Reversal of ultraviolet radiation-induced immune suppression by recombinant interleukin-12: suppression of cytokine production.

Exposure to ultraviolet (UV) radiation, a complete carcinogen, suppresses the immune response. Data from a number of laboratories have indicated that one consequence of UV exposure is suppressed T helper type 1 (Th1) cell function with normal Th2 cell activation, resulting in a shift to a Th2-like phenotype. The reversal of UV-induced immune suppression and tolerance induction by recombinant interleukin-12 (rIL-12) supports this observation. The focus of this study was to determine the mechanism(s) by which rIL-12 reverses UV-induced immune suppression. Two possibilities were considered: up-regulation of interferon-gamma (IFN-gamma) secretion by rIL-12 and suppression of UV-induced cytokine secretion by rIL-12. To our surprise we found that the ability of rIL-12 to overcome UV-induced immune suppression was independent of its ability to up-regulate IFN-gamma secretion. Rather, rIL-12 suppressed the production of cytokines that are known to be important in UV-induced immune suppression. Injecting UV-irradiated mice with rIL-12, or adding rIL-12 to UV-irradiated keratinocyte cultures suppressed IL-10 secretion, in part by affecting the transcription of the IL-10 gene. Furthermore, we found that rIL-12 suppressed UV-induced tumour necrosis factor-alpha (TNF-alpha) production. Because IL-10 is involved in the UV-induced suppression of delayed-type hypersensitivity and TNF-alpha in the UV-induced suppression of contact allergy, these findings provide a mechanism to explain how rIL-12 overcomes UV-induced immune suppression in these related but different immune reactions. In addition, they suggest a novel mechanism by which rIL-12 alters immune reactivity, direct suppression of cytokine secretion induced by UV radiation.

CD40-CD40 ligand interactions in vivo regulate migration of antigen-bearing dendritic cells from the skin to draining lymph nodes.

Whereas CD40-CD40 ligand interactions are important for various dendritic cell (DC) functions in vitro, their in vivo relevance is unknown. We analyzed the DC status of CD40 ligand -/- mice using a contact hypersensitivity (CHS) model system that enables multiple functions of DCs to be assessed in vivo. Immunohistochemistry of skin sections revealed no differences in terms of numbers and morphology of dendritic epidermal Langerhans cells (LCs) in unsensitized CD40 ligand -/- mice as compared with wild-type C57BL/6 mice. However, after contact sensitization of CD40 ligand -/- mice, LCs failed to migrate out of the skin and substantially fewer DCs accumulated in draining lymph nodes (DLNs). Furthermore, very few antigen-bearing DCs could be detected in the paracortical region of lymph nodes draining sensitized skin. This defect in DC migration after hapten sensitization was associated with defective CHS responses and decreased cutaneous tumor necrosis factor (TNF)-alpha production and was corrected by injecting recombinant TNF-alpha or an agonistic anti-CD40 monoclonal antibody. Thus, CD40-CD40 ligand interactions in vivo regulate the migration of antigen-bearing DCs from the skin to DLNs via TNF-alpha production and play a vital role in the initiation of acquired T cell-mediated immunity.

Development of a chaperone-deficient system by fractionation of a prokaryotic coupled transcription/translation system.

A coupled transcription/translation system from Escherichia coli has been developed that is very active for protein synthesis but deficient in chaperone proteins. The chaperones GroEL and DnaK distribute during the first ultracentrifugation of the E. coli extract partially with the ribosomes and partially in a liquid, viscous fraction above the ribosomes. Gel filtration chromatography of this latter fraction separates GroEL and DnaK as high-molecular-weight components from the peak of activity of the factors required for protein synthesis. Thus, a chaperone-deficient transcription/translation system can be reconstituted with salt-washed ribosomes. This chaperone-deficient system was used to study synthesis and folding of bacterial dihydrofolate reductase and of rhodanese, a eukaryotic mitochondrial enzyme. Both enzymes were synthesized from nonlinearized plasmids that had the respective coding sequence under the SP6 promoter. Both enzymes were synthesized in active form and with high specific activity in the chaperone-deficient system. A high proportion, about 20% of newly synthesized dihydrofolate reductase and about 50% of rhodanese, stayed with the ribosomes after coupled transcription/translation. No enzymatic activity was detected in this fraction. Addition of the chaperones GroEL/ES and DnaK resulted in a shift of rhodanese molecules from the ribosomes into the supernatant fraction. Nearly all molecules in the supernatant were enzymatically active.