Both free-living and parasitic nematodes induce a characteristic Th2 response that is dependent on the presence of intact glycans

Abstract

Infection with parasitic nematodes is characterized by the induction of a profound type 2 immune response. We have studied the role of glycans in the induction of the skewed type 2 response by antigens of the parasitic nematode Brugia malayi as well as the free-living nematode Caenorhabditis elegans. Lymph node cells from BALB/c mice immunized with soluble extracts of the two nematodes showed distinct antigen-specific proliferation and cytokine production; however, both nematodes induced antigen-specific interleukin 4 (IL-4) production, demonstrating that the induction of a biased type 2 response is not unique to parasitic nematodes. Sodium periodate-treated soluble extracts of both nematodes consistently induced significantly less IL-4 production than the respective mock-treated extracts, indicating that glycans play a critical role in the induction of the Th2 immune response by these nematodes. The glycan-dependent induction of the Th2-potentiating cytokine IL-4 occurs by 72 h postinoculation. Our data suggest that glycan determinants common to nematodes act as ligands, displaying distinct molecular patterns that trigger the immune system to launch a biased Th2 immune response upon exposure to these organisms or their products. Further, the similarity of our findings to those for Schistosoma mansoni egg antigen is striking considering the enormous phylogenetic distance between nematodes and trematodes. These data thus have important implications for how the mammalian host responds to widely divergent metazoan invaders and suggest that the powerful C. elegans model system can be used to address these questions.

FIG. 1.

Proliferation of lymph node cells from BALB/c mice immunized with nematode extracts. Lymph node cells from mice immunized 10 days previously with CFA alone or with a 1:1 suspension of CFA and BmA or CeA were stimulated in vitro with media, native BmA or CeA, or PPD. Proliferation is expressed as counts per minute (c.p.m.). The data represent the arithmetic mean and standard deviation for five animals in each group. One of four independent experiments with similar results is shown.

FIG. 2.

Antigen-specific CD4⁺ lymphocytes proliferate in response to antigen and produce IL-4. Lymph node cells from mice immunized 7 days previously with CFA alone or with a 1:1 suspension of CFA and BmA or CeA were stained ex vivo with CFSE and stimulated in vitro with BmA, CeA, or PPD for 5 days. Cells stained with anti-CD4 (Cy-chrome) and anti-IL-4 (phycoerythrin) were analyzed by flow cytometry. CFSE staining is shown on the x axis (dividing cells lose fluorescence), and IL-4-positive cells are shown on the y axis. The percentages of IL-4-producing, dividing cells that were CD4⁺ are indicated.

FIG. 3.

Western blot analysis of sodium periodate-treated and mock-treated soluble extracts of B. malayi. pBmA (lanes 1) and mBmA (lanes 2) prepared as detailed in Materials and Methods were separated by SDS-PAGE and transferred to NC sheets; the NC sheets were used in parallel to detect N-linked glycans with horseradish peroxidase (HRP)-conjugated ConA or the B. malayi ladder protein gp15/400, a polyclonal rabbit anti-gp15/400 antibody, and an HRP-conjugated goat anti-rabbit secondary antibody. A change in protein mobility is a common feature after treatment with sodium periodate (53).

FIG. 4.

Proliferation of lymph node cells from BALB/c mice immunized with chemically treated nematode extracts. Lymph node cells from mice immunized with CFA alone or with a 1:1 suspension of CFA and either mBmA, pBmA, mCeA, or pCeA were stimulated in vitro with medium, native BmA, or PPD (top) or with medium, native CeA, or PPD (bottom). Proliferation is expressed as counts per minute (c.p.m.). The data represent the arithmetic mean and standard deviation for five animals in each group. One of four independent experiments with similar results is shown.

FIG. 5.

IL-4 production by lymph node cells from BALB/c mice immunized with chemically treated nematode extracts. (A) IL-4 production in culture supernatants of lymph node cells from mice immunized with CFA, mBmA, or pBmA in response to in vitro stimulation with medium, native BmA, or PPD. Significantly more IL-4 (P < 0.05) was detected in culture supernatants of lymph node cells from mice immunized with mBmA than in those immunized with pBmA. (B) IL-4 production in culture supernatants of lymph node cells from mice immunized with CFA, mCeA, or pCeA in response to in vitro stimulation with medium, native CeA, or PPD. Significantly more IL-4 (P < 0.05) was detected in culture supernatants of lymph node cells from mice immunized with mCeA than in those immunized with pCeA. The data represent the arithmetic means and standard deviations of results for five animals in each group. One of three independent experiments with similar results is shown.

FIG. 6.

Antigen-specific IgG1 antibodies in sera from mice immunized with CFA, mBmA, or pBmA. Significantly more IgG1 (P < 0.01) was detected in sera from BALB/c mice immunized with mBmA than in those immunized with pBmA (n = 5). The horizontal line represents the cutoff line calculated as the arithmetic mean (M) of the optical densities at 450 nm (OD 405) for the control group ±2 standard deviations (SD). One of two independent experiments with similar results is shown.

FIG. 7.

Real-time RT-PCR analysis of IL-4, IL-10, and IFN-γ mRNAs in lymph node cells from BALB/c mice 72 h (A) and 10 days (B) after immunization with CFA, BmA, or CeA. The data represent the arithmetic mean and standard deviation for five mice in each group. Cytokine RT-PCR transcripts are represented in arbitrary units (A.U.) relative to the reference Th-cell clone. BALB/c mice immunized with BmA or CeA showed significantly more IL-4 mRNA in lymph node cells than control mice immunized with CFA alone but showed comparable IL-10 and IFN-γ mRNA levels. One of two independent experiments with similar results is shown.

FIG. 8.

Real-time RT-PCR analysis of IL-4, IL-10, and IFN-γ mRNAs in lymph node cells from BALB/c mice 72 h after immunization with chemically treated nematode extracts. The data represent the arithmetic mean and standard deviation for five mice in each group. Cytokine RT-PCR transcripts are represented in arbitrary units (A.U.) relative to the reference Th-cell clone. One of three independent experiments with similar results is shown.