The anti-inflammatory properties of soluble CD83 in a mouse-model of allergic contact dermatitis

Background and objectives: Allergic contact dermatitis (ACD) is a common inflammatory skin disease with a great socioeconomic impact in industrialized countries. No causal therapy for ACD has been implemented, yet. Based on the pathophysiology, irritant contact dermatitis (ICD) and ACD are distinguished. While ICD is a non-specific inflammatory skin reaction involving the innate immune system, ACD, by contrast, is a delayed-type T cell-mediated inflammatory reaction of the skin occurring after repeated exposure of sensitized individuals to a contact allergen. Insights into pathophysiology of ACD have principally been gained from experiments with mouse-models, also referred to as contact hypersensitivity (CHS). Human CD83 is a 45 kDa integral membrane glycoprotein which – as a member of the immunoglobulin superfamily – was highly conserved throughout mammalian evolution. CD83 exists in two isoforms with contrary biological effects: While membrane-bound CD83 (mCD83), the full-length variant, is a well-known surface marker for mature DC that is functioning as a co-stimulator of the immune system, the existence of soluble CD83 (sCD83), a variant consisting only of the extracellular domain that exerts immunosuppressive functions and is released by activated DC and B cells, was unknown until 2001. Since then, low levels of sCD83 have been detected in normal human (but not in murine) sera, whereas elevated sCD83 concentrations have been found in several hematological malignancies and autoimmune diseases. In this thesis, sCD83 was purified and subsequently tested in a mouse-model of ACD with the aim to investigate the potential impact of sCD83 on the inflammatory response in ICD as well as on sensitization phase and elicitation phase in ACD. Methods (material and investigation methods): The external immunoglobulin domain of human CD83 (soluble CD83), along with glutathione S-transferase (GST) for further use as a negative control, was expressed and purified as a hCD83ext-GST fusion protein in E. coli bacteria. The influence of sCD83 on irritant (ICD) and allergic (ACD) skin responses was tested in a mouse-model of ACD, also referred to as CHS, using C57BL/6 mice between 6 and 26 weeks of age. Skin inflammation was triggered with the hapten 2,4-dinitrofluorobenzene (DNFB), diluted in acetone/olive oil (AOO) vehicle. For the investigation of ICD, mice received an application of DNFB on the left ear (ear painting) while AOO was applied on the right ear (as a negative control). In ACD experiments, mice were additionally sensitized with DNFB on the shaved abdomen five days prior to ear painting. Groups of mice received intraperitoneal (i.p.) injections of either sCD83, diluted in phosphate-buffered saline (PBS), or pure PBS at different time points around abdominal sensitization and ear painting. The DNFB-triggered skin inflammation was evaluated macroscopically with the mouse ear swelling test (MEST), while the effect of sCD83 on the molecular level was tested with several independent assays: IFN-γ and IL-2 ELISPOT assays, T cell proliferation assay, DC migration assay using FACS and analysis of cytokine mRNA expression (IL-1β, IL-6, IL-10) via qRT-PCR. Experiments with transgenic C57BL/6 mice (RAG-2-/- mice, CD3ε-/- mice, μMT mice, IL-10-/- mice) served the investigation of the mediating cell population. For further exploration of the underlying signaling pathway, IL-10 ELISAs were performed with the blood serum. Results and observations: Within this thesis project it was demonstrated that i.p.-injections of sCD83 inhibit the development of DNFB-induced irritant and allergic responses in CHS, the equivalent of ACD in mice: In sCD83-treated mice, skin irritation after hapten painting is diminished both at the clinical level (reduced ear swelling) and at the molecular level (lower local mRNA expression of the proinflammatory cytokine IL-6). Soluble CD83 reduces DC migration to the draining lymph nodes upon hapten painting and inhibits DNFB-specific T cell priming in the draining lymph nodes. In experiments with transgenic C57BL/6 mice it was observed that i.p.-injections of sCD83 fail to inhibit skin irritation in B cell-deficient or IL-10-deficient mice (RAG-2-/- mice, μMT mice, IL-10-/- mice), but not in T cell-deficient animals (CD3ε-/- mice). In the serum of sCD83-treated mice, an elevated IL-10 concentration was found shortly after hapten painting. (Practical) conclusions and discussion: The results show for the first time that sCD83 has an impact on skin inflammation in a murine model of ACD, both on the clinical and on the molecular level. Experiments with transgenic mice have revealed that B cells might play a crucial role in the mediation of the immunomodulatory effects of sCD83, potentially via the release of IL-10. These insights open up new avenues with regard to further exploration of a potential utility of sCD83 in the treatment or prevention of ACD.