Chemokine Macrophage Inflammatory Protein-3alpha Research Articles

Antitumor effects of combined granulocyte macrophage colony stimulating factor and macrophage inflammatory protein‐3 alpha plasmid DNA

Dendritic cells (DC) are critical for priming adaptive immune responses to foreign antigens. However, the feasibility of harnessing these cells in vivo to optimize the antitumor effects has not been fully explored. The authors investigated a novel therapeutic approach that involves delivering synergistic signals that both recruit and expand DC populations at sites of intratumoral injection. More specifically, the authors examined whether the co-administration of plasmids encoding the chemokine macrophage inflammatory protein-3 alpha (pMIP3α) and plasmid encoding the granulocyte macrophage colony stimulating factor (pGM-CSF; a DC-specific growth factor) can recruit, expand and activate large numbers of DC at sites of intratumoral injection. It was found that the administration of pGM-CSF and pMIP3α resulted in dramatic recruitment and expansion of DC at these sites and in draining lymph nodes. Furthermore, treatment with pGM-CSF and pMIP3α generated the strongest MUC1-associated CD8+ T-cell immune responses in draining lymph nodes and in tumors, produced the greatest antitumor effects and enhanced survival rates more than pcDNA3.1, pGM-CSF alone and pMIP3α alone. It was also found that pGM-CSF plus pMIP3α generated the strongest MUC1-associated CD4+ T-cell immune responses in draining lymph nodes and in tumors. The findings of the present study suggest that the recruitment and activation of DC in vivo due to the synergistic actions of pGM-CSF and pMIP3α presents a potentially feasible means of controlling immunogenic malignancies and provides a basis for the development of novel immunotherapeutic treatments.

Structure–function studies of chemokine-derived carboxy-terminal antimicrobial peptides

Recent reports which show that several chemokines can act as direct microbicidal agents have drawn renewed attention to these chemotactic signalling proteins. Here we present a structure-function analysis of peptides derived from the human chemokines macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20), interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) and thrombocidin-1 (TC-1). These peptides encompass the C-terminal alpha-helices of these chemokines, which have been suggested to be important for the direct antimicrobial activities. Far-UV CD spectroscopy showed that the peptides are unstructured in aqueous solution and that a membrane mimetic solvent is required to induce a helical secondary structure. A co-solvent mixture was used to determine solution structures of the peptides by two-dimensional (1)H-NMR spectroscopy. The highly cationic peptide, MIP-3alpha(51-70), had the most pronounced antimicrobial activity and displayed an amphipathic structure. A shorter version of this peptide, MIP-3alpha(59-70), remained antimicrobial but its structure and mechanism of action were unlike that of the former peptide. The NAP-2 and TC-1 proteins differ in their sequences only by the deletion of two C-terminal residues in TC-1, but intact TC-1 is a very potent antimicrobial while NAP-2 is inactive. The corresponding C-terminal peptides, NAP-2(50-70) and TC-1(50-68), had very limited and no bactericidal activity, respectively. This suggests that other regions of TC-1 contribute to its bactericidal activity. Altogether, this work provides a rational structural basis for the biological activities of these peptides and proteins and highlights the importance of experimental characterization of peptide fragments as distinct entities because their activities and structural properties may differ substantially from their parent proteins.

Seminal plasma promotes the attraction of Langerhans cells via the secretion of CCL20 by vaginal epithelial cells: involvement in the sexual transmission of HIV

Heterosexual human immunodeficiency virus (HIV) transmission implies the crossing of the vaginal mucosa by virions present in the semen, potentially using Langerhans cells as transporters. The recruitment of these cells in the mucosa is mediated by the chemokine macrophage inflammatory protein 3alpha (CCL20). The aim of this study was to evaluate the capacity of the semen to induce Langerhans cell recruitment via the production of CCL20 by vaginal epithelial cells. Using a vaginal epithelium model based on the SiHa cell line and human seminal plasma, we demonstrated that semen enhanced the production of CCL20. This secretion was regulated by the nuclear factor-kappaB intracellular signalling pathway. Fractionation of the seminal plasma indicated that the secretion of CCL20 was stimulated by high molecular weight compounds present in semen. Migration assays demonstrated that secreted CCL20 was able to promote the recruitment of Langerhans cell precursors (LCps), which remain permissive to X4 and R5 HIV infection. Our results demonstrate that epithelial cells respond to factors present in semen by secreting CCL20, leading to the enhancement of LCp recruitment. These data argue in favour of the implication of epithelial cells in the heterosexual transmission of HIV.

