Resistance/susceptibility to Echinococcus multilocularis infection and cytokine profile in humans. II. Influence of the HLA B8, DR3, DQ2 haplotype

Abstract

Differences have been shown between HLA characteristics of patients with different courses of alveolar echinococcosis (AE). Notably the HLA B8, DR3, DQ2 haplotype was associated with more severe forms of this granulomatous parasitic disease. We compared IL-10, IL-5, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) secretion by peripheral blood mononuclear cells (PBMC) isolated from eight HLA-DR3+, DQ2+, B8+ AE patients and from 10 HLA-DR3-, DQ2-, B8- patients after non-specific mitogenic and specific Echinococcus multilocularis antigenic in vitro stimulation. PBMC from seven HLA-DR3+, DQ2+, B8+ healthy subjects and nine HLA-DR3-, DQ2-, B8- subjects were also studied as controls. PBMC from AE patients with HLA DR3+, DQ2+ haplotype secreted higher levels of IL-10 without any stimulation and after specific antigenic stimulation than did patients without this haplotype. Higher levels of IL-5 and IFN-gamma were also produced by these patients' PBMC after stimulation with non-purified parasitic antigenic preparations; however, the specific alkaline phosphatase antigen extracted from E. multilocularis induced only Th2-type cytokine secretion. A spontaneous secretion of TNF by HLA DR3+, DQ2+ B8+ AE patients was also found. These results suggest that HLA characteristics of the host can influence immune-mediated mechanisms, and thus the course of AE in humans; specific antigenic components of E. multilocularis could contribute to the preferential Th2-type cytokine production favoured by the genetic background of the host.

Fig. 1

Secretion of IL-10 (a) and IL-10 mRNA expression (b) in peripheral blood mononuclear cell (PBMC) cultures. IL-10 protein and mRNA levels were assessed in PBMC cultures from alveolar echinococcosis (AE) patients and healthy subjects with or without the HLA-DR3, DQ2, B8 haplotype, after cell stimulation with phytohaemagglutinin (PHA) or Echinococcus multilocularis antigens. Cultures without any stimulation served as controls. (a) Cytokine secretion was measured using an ELISA assay. Values are means ± SEM. (b) IL-10 mRNA expression was semiquantified by densitometric analysis after polymerase chain reaction amplification of cDNA with specific primer pairs. Results are expressed as arbitrary units as described in PATIENTS and METHODS. *P < 0·05: statistically significant difference for cytokine protein or cytokine mRNA level between non-stimulated and stimulated cells in each group (Wilcoxon signed rank test); **P < 0·05: statistically significant difference for cytokine protein or mRNA level between cell cultures from DR3⁺ and DR3⁻ AE patients (Mann–Whitney U-test). Control medium, no stimulation; Emc Ag, stimulation with the crude extract antigen of E. multilocularis; Emf Ag, stimulation with the vesicular fluid purified antigen; EmAp Ag, stimulation with the alkaline phosphatase of E. multilocularis; PHA, stimulation with the non-specific mitogen, phytohaemagglutinin.

Fig. 2

Secretion of IL-5 (a) and IL-5 mRNA expression (b) in peripheral blood mononuclear cell (PBMC) cultures. IL-5 protein and mRNA levels were assessed in PBMC cultures from alveolar echinococcosis (AE) patients and healthy subjects with or without the HLA-DR3, DQ2, B8 haplotype, after cell stimulation with phytohaemagglutinin (PHA) or Echinococcus multilocularis antigens. Cultures without any stimulation served as controls. (a) Cytokine secretion was measured using an ELISA assay. Values are means ± s.e.m. (b) IL-5 mRNA expression was semiquantified by densitometric analysis after polymerase chain reaction amplification of cDNA with specific primer pairs. Results are expressed as arbitrary units as described in PATIENTS and METHODS. *P < 0·05: statistically significant difference for cytokine protein or cytokine mRNA level between non-stimulated and stimulated cells in each group (Wilcoxon signed rank test); **P < 0·05: statistically significant difference for cytokine protein or mRNA level between cell cultures from DR3⁺ and DR3⁻ AE patients (Mann–Whitney U-test). Control medium, no stimulation; Emc Ag, stimulation with the crude extract antigen of E. multilocularis; Emf Ag, stimulation with the vesicular fluid purified antigen; EmAp Ag, stimulation with the alkaline phosphatase of E. multilocularis; PHA, stimulation with the non-specific mitogen, phytohaemagglutinin.

Fig. 3

Secretion of IFN-γ (a) and IFN-γ mRNA expression (b) in peripheral blood mononuclear cell (PBMC) cultures. IFN-γ protein and mRNA levels were assessed in PBMC cultures from alveolar echinococcosis (AE) patients and healthy subjects with or without the HLA-DR3, DQ2, B8 haplotype, after cell stimulation with phytohaemagglutinin (PHA) or Echinococcus multilocularis antigens. Cultures without any stimulation served as controls. Cytokine secretion was measured using an ELISA assay. Values are means ± s.e.m. IFN-γ mRNA expression was semiquantified by densitometric analysis after polymerase chain reaction amplification of cDNA with specific primer pairs. Results are expressed as arbitrary units as described in PATIENTS and METHODS. *P < 0·05: statistically significant difference for cytokine protein or cytokine mRNA level between non-stimulated and stimulated cells in each group (Wilcoxon signed-rank test); **P < 0·05: statistically significant difference for cytokine protein or mRNA level between cell cultures from DR3⁺ and DR3⁻ AE patients (Mann–Whitney U-test). Control medium, no stimulation; Emc Ag, stimulation with the crude extract antigen of E. multilocularis; Emf Ag, stimulation with the vesicular fluid purified antigen; EmAp Ag, stimulation with the alkaline phosphatase of E. multilocularis; PHA, stimulation with the non-specific mitogen, phytohaemagglutinin.

Fig. 4

Secretion of tumour necrosis factor (TNF) in peripheral blood mononuclear cell (PBMC) cultures. TNF protein was assessed in PBMC cultures from alveolar echinococcosis (AE) patients and healthy subjects with or without the HLA-DR3, DQ2, B8 haplotype after cell stimulation with phytohaemagglutinin (PHA) or Echinococcus multilocularis antigens. Cultures without any stimulation served as controls. Cytokine secretion was measured using an ELISA assay. Values are means ± s.e.m. *P < 0·05: statistically significant difference for cytokine protein level between non-stimulated and stimulated cells in each group (Wilcoxon signed-rank test); †P < 0·05: statistically significant difference for cytokine protein level between cell cultures from DR3⁺ and DR3⁻ healthy control subjects (Mann–Whitney U-test). Control medium, no stimulation; Emc Ag, stimulation with the crude extract antigen of E. multilocularis; Emf Ag, stimulation with the vesicular fluid purified antigen; EmAp Ag, stimulation with the alkaline phosphatase of E. multilocularis; PHA, stimulation with the non-specific mitogen, phytohaemagglutinin.