Eosinophil-derived neurotoxin (EDN) is an eosinophil granule–derived secretory protein with ribonuclease and antiviral activity. We have previously shown that EDN can induce the migration and maturation of dendritic cells (DCs). Here, we report that EDN can activate myeloid DCs by triggering the Toll-like receptor (TLR)2–myeloid differentiation factor 88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independently of TLR1 or TLR6. When mice were immunized with ovalbumin (OVA) together with EDN or with EDN-treated OVA-loaded DCs, EDN enhanced OVA-specific T helper (Th)2-biased immune responses as indicated by predominant production of OVA-specific interleukin (IL)-5, IL-6, IL-10, and IL-13, as well as higher levels of immunoglobulin (Ig)G1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen-specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen-specific Th2 immune responses.

Mammalian antimicrobial proteins, such as defensins and cathelicidin, have stimulating effects on host leukocytes. Cathelin-related antimicrobial peptide (CRAMP), the orthologue of human cathelicidin/LL-37, is the sole identified murine cathelicidin. CRAMP has been shown to have both antimicrobial and angiogenic activities. However, whether CRAMP, like human cathelicidin/LL-37, also exhibits a direct effect on the migration and function of leukocytes is not known. We have observed that CRAMP, like LL-37, was chemotactic for human monocytes, neutrophils, macrophages, and mouse peripheral blood leukocytes. CRAMP also induced calcium mobilization and the activation of MAPK in monocytes. CRAMP-induced calcium flux in monocytes was desensitized by MMK-1, an agonistic ligand specific for formyl peptide receptor-like-1 (FPRL1), and vice versa, suggesting the use of FPRL1 by CRAMP as a receptor. Furthermore, CRAMP induced the chemotaxis of human embryonic kidney 293 cells transfected with either FPRL1 or mouse formyl peptide receptor-2, the mouse homologue of FPRL1, but not by untransfected parental human embryonic kidney 293 cells, confirming the use of FPRL1/mouse formyl peptide receptor-2 by CRAMP. Injection of CRAMP into mouse air pouches resulted in the recruitment predominantly of neutrophils and monocytes, indicating that CRAMP acts as a chemotactic factor in vivo. Finally, simultaneous administration of OVA with CRAMP to mice promoted both humoral and cellular Ag-specific immune responses. Thus, CRAMP functions as both a chemoattractant for phagocytic leukocytes and an enhancer of adaptive immune response.

IntroductionAntimicrobial proteins of neutrophil granules have long been considered as important elements of innate host defense. 1 In recent years, evidence has been accumulating indicating that some neutrophil granule-derived antimicrobial proteins also act as chemoattractants for various types of leukocytes. For example, neutrophil-derived ␣-defensins have been shown to be chemotactic for human monocytes, T cells, and immature dendritic cells (iDCs). [2][3][4] Azurocidin and cathelicidins, 2 antimicrobial proteins predominantly stored in neutrophil granules, are chemotactic for neutrophils, monocytes, T cells, and mast cells. 3,[5][6][7][8][9] Cathepsin G, on the other hand, is chemotactic for neutrophils and monocytes. 5 Furthermore, in mouse experimental models, neutrophil granulederived defensins are capable of enhancing antigen-specific immune responses when administered simultaneously with antigens. 10,11 Thus, these neutrophil granule-derived antimicrobial proteins have the capacity to participate in mobilizing and amplifying adaptive immunity by functioning as leukocyte chemoattactants and/or activators. 12 In addition to neutrophil granule-derived antimicrobial proteins, chymase, a serine protease found in the granules of basophils and mast cells, has been shown to induce neutrophil and monocyte chemotaxis. 13 Human eosinophil granules contain several antimicrobial proteins including eosinophil-derived neurotoxin (EDN), 14,15 which together with eosinophil cationic protein belongs to what has come to be recognized as the RNase A superfamily. 16 EDN was purified in 1981 14 and its gene was cloned in 1989. 17 Its 3-dimensional structure has also been solved by X-ray crystallography. 18 Aside from its neurotoxic effect, 14 EDN has been shown to have antiviral activity, in particular against respiratory syncytial virus in vitro, 19,20 suggesting that EDN may contribute to host antiviral defense against single-strand RNA viruses. 21 Very recently, EDN has also been shown to be responsible in part for the HIV-1 inhibitory activities in the supernatant of allogeneic mixed lymphocyte reaction. 22 However, it is not known whether EDN, like several other antimicrobial proteins derived from the granules of neutrophils and basophils/mast cells, may also have chemotactic activity for leukocytes. To test this possibility, we have investigated the capacity of EDN to induce the migration of various types of human leukocytes and found that EDN and its divergent mouse ortholog, mouse eosinophil-associated RNase 2 (mEAR2), 23 can act as selective chemoattractants for DCs. Materials and methods ReagentsRecombinant human (rh) stromal cell-derived factor-1␣ (SDF-1␣), tumor necrosis factor ␣ (TNF␣), granulocyte-macrophage colony-stimulating The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. The publisher or recipient acknowledges right ...

