β-defensins play a dual role during immune response. Their direct antimicrobial properties contribute to the local innate immune response by combating microbial invasions. Furthermore, previous studies revealed the capacity of certain β-defensin family members to chemoattract immature dendritic cells and CD45RO+ CD4+ T cells through chemokine receptor CCR6. However, because β-defensins also chemoattract macrophages and monocytes, which do not express CCR6, efforts have been made to identify other receptors for these polypeptides. In this study, we demonstrate the capacity of human β-defensin (hBD)2 and 3 and their mouse orthologs, β-defensin 4 and 14, to interact with CCR2, a chemokine receptor expressed on monocytes, macrophages, and neutrophils. These β-defensins, fused to the Fc region of human IgG1, showed binding to CCR2-transfected HEK293 cells, as revealed by flow cytometry. The β-defensin fusion proteins also induced CCR2-specific chemotaxis of transfected HEK293 cells, human peripheral blood monocytes, and mouse peritoneal exudate cells in a dose-dependent manner. Preincubation of human monocytes with CCL2/MCP-1, the chemokine ligand for CCR2, abolished migration induced by β-defensins. Conversely, pre-incubation with hBD2:Ig or hBD3:Ig inhibited MCP-1 induced migration. Peritoneal exudate cells from CCR2-deficient mice failed to migrate toward these fusion proteins. In conclusion, the β-defensins used in this study contribute to the innate and adaptive immune response in their role as chemoattractants. Our data indicate that hBD2 and hBD3, together with their mouse orthologs (β-defensin 4 and 14), are chemotactic for a broad spectrum of leukocytes in a CCR6- and CCR2-dependent manner.

␤-Defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, it became evident that certain members of the ␤-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses by interacting with the CC-chemokine receptor CCR6. We have identified mouse ␤-defensin 14 (mBD14, Defb14) as an orthologue of human ␤-defensin 3 (hBD3 or DEFB103). Based on primary structural analysis, mBD14 demonstrates greater (68%) homology to its human orthologue, containing three conserved cystein linkages, characteristic for the ␤-defensin super family. mBD14 is expressed in a wide variety of tissues including spleen, colon, and tissues of the upper and lower respiratory tract. Interestingly, we also detected mBD14 expression in immature CD11c؉ bone marrow-derived dendritic cells. The expression of mBD14 can be induced by Toll-like receptor agonists such as lipopolysaccharide and poly(I:C) and by pro-inflammatory stimuli e.g. tumor necrosis factor and interferon-␥. Furthermore, expression of mBD14 seems to be regulated by activation of the intracellular pattern recognition receptor NOD2/CARD15 as revealed by reporter gene analysis. We prepared a recombinant mBD14-Ig fusion protein that retained potent antimicrobial activity against several Escherichia coli strains but not against various Gram-positive Staphylococcus aureus strains. hBD3 and also the newly identified mBD14 were chemotactic for cells expressing the mouse CC-chemokine receptor CCR6. In addition, both hBD3 and mBD14 were chemotactic for freshly isolated mouse resident peritoneal cells. Thus, mBD14, based on structural and functional similarities, appears to be an orthologue of hBD3.

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Our previous studies indicated that lymphotoxin β receptor (LTβR) activation controls and downregulates inflammatory reactions. In this study, we report that LTβR activation on primary mouse macrophages results in induction of tripartite motif containing (TRIM) 30α, which negatively regulates NF-κB activation induced by TLR signaling. LTβR activation results in a downregulation of proinflammatory cytokine and mediator expression upon TLR restimulation, demonstrating that LTβR signaling is involved in the induction of TLR cross-tolerance. Specific knockdown experiments using TRIM30α-specific small interfering RNA abolished the LTβR-dependent induction of TRIM30α and LTβR-mediated TLR cross-tolerance. Concordantly, LTβR activation on bone marrow-derived macrophages induced cross-tolerance to TLR4 and TLR9 ligands in vitro. Furthermore, we have generated cell type-specific LTβR-deficient mice with ablation of LTβR expression on macrophages/neutrophils (LTβRflox/flox × LysM-Cre). In bone marrow-derived macrophages derived from these mice LTβR-induced cross-tolerance to TLR4 and TLR9 ligands was impaired. Additionally, mice with a conditional ablation of LTβR expression on macrophages (LTβRflox/flox × LysM-Cre) are resistant to LTβR-induced TLR4 tolerance in vivo. Collectively, our data indicate that LTβR activation on macrophages by T cell-derived lymphotoxin α1β2 controls proinflammatory responses by activation of a TRIM30α-controlled, counterregulatory signaling pathway to protect against exacerbating inflammatory reactions.