Primary structures of six antimicrobial peptides of rabbit peritoneal neutrophils

Six microbicidal peptides, NP-1, NP-2, NP-3a, NP-3b, NP-4, and NP-5, from rabbit peritoneal neutrophils were characterized. As a family, the peptides were 32-34 residues in length, were cystine- and arginine-rich, and each contained three intramolecular disulfide bonds. Within their sequences, the six peptides shared 11-residue positions, which included the six half-cystines contained in each peptide. NP-1 and NP-2 differed by a single residue and were identical in their respective sequences to MCP-1 and MCP-2, the peptide analogs from rabbit alveolar macrophages. NP-4 and NP-5 were homologous in 27 of their residues, but NP-3a and NP-3b shared little more than the 11-residue backbone common to all members of this peptide family.     

Primary structures of MCP-1 and MCP-2, natural peptide antibiotics of rabbit lung macrophages

The microbicidal peptides, MCP-1 and MCP-2, of rabbit alveolar macrophages were purified by an improved procedure that employed preparative gel electrophoresis and high performance liquid chromatography. The peptides were arginine- and cystine-rich and lacked free sulfhydryl groups and detectable levels of carbohydrate. Complete sequence determinations revealed that MCP-1 differed from MCP-2 only by the substitution of arginine for leucine at residue 13 from the NH2 terminus and that the molecules were each single chain polypeptides of 33 amino acid residues containing three intramolecular disulfide bonds. The complete amino acid sequences of MCP-1 and MCP-2 are: (sequence in text)     

Inhibition of the angiogenesis by the MCP-1 (monocyte chemoattractant protein-1) binding peptide

The CC chemokine, monocyte chemoattractant protein-1 (MCP-1), plays a crucial role in the initiation of atherosclerosis and has direct effects that promote angiogenesis. To develop a specific inhibitor for MCP-1-induced angiogenesis, we performed in vitro selection employing phage display random peptide libraries. Most of the selected peptides were found to be homologous to the second extracellular loops of CCR2 and CCR3. We synthesized the peptide encoding the homologous sequences of the receptors and tested its effect on the MCP-1 induced angiogenesis. Surface plasmon resonance measurements demonstrated specific binding of the peptide to MCP-1 but not to the other homologous protein, MCP-3. Flow cytometry revealed that the peptide inhibited the MCP-1 binding to THP-1 monocytes. Moreover, CAM and rat aortic ring assays showed that the peptide inhibited MCP-1 induced angiogenesis. Our observations indicate that the MCP-1-binding peptide exerts its anti-angiogenic effect by interfering with the interaction between MCP-1 and its receptor.     

Direct inactivation of viruses by human granulocyte defensins.

Human neutrophils contain a family of microbicidal peptides known as defensins. One of these defensins, human neutrophil peptide (HNP)-1, was purified, and its ability to directly inactivate several viruses was extensively tested. Herpes simplex virus (HSV) types 1 and 2, cytomegalovirus, vesicular stomatitis virus, and influenza virus A/WSN were inactivated by incubation with HNP-1. Two nonenveloped viruses, echovirus type 11 and reovirus type 3, were resistant to inactivation. Purified homologous peptides HNP-2 and HNP-3 were found to have HSV-1-neutralizing activities approximately equal to that of HNP-1. Inactivation of HSV-1 by HNP-1 depended on the time, temperature, and pH of incubation. Antiviral activity was abrogated by low temperature or prior reduction and alkylation of the defensins. Addition of serum or serum albumin to the incubation mixtures inhibited neutralization of HSV-1 by HNP-1. We used density gradient sedimentation techniques to demonstrate that HNP-1 bound to HSV-1 in a temperature-dependent manner. We speculate that binding of defensin peptides to certain viruses may impair their ability to infect cells.     

Inhibition of virus multiplication by immunoactive peptides

In the present study we show that peritoneal macrophages obtained from the mice treated with the immunoactive peptides inhibit the multiplication of Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), but not that of vesicular stomatitis virus (VSV), and that the intraperitoneal administration of the peptides suppresses the infection with HSV-1 in mice.     

