Various types of Dirofilaria immitis polyproteins selectively induce a Th2-Type immune response

Abstract

Dirofilaria immitis polyproteins (DiAgs) are found as 15-kDa monomeric and 30-kDa dimeric forms in excretory-secretory products of the adult worm. We evaluated the ability of various types of recombinant DiAg (rDiAg; V1 and V2 as monomers and V1V2, V2V1, V1V1, and V2V2 as dimers) to influence Th1/Th2 immune responses. V1-, V1Vx- and V2-, V2Vx-driven nonspecific immunoglobulin E (IgE) production peaked at 21 and 14 days after administration, respectively. Dimer-induced IgE response was an interesting biphasic pattern with the second peaks on days 35 (V2Vx) or 42 (V1Vx). Absolute amounts of nonspecific IgE production induced with monomers were larger than those observed with dimers at the first peak. The magnitude of cell expansion and interleukin-10 (IL-10) production in mesenteric lymph node (MLN) B-cell induced with rDiAgs was linked to the levels of the first IgE peak in vivo and IgE produced by rDiAg plus IL-4-stimulated B cells in vitro. All rDiAgs failed to augment IgG2c production. V2 and V2Vx elicited IL-4 production by MLN cells more rapidly than V1 and V1Vx. The inhibitory effect of rDiAg on gamma interferon (IFN-gamma) production was stronger in monomers than in dimers. Neutralization of IL-10 restored IFN-gamma production, whereas the expression of IL-4 and IgE was partly prevented by depletion of IL-10. These results indicate that monomer rather than dimer is an efficient form of DiAg and suggest that the difference of IgE-inducing capacity among these DiAgs is closely associated with the pattern of both B-cell activation and IL-4 production.

FIG. 1.

(A) Alignment of rDiAg V1 and V2 amino acid sequences. Asterisks and dots designate amino acid identity and similarity, respectively. Gaps were introduced in sequences for optimal alignment. DiAg (D. immitis; GenBank accession no. D88757). (B) Map and expression of rDiAgs used to evaluate immune responses. Recombinant proteins were separated by SDS-14% PAGE and detected by staining with Coomassie brilliant blue and immunoblotting with rabbit anti-DiAg antiserum.

FIG. 2.

Time course of immunoglobulin synthesis in rDiAg-treated mice. Mice were administrated several types of DiAg, plasmas were collected weekly. Total IgE (A to C) and IgG2c (D) levels were quantitated by ELISA. The data represent the mean values and standard deviations (error bars) for five mice.

FIG. 3.

rDiAg dimers do not induce specific IgE antibody. Mice were administered rDiAg dimers (V2V1 or V1V1), and plasma samples were collected weekly. At second peaks (V2V1 [on day 35] and V1V1 [on day 42]), specific IgE antibodies to dimer (V2V1 and V1V1) and monomer (V1 and V2) were detected by ELISA (A and B) and dot blot analysis (C to E). rDiAg-blotted membranes were treated with serum from mice or rabbits and then reacted with HRP-conjugated anti-mouse IgG (C and E) or anti-rabbit IgG (E). The data represent the mean values for five mice (A and B). The data represent one typical experiment out of five (C to E).

FIG. 4.

rDiAgs induce B-cell activation. MLN B cells (2 × 10⁶/ml) from naive C57BL/6 (A and C) and C3H/HeJ (B) mice were cultured with each rDiAg (3.3 μg/ml), rCont (3.3 μg/ml), anti-CD40 MAb (1 μg/ml), or LPS (2 μg/ml) for 48 h. Cell proliferation (A and B) and IL-10 production (C) were measured by the BrdU incorporation assay and ELISA, respectively. The data represent the mean values and standard deviations (error bars) from five experiments. ∗∗ and ∗, P < 0.01 and P < 0.05, respectively, compared to the use of medium alone.

FIG. 5.

rDiAgs upregulate IL-4 production. Whole MLN cells (5 × 10⁶/ml) from naive mice were cultured with each rDiAg (3.3 μg/ml) in the absence (A) or presence (B) of ConA (1 μg/ml) for 24 (A) or 72 h (A and B). IL-4 levels in the culture supernatants were quantitated by ELISA. The data represent the mean values and standard deviations (error bars) from five experiments. ∗∗ and ∗, P < 0.01 and P < 0.05, respectively, compared to the use of medium alone.

FIG. 6.

rDiAgs downregulate IFN-γ production. Whole MLN cells (5 × 10⁶/ml) from naive mice were cultured with each rDiAg (3.3 μg/ml) in the absence (A and C) or presence (B and D) of ConA (1 μg/ml) for 72 h. IFN-γ levels in the culture supernatants were quantitated by ELISA (A and B). The data represent the mean values and standard deviations (error bars) from five experiments. ∗∗ and ∗, P < 0.01 and P < 0.05, respectively, compared to the use of medium alone. Cell injury was estimated by LDH assay, and the data represent the relative ratio, which is equal to the mean optical density value of rDiAg-treated cells divided by the mean value of untreated cells and standard deviations (error bars) from five experiments (C and D).

FIG. 7.

Effect of endogenous IL-10 on DiAg-induced immnue responses. MLN cells were cultured with anti-IL-10 MAb or isotype-mached control MAb (5 μg/ml) in the presence of each rDiAg (3.3 μg/ml) for 72 h. IL-4 (A) and IFN-γ (B) levels were measured by ELISA. The data represent the mean values and standard deviations (error bars) from five experiments. ∗∗ and ∗, P < 0.01 and P < 0.05, respectively, compared to the use of control MAb.

FIG. 8.

Effect of cleaved DiAg dimer on IgE synthesis. (A) A total of 25 μg of DiAg dimer (25 μl) was treated with 1 μg of native or heat-inactivated trypsin (1 μl) for 15 or 30 min. Immunoblot of trypsin-digested DiAg dimer (V1V1) probed with anti-DiAg antiserum. The data represent one typical experiment out of five. (B) MLN B cells were cultured with 10 μl of each trypsin-treated V1V1 in the presence of IL-4 (200 U/ml) and polymyxin B (10 μg/ml) for 8 days. IgE levels in culture supernatants were measured by ELISA. The data represent the relative ratio, which is equal to the mean value of rDiAg-treated cells divided by the mean value of untreated cells, and standard deviations (error bars) from five experiments. ∗∗ and ∗, P < 0.01 and P < 0.05, respectively, compared to the use of heat-inactivated trypsin.