Low-dose synergistic immunosuppression of T-dependent antibody responses by polycyclic aromatic hydrocarbons and arsenic in C57BL/6J murine spleen cells

Abstract

Polycyclic aromatic hydrocarbons (PAHs) and arsenic are both environmental agents that are known to have significant immunotoxicity. Previous studies have shown that PAH exposure of spleen cells in vitro produces significant immune suppression of humoral immunity, especially when P450 activation products are examined. Exposure to arsenic, particularly sodium arsenite, has also been found to be suppressive to antibody responses in vitro and in vivo. The purpose of the present studies was to examine the immunotoxicity of PAHs and arsenite following coexposures with the theory being that the agents may exert synergistic actions, which might be based on their different mechanisms of action. Spleen cells were isolated from male C57BL/6J wild-type mice and treated with PAHs and/or arsenic (arsenite or arsenate). Immunotoxicity assays were used to assess the T-dependent antibody response (TDAR) to sheep red blood cells (SRBCs), measured by a direct plaque-forming cell (PFC) assay. Cell viability was measured by trypan blue staining. Spleen cell viability was not altered following 4 days of PAH and/or arsenic treatment. However, the TDAR demonstrated suppression by both PAHs and arsenic in a concentration-dependent manner. p53 was also induced by NaAsO(2) (As(3)(+)) and PAHs alone or in combination. The PAHs and their metabolites investigated included benzo[a]pyrene (BaP), BaP-7,8-diol, BaP-7,8-diol-9,10-epoxide (BPDE), 7,12-dimethylbenz[a]anthracene (DMBA), DMBA-3,4-diol, dibenzo[a,l]pyrene (DB[a,l]P). PAH metabolites were found to be more potent than parent compounds in producing immunosuppression and inducing p53 expression. Interestingly, DB[a,l]P, a potent carcinogenic PAH not previously characterized for immunotoxicity, was also found to be strongly immunosuppressive. Arsenite (NaAsO(2), As(3)(+)) was found to produce immunosuppression at concentrations as low as 0.5 microM and was immunosuppressive at a 10-fold lower concentration than sodium arsenate (Na(2)HAsO(4), As(5)(+)). Coexposure of spleen cell cultures to PAHs and As(3)(+), both at individual low-effect concentrations, was found to produce profound suppression of the TDAR demonstrating synergy between these two chemical classes of agents.

Fig. 1

Suppression of the TDAR by BaP, BaP-diol and BPDE in male C57BL/6J mice examined in vitro. Spleen cells were immunized with SRBC in vitro at the same time of treatment with DMSO, BaP, BaP-diol, or BPDE. The number of antibody producing spleen cells was determined by a modified Jerne and Nordin PFC assay as described in Materials and Methods. No cytotoxicity was observed in these cultures. Data from a pool of three mouse spleens that were assayed in quadruplicate are shown as mean ± S.E.M. Statistically significant differences compared with 0.1% DMSO are indicated (*, p< 0.05).

Fig. 2

Suppression of the SRBC TDAR by DMBA, DMBA-diol, or DB[a,l]P. No cytotoxicity was observed in these cultures. Data from a pool of three mouse spleens that were assayed in quadruplicate are shown as mean ± S.E.M. Statistically significant differences compared with 0.1% DMSO are indicated (*, p< 0.05).

Fig. 3

Sodium arsenite (As⁺³) and sodium arsenate (As⁺⁵) treatment on the primary spleen cell humoral immune (IgM) response to SRBC TDAR as determined by PFC assay in male C57BL/6J mice examined in vitro. Spleen cells were immunized in vitro at the same time of treatment with H₂O or As⁺³, As⁺⁵. No cytotoxicity was observed in these cultures. Data from a pool of three mouse spleens that were assayed in quadruplicate are shown as mean ± S.E.M. Statistically significant differences compared with 0.1% H₂O are indicated (*, p< 0.05).

Fig. 4

Effect of As⁺³ co-treatment with BaP, BaP-diol and BPDE on the in vitro primary spleen cell TDAR. Spleen cells were immunized in vitro at the same time of treatment with DMSO, As⁺³, and/or BaP, BaP-diol and BPDE. Data from a pool of three mouse spleens that were assayed in quadruplicate are shown as mean ± S.E.M. No cytotoxicity was observed in these cultures. *indicates a statistically significant difference compared with 0.1% H₂O+ 0.1% DMSO (p< 0.05). ^#indicates a statistically significant effect of arsenite when added to PAHs compared to the PAH alone (p < 0.05). Data are representative of two experiments.

Fig. 5

Effect of As⁺³ co-treatment with DMBA, DMBA-diol and DB[a,l]P on the primary spleen cell TDAR response to SRBC examined in vitro. Spleen cells were immunized in vitro at the same time of treatment with DMSO or As⁺³ and/or DMBA, DMBA-diol and DB[a,l]P. No cytotoxicity was observed in these cultures. Data from a pool of three mouse spleens that were assayed in quadruplicate are shown as mean ± S.E.M. *indicates a statistically significant difference compared with 0.1% H₂O or 0.1% DMSO (p< 0.05). ^#indicates a statistically significant effect of arsenite when added to PAHs compared to the PAH alone (p < 0.05). Data are representative of two experiments.

Fig. 6

Effect of As⁺³ co-treatment with DB[a,l]P on the primary spleen cell humoral immune (IgM) response to TDAR SRBC examined in vitro. Spleen cells were immunized in vitro at the same time of treatment with As⁺³ (0.5 or 5 µM in H₂O) and/or DB[a,l]P (0.001 – 1 µM in DMSO). No cytotoxicity was observed in these cultures. Data from a pool of three mouse spleens that were assayed in quadruplicate are shown as mean ± S.E.M. *indicates a statistically significant effect of As⁺³ or PAHs compared to water control for As⁺³ or DMSO control for PAHs (p < 0.05). ^#indicates the synergistic interaction between As⁺³ and PAH as the suppression is greater than an additive effect.

Fig. 7

Effect of As⁺³ co-treatment with DMBA-diol on the primary spleen cell humoral immune (IgM) response to SRBC examined in vitro. Spleen cells were immunized in vitro at the same time of treatment with As⁺³ (0.5 or 5 µM in H₂O) and/or DMBA-diol (0.001 – 1 µM in DMSO). No cytotoxicity was observed in these cultures. Data from a pool of three mouse spleens that were assayed in quadruplicate are shown as mean ± S.E.M. *indicates a statistically significant effect of As⁺³ or PAHs compared to water control for As⁺³ or DMSO control for PAHs (p < 0.05). ^#indicates the synergistic interaction between As⁺³ and PAH as the suppression is greater than an additive effect.

Fig. 8

Western Blot analysis of As⁺³ and PAHs effects on whole cell lysate p53 protein levels in murine spleen cells treated in vitro at the indicated concentrations for 4, 8, or 18 hrs. Numbers shown at the bottom of each gel lane are the fold inductions for p53 protein normalized to actin and compared to controls (H₂O for As⁺³ and DMSO for PAHs) and treated cells. The boxed numbers show the comparison for 5 µM As⁺³ induction (L4) of p53 at 8 hrs, as compared to 0.01 µM DB[a,l]P (L6) and the combination of both agents (L11).