INTRODUCTION

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Naive T helper cells can differentiate into one of two subsets, known as Th1 or Th2, depending on the context of the initial stimulation they receive (10). In the presence of interleukin-12 (IL-12) Th1 cells, which secrete IL-2, gamma interferon (IFN-), and tumor necrosis factor beta (TNF-), develop. On the other hand, induction of IL-4 favors the development of Th2 cells, which produce IL-4, IL-5, IL-6, IL-10, and IL-13 (11, 25). Antibody class switching in the mouse is influenced by T-cell cytokines such that IFN- promotes secretion of immunoglobulin G2a (IgG2a) antibodies, and IL-4 promotes secretion of IgG1 and IgE (1, 4). T-cell subsets are also capable of cross-regulation: IFN- inhibits the actions of IL-4 on resting B cells, including secretion of IgE and IgG1 isotypes (18). IL-4, on the other hand, down-regulates the secretion of IFN- by CD4⁺ T cells (14). Antigen-specific responses have not been studied in detail at early stages of immune development.

Host protection against intracellular infections, such as Brucella abortus infections, involves a Th1-like response (9). Heat-killed B. abortus (HKBA) has been shown to up-regulate the secretion of IL-12 and IFN- (2, 12, 21) and is considered a strong Th1 stimulus (2, 19). We have recently demonstrated that a single injection of B. abortus, when injected together with ovalbumin adsorbed to alum (OVA-A) into adult mice, has the ability to abolish antigen-specific IgE-mediated allergic responses to repeated OVA-A challenge and to increase production of IgG2a in vivo (12, 13). This effect of B. abortus on isotype switching correlated with an increase in IFN- and a decrease in IL-4-secreting-cells (12). Thus, B. abortus has the ability to alter the antigen-specific cytokine profile from Th2- to Th1-like when injected together with OVA-A in adult mice. This has implications for mounting effective immune responses against B. abortus, as well as enabling B. abortus to be used as a vaccine adjuvant or carrier in situations where Th1 reactivity is beneficial.

In this report we examine the ontogeny of this Th1-to-Th2 switch in young mice using OVA conjugated to HKBA (HKBA-OVA) as a Th1 stimulus. The results have implications for immunization and protection against bacterial infections. In addition, they provide insights into approaches that could prevent allergic disease.

	MATERIALS AND METHODS

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Animals. BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, Maine) and bred at the Food and Drug Administration (FDA) animal quarters. Pregnant females were observed daily, and the day of birth was recorded as the day the litter was found. Groups for different treatments comprised 7 to 16 mice, depending on the size of the litters. All animals were used in accordance with the National Institutes of Health (NIH) guidelines for animal use and care.

Preparation of OVA-A. The protocol for OVA-A preparation was modified from the work of Hayglass and Stefura (8). Aluminum potassium sulfate [AlK(SO₄)₂] was prepared at a concentration of 10% in pyrogen-free water. Twenty milliliters was transferred to a fresh container, and 25.4 ml of 0.5 M NaOH was added in a dropwise fashion with stirring. The precipitate was washed six times in phosphate-buffered saline (PBS) and resuspended in 40 ml of PBS, and pH was adjusted to 7.2 to 7.4. The solution was stored at 4°C. Immediately prior to injection, OVA (Sigma Chemical Co., St. Louis, Mo.) was added to the alum solution at 8 µg/ml, mixed, and left standing for 10 min at room temperature. OVA-A was diluted twofold with PBS and injected into mice.

Immunization procedures. Mice were immunized intraperitoneally (i.p.) at the age of 1 day or 1 week and received one of the following treatments: (i) PBS, (ii) HKBA (B. abortus strain 1119.3, obtained from the U.S. Department of Agriculture, Ames, Iowa), (iii) HKBA conjugated to OVA (HKBA-OVA) (6), or (iv) OVA. All immunizations of neonates were given in a final volume of 30 µl. One-day-old mice received 10⁷ HKBA organisms/mouse in PBS, or 10⁷ organisms/mouse as HKBA-OVA conjugate, which contains 0.2 µg of OVA. One-week-old mice received 5 × 10⁷ HKBA organisms/mouse or 5 × 10⁷ organisms of HKBA-OVA, containing 1 µg of OVA, per mouse. The OVA dose was the same as the contents of OVA in the HKBA-OVA conjugate, i.e., 0.2 µg for 1-day-old mice and 1 µg for 1-week-old mice. At the age of 4 weeks, all mice were injected i.p. with OVA-A [2 µg of OVA in 0.5 ml of Al(OH)₃/mouse] (8) and bled after 2 weeks for assessment of T- and B-cell responses. At 2 and 3 months all mice were boosted i.p. with OVA-A; they were bled 10 days after each boost. On the day of the last bleed, mice were boosted i.p. with 15 µg of OVA and they were sacrificed 2 days later for determination of cytokine secretion by enzyme-linked immunospot (ELISPOT).

