IL‐10 plays an important role in the regulation of immune responses. We and others have demonstrated recently that cyclic adenosine monophosphate (cAMP)‐elevating substances up‐regulate monocytic IL‐10 expression in vitro and in vivo. Computer analysis of the IL‐10 promoter/enhancer region localized four putative cAMP‐responsive elements (CRE1– 4) with homology to the CRE consensus motif. In electrophoretic mobility shift assays CRE1 and CRE4 bound protein complexes consisting of transcription factors CREB‐1 and ATF‐1, while CRE3 bound only marginal amounts of CREB‐1/ATF‐1 in combination with unknown protein(s). CRE2 showed no protein binding activity. In vitro mutation of CRE1 and CRE4 reduced the level of cAMP‐stimulated transactivation in reporter gene assays in comparison to the wild‐type promoter by 20 % and 50 %, respectively, while mutation of CRE3 had no effect. The main action of CRE4 on cAMP‐dependent stimulation is probably based on its adjacent localization to the TATA box and its sequence comprising a perfect half site. Experiments with double and triple mutants and with deleted promoter fragments indicated the participation of additional elements beside the CRE motifs in the cAMP‐dependent stimulation. Our data suggest that intracellular cAMP may directly affect expression of the immunoregulatory cytokine IL‐10 in monocytic cells via activation of the eukaryotic transcription factors CREB‐1 and ATF‐1 and their binding to CRE1 and CRE4 in the upstream enhancer of the IL‐10 promoter.

Migration of leukocytes to the site of microbial infection is important for the development of effective host immunity. Recombinant modified vaccinia virus Ankara is frequently used as a viral vector vaccine in preclinical and clinical studies. In comparison to other vaccinia virus strains, modified vaccinia virus Ankara robustly induces chemokine expression and rapid attraction of leukocytes. In particular, chemokine (C-C motif) ligand 2 (CCL2) has been shown to be critical for leukocyte recruitment to the lung. In this study, MVA-induced CCL2 expression in murine macrophages was dependent on type I interferon receptor and not Toll-like receptor-2. The critical role of type I interferon receptor signaling for CCL2 production in the lung was confirmed in type I interferon receptor–deficient mice (Ifnar1^−/−). In addition, comparing Ifnar1^−/− and Ccl2^−/− mice with wild-type mice, we observed a similar impairment in the recruitment of natural killer and T cells to the lung after intranasal infection with modified vaccinia virus Ankara. Conversely, neutrophil recruitment was not affected in Ifnar1^−/− and Ccl2^−/− mice. We conclude that type I interferons, besides their known antiviral properties, can initiate the recruitment and activation of leukocytes via induction of chemokine expression including CCL2.

Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4⁺lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.