Nitric oxide-mediated immunosuppression following murine Echinococcus multilocularis infection

Abstract

In some parasitic infections immunosuppression is a prominent characteristic of the host-parasite interplay. We have used a murine alveolar echinococcosis (AE) model in susceptible C57BL/6 mice to document a suppressed splenocyte proliferative response to concanavalin A (Con A) at the early (1-month) stage and to Echinococcus multilocularis-crude antigen (Emc-antigen) at the late (4-6-month) stage of chronic infection. Despite proliferative suppression, splenic cytokine production [interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma)] in response to Con A or Emc-antigen stimulation was not suppressed at 1 month postinfection (p.i.). Infection resulted in a strong Mac-1+ cell infiltration of the peritoneal cavity and spleen. Peritoneal cells (PEC) from mice infected at the 1-month stage were rich in macrophages and expressed significantly higher levels of transcripts for the inflammatory cytokine IL-1beta and for tumour necrosis factor-alpha and inducible nitric oxide synthase (iNOS), when compared with PEC from non-infected control mice. Conversely, the IL-10 transcript level remained low and did not change during infection. Spleen cells supplemented with PEC from infected mice induced a marked increase in the levels of nitrite in response to Con A and Emc-antigen stimulation, and also a complete suppression of splenic proliferation. The spleen cells from late-stage infected mice expressed only background levels of IL-10 but greatly increased levels of iNOS, when compared with normal spleen cells. This observation correlated with the immunosuppression demonstrated at the late stage of murine AE. Furthermore, the suppressed splenic proliferative responses observed at the early and late stage were reversed to a large extent by the addition of NG-monomethyl-l-arginine and partially by anti-IFN-gamma. Thus, our results demonstrated that the immunosuppression observed in chronic AE was not primarily dependent on IL-10 but rather on nitric oxide production by macrophages from infected animals.

Figure 1

C57BL/6 mice (five mice per group) were infected intraperitoneally with 50–100 freshly isolated vesicular cysts of E. multilocularis. The proliferative responses to Con A and Emc-antigen stimulation of pooled spleen cells from infected (solid bars) and non-infected control (open bars) mice were determined at different time-points p.i. (1 month, 3 months and 6 months). *Indicates the statistically significant differences (P < 0·05). This is one representative out of two independent experiments exhibiting identical results. The suppression of Con A-derived proliferation at 1 month was repeated in four independent experiments with similar results.

Figure 2

Effect on Con A- and Emc-antigen-stimulated proliferation and NO production of supplementing spleen cells with PEC from control or 1-month infected mice. Spleen cells from 1-month infected mice were supplemented either with medium alone (open bars) or with 5×10⁴ peritoneal cells from either normal (stippled bars) or 1-month infected mice (solid bars) and stimulated with Con A for 72 hr or Emc-antigen for 96 hr. (a) Incorporation of [³H]thymidine was determined in quadruplicate wells. Data represent the mean c.p.m.±SEM of quadruplicate wells. Similar results were obtained in five independent experiments. (b) Simultaneously, 50 μl supernantants from wells kept in parallel were collected at 96 hr for assessing NO production. One representative out of two independent experiments with similar results is shown.

Figure 3

Determination of cytokine-and iNOS-mRNA expression in pooled PEC from control (PEC⁻) and infected (AE-PEC) mice. At 1 month p.i. levels of the cytokine-and iNOS-mRNA transcripts were determined by competitive quantitative RT-PCR after standardization for the expression of the constitutively transcribed β₂-microglobulin gene. Appropriate amounts of cDNA were amplified in the presence of serial fourfold dilutions of a multispecific internal plasmid control ranging from point 1 (5×10⁶ molecules) to point 9 (7·5×10¹ molecules). Numbers reflect to the dilution step. The arrows indicate equal amounts of PCR products of cDNA compared with mimic plasmid controls; c, cDNA amplification alone; −, negative control; m, DNA size marker. * Indicates the cDNA band sites. This is one representative out of two independent experiments with similar results.

Figure 4

Recovery by NMMA and anti-IFN-γ of the suppressed proliferative response of spleen cells from 1-month AE-infected mice. Spleen cells were isolated from four mice and subsequently pooled, then cultivated alone (solid bars) or cocultivated with AE-PEC (cross-hatched bars) and stimulated in vitro with Con A and Emc-antigen, or left unstimulated (background). The cell cultures were supplemented with 500 μm NMMA or 20 μg/ml anti-IFN-γ, or with an appropriate amount of medium (control). The [³H]thymidine uptake results were expressed as geometric mean minus background (▵c.p.m.). Background was determined by unstimulated cells kept in medium alone. A representative out of three independent experiments is shown.

Figure 5

(a) Expression of iNOS and IL-10 in spleen cells of four 4-month infected mice. In this experiment, two out of six mice died from AE infection at 121 days p.i. The iNOS and IL-10 mRNA transcripts in spleens from four control mice (Sp⁻) and four 1-month infected (Sp⁺ 1M) and 4-month infected mice (Sp⁺ 4M) were determined by competitive quantitative RT-PCR as described in Fig. 3. (b) Recovery by NMMA and anti-IFN-γ of the suppressed proliferative response of spleen cells from 6-month infected mice. Spleen cells were isolated from four uninfected mice (Sp⁻), four 3-month infected mice (Sp⁺3M) and three 6-month infected mice (Sp⁺6M). The spleen cells from 6-month infected mice were treated with NMMA (Sp⁺6M NMMA) and anti-IFN-γ (Sp⁺6M anti-IFN-γ) in the same manner as shown in Fig. 4 and stimulated with Con A or Emc-antigen. The [³H]thymidine-uptake results were expressed as geometric mean minus background (▵c.p.m.).