Detection of antigen-specific immunoglobulins in serum by ELISA. The enzyme-linked immunosorbent assay (ELISA) protocol for determination of OVA-specific IgE was modified from the work of Scott et al. (12) and Hayglass et al. (7). Serum samples were first incubated with protein G-Sepharose (Pharmacia, Piscataway, N.J.) in order to preadsorb IgG. Samples were diluted 1/100 in 1% bovine serum albumin (BSA)-PBS and combined with a 50% slurry of protein G-Sepharose at a volume ratio of 4:1. Samples were rotated for 1 h at room temperature. This procedure removes IgG antibodies, which compete with IgE for binding to OVA on the ELISA plates. Protein G-Sepharose was removed by spinning the samples and transferring the supernatant to new tubes. Immulon 2 plates (Dynatech, Chantilly, Va.) were coated overnight with 0.2 mg of OVA/ml in PBS. Plates were washed and blocked with 1% BSA-PBS for 1 h at 37°C. Preadsorbed serum samples were added in twofold serial dilutions, and plates were incubated overnight at 4°C. Plates were washed, and biotin-conjugated anti-mouse IgE (PharMingen, San Diego, Calif.) was added at a 1/500 dilution and incubated for 1 h at 37°C. Plates were washed and incubated as before, with streptavidin-alkaline phosphatase conjugate (PharMingen) at a 1/1,500 dilution. Binding was detected with diethanolamine buffer and phosphatase substrate tablets (Pierce Rockford, Ill.). Results were considered positive if the optical density (OD) exceeded the mean plus 2 standard deviations (SD) of the OD of control samples on each plate. GraphPad Prism, version 3.00, for Windows (GraphPad Software, San Diego, Calif. [www.graphpad.com]) was used for statistical comparisons among groups for ELISA data.

Enumeration of cytokine-producing cells by ELISPOT. Th1 and Th2 memory T-cell responses were assessed by measuring the frequencies of IFN-- and IL-4-secreting cells, respectively, in modified ELISPOT assays (22). Immulon 2 plates (Dynatech) were coated overnight at 4°C with 50 µl of the anti-IL-4 antibody RVD4 (PharMingen) or with anti-IFN- antibody (Biosource International, Camarillo, Calif.) at 5 µg/ml in PBS. After washing, wells were blocked for 1 h at 37°C with 200 µl of 1% BSA in PBS/well. Single-cell suspensions were prepared from spleens of individual mice that were immunized as described above and injected with soluble OVA (15 µg i.p.) 2 days before the ELISPOT assay. Cells were plated for the ELISPOT assay in duplicate wells at 5 × 10⁵/well and serially diluted twofold. Plates were incubated for 4 h at 37°C. Wells were then washed four times with PBS, followed by four washes with PBS containing 0.05% Tween (PBS-0.05% Tween). Biotinylated anti-IL-4 or anti-IFN- (PharMingen) was added at 4 or 2 µg/ml, respectively, in 100 µl of PBS-0.05% Tween and 5% fetal calf serum (FCS). Plates were incubated overnight at 4°C. Plates were washed as before, streptavidin-alkaline phosphatase (PharMingen) was diluted 1/1,500, and 100 µl was added per well. After a 2-h incubation at 37°C, spots were developed with 200 µl of the substrate 5-bromo-4-chloro-indolyl phosphate (Sigma)/well at 1 mg/ml, dissolved in 0.1 M 2-amino-2-methyl-1-propanol buffer (Sigma) and 0.6% SeaPlaque agarose (FMC Bioproducts, Rockland, Maine). Agarose and buffer were boiled and then cooled to 50°C before addition of the substrate, which was dissolved gradually in a 50°C water bath. Plates were left covered and stationary overnight. On the following day, spots in each well were enumerated by a blinded observer using a dissecting microscope. As a positive control, cells were stimulated with concanavalin A at a concentration of 4 µg/well. GraphPad Prism, version 3.00, for Windows (GraphPad Software) was used for statistical comparisons among groups for ELISPOT data.

