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The Regulatory Role of MRP8 (S100A8) and MRP14 (S100A9) in the Transendothelial Migration of Human Leukocytes

The hallmark of developing inflammatory lesions is the excess migration of recruited phagocytes together with the enhanced cell surface expression of adhesion molecules. Recent investigations give evidence that the two myeloid-related proteins MRP8 (S100A8) and MRP14 (S100A9), which are abundant in activated or recruited phagocytes, may have a modulatory role in inflammatory responses. S100A9 displays a regulatory role in the transendothelial migration of human monocytes, and the secreted S100A8/A9 complex may serve as a transport protein to move arachidonic acid to its target cells.

Isolation and characterization of earthworm hemolysins and agglutinins.

Hemolytic activity in coelomic fluid of Eisenia fetida (ECF) is due to three proteins H1, H2, H3 with molecular weights of 46, 43 and 40 kD, respectively. These proteins were isolated by preparative PAGE. H1 and H2 were shown to be stable in SDS and alpha-2-ME whereas H3 splits into two fragments with molecular weights of 18 and 21 kD after SDS treatment. IEF indicates that each protein consists of different isoforms with pIs between 5.1 and 6.2 H3 was demonstrated to be a bifunctional protein that can lyse and agglutinate erythrocytes. At 56 degrees C hemolytic activity of all three proteins was inactivated, but the agglutination activity of H3 was stable. Intracoelomic injection of erythrocytes reduced the number of hemolysins from three to two. Monospecific antisera were raised against the isolated hemolysins H1,2 and 3. The use of these antibodies and of carbohydrates as inhibitors of the biological activity of the molecules demonstrates the close structural relationship of agglutinins and hemolysins in the CF of E. fetida.

Evidence for perforin-like activity associated with earthworm leukocytes

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.

Myeloid related protein (MRP) 14 expressing monocytes infiltrate atherosclerotic lesions of ApoE null mice

The consequences of atherosclerotic plaque formation result in a number of diseases of the cardiovascular system which represent a serious health problem and major cause of death in the western world. Despite a growing research interest the pathogenesis of atherosclerosis especially regarding the initial steps of the disease and the participation of immune cells is not fully understood yet. Since these investigations are hard to manage in humans the development of animal models which closely resemble human conditions has long been an important research goal. Mice deficient in production of apolipoprotein (Apo) E are a useful model since they develop advanced atherosclerotic lesions at several locations throughout the arterial tree which show the typical features of those in man: early fatty streak formation, later calcification and development of necrotic cores and fibrous caps [1–3]. ApoE −/− mice were generated by gene targeting [4–6] and develop advanced atherosclerotic lesions and hypercholesterolaemia under a normal chow diet at about 6–8 months age. This animal model is widely used in recent studies about the pathomechanisms of atherosclerosis [6–13]. The interest of this study focused on the cellular infiltration of atherosclerotic lesions at different time points and the functional characterization of these cells. The appearance of S100 protein expressing immune cells in atherosclerotic lesions has been reported in recent literature for mice, rats and humans [14–16] and raises the question whether these cells participate in proatherogenic mechanisms. Myeloid related proteins (MRP) 8 and 14 belong to the class of S100 calcium binding proteins (S100A8 and S100A9) and represent peripheral marker proteins for acute and chronic inflammation. In a number of inflammatory diseases and animal models a subpopulation of monocytes expressing MRP8 and MRP14 were shown to be among the first cells infiltrating inflammatory lesions [17,18], implying a function in the initial steps of inflammation. Leukocytes positive for MRP8 and 14 have been found adjacent to activated endothelium [19] and, as was recently shown, the proteins are released from monocytes after contact to activated endothelium. MRP8 and MRP14 are cytosolic proteins occurring both in granulocytes and monocytes. They are translocated to the cell membrane after activation as it occurs under inflammatory conditions where they form a heterodimer complex [20]. Secreted MRP proteins may modulate adhesion and transendothelial migration properties of leukocytes to activated endothelium by regulating Mac-1 (CD11b) affinity [21]. The development of atherosclerotic lesions results from an excessive response to various forms of endothelium injury and smooth muscle proliferation of the arterial wall, whereas a number of cytokines, growth factors and vasoactive molecules are involved which are also typical for early reactions during acute inflammations. From the authors’ knowledge about the migration behavior of MRP expressing cells in inflammatory models a function of these cells was proposed during atherogenesis as well. That is why one was interested in the time course and phenotypic analysis of the infiltrating MRP expressing neutrophils. The aorta ascendence was prepared from the animals under a dissection microscope as described earlier [22]. The tissue was thoroughly washed in phosphate buffered saline, mounted in OCT compound (Miles, Elkhart, Ind) and shock frozen in liquid nitrogen. Sections (5 mm) were prepared and acetone fixed for 10 min. The sections were stained with the appropriate antibodies in a concentration of 1 mg/ml and counter stained with Mayer’s Hämalaun. Light microscopy was performed for morphological observations and cellular infiltration was quantified by counting using a microscopic grid. Interestingly enough, mainly MRP14 expressing cells were found in the atherosclerotic lesions of 6 months old ApoE −/− mice, whereas MRP8 positive cells very rarely occured in the plaques (Fig. 1). Equivalent binding of both antibodies to mononuclear cells have been checked in control experiments so that the real

