Mechanisms of Hexachlorobenzene-induced Adverse Immune Effects

Abstract

Hexachlorobenzene (HCB) is an environmental pollutant that can induce adverse immune effects in humans and rats. Brown Norway rats (BN) appeared to be very susceptible to HCB-induced immune effects. Oral exposure causes inflammatory skin and lung lesions, enlarged spleen and lymph nodes (LN) and elevated humoral responses. Although the immunopathological effects induced by HCB are investigated comprehensively, the exact mechanisms involved are unknown. Experiments described in this thesis were performed to obtain more insight in the mechanisms underlying HCB-induced adverse immune effects, both on a cellular and molecular level. The immunomodulatory effects of the oxidative metabolites tetrachlorohydroquinone (TCHQ) and tetrachlorobenzoquinone (TCBQ) were assessed. Therefore, these metabolites were studied in the mouse reporter antigen popliteal lymph node assay (RA-PLNA) with TNP-Ficoll as a RA. In this assay an isotype switch to IgG1 indicates that the chemical injected is able to form neoantigens that can stimulate T cells. Both TCHQ and TCBQ induced an isotype switch to IgG1 and were thus capable of providing neoantigen-specific T cell help. Furthermore, this response was compound-specific as was shown in a secondary PLNA. In addition, gene expression profiles with DNA microarray analysis in BN rats were obtained. Transcriptome profiling was done in spleen, mesenteric lymph node (MLN), thymus, blood, liver and kidney. Data confirmed known effects of HCB such as stimulatory effects on the immune system and induction of enzymes involved in drug metabolism, porphyria and the reproductive system. New findings include the upregulation of genes encoding pro-inflammatory cytokines, antioxidants, acute phase proteins, mast cell markers, complement, chemokines, and cell adhesion molecules. In general, gene expression data provide evidence that HCB induces a systemic inflammatory response, accompanied by oxidative stress and an acute phase response. The role of T cells in HCB-induced immunotoxic effects was investigated further by using the Cyclosporin A (CsA). This immunosuppressive drug decreases peripheral T cell numbers and inhibit T cell activation. CsA treatment delayed the development of HCB-induced skin lesions and prevented the increase in spleen weight slightly. Furthermore, increase in ALN weight, lung eosinophilia and humoral responses were prevented completely by in HCB-exposed rats treated with CsA. Macrophage infiltration was independent of T cells and macrophages seem also important. Therefore, the relationship between macrophages and T cells was further investigated. Co-administration of clodronate-liposomes was used to eliminate macrophages in HCB-exposed rats. Results indicated that macrophages played an important role in HCB-induced skin lesions, in particular in the aggravation of skin lesions. Also, HCB-induced lung eosinophilia and elevation of anti-ssDNA IgM antibodies were less pronounced after depletion of macrophages. To obtain information on events that occur in the initiation phase a kinetic study was performed. This revealed that both skin and lung pathology were early inflammatory events. Furthermore, macrophage infiltrations in the spleen seemed to precede the increase in spleen weight. Remarkably, studies performed to further assess the functional role and specificity of T cells induced by HCB did not provide any evidence for the presence of T cells specific for TCBQ. Moreover, HCB-induced immune effects could not be adoptively transferred to naive recipients.