MCP-1 derived from stromal keratocyte induces corneal infiltration of CD4+ T cells in herpetic stromal keratitis

Herpetic stromal keratitis (HSK) is an inflammatory disorder induced by HSV-1 infection and characterized by T cell-dependent destruction of corneal tissues. It is not known what triggers CD4(+) T cell migration into the stroma of HSV-1-infected corneas. The keratocyte is a fibroblast-like cell that can function as an antigen-presenting cell in the mouse cornea by expressing MHC class II and costimulatory molecules after HSV-1 infection. We hypothesized that chemokines produced by stromal keratocytes are involved in CD4(+) T cell infiltration into the cornea. We found that keratocytes produce several cytokines and chemokines, including MCP-1, RANTES, and T cell activation (TCA)-3. HSV-1 infection increased the production of MCP-1 and RANTES by keratocytes, and these acted as chemoattractants for HSV-1-primed CD4(+) T cells expressing CCR2 and CCR5. Expression of MCP-1 in the corneal stroma was confirmed in vivo. Finally, when HSV-1-primed CD4(+) T cells were adoptively transferred into wild type and MCP-1-deficient mice that had been sublethally irradiated to minimize chemokine production from immune cells, infiltration of CD4(+) T cells was markedly reduced in the MCP-1-deficient mice, suggesting that it is the MCP-1 from HSV-1-infected keratocytes that attracts CD4(+) T cells into the cornea.     

NMR studies of defensin antimicrobial peptides. 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1.

Two-dimensional nuclear magnetic resonance spectroscopy has been used to make resonance assignments of the proton spectra of two defensin antimicrobial peptides, human neutrophil peptide HNP-1 and rabbit neutrophil peptide NP-2. The secondary structures of these peptides were determined from analysis of the proton-proton NOEs and from the positions of slowly exchanging amide protons. Both peptides contain a long stretch of a double-stranded antiparallel beta-sheet in a hairpin conformation that contains a beta-bulge, a short region of triple-stranded beta-sheet, and several tight turns. The NMR results clearly show that HNP-1 forms a dimer or higher order aggregate in solution and that Pro8 exists as a cis peptide bond. The NMR data on these peptides are compared with NMR data for a homologous peptide NP-5 [Bach, A. C., Selsted, M. E., & Pardi, A. (1987) Biochemistry 26, 4389-4397]. Analysis of the conformation-dependent proton chemical shifts shows that it is not possible to confidently judge the structural similarity of the three defensins from chemical shift data alone. However, comparison of the 3JHN alpha coupling constants in NP-2 and NP-5 indicates that the backbone conformations for these peptides are very similar. A more detailed comparison of the solution conformations of the defensins peptides is made in the following paper in this issue where the NMR data are used as input for distance geometry and molecular dynamics calculations to determine the three-dimensional structures of HNP-1 and NP-2.     

Modulatory effect of HHV-6 on MCP-1 production by human monocytes

Chemokines represent a large family of proinflammatory proteins that orchestrate leukocyte trafficking to sites of viral infection. Human Herpes virus type 6 (HHV-6) is a typical immunosuppressive agent, as suggested by its tropism. In this study the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) by human peripheral blood monocytes was evaluated during HHV-6 infection. Our results demonstrate that HHV-6 infection triggers monocytes to release MCP-1 and IL-10. The addition of exogenous recombinant MCP-1 upregulates the release of extracellular virus, whereas does not influence the percentage of viral-antigen positive cells. Furthermore, the addition of monoclonal antibodies anti-IL-10 down-regulates MCP-1 release induced by HHV-6. These findings indicate that IL-10 and MCP-1 production was closely related and that the marked amounts of MCP-1 were supported not only by virus but also by virus-induced IL-10.     