Semiquantitative RT-PCR. Coupled reverse transcriptase (RT)-PCR was performed as previously described (5, 12, 20). Briefly, spleens were removed from mice 6 h after injection, pooled, and homogenized immediately in RNAzol (Tel-Test, Friendswood, Tex.). Due to the small size of spleens from 1-day-old mice, it was not possible to analyze the mice individually, and mRNA analysis was performed on groups rather than individual mice. RNA samples (3 µl of 1.2-µg/µl sample, diluted in water) were reverse transcribed with Superscript RT (Bethesda Research Laboratories, Bethesda, Md.), and cytokine-specific primers were used to amplify selected cytokines (12, 20). Primers for a housekeeping gene, hypoxanthine phosphoribosyltransferase (HPRT) were used in each experiment to standardize amounts of RNA that were added in each PCR. The following number of cycles was used for each gene: for HPRT, 19; for IFN-, 27; for IL-12, 31; for IL-4, 28. Amplified PCR products were detected by Southern blot analysis using ³²P-labeled oligonucleotide probes (12, 20) and were developed on X-ray film at 70°C. Films were analyzed by densitometry, using the NIH image program for Macintosh.

	RESULTS

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Conversion of OVA-A responses from Th2- to Th1-like is effective following a single administration of HKBA-OVA in 1-week-old but not in 1-day-old mice. The age of onset of Th1 responses has implications for disease susceptibility and the development of vaccines against intracellular infections. To determine the ontogeny of Th1 responses in mice, 1-day-old and 1-week-old mice were immunized with either PBS, HKBA, HKBA-OVA, or OVA and then challenged with OVA-A 30, 60, and 90 days later. After each exposure to OVA-A, the mice were bled and the sera were assayed for OVA-specific IgE antibodies. The results show that a single immunization with HKBA-OVA given at the age of 1 week greatly reduced the subsequent OVA-specific IgE responses, as seen after three OVA-A boosts (Fig. 1B). In contrast, mice that were given HKBA only did not exhibit decreased IgE responses when challenged later with OVA-A. As seen previously for adult mice, increases in IgE levels are clearly seen only after several OVA-A boosts (12). The differences between the treatment groups were not distinct following the first and second boosts. The differences seen after the third boost indicated that a temporal association between the antigen (OVA) and the Th1 inducer (HKBA) is required for a long-term effect on antigen-specific T-cell responses. A similar effect was previously seen with adult mice (12).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Conversion of OVA-A responses from Th2- to Th1-like by HKBA is dependent on (i) the presence of OVA and (ii) the age of the mice. Mice were injected at the age of 1 day or 1 week with either PBS, HKBA, OVA, or HKBA-OVA. All mice were challenged with OVA-A after 1, 2, and 3 months, and serum IgE levels were measured after the third OVA-A challenge. (A) Mice treated at 1 day; (B) mice treated at 1 week. Horizontal lines indicate medians. One-way analysis of variance was performed using GraphPad Prism.

The ratio of OVA-specific IFN-- to IL-4-secreting cells is increased by HKBA-OVA given to 1-week-old but not 1-day-old mice. The reduced levels of OVA-specific IgE in 1-week-old mice compared to 1-day-old mice. suggested that long-term OVA-specific Th1 memory cells were generated in 1-week-old but not in 1-day-old mice. Experiments were performed to test this interpretation. One-day- or 1-week-old mice were immunized as before, and after the third OVA-A challenge, all mice were boosted with OVA, and numbers of OVA-specific IFN-- and IL-4-secreting cells in the individual spleens were determined by ELISPOT. The response generated in these mice was a mixed response and comprised cells producing Th1 cytokines as well as cells secreting Th2 cytokines. The ratio of IFN-- to IL-4-secreting cells was calculated for each individual mouse as a measure of the cytokine balance, since this could influence the isotype-switching outcome. Mice treated at 1 day with PBS, HKBA, HKBA-OVA, or OVA had no significant differences in the ratio of IFN-- to IL-4-secreting cells (Fig. 2). In contrast, the ratio of IFN-- to IL-4-secreting cells for mice treated at the age of 1 week was higher in the group treated with HKBA-OVA than in all other groups. The HKBA-OVA group had four out of five mice with more IFN-- than IL-4-secreting cells, whereas in the OVA group, seven out of eight mice had more IL-4-secreting cells. Thus the 1-week-old mice receiving HKBA-OVA generated long-term memory predominated by Th1 rather than Th2 against OVA. This result correlated with the lower level of antigen-specific IgE observed for that group.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   The ratio of IFN-- to IL-4-secreting-cells is higher after treatment with HKBA-OVA at the age of 1 week. Mice were treated as described in Materials and Methods, boosted with 15 µg of OVA, and sacrificed. Spleens were removed, and cells were analyzed for cytokine secretion by ELISPOT. Cytokine-secreting cells were counted per 5 × 105 spleen cells, and the ratio of IFN-- to IL-4-secreting cells was determined. (A) Mice treated at 1 day; (B) mice treated at 1 week. Horizontal lines indicate medians. One-way analysis of variance was performed using GraphPad Prism.

