Characterization of mercuric mercury (Hg2+)-induced lymphoblasts from patients with mercury allergy and from healthy subjects

Abstract

Hg2+ induces lymphocyte proliferation when added to cell cultures from both healthy and mercury-allergic subjects. Consequently, when measuring DNA synthesis a possible Hg2+-specific response, resulting from proliferating memory cells, cannot be discriminated from a non-allergic response. The mechanism behind this non-allergic response is unknown but a superantigenic effect of Hg2+ has been suggested. In this study, five mercury-allergic patients, with oral lichen planus (OLP) lesions adjacent to dental amalgam and a positive patch test to Hg0, and five healthy subjects without amalgam were examined. The immunophenotype and the T cell receptor Vbeta (TCR Vbeta) repertoire of Hg2+-induced lymphoblasts as well as the expression of the lymphocyte activation markers CD23 and CD134 were analysed for possible differences between healthy and allergic subjects. The mechanism of Hg2+-induced proliferation was examined by comparing the TCR Vbeta expression of Hg- and staphylococcal enterotoxin B (SEB)-activated lymphoblasts, the latter used as a positive superantigen control. It was not possible to discriminate between mercury-allergic and healthy subjects using the immunophenotype or the TCR Vbeta profile of the Hg2+-induced lymphoblasts or the expression of CD23 and CD134. However, Hg2+-induced CD4+ lymphoblasts showed a skewing towards Vbeta2. This relative increase in Vbeta2 was only detected in the CD4+ but not in the CD8+ lymphoblast population. In conclusion, Hg2+ induced a proliferation-dependent skewing towards CD4+ but not CD8+ lymphocytes expressing Vbeta2. In this respect Hg2+ differs from the classical bacterial superantigen SEB, which also stimulates unique TCR Vbeta families among CD8+ cells.

Fig. 1

Flow cytometric gating of cells treated for 5 days with 4 μ g/ml HgCl₂ using a forward scatter (size)/side scatter (granularity) plot. R1, Resting cells; R2, lymphoblasts. Approximately 6 × 10⁴ events (2 × 10⁴ lymphocytes) were collected from each sample.

Fig. 2

Mean fluorescence intensity (MFI) ± s.d. of indicated activation marker after 5 days in untreated-, staphylococcal enterotoxin B (SEB)-(10 ng/ml) or HgCl₂ (4 μ g/ml)-treated cultures. A significant increase in treated compared with untreated 5-day cultures is given as *P < 0·05; **P < 0·01; ***P < 0·001. ▪, Oral lichen planus (OLP) patients; □, healthy subjects. (a) CD23 expression on CD19⁺/CD20⁺ lymphocytes. (b) CD134 expression on CD4⁺ lymphocytes.

Fig. 3

Percentage T cell receptor (TCR) Vβ expression of Hg²⁺-treated lymphoblasts minus percentage TCR Vβ expression of resting lymphocytes in untreated cultures. •, Oral lichen planus (OLP) patients; ○, healthy subjects.

Fig. 4

Changes in T cell receptor (TCR) Vβ expression as an effect of 5 days stimulation with Hg²⁺ or staphylococcal enterotoxin B (SEB). The relative increase of the indicated Vβ families are given as percentage among the resting untreated lymphocytes compared with percentage among the lymphoblast population. ▪, Oral lichen planus (OLP) patients; ○, healthy subjects. (a) Percent Vβ3⁺, + Vβ12⁺, + Vβ14⁺, + Vβ17⁺, + Vβ20⁺ CD4⁺ and CD8⁺ lymphoblasts in SEB-treated cultures and corresponding spontaneous lymphoblasts in untreated cultures. (b) Percent Vβ2⁺ CD4⁺ and CD8⁺ lymphoblasts in Hg-treated cultures and corresponding spontaneous lymphoblasts in untreated cultures.

Fig. 5

T cell receptor (TCR) Vβ profiles of Hg²⁺-induced CD4⁺ blasts (▪) and CD8⁺ blasts (□) from two patients with oral lichen planus (OLP) and one healthy subject showing the highest proliferation intensities with Hg²⁺ obtained in this study. (a) Patient OLP 3. Stimulation index (SI) in Hg²⁺-exposed culture = 39 (65 853 ct/min). (b) Patient OLP 5. SI in Hg²⁺-exposed culture = 120 (65 608 ct/min). (c) Healthy subject AF 4. SI in Hg²⁺-exposed culture = 42 (52 175 ct/min).