Requirement for PI 3-kinase gamma in macrophage migration to MCP-1 and CSF-1

Phosphoinositide 3-kinases (PI3Ks) are important regulators of cell migration. The PI3K isoform gamma is primarily expressed in haematopoietic cells, and is activated by G protein-coupled receptors (GPCRs). Here, we investigate the contribution of PI3Kgamma to macrophage responses to chemoattractants, using bone marrow-derived macrophages from wild-type and PI3Kgamma-null mice. We observe that early membrane ruffling induced by MCP-1, which activates a GPCR, or by CSF-1, which activates a tyrosine kinase receptor, is unaltered in PI3Kgamma(-/-) mice, although by 30 min MCP-1-induced cell polarization was strongly reduced in PI3Kgamma(-/-) compared to wild-type macrophages. The migration behaviour of the macrophages was analysed by time-lapse microscopy in Dunn chemotaxis chambers. PI3Kgamma(-/-) macrophages showed reduced migration speed and translocation, and no chemotaxis to MCP-1. Interestingly, there was also a reduction in migration efficiency in PI3Kgamma(-/-) macrophages stimulated with CSF-1 although early CSF-1R signalling was normal. These results indicate that the initial actin reorganization induced by either a GPCR or tyrosine kinase receptor agonist is not dependent on PI3Kgamma, whereas PI3Kgamma is needed for optimal migration of macrophages to either agonist.     

Pseudogenization of the MCP-2/CCL8 chemokine gene in European rabbit (genus Oryctolagus), but not in species of Cottontail rabbit (Sylvilagus) and Hare (Lepus)

ABSTRACT: BACKGROUND: Recent studies in human have highlighted the importance of the monocyte chemotactic proteins (MCP) in leukocyte trafficking and their effects in inflammatory processes, tumor progression, and HIV-1 infection. In European rabbit (Oryctolagus cuniculus) one of the prime MCP targets, the chemokine receptor CCR5 underwent a unique structural alteration. Until now, no homologue of MCP-2/CCL8a, MCP-3/CCL7 or MCP-4/CCL13 genes have been reported for this species. This is interesting, because at least the first two genes are expressed in most, if not all, mammals studied, and appear to be implicated in a variety of important chemokine ligand-receptor interactions. By assessing the Rabbit Whole Genome Sequence (WGS) data we have searched for orthologs of the mammalian genes of the MCP-Eotaxin cluster. RESULTS: We have localized the orthologs of these chemokine genes in the genome of European rabbit and compared them to those of leporid genera which do (i.e. Oryctolagus and Bunolagus) or do not share the CCR5 alteration with European rabbit (i.e. Lepus and Sylvilagus). Of the Rabbit orthologs of the CCL8, CCL7, and CCL13 genes only the last two were potentially functional, although showing some structural anomalies at the protein level. The ortholog of MCP-2/CCL8 appeared to be pseudogenized by deleterious nucleotide substitutions affecting exon1 and exon2. By analyzing both genomic and cDNA products, these studies were extended to wild specimens of four genera of the Leporidae family: Oryctolagus, Bunolagus, Lepus, and Sylvilagus. It appeared that the anomalies of the MCP-3/CCL7 and MCP-4/CCL13 proteins are shared among the different species of leporids. In contrast, whereas MCP-2/CCL8 was pseudogenized in every studied specimen of the Oryctolagus - Bunolagus lineage, this gene was intact in species of the Lepus - Sylvilagus lineage, and was, at least in Lepus, correctly transcribed. CONCLUSION: The biological function of a gene was often revealed in situations of dysfunction or gene loss. Infections with Myxoma virus (MYXV) tend to be fatal in European rabbit (genus Oryctolagus), while being harmless in Hares (genus Lepus) and benign in Cottontail rabbit (genus Sylvilagus), the natural hosts of the virus. This communication should stimulate research on a possible role of MCP-2/CCL8 in poxvirus related pathogenicity.     

