RESUMO
Emerging evidence suggests a role of the cytokine midkine (MK) in inflammation. In this study, its functional relevance for recruitment of polymorphonuclear neutrophils (PMNs) during acute inflammation was investigated. Intravital microscopy and histologic analysis of tumor necrosis factor-α-stimulated cremaster muscle venules revealed severely compromised leukocyte adhesion and extravasation in MK(-/-) mice compared with MK(+/+) animals. Systemic administration of recombinant MK completely rescued the adhesion defect in MK(-/-) mice. In a hind limb ischemia model, leukocyte accumulation in MK(-/-) mice was significantly diminished compared with MK(+/+) animals. However, MK did not lead to an inflammatory activation of PMNs or endothelial cells suggesting that it does not serve as classical proinflammatory cytokine. Unexpectedly, immobilized MK mediated PMN adhesion under static and flow conditions, whereas PMN-derived MK was dispensable for the induction of adhesion. Furthermore, adhesion strengthening remained unaffected by MK. Flow cytometry revealed that immobilized, but not soluble MK, significantly promoted the high affinity conformation of ß2 integrins of PMNs. Blocking studies of low-density lipoprotein receptor-related protein 1 (LRP1) suggested that LRP1 may act as a receptor for MK on PMNs. Thus, MK seems to support PMN adhesion by promoting the high affinity conformation of ß2 integrins, thereby facilitating PMN trafficking during acute inflammation.
Assuntos
Antígenos CD18/fisiologia , Inflamação/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neutrófilos/fisiologia , Animais , Antígenos CD11/fisiologia , Antígenos CD18/genética , Adesão Celular/imunologia , Adesão Celular/fisiologia , Citocinas/imunologia , Citocinas/fisiologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/imunologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Midkina , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/fisiologia , Neutrófilos/imunologia , Neutrófilos/patologia , Receptores de LDL/imunologia , Receptores de LDL/fisiologia , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/fisiologiaRESUMO
The cytokine midkine (MK) promotes tumor growth mainly by inducing angiogenesis. Here, we identified the source of MK in the vascular system under hypoxic conditions and demonstrated the relevance of MK during ischemia of normal tissue. Hypoxia increased MK protein expression in human polymorphonuclear neutrophils (PMN), monocytes, and human umbilical vein endothelial cells (HUVEC) compared with normoxia. Immunoelectron microscopy showed elevated cell surface expression of MK in PMN and monocytes during hypoxia. However, only HUVEC released significant amounts of soluble MK during hypoxia compared with normoxia (301 ± 81 pg/ml vs. 158 ± 45 pg/ml; P < 0.05). Exogenous MK induced neovascularization in a chorioallantoic membrane (CAM) assay compared with negative control as measured by counting the number of branching points per visual field (1,074 ± 54 vs. 211 ± 70; P < 0.05). In a hind limb ischemia model, the angiogenic response was almost completely absent in MK-deficient mice, whereas control animals showed a profound angiogenic response measured as proliferating endothelial cells per visual field (45 ± 30 vs. 169 ± 34; P < 0.01). These unanticipated results identified endothelial cells as the source of soluble MK in the vascular system during hypoxia and defined MK as a pivotal player of angiogenesis during ischemia in nonmalignant tissue.
Assuntos
Proteínas Angiogênicas/metabolismo , Membrana Corioalantoide/irrigação sanguínea , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Proteínas Angiogênicas/administração & dosagem , Proteínas Angiogênicas/deficiência , Proteínas Angiogênicas/genética , Animais , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Embrião de Galinha , Citocinas/administração & dosagem , Citocinas/deficiência , Citocinas/genética , Modelos Animais de Doenças , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Isquemia/genética , Isquemia/patologia , Isquemia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Imunoeletrônica , Midkina , Monócitos/metabolismo , Fatores de Crescimento Neural/administração & dosagem , Fatores de Crescimento Neural/metabolismo , Neutrófilos/metabolismo , Fatores de Tempo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
The first effectively achieved germ-line transformations of non-drosophilid insects were based on mutant rescue of eye color phenotypes. However, for most insect species neither visible mutants nor corresponding cloned genes are available. Therefore, the development of broadly applicable and reliable transformation markers will be of great importance to fully exploit the enormous potential transgenic insect technology has to offer. Here we review transposon-mediated germ-line transformation approaches that employ green fluorescent protein (GFP) variants to identify successful gene transfer. Furthermore, we provide novel data on the use of DsRed as an additional red fluorescent transformation marker for insect transgenesis. In conclusion, fluorescent proteins controlled by suitable strong promoters possess ideal characteristics to serve as transformation markers for a wide range of insect species.
