Extract of Nippostrongylus brasiliensis stimulates polyclonal type-2 immunoglobulin response by inducing De novo class switch

Abstract

Infection with the nematode parasite Nippostrongylus brasiliensis induces a pronounced type-2 T-cell response that is associated with marked polyclonal immunoglobulin E (IgE) and IgG1 production in mice. To examine the differential roles of the infection and products produced by nematodes, we investigated a soluble extract of N. brasiliensis for the ability to mediate this type-2 response. We found that the extract induced a marked increase in IgE and IgG1 levels, similar to that induced by the infection. The extract did not affect the level of IgG2a in serum, showing that the effect was specific to IgE and IgG1 (type-2-associated immunoglobulin) rather than inducing a nonspecific increase in all immunoglobulin isotypes. This response was also associated with increased interleukin-4 production in vitro. These results confirm that the extract, like infection, is a strong inducer of polyclonal type-2 responses and a reliable model for investigating the regulation of nematode-induced responses. The extract induced the production of IgG1 when added to in vitro cultures of lipopolysaccharide-stimulated B cells. This provides evidence for the induction of class switch. It did not induce upregulation of IgG1 in naive (unstimulated) B cells or expand B cells in in vitro cultures. Analysis of DNA from the spleens of mice treated with the extract by digestion-circularization PCR demonstrated a marked increase in the occurrence of gamma1 switch region gene recombination in the cells in vivo. These results provide strong evidence that soluble worm products are able to mediate the marked polyclonal gamma1/epsilon response and that infection is not required to mediate this response. Furthermore, these data provide evidence that the soluble nematode extract induces this effect by causing de novo class switch of B cells and not by an expansion of IgG1 B cells or an increase in antibody production by IgG1 plasma cells.

FIG. 1

AWH induces IgE and IgG1 production in the serum of BALB/c mice. Female BALB/c mice in groups of three were injected subcutaneously at the back of the neck with AWH emulsified in FIA (100 to 200 μg of protein in a 200-μl volume). The control group was injected with the vehicle alone (FIA). Each group of mice was bled for serum each week for 3 weeks, and the sera within the groups were pooled. The pooled serum samples were assayed for IgE (A), IgG1 (B), and IgG2a (C) levels by capture ELISA. The data are from one experiment and are representative of three (IgE), five (IgG1), and four (IgG2a) independent experiments.

FIG. 2

Increased IgE and IgG1 response is specific to AWH treatment. BALB/c mice in groups of three were injected with killed mycobacteria (Myc) or AWH emulsified in FIA (vehicle). The third group received vehicle alone (FIA). Mice in each group were bled for serum at 14 and 21 days posttreatment. Serum samples were pooled within each group. Serum samples were assayed for IgE, IgG1, and IgG2a levels by capture ELISA. Data shown are the IgE (A), IgG1 (B), and IgG2a (C) levels at 21 days posttreatment. Results are expressed as mean ± SD of triplicate wells and are representative of three experiments (A) ∗∗∗, P < 0.0004, two-tailed, unpaired Student's t test; (B and C) ∗∗∗, P < 0.001; NS, not significant (P > 0.05), one-way ANOVA.

FIG. 3

IL-4 and IL-13 mRNA induction by AWH. Spleen cells were isolated from mice 21 days after treatment with AWH or N. brasiliensis (Nb). For assessment of IL-4 (A) and IL-13 (B) mRNA expression, total cellular RNA was isolated with TRI_zol and reverse-transcribed into single-stranded cDNA using M-MLV reverse transcriptase and random hexamers as described in Materials and Methods. The resulting cDNA template was used in a PCR with primers specific for either IL-4 or IL-13. β-Actin mRNA levels were also determined by RT-PCR to control for equal RNA loading. PCR amplicons were resolved on a 1.5% agarose gel with ethidium bromide staining. Results are representative of three experiments. Nv, naive (untreated). The negative controls in panel B were no enzyme in RT reaction, no RNA in RT reaction, no cDNA in PCR, and no primers in PCR (left to right, respectively).

FIG. 4

Increased IL-4 response in spleen cell cultures is specific to AWH treatment. For assessment of IL-4 protein levels, spleen cells isolated from naive (untreated) mice or mice treated with AWH or worms (Nb) (A), killed mycobacteria (Myc), or AWH emulsified in FIA (B) 21 days posttreatment were stimulated with ConA (5 μg/ml) for 24 or 48 h. Culture supernatants were then analyzed by ELISA for IL-4 levels as described in Materials and Methods. Results are expressed as the mean concentration of IL-4 ± SD of three replicate wells. ∗∗∗, P < 0.001, one-way ANOVA. Data in panel B were normalized for vehicle. ND, below detection limit (15 pg/ml). Results are representative of three experiments.

FIG. 5

Influence of AWH on immunoglobulin production in in vitro B-cell culture. Highly purified naive B cells from uninfected mice (8 to 12 weeks old) isolated as described in Materials and Methods were not stimulated (NS) or stimulated in culture containing AWH alone (20 μg/ml), LPS alone (10 μg/ml), or LPS in combination with AWH (20 μg/ml) or IL-4 (5 ng/ml). Culture supernatants were harvested 7 days later and then analyzed for IgG1 (A) and IgM (B) levels using antibody capture ELISA as described in Materials and Methods. Results are expressed as the mean concentration ± SD of three replicate wells and are representative of six separate experiments. ∗∗∗, P < 0.001, one-way ANOVA.

FIG. 6

AWH does not expand B cells. B cells from BALB/c mice were stimulated in culture containing LPS alone (5 μg/ml) or LPS in combination with AWH (10 or 50 μg/ml). After 72 h of incubation at 37°C, the cultures were pulsed with [³H]thymidine, and the cells were assayed for incorporation 18 h later. Data shown are expressed as mean disintegrations per minute (dpm) of triplicate wells ± SD and are representative of seven experiments. ∗∗∗, P < 0.001, one-way ANOVA.

FIG. 7

AWH-induced IgG1 production is associated with an increase in the number of IgG1-switched cells. Genomic DNA was isolated from either the TSI-18 and IB4 hybridomas (A) or the spleen cells of mice treated with either AWH, worms (Nb), or FIA (B) as described in Materials and Methods. The DNA was digested with EcoRI, ligated with T4 DNA ligase, and amplified by PCR using primers specific for the recombined switch regions. nAChRe levels in all samples were also determined by DC-PCR to control for equal template loading and allow semiquantitation (comparison) of the Sμ-Sγ1 product. PCR amplicons were resolved on a 1.5% agarose gel with ethidium bromide staining. Results are representative of six experiments. (A) Lane 1, TSI-18 (IgG1-producing hybridoma); lane 2, IB4 (IgG2a-producing hybridoma); lane 3, no DNA (control for PCR contamination); lane 4, TSI-18 (nAChRe amplicon from IgG1-producing hybridoma).