MCP-1 targeting inhibits muscularis macrophage recruitment and intestinal smooth muscle dysfunction in colonic inflammation

Upregulation of muscularis macrophage numbers and activities plays an important role in the intestinal dysmotility associated with intestinal inflammation. The present study aimed to clarify changes in population dynamics of intestinal muscularis macrophages during colonic inflammation and to test possible inhibitory actions of agents targeting monocyte chemoattractant protein-1 (MCP-1) on muscularis macrophage dynamics and motility disorder in the colonic inflammation elicited by 2,4,6-trinitrobenzene sulfonic acid. In the inflamed muscle layer, ED1 antibody-positive monocytes and monocyte-derived macrophages were increased, followed by increasing resident macrophages positively staining for ED2 antibody. Initiation of the ED1-positive macrophage dynamic is associated with MCP-1 mRNA expression. MCP-1 was expressed in both ED1- and ED2-positive macrophages after inflammation. Electromicroscopic analysis revealed that the cell-division phase of muscularis macrophages was seen only in the early stages of inflammation. In addition, ED1 and ED2 double-positive macrophages can be detected during inflammation. Treatment with dominant negative MCP-1 or neutralizing MCP-1 antibodies markedly inhibited numbers of both ED1- and ED2-positive macrophages. Inflammation-mediated dysmotility was partially recovered by treatment with neutralizing MCP-1 antibodies. These results suggest that the inflamed muscle layer is initially infiltrated by monocytes, which then differentiate and develop into muscularis-resident macrophages. These macrophages express MCP-1 for further recruitment of monocytes. MCP-1 may be one potential therapeutic target for inhibiting intestinal motility disorders in gut inflammation.     

Synthetic glycoprotein D-related peptides protect mice against herpes simplex virus challenge.

Glycoprotein D (gD) of herpes simplex virus (HSV) protects mice from a lethal challenge by either HSV type 1 (HSV-1; oral) or HSV-2 (genital). We evaluated whether synthetic peptides representing residues 1 through 23 of gD (mature protein) can be used as a potential synthetic herpesvirus vaccine. The immunogenicity of the peptides was demonstrated by the biological reactivity of antipeptide sera in immunoprecipitation and neutralization assays. All sera which immunoprecipitated gD had neutralizing against both HSV-1 and HSV-2. The highest titers were found in animals immunized with the longest peptides. The region of residues 1 through 23 was immunogenic regardless of whether the type 1 or type 2 sequence was presented to the animal. Immunization of mice with gD or synthetic peptides conferred solid protection against a footpad challenge with HSV-2. However, the peptides were not as effective as gD in protection against an intraperitoneal challenge. The results suggested that synthetic vaccines based on gD show promise and should be more rigorously tested in a variety of animal models.     

Identification of herpes simplex virus type 1 glycoproteins interacting with the cell surface.

To investigate the interaction of herpes simplex virus type 1 (HSV-1) with the cell surface, we studied the formation of complexes by HSV-1 virion proteins with biotinylated cell membrane components. HSV-1 virion proteins reactive with surface components of HEp-2 and other cells were identified as gC, gB, and gD. Results from competition experiments suggested that binding of gC, gB, and gD occurred in a noncooperative way. The observed complex formation could be specifically blocked by monospecific rabbit antisera against gB and gD. The interaction of gD with the cell surface was also inhibited by monoclonal antibody IV3.4., whereas other gD-specific monoclonal antibodies, despite their high neutralizing activity, were not able to inhibit this interaction. Taken together, these data provide direct evidence that at least three of the seven known HSV-1 glycoproteins are able to form complexes with cellular surface structures.     

The structure of the rabbit macrophage defensin genes and their organ-specific expression

Defensins are a family of microbicidal and cytotoxic peptides abundant in the lysosomal granules of mammalian phagocytes. We present the cDNA and genomic sequences of two rabbit defensins, macrophage cationic peptides MCP-1 and MCP-2. Their cDNA and genomic sequences are highly homologous, reflecting the homology between the two defensins (32 of 33 amino acids). The MCP genes are closely linked (within 13 kb) suggesting that they evolved by a recent tandem gene duplication. Their cDNA sequences indicate that the peptides are synthesized as 95 amino acid prepro-MCPs, consistent with their lysosomal location. The MCP genes are separated into three exons encoding distinct domains: the 5' untranslated region, the prepropeptide domain, and the mature defensin sequence. Fully developed polymorphonuclear leukocytes, short-lived phagocytes with limited capacity for protein and nucleic acid synthesis, contained MCPs but lacked MCP mRNA. MCP mRNA was found in bone marrow and spleen, organs which contained immature polymorphonuclear leukocytes. MCP and MCP mRNA were detected in lung macrophages, but not in macrophages from other organs, nor in monocytes, the putative macrophage precursors. In macrophages, the expression of MCPs appears to be a marker of lung-specific differentiation.     

Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system

After corneal infection, herpes simplex virus type 1 (HSV-1) invades sensory neurons with cell bodies in the trigeminal ganglion (TG), replicates briefly, and then establishes a latent infection in these neurons. HSV-1 replication in the TG can be detected as early as 2 days after corneal infection, reaches peak titers by 3-5 days after infection, and is undetectable by 7-10 days. During the period of HSV-1 replication, macrophages and gammadelta TCR+ T lymphocytes infiltrate the TG, and TNF-alpha, IFN-gamma, the inducible nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-alpha, IFN-gamma, and the iNOS product nitric oxide (NO) all inhibit HSV-1 replication in vitro. Macrophage and gammadelta TCR+ T cell depletion studies demonstrated that macrophages are the main source of TNF-alpha and iNOS, whereas gammadelta TCR+ T cells produce IFN-gamma. Macrophage depletion, aminoguanidine inhibition of iNOS, and neutralization of TNF-alpha or IFN-gamma all individually and synergistically increased HSV-1 titers in the TG after HSV-1 corneal infection. Moreover, individually depleting macrophages or neutralizing TNF-alpha or IFN-gamma markedly reduced the accumulation of both macrophages and gammadelta TCR+ T cells in the TG. Our findings establish that after primary HSV-1 infection, the bulk of virus replication in the sensory ganglia is controlled by macrophages and gammadelta TCR+ T lymphocytes through their production of antiviral molecules TNF-alpha, NO, and IFN-gamma. Our findings also strongly suggest that cross-regulation between these two cell types is necessary for their accumulation and function in the infected TG.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. III. Analysis of viral glycoproteins recognized by CTL clones by using recombinant herpes simplex viruses

Human cytotoxic T cell (CTL) clones specific for herpes simplex virus (HSV) type 1- and type 2-infected cells were generated and were analyzed with regard to the viral glycoproteins they recognize on autologous HSV-infected cells. By use of target cells infected with wild-type HSV strains, a gC deletion mutant of HSV-1, and HSV-1 X HSV-2 intertypic recombinants, some HSV-1-specific CTL clones were found to be directed against L region-encoded gA/B-1, and others against S region-encoded glycoproteins (gD-1 or gE-1). Some HSV-2-specific clones were found to be directed against L region-encoded gC-2, whereas others were directed against S region-encoded glycoproteins (gD-2, gE-2, or gG). These findings provide direct evidence that several HSV glycoproteins that are expressed on the surface of HSV-infected cells serve as recognition structures for human HSV-specific CTL.     

Triglyceride (TG) down-regulates expression of MCP-1 and CCR2 in PMA-derived THP-1 macrophages

Triglycerides (TGs) are implicated in the development of atherosclerosis. A key contributing factor for atherosclerosis is the migration of macrophages to atherosclerotic lesions. MCP-1 is a major chemoattractant for macrophages to atherosclerotic lesions. We examined the expression profile of MCP-1 and CCR2 in THP-1 macrophages in response to TG treatment by RT-PCR analysis. Chemical inhibitors were used to identify cell signaling pathway(s) involved in regulation of MCP-1 and CCR2 expression. We found that treatment of THP-1 macrophages with TG down-regulated MCP-1 expression in a time and dose-dependent manner. PMA treatment alone did not affect MCP-1 expression. Using chemical inhibitors of cell signaling pathways, we found that the NF-κB inhibitor inhibited TG-induced down-regulation of MCP-1. CCR2 expression decreased after TG treatment in THP-1 macrophages and the PKC inhibitor alleviated TG-induced down-regulation of CCR2. Our results provide further insights into the role of TG on macrophages during atherosclerosis.     