Cytokine mRNA patterns following primary immunization with HKBA-OVA differ between 1-day-old and 1-week-old mice. The different results for 1-day-old and 1-week-old mice in terms of memory T cells and antibody responses could be explained by differences in the cytokine milieu evoked at or shortly after the single HKBA-OVA administration. In order to examine this possibility, 1-day- and 1-week-old mice were sacrificed 6 h after the primary immunization and spleens were assayed for cytokine gene expression by RT-PCR. IL-12 is released by antigen-presenting cells (APC) and is pivotal for promoting Th1-like differentiation. IL-12 p35 was expressed at similarly high levels in 1-day-old and 1-week-old mice in the absence or presence of HKBA or HKBA-OVA stimulation (data not shown). This is in accordance with findings of other studies showing constitutive IL-12 p35 expression (24). In contrast, HKBA and HKBA-OVA elicited high IL-12 p40 mRNA levels in both 1-day-old and 1-week-old mice (Fig. 3), suggesting that APC were able to mature in these age groups. However, induction of IFN-, which is critical for Th1 differentiation, was different between 1-day-old and 1-week-old mice treated with HKBA-OVA. As seen in Fig. 3, IFN- mRNA was expressed at significantly higher levels in 1-week-old mice than in 1-day-old mice after treatment with HKBA-OVA. This difference in IFN- induction following the initial HKBA-OVA probably explains the Th2-to-Th1 shift observed later in life as evidenced by increased numbers of Th1 memory cells and suppressed IgE levels in response to OVA-A in the mice that were immunized at 1 week but not in those immunized at 1 day.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   IFN- levels are higher in 1-week-old mice than in 1-day-old mice. Mice were injected at the age of 1 day or 1 week as before, and sacrificed after 6 h. mRNA was extracted from four to six pooled spleens and amplified in duplicate for specific cytokines. (A) Mice treated at 1 day; (B) mice treated at 1 week. Positive controls for mRNA levels were obtained by injecting adult mice with anti-CD3 for IFN- and IL-4, and with HKBA for IL-12 p40. (C) Cytokine levels were normalized to HPRT levels for 1-day- and 1-week-old mice. Values for the control group (PBS) were subtracted from those for the other experimental groups to show increase above background. These results are representative of three experiments.

	DISCUSSION

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HKBA has been shown to deliver a strong Th1 stimulus in adult mice. When HKBA-OVA was given as a single injection to adult mice, it induced IL-12 and IFN- mRNA expression shortly thereafter. This treatment facilitated the generation of long-term OVA-specific memory responses, predominated by IFN--secreting cells and IgG2a (12). The goal of the present study was to determine how early during development a strong Th1 stimulus could generate long-term Th1 memory cells and shift subsequent antigen-specific responses from Th2 to Th1. With that purpose in mind, we inoculated 1-day-old and 1-week-old mice with a single injection of HKBA-OVA followed by three subsequent injections of OVA-A given after these mice reached maturity.

One-week-old mice responded to a single injection of HKBA-OVA like adult mice (12) in that they expressed IL-12 and IFN- mRNA within 6 h of injection and generated long-term OVA-specific Th1-like responses. When challenged with OVA-A as adults, these mice exhibited higher ratios of IFN-- to IL-4-secreting-cells and suppression of IgE. In contrast, 1-day-old mice failed to generate long-term Th1-like OVA-specific memory. Instead, their response to OVA-A as adults was Th2-like, that is, predominated by IL-4-secreting cells and elevated IgE levels. The response generated by the mice in the different treatment groups appears to be more of a mixed response, generated by the presence of both Th1 and Th2 cytokines. When the mice receive their initial treatment at a very early age (1 day or 1 week), the scarcity of the T cells in the spleen suggests that new T cells will be generated after the treatment is given, and these will respond differently to subsequent boosts. However, the T cells that are present during the initial treatment will influence any later response. Therefore, the final outcome will be determined mostly by the dominance of one type of response over the other, i.e., as expressed by the ratio. In analysis of the ELISPOT results, the diversity between individual mice within the same group was quite large: some mice had high numbers of cells that were making IL-4, and some had very low numbers, Nevertheless, the balance between IFN-- and IL-4-secreting cells will most likely determine the outcome as measured by the antibody responses that will dominate (11).