Suppression of tumorigenicity and metastasis in murine UV-2237 fibrosarcoma cells by infection with a retroviral vector harboring the interferon-beta gene

Abstract In this study, we endeavored to determine the effectiveness of interferon β (IFNβ) gene therapy against highly metastatic murine UV-2237m fibrosarcoma cells. UV-2237m cells were engineered to produce murine IFNβ constitutively following infection by a retroviral vector harboring the murine IFNβ gene. Parental (UV-2237m-P), control-vector-transduced (UV-2237m-Neo), and IFNβ-transduced (UV-2237m-IFNβ) cells were injected subcutaneously (s.c.) or intravenously (i.v.) into syngeneic mice. Parental and control-transduced cells produced rapidly growing tumors, whereas IFNβ-transduced cells did not. The tumorigenicity of IFNβ-sensitive or -resistant parental cells was significantly suppressed when they were injected s.c. together with IFNβ-transduced cells. The IFNβ-transduced cells did not inhibit growth of parental cells injected s.c. at a distant site. UV-2237m-IFNβ cells produced s.c. tumors in nude, SCID/Beige, and natural killer(NK)-cell-compromised syngeneic mice. The IFNβ-transduced cells were more sensitive to in vitro splenic cell-mediated lysis than were the parental or control-transduced cells. Pretreatment of C3H/HeN mice with the NK-cell-selective antiserum (anti-asialoGM1) partially abrogated the cytotoxic activity of the cells. Cytotoxic activity was not observed in mixed culture of UV-2237m-IFNβ cells and splenic cells from SCID/Beige mice. Significant cytotoxicity against UV-2237m-IFNβ cells was mediated by macrophages activated by either IFNγ, lipopolysaccharide, or a combination of both. Our data led us to conclude that the constitutive expression of IFNβ can suppress tumorigenicity and metastasis of UV-2237m cells, which is due, in part, to activation of host effector cells.

The Transcription Factors c-myb and C/EBPα Regulate the Monocytic/Myeloic Gene MRP14

Abstract The entry of microorganisms into the body induces inflammatory processes.During this process a sequence of cellular, humoral, non-specific and specific actions are evoked to combat the infection.Macrophages and granulocytes, which are developed from a common progenitor cell, are the cellular components of the specific and non-specific immunoreaction.MRP14 (Macrophage migration inhibitory related protein) and MRP8, two S-100 proteins contained in high concentrations in these cells are obviously essential for adhesion and migration of monocytes and granulocytes.To investigate the transcriptional regulation of these genes we cotransfected constructs expressing CAT under control of the MRP14 promoter and expression constructs of C/EBPoc and v-myb, two transcription factors involved in myeloid/monocytic differentiation.Transfection with C/EBPoc revealed a massive enhancement of the MRP14 promoter in both, HL 60 cells (granulocytic differentiated) and L132 fibroblasts.In contrast, v-myb reduces MRP14 promoter activity.Northern blot analysis of L132 cells transfected with the C/EBPoc expression vector demonstrate that C/EBPcc is sufficient to enhance MRP14 expression in the context of the whole genome.

Growth inhibition of human mammary carcinoma by liposomal hexadecylphosphocholine: Participation of activated macrophages in the antitumor mechanism

This study was undertaken to investigate the antitumor effect of liposomal hexadecylphosphocholine (L‐HPC), a synthetic phospholipid encapsulated into multilamellar vesicles (MLV). The effect of these liposomes was tested in an orthotopic nude mouse model using the human mammary carcinomas MDA‐MB 435 and 231. The main interest of the investigation was to study whether activated macrophages are substantially involved in the tumor growth inhibition mechanism. The growth of both MDA‐MB 435 and 231 tumors in the mammary fat pad was significantly inhibited by a 14‐day intraperitoneal therapy with L‐HPC. The remaining tumors were shown to be heavily infiltrated with macrophages. In vitro studies of mPEM demonstrated a significant induction of macrophage‐mediated tumor cytotoxicity (MMCTX) against the 2 cell lines by L‐HPC. The L‐HPC‐mediated activation mechanism was characterized to be IL‐6 and TNFα dependent but rather independent of IL‐1α and nitric oxide (NO). NMA, a specific inhibitor of NO production, did not inhibit L‐HPC‐induced MMCTX. Furthermore, L‐HPC was shown to upregulate the matrixmetalloproteinases MMP‐9 and MMP‐2 secretion into the supernatant. Considering cytokine release and production of collagenases, the L‐HPC‐induced macrophage activation cascade is assumed to be comparable with that of classical activators such as lipopolysaccharide (LPS) and interferon (IFN) γ. As far as NO production is considered, the L‐HPC activation mechanism differs from that caused by LPS and IFN γ.

