cells with reactivity to the Id-recognized Ag have recently been demonstrated for an Id that recognizes mutant p53 (54).

We do not believe that our observations are simply the result of minute amounts of SEA that have leached from our affinity column and contaminated the Id preparations. To counter this possibility, all Id preparations for a given experiment were purified using the same column. Thus, any leached SEA would be present in all Id preparations in similar amounts and neonatal injection of HSS Id would have given us the same results as injection with MSS Id or 8WkId. Because HSS Id preparations were obtained from the same SEA-column, showed no contaminating SEA on silver stained SDS polyacrylamide gels (Ref. 34 and data not shown), and did not induce SEA responsiveness in neonatally injected mice, we conclude that the stimulatory activity of MSS Id as well as 8WkId preparations was not due to SEA contamination.

One difference that does exist between the CRI+ and the CRI- Id preparations is their istoypic makeup. We have previously demonstrated that 8WkId and MSS Id preparations have higher levels of SEA-specific IgG2a and IgG2b than do HSS Id preparations (14). However, these differences are a function of the sera used to make the Id rather than differences in the physical preparation as sera from MSS animals likewise have higher levels of these isotypes than do sera from HSS animals (7). Nevertheless, it is possible that in addition to the presence of CRI, these isotypic differences could contribute to the immunologic and pathology differences we observe in animals that were neonatally injected. For example, Jankovic et al. have demonstrated that mice deficient for Fc IgG receptor (FcR{gamma}) have increased liver pathology and fail to down-regulate granulomatous responses during the chronic phase of infection (55). It may be possible that IgG2a bound to Fc{gamma}RI or Id/anti-Id immune complexes bound to Fc{gamma}RIII could effect specific immunoregulation by signals delivered through these receptors. This group has also demonstrated a role for Fc{epsilon}RI, the high-affinity receptor for IgE, in immunoregulation of schistosomiasis immunopathology (56). However, although there is IgE in the sera of 8-wk-infected MSS and HSS mice, we have been unable to detect IgE in Id preparations from these animals (data not shown). This is most likely due to the extensive manipulation needed to purify Id preparations and the lability of IgE.

An additional interesting aspect of this study is how these data may relate to observations in individuals who live in endemic areas and have contact with infectious waters but that have no history or evidence of infection despite repeated stool examinations. These "endemic normal" individuals, who may well be children of mothers with schistosomiasis, have high Ab titers, display strong PBMC proliferative responses, and produce elevated IFN-{gamma} levels to schistosome Ags (57, 58). While it is not possible to rule out the possibility that these individuals were infected at one time and self-cured, it is tempting to speculate that the similarity of their schistosome-specific immune response profile to the mice neonatally injected with CRI+ anti-SEA Abs could be related to idiotypic exposure from their mothers.

Further studies are needed to fully establish the mechanisms involved in the priming and responses seen and their roles in ameliorating morbidity and mortality due to experimental schistosomiasis. However, it is clear that neonatal exposure to CRI+ anti-SEA Abs leads to anti-SEA humoral and cellular responsiveness. This Ag-specific reactivity appears to have been elucidated via related Id and anti-Id in a functional idiotypic network that is expressed whether or not the mice are later exposed to schistosome Ags.


   Acknowledgments
 
We thank Dr. Patrick J. Lammie for critical reading of the manuscript and helpful discussions.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. W. Evan Secor, Immunology Branch, Division of Parasitic Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, NE, MS-F13, Atlanta, GA 30341. E-mail address: Back

2 Abbreviations used in this paper: SEA, schistosome soluble egg Ag; MSS, moderate splenomegaly syndrome; HSS, hypersplenomegaly syndrome; CRI, cross-reactive Id; NoMoIgG, normal mouse Ig; TMB, 3,3',5,5'-tetramethylbenzidine peroxidase substrate; 8WkId, 8-wk-infected mice. Back

Received for publication February 5, 1999. Accepted for publication May 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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