Although the single HKBA-OVA injection in 1-week-old mice favored long-term Th1-like responses to OVA-A in terms of ratios of IFN-- to IL-4-secreting cells and inhibition of IgE, this was not sufficient to impact the IgG1/IgG2a ratios significantly. This was unlike the effect seen in adult mice (12), in which IgG2a levels and IgG2a/IgG1 ratios were increased over those for mice receiving OVA-A as the initial injection. This difference between adult and younger mice may be attributed to weaker cytokine (IL-12 and IFN-) responses in younger mice. The difference can be explained by the observations that IgE induction is more dependent on IL-4 than IgG1 (3). The difference in cytokine levels between mature and young mice may relate to the complement of mature antigen-specific cells in the periphery at the time of the initial HKBA-OVA or OVA-A immunization. In the young mice it is likely that more naïve OVA-specific cells reach the periphery after the initial treatment, when very few T cells are present in the spleen. Therefore, at the time the OVA-A boost is given in adulthood, there will be Th1 memory cells, from the HKBA-OVA injection, but a relatively larger pool of naïve cells that can differentiate into Th2 cells than in adults. This would explain why the ratio of IFN- to IL-4 is higher in adult mice than in 1-week-old mice following a single injection of HKBA-OVA.

The disparity between 1-week-old and 1-day-old mice can probably be explained by the finding that 1-day-old mice, unlike 1-week-old mice, failed to express IFN- mRNA 6 h after the HKBA-OVA injection. This was despite the fact that IL-12 mRNA expression was strongly induced in both 1-day-old and 1-week-old mice. Preliminary immunohistochemistry data from 1-day-old mice following HKBA-OVA inoculation show few T cells in the spleen, and dendritic cells secreting IL-12 are not forming tight clusters in the T-cell areas, unlike data from 1-week-old and adult mice. Thus, the inability of neonatal mice to respond to a Th1 stimulus, such as HKBA, stems from the immaturity of the neonatal mouse immune system, resulting in a reduced or absent interaction between dendritic cells and T cells in the spleens of these mice. The failure of 1-day-old mice to mount a long-term Th1 response to HKBA implies that these mice could not be successfully vaccinated against microorganisms that require a Th1 response for protection.

These results have implications for immunization and protection against bacterial infections. One-week-old mice are analogous to human newborns in terms of immune function, whereas 1-day-old mice represent human fetuses in the last trimester (17). Thus, our results, when extrapolated to human responses, suggest that human newborns would be expected to generate memory responses to Th1 stimuli such as intracellular pathogens. On the other hand, infections in utero would probably not generate Th1, but would rather be associated with Th2, memory responses.

Observations made in this study also impact on our understanding of the development of allergic responses in young animals and suggest possible ways to prevent them. This is potentially important because of the increase in allergic diseases in developed countries, which has been attributed to a decrease in Th1 stimuli in the form of microbial infections during the early years of development of the immune system. Epidemiologic studies in humans suggest that exposure to strong Th1 stimuli at an early age may suppress atopic reactions later in life (15, 16, 23). However, our data suggest that injection of a Th1 stimulus alone in the absence of the allergen, in this case unconjugated HKBA, did not prevent an allergen from inducing a Th2 response. On the other hand, a single injection of HKBA-OVA reversed the ratio of OVA-specific IFN-- to IL-4-secreting cells from <0.5 to 2 and decreased anti-OVA IgE levels significantly. This implies that a generalized non-antigen-specific Th1 stimulus alone may not be sufficient to imprint the immune system against allergens. However, a Th1 stimulus provided at the time of the first allergenic challenge can switch the phenotype of subsequent responses to the allergen from Th2-like to Th1-like. This raises the possibility of preventing allergies by treating individuals from families with atopic tendencies in a particular locale with a strong Th1 stimulus at the time of exposure to allergens associated with that area.

In summary, this study shows that the ability of the immune system to respond to a Th1 stimulus depends on the state of maturity of the immune system. In particular, Th1 memory can be generated only if the initial response to the Th1 stimulus is associated with elaboration of both IL-12 and IFN-. Furthermore, a strong Th1 stimulus can imprint the immune system to a nominal antigen only if the antigen is present at the time the Th1 stimulus is administered, so that the cytokine milieu for induction of antigen-specific memory coincides with triggering of antigen-specific T cells.