Calcium triggers β‐defensin (hBD‐2 and hBD‐3) and chemokine macrophage inflammatory protein‐3α (MIP‐3α/CCL20) expression in monolayers of activated human keratinocytes

The inducible epidermal beta-defensins and the chemokine macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20) are important mediators involved in innate and adaptive immunity and in the recruitment of immune cells. The aim of our study was to determine whether calcium could trigger the induction of beta-defensins (hBD-2 and hBD-3) mRNA and the release of MIP-3alpha by normal human keratinocyte monolayers. Epidermal cells derived from foreskin were cultured in defined medium supplemented with different calcium levels (0.09, 0.8 and 1.7 mM) and were stimulated or not with the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha 1-500 ng/ml) or interferon-gamma (INF-gamma 1-100 ng/ml). A high calcium concentration (1.7 mM) alone applied in culture medium for 4 days was sufficient to induce hBD-2 and hBD-3 mRNA expression. Whatever interindividual variability in the expression of hBD-2 and hBD-3 mRNA and MIP-3alpha secretion, the addition of TNF-alpha for a short duration (26h), initiated a dose-dependent and coordinated up-regulation of hBD-2 and hBD-3 mRNA and MIP-3alpha release in keratinocyte cultures. Unlike hBD-2 and hBD-3 mRNA was preferentially stimulated by IFN-gamma rather than TNF-alpha. In our experimental conditions, L-isoleucine, described to stimulate beta-defensin in bovine epithelial cells, did not exert any effect either on hBD-2 and hBD-3 transcripts or MIP-3alpha protein. Taken together, these results confirm the major role of the maturation/differentiation process of normal human keratinocytes in the induction of inducible beta-defensins and MIP-3alpha chemokine, which contribute in vivo to the immunosurveillance of the skin barrier function.

MIP-3alpha induces human eosinophil migration and activation of the mitogen-activated protein kinases (p42/p44 MAPK).

The CC chemokine macrophage inflammatory protein-3alpha (MIP-3alpha) is the product of recent electronic cloning efforts, however, little characterization of its spectrum of biological effects has been undertaken. Human eosinophils exhibited pertussis-toxin-sensitive migration in response to human recombinant (hr)MIP-3alpha. Messenger RNA for the MIP-3alpha receptor, CCR-6, and low levels of surface expression were demonstrated by reverse transcriptase-polymerase chain reaction and FACS analysis. Analyses of cell signaling revealed dose-dependent increases in intracellular calcium mobilization, calcium transients that were, however, greatly reduced when compared with MCP-3-induced responses. Further investigations of MIP-3alpha-induced signal transduction revealed time- and dose-dependent, partially pertussis toxin-dependent, increases in phosphorylation of the p42/p44 mitogen-activated protein kinases (MAPK) that occurred at 10- to 100-fold lower concentrations, and that were linked to a phosphoinositide 3-kinase pathway. These results suggest that MIP-3alpha can regulate multiple, parallel signal transduction pathways in eosinophils, and suggest that MAPK activation by MIP-3alpha in eosinophils is a significant signaling pathway for migration induction.

MIP-3alpha, MIP-3beta and fractalkine induce the locomotion and the mobilization of intracellular calcium, and activate the heterotrimeric G proteins in human natural killer cells.

We demonstrate here that the CC chemokines macrophage inflammatory protein-3alpha (MIP-3alpha), macrophage inflammatory protein-3beta (MIP-3beta) and the CX3C chemokine fractalkine induce the chemotaxis of interleukin-2 (IL-2)-activated natural killer (IANK) cells. In addition, these chemokines enhance the binding of [gamma-35S]guanine triphosphate ([gamma-35S]GTP) to IANK cell membranes, suggesting that receptors for these chemokines are G protein-coupled. Our results show that MIP-3alpha receptors are coupled to Go, Gq and Gz, MIP-3beta receptors are coupled to Gi, Gq and Gs, whereas fractalkine receptors are coupled to Gi, and Gz. All three chemokines induced a robust calcium response flux in IANK cells. Cross-desensitization experiments show that MIP-3alpha, MIP-3beta or fractalkine use receptors not shared by each other or by the CC chemokine regulated on activation, normal, T-cell expressed, and secreted (RANTES), the CXC chemokines stromal-derived factor-1alpha (SDF-1alpha) and interferon-inducible protein-10 (IP-10), or the C chemokine lymphotactin.