A novel 62-kilodalton egg antigen from Schistosoma mansoni induces a potent CD4(+) T helper cell response in the C57BL/6 mouse - PubMed (original) (raw)

A novel 62-kilodalton egg antigen from Schistosoma mansoni induces a potent CD4(+) T helper cell response in the C57BL/6 mouse

H Asahi et al. Infect Immun. 1999 Apr.

Abstract

In infection with Schistosoma mansoni, hepatic granuloma formation is mediated by CD4(+) T helper (Th) cells sensitized to schistosomal egg antigens. There is considerable variation among infected individuals with respect to both severity of disease and the T-cell response to egg antigens. In the BL/6 mouse, the egg granulomas are relatively small and the relevant sensitizing egg antigens are largely unknown. We investigated the CD4(+) Th cell response of infected BL/6 mice to egg antigens fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found a prominent lymphoproliferative response to be directed against a 62-kDa component. With the aid of a specific T-cell hybridoma, 4E6, the 62-kDa antigen was isolated; following partial digestion with endoproteinase Glu-C, an internal amino acid sequence was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK) of the organisms Caenorhabditis elegans and Treponema pallidum and to differ by one residue from PEPCK of various other species. In CD4(+) Th cells from 7.5- 8.5-week-infected BL/6 mice, the purified 62-kDa molecule elicited a potent proliferative response which, based on cytokine analysis, was of a mixed Th-1 and Th-2 type. Our results reveal a novel egg antigen of particular prominence in the BL/6 mouse and suggest that the immune response in schistosomiasis is a product of sensitization to egg antigens that may vary considerably in immunogenicity from strain to strain.

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Figures

FIG. 1

Representative SDS-PAGE (7.5% gel) profile of SEA on immunoblots stained with amido black. Molecular weight marker standards are indicated on the left. The prominent band of fraction 15 represents the Sm-p40 antigen. Frs., fractions.

FIG. 2

CD4+ Th cell responses (A) and 4E6 T-cell hybridoma responses (B) to SDS-PAGE fractions on immunoblots of SEA. CD4+ Th cells were isolated from mesenteric lymph nodes of 8.5-week-infected BL/6 mice. Cell culture conditions in each case were as described in Materials and Methods. [3H]dThd incorporation was assessed by liquid scintillation spectroscopy. Data are expressed as means ± 1 SD. Background radioactivity from cultures in the absence of antigen is subtracted. Panels on the right reflect corresponding CD4+ Th cell or hybridoma stimulation in the presence of NC, NC coated with 50 μg of SEA (SEA-NC), or the indicated amounts of SEA. Experiments with CD4+ Th cells as well as with the T-cell hybridoma were repeated twice, with similar results. Frs., fractions.

FIG. 3

Identification of a 62-kDa antigen recognized by T-cell hybridoma 4E6. (A) Eluted SDS-PAGE gel fraction 7 (Fr.7) (Fig. 1) from 20 μg of SEA, examined for purity on a 10% silver-stained SDS-polyacrylamide gel, shown next to total SEA and molecular weight marker standards. (B) Dose response of T-cell hybridoma 4E6 to 62-kDa antigen as measured by HT-2 indicator cell proliferation. Data are expressed as mean ± 1 SD. Background radioactivity from hybridoma cultures in the absence of antigen is subtracted.

FIG. 4

Stimulation of the 4E6 T-cell hybridoma with V8 protease fragments of the 62-kDa antigen. (A) Stimulation of T-cell hybridoma 4E6 by, from left to right, the intact 62-kDa antigen, the fragments of the 62-kDa antigen derived from digestion with 2 μg of V8 protease, and the most stimulatory fragment g. Each of these stimulatory activities was obtained from an initial 450 μg of SEA. Data are expressed as mean ± 1 SD. Background radioactivity from hybridoma cultures in the absence of antigen is subtracted. (B) Corresponding silver-stained SDS-PAGE profile of, from left to right, the 62-kDa antigen, the combined V8 protease fragments of the 62-kDa antigen, and fragment g. Five percent of each antigen preparation used for stimulation was examined on the SDS-polyacrylamide gel. Part of the V8 protease migrated close to fragment g but had no stimulatory activity (not shown). The position of intact V8 protease (28 kDa) is shown.

FIG. 5

Internal amino acid sequence obtained from fragment g and related sequences in other species corresponding to PEPCK. The 10-amino-acid sequence is shown at the top. Vertical lines indicate identical residues; points indicate mismatched residues. The location (∗) of the 10-mer together with the deduced Glu (E) residue (the site of V8 protease cleavage) within PEPCK is indicated for each species. The total number of amino acids (aa) in each PEPCK is indicated in parentheses.

FIG. 6

Proliferative responses of CD4+ Th cells from BL/6 (A) and CBA (B) mice to the 62-kDa antigen. CD4+ Th cells were isolated from mesenteric lymph nodes of 8.5-week-infected mice. Culture conditions were as described in Materials and Methods. [3H]dThd incorporation was assessed by liquid scintillation spectroscopy. Data are expressed as mean ± 1 SD. Also shown for comparison are responses to Sm-p40 and SEA. Background radioactivity from cultures in the absence of antigen is subtracted. The same pattern of stimulation was observed when cells from 7.5-week-infected mice were used (not shown).

FIG. 7

Cytokine production by CD4+ Th cells from BL/6 and CBA mice stimulated with the 62-kDa antigen. CD4+ Th cells were isolated from mesenteric lymph nodes of 8.5-week-infected mice. Culture conditions were as described in Materials and Methods. The cytokines IFN-γ, IL-2, IL-4, and IL-5 were measured in culture supernatants by ELISA. Data are expressed as mean ± 1 SD. Also shown for comparison are responses to Sm-p40 and SEA. The same pattern of cytokine production was observed when cells from 8-week-infected mice were used (not shown).

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A novel 62-kilodalton egg antigen from Schistosoma mansoni induces a potent CD4(+) T helper cell response in the C57BL/6 mouse - PubMed (original) (raw)