Apoptotic cells are phagocytosed as soon as they appear in vivo. In this study, we first determined precisely at what stage apoptotic cells are phagocytosed by macrophages, and then examined the subsequent cytokine production. Phagocytosis was confirmed by flow cytometry and confocal laser microscopy, whereas the subsequent response was examined by ELISA and RT-PCR for quantitative and semiquantitative measurement of the protein and mRNA levels of cytokines, respectively. Even the cell populations containing very early apoptotic cells, such as IL-2-dependent CTLL-2 cells cultured in the absence of IL-2 for 4 h and a murine leukemic cell line, P388 cells, treated with etoposide for 5 h, were phagocytosed by macrophages. Although the cell populations containing the very early apoptotic cells used in this study were FITC-Annexin V-negative and did not show a decrease in cell size as compared with untreated cells, they showed a very small increase in phosphatidylserine on the cell surface, as detected with Cy3-Annexin V, and a decrease in mitochondrial membrane potential, indicating that the cell populations had already started the apoptotic process. Phagocytosis of such populations containing very early apoptotic cells was inhibited by phospho-l-serine much more significantly than Arg-Gly-Asp-Ser. In addition, macrophages hardly produced either proinflammatory or anti-inflammatory cytokines after phagocytosis, thus being an almost null response. These results are contrary to the generally accepted concept that the phagocytosis of apoptotic cells leads to the production of anti-inflammatory cytokines, suggesting instead that cells starting to undergo apoptosis are quickly phagocytosed by macrophages without any inflammation in vivo.

Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ESlike cells (E-1 and E-2) appear to maintain a normal diploid karyotype inde¢nitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-speci¢c embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells di¡erentiated to neural precursor cells in the presence of basic ¢broblast growth factor (bFGF), epidermal growth factor and platelet-derived growth factor. We also developed a protocol that resulted in the di¡er-entiation of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell factor and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.

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Chemotherapy and radiotherapy are performed for cancer patients with the hope that dying cancer cells are safely scavenged by phagocytic cells such as macrophages. In this study, we examined cytokine production by macrophages during and after the phagocytosis of etoposide-treated P388 cells in vitro and in vivo. Etoposide caused apoptosis as early as 5 h after treatment, as assessed as to the exposure of phosphatidylserine, increase in membrane permeability and DNA ladder formation. Phagocytosis by phorbol myristate acetate (PMA)-treated THP-1 cells occurred marginally when P388 cells were treated with etoposide for 10 h, while it occurred significantly with P388 cells treated for 24 h, as evidenced by flow cytometry and confocal microscopy. PMA-treated THP-1 cells produced pro-inflammatory cytokines, such as interleukin (IL)-1alpha, IL-8 and macrophage migration inhibitory factor (MIF), but not anti-inflammatory cytokines among those tested at the mRNA level during and after the phagocytosis of apoptotic cells. IL-8 and MIF were also produced at the protein level, and the IL-8 production was dependent on cell-to-cell contact when the plasma membranes of apoptotic cells were intact enough not to leak one of the cytoplasmic enzymes, lactate dehydrogenase. In addition, etoposide-treated P388 cells induced neutrophil infiltration as well as MIP-2 production upon injection into the peritoneal cavity of either normal mice or mice with sterile peritonitis. When macrophages ingesting and/or binding apoptotic P388 cells were isolated from the mice with sterile peritonitis using a cell sorter, they were found to produce MIP-2 upon culture.

Many mammalian antimicrobial peptides (AMPs) have multiple effects on antimicrobial immunity. We found that temporin A (TA), a representative frog-derived AMP, induced the migration of human monocytes, neutrophils, and macrophages with a bell-shaped response curve in a pertussis toxin-sensitive manner, activated p44/42 MAPK, and stimulated Ca2+ flux in monocytes, suggesting that TA is capable of chemoattracting phagocytic leukocytes by the use of a Giα protein-coupled receptor. TA-induced Ca2+ flux in monocytes was cross-desensitized by an agonistic ligand MMK-1 specific for formyl peptide receptor-like 1 (FPRL1) and vice versa, suggesting that TA uses FPRL1 as a receptor. This conclusion was confirmed by data showing that TA selectively stimulated chemotaxis of HEK 293 cells transfected with human FPRL1 or its mouse ortholog, murine formyl peptide receptor 2. In addition, TA elicited the infiltration of neutrophils and monocytes into the injection site of mice, indicating that TA is also functionally chemotactic in vivo. Examination of two additional temporins revealed that Rana-6 was also able to attract human phagocytes using FPRL1, but temporin 1P selectively induced the migration of neutrophils using a distinct receptor. Comparison of the chemotactic and antimicrobial activities of several synthetic analogues suggested that these activities are likely to rely on different structural characteristics. Overall, the results demonstrate that certain frog-derived temporins have the capacity to chemoattract phagocytes by the use of human FPRL1 (or its orthologs in other species), providing the first evidence suggesting the potential participation of certain amphibian antimicrobial peptides in host antimicrobial immunity.