Mass spectrometric analyses of CL39, CL41 and H1, H2, H3 confirm identity with fetidin and lysenin produced by earthworm leukocytes

Antibacterial proteins realize effective immunological mechanisms against invading pathogens. Some of them exert hemolytic and agglutinating properties. Here, we analyzed two hemolysins isolated from cell lysate (CL(39) and CL(41)) and three hemolytic proteins isolated from coelomic fluid (H(1), H(2) and H(3)) of the annelid Eisenia fetida using mass spectrometry and bioinformatics. We demonstrated the identity of CL(39,41) with fetidin and lysenin; these have been described earlier. H(1-3) share sequence components with fetidin but they seem to be glycosylated as shown for H(1). The results help to resolve a long debate concerning nomenclature and identity of these hemolytic proteins. They support: (1). the concept that the hemolytic proteins originate from chloragocytes; (2). their origin to some extent from large coelomocytes; and (3). the view that they are secreted into CF.

INDUCTION OF NITRIC OXIDE PRODUCTION AND TUMORICIDAL PROPERTIES IN MURINE MACROPHAGES BY A NEW SYNTHETIC LIPOPEPTIDE JBT3002 ENCAPSULATED IN LIPOSOMES

We studied activation to the tumoricidal state of murine peritoneal macrophages by liposomes containing a new synthetic analogue, JBT3002, of a lipoprotein from the outer wall of a gram-negative bacterium. The liposomes containing JBT3002 or CGP31362 were superior to liposomes containing muramyl tripeptide phosphati-dylethanolamine (MTP-PE) for tumoricidal activation in three ways. First, efficient macrophage activation required lower concentrations of JBT3002 or CGP31362 than MTP-PE. Second, macrophage activation by JBT3002 was less dependent on priming by interferon-γ. Third. MLV-JBT3002 activated tumoricidal properties in both lipo-polysaccharide (LPS)-responsive and LPS-nonresponsive macrophages. The activation of tumoricidal properties by MLV-JBT3002 depended on protein tyrosine kinase (PTK) activity associated with phosphorylation of tyrosine. The major mechanism for tumoricidal activity in macrophages incubated with MLV-JBT3002 was due to increased activity of inducible nitric oxide synthase (iNOS) and, hence, production of nitric oxide (NO). We base this conclusion on the results of several experiments. First, MLV-JBT3002 was not directly toxic to tumor target cells. Second, the specific iNOS inhibitor NG-monomethyl-L.-arginine abrogated tumor cell lysis by MLV-JBT3002-treated macrophages. Third, macrophages from iNOS knockout mice did not lyse tumor cells, even after incubation with high concentrations of MLV-JBT3002. These data suggest that liposomes containing the synthetic bacterial lipopeptide JBT3002 are potent activators of macrophage tumoricida properties.

Crossreactivity of hemolytic and hemagglutinating proteins in the coelomic fluid of lumbricidae (Annelida).

Oligospecific antisera against the hemolytic and hemagglutinating compounds of the coelomic fluid (CF) of the earthworm species Eisenia fetida, Lumbricus terrestris and Aporrectodea caliginosa were prepared. The proteins were bound to the membranes of erythrocytes and injected into rabbits. By the use of these antisera the following results were obtained: 1) The antisera RaL T and RaAC reached a titer of 1:64,000, the detection limit of RaEF was 1:512,000. RaEF was demonstrated to be oligospecific only against three hemolytic proteins by Western blotting. 2) RaEF is able to inhibit the biological activity not only of hemolysins (E. fetida, A. caliginosa) but also of hemagglutinins (L. terrestris, L. rubellus, D. rubidus). 3) Complex carbohydrates, but not simple sugar compounds, are able to inhibit hemolytic and agglutinating activities in the CF of the investigated species. Fet. and alpha 1ac. GP were demonstrated to be strong inhibitors both of the hemolytic and of the hemagglutinating activity, whereas N-acetylglucosamine was only able to inhibit the hemagglutinating activity. 4) The investigated compounds were shown to crossreact in ELISA experiments. 5) By the use of Western blotting, the crossreacting molecules in the CF of the investigated lumbricid species were identified.