Cytotoxic and mitogenic effect of antimicrobial peptides from neutrophils on cultured cells

Cytotoxic and mitogenic activities of human and rabbit defensines (HNP and NP-2, resp.) and pig antimicrobial peptides from leukocytes (PR-39, prophenin PF-2 and protegrin PG-2) were studied. The above peptides were added to serum-free cell culture medium of the target cell lines K562, L929 and Hep22a. Cytotoxicity was estimated within 1, 3, 6, 24 and 48 h of cell incubation with the tested peptides in concentrations 1, 10, 25 or 100 micrograms/ml. All the examined peptides exhibited a distinct time- and concentration-dependent cytotoxicity. Moreover, by contrast to pig peptides, defensines could induce proliferation in cell subpopulations from cell lines L929 amd Hep22a, or L929 (defensines HNP and NP-2, resp.), keeping resistance to their cytotoxic action.     

RANTES and MCP-3 inhibit the replication of T-cell-tropic human immunodeficiency virus type 1 strains (SF-2, MN, and HE).

The effects of the C-C chemokines RANTES (regulation upon activation normal T-cell expressed and secreted) and MCP-3 (monocyte chemotactic protein 3) on human immunodeficiency virus (HIV) replication in normal human peripheral blood mononuclear cells (PBMC) activated in vitro with phytohemagglutinin (PHA) were investigated. The following T-cell line-tropic (T-tropic) HIV strains were tested: HIV type 1 (HIV-1) SF-2, HIV-1 IIIB, HIV-1 MN, HIV-1 NDK, HIV-1 HE, HIV-1 NL4-3, HIV-2 ROD, and HIV-2 EHO. The strain most sensitive to the antiviral effects of RANTES and MCP-3 appeared to be HIV-1 SF-2. A 50% inhibitory concentration for HIV-1 SF-2 of 4 ng of RANTES per ml was obtained, and that of MCP-3 was about 1 ng/ml. However, MCP-3 was inactive at 100 ng/ml. Other HIV-1 strains, such as MN and HE, were less sensitive to the antiviral effects of RANTES and MCP-3, whereas all the other HIV strains tested were insensitive. Although the ratio of CD3+ CD4+ to CD3+ CD8+ T cells was the same in HIV-infected PBMC cultures treated or untreated with the chemokines, RANTES and MCP-3 interfered with the binding of monoclonal antibody (MAb) OKT4 to the CD4 receptor on T cells but not with the binding of MAb OKT4A. Therefore, RANTES and MCP-3 not only interfere with the HIV-induced fusion process but also have some modulating effect on the CD4 cell receptor. The chemokines did not affect HIV-1 binding to PHA-stimulated PBMC. Taken together, our observations point to the important role that both RANTES and MCP-3 may play in inhibiting HIV-1 replication of certain T-tropic strains in primary PBMC cultures. This may have important implications for immunotherapeutic strategies designed to slow down disease progression in AIDS.     

Deoxycytidine kinase from rabbit kidney cells infected with herpes simplex virus type 1 or 2.

When rabbit kidney cells were infected with herpes simplex virus type 1 (strain Seibert) or herpes simplex virus type 2 (strain 316D), deoxycytidine kinase (CdR kinase) activity, assayed at 38 degrees, increased 5- to 15-fold relative to controls. The CdR kinase activity induced by type 2 virus was more thermolabile than the enzyme activity induced by type 1 virus. When CdR kinase activity was assayed at various temperatures between 0.5 and 38 degrees, maximum activity for type 1 enzyme was obtained at 16 degrees while maximum activities for host and type 2 enzymes were obtained at 38 degrees. Both type 1 and type 2 induced CdR kinase activities eluted at the same positions as deoxythymidine kinase activities on a Sephadex G-100 column. The estimated mol wt for HSV-1 (Seibert) and HSV-2 (316D) induced CdR kinases are 67,000 and 60,000, respectively.