RESUMO
Long-term hematopoietic stem cells (LT-HSC) and short-term hematopoietic stem cells (ST-HSC) have been characterized as having markedly different in vivo repopulation, but similar in vitro growth in liquid culture. These differences could be due to differences in marrow homing. We evaluated this by comparing results when purified ST-HSC and LT-HSC were administered to irradiated mice by three different routes: intravenous, intraperitoneal, and directly into the femur. Purified stem cells derived from B6.SJL mice were competed with marrow cells from C57BL/6J mice into lethally irradiated C57BL/6J mice. Serial transplants into secondary recipients were also carried out. We found no advantage for ST-HSC engraftment when the cells were administered intraperitoneally or directly into femur. However, to our surprise, we found that the purified ST-HSC were not short-term in nature but rather gave long-term multilineage engraftment out to 387 days, albeit at a lower level than the LT-HSC. The ST-HSC also gave secondary engraftment. These observations challenge current models of the stem cell hierarchy and suggest that stem cells are in a continuum of change.
Assuntos
Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas/métodos , Animais , Medula Óssea , Proliferação de Células , Fêmur , Camundongos , Fatores de TempoRESUMO
Microvesicles have been shown to mediate varieties of intercellular communication. Work in murine species has shown that lung-derived microvesicles can deliver mRNA, transcription factors, and microRNA to marrow cells and alter their phenotype. The present studies evaluated the capacity of excised human lung cancer cells to change the genetic phenotype of human marrow cells. We present the first studies on microvesicle production by excised cancers from human lung and the capacity of these microvesicles to alter the genetic phenotype of normal human marrow cells. We studied 12 cancers involving the lung and assessed nine lung-specific mRNA species (aquaporin, surfactant families, and clara cell-specific protein) in marrow cells exposed to tissue in co-culture, cultured in conditioned media, or exposed to isolated lung cancer-derived microvesicles. We assessed two or seven days of co-culture and marrow which was unseparated, separated by ficoll density gradient centrifugation or ammonium chloride lysis. Under these varying conditions, each cancer derived from lung mediated marrow expression of between one and seven lung-specific genes. Microvesicles were identified in the pellet of ultracentrifuged conditioned media and shown to enter marrow cells and induce lung-specific mRNA expression in marrow. A lung melanoma and a sarcoma also induced lung-specific mRNA in marrow cells. These data indicate that lung cancer cells may alter the genetic phenotype of normal cells and suggest that such perturbations might play a role in tumor progression, tumor recurrence, or metastases. They also suggest that the tissue environment may alter cancer cell gene expression.
Assuntos
Células da Medula Óssea/metabolismo , Comunicação Celular/genética , Neoplasias Pulmonares/genética , Pulmão/metabolismo , Células da Medula Óssea/química , Células da Medula Óssea/citologia , Técnicas de Cocultura , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/química , Pulmão/patologia , Neoplasias Pulmonares/patologia , Fenótipo , Proteínas/genética , RNA Mensageiro/análiseRESUMO
Biliary obstruction results in a well-characterized cholestatic inflammatory and fibrogenic process; however, the mechanisms and potential for liver repair remain unclear. We previously demonstrated that Kupffer cell depletion reduces polymorphonuclear cell (neutrophil) (PMN) and matrix metalloproteinase (MMP)8 levels in repairing liver. We therefore hypothesized that PMN-dependent MMP activity is essential for successful repair. Male Sprague-Dawley rats received reversible biliary obstruction for 7 days, and the rat PMN-specific antibody RP3 was administered 2 days before biliary decompression (repair) and continued daily until necropsy, when liver underwent morphometric analysis, immunohistochemistry, quantitative RT-PCR, and in situ zymography. We found that RP3 treatment did not reduce Kupffer cell or monocyte number but significantly reduced PMN number at the time of decompression and 2 days after repair. RP3 treatment also blocked resorption of type I collagen. In addition, biliary obstruction resulted in increased expression of MMP3, MMP8, and tissue inhibitor of metalloproteinase 1. Two days after biliary decompression, both MMP3 and tissue inhibitor of metalloproteinase 1 expression declined toward sham levels, whereas MMP8 expression remained elevated and was identified in bile duct epithelial cells by immunohistochemistry. PMN depletion did not alter the hepatic expression of these genes. Conversely, collagen-based in situ zymography demonstrated markedly diminished collagenase activity following PMN depletion. We conclude that PMNs are essential for collagenase activity and collagen resorption during liver repair, and speculate that PMN-derived MMP8 or PMN-mediated activation of intrinsic hepatic MMPs are responsible for successful liver repair.
Assuntos
Colestase/patologia , Colágeno/metabolismo , Procedimentos de Redução de Leucócitos , Fígado/patologia , Neutrófilos/metabolismo , Processamento de Proteína Pós-Traducional , Cicatrização , Animais , Contagem de Células Sanguíneas , Colestase/enzimologia , Colestase/genética , Gelatinases/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Inflamação/patologia , Fígado/enzimologia , Cirrose Hepática/patologia , Masculino , Metaloproteinase 8 da Matriz/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Biliary atresia is characterized by extrahepatic bile duct obliteration along with persistent intrahepatic portal inflammation. Steroids are standard in the treatment of cholangitis following the Kasai portoenterostomy, and were advocated for continued suppression of the ongoing immunologic attack against intrahepatic ducts. Recent reports, however, have failed to demonstrate an improved patient outcome or difference in the need for liver transplant in postoperative patients treated with a variety of steroid regimes compared with historic controls. In the wake of progressive liver disease despite biliary decompression, steroids are hypothesized to suppress inflammation and promote bile flow without any supporting data regarding their effect on the emerging cellular and molecular mechanisms of liver repair. We have previously shown in a reversible model of cholestatic injury that repair is mediated by macrophages, neutrophils, and specific matrix metalloproteinase activity (MMP8); we questioned whether steroids would alter these intrinsic mechanisms. METHODS: Rats underwent biliary ductal suspension for 7 d, followed by decompression. Rats were treated with IV dexamethasone or saline at the time of decompression. Liver tissue obtained at the time of decompression or after 2 d of repair was processed for morphometric analysis, immunohistochemistry, and quantitative RT-PCR. RESULTS: There was a dramatic effect of dexamethasone on the inflammatory component with the initiation of repair. Immunohistochemistry revealed a reduction of both ED1+ hepatic macrophages and ED2+Kupffer cells in repair compared with saline controls. Dexamethasone treatment also reduced infiltrating neutrophils by day 2. TNF-alpha expression, increased during injury in both saline and dexamethasone groups, was markedly reduced by dexamethasone during repair (day 2) whereas IL-6, IL-10, and CINC-1 remained unchanged compared with saline controls. Dexamethasone reduced both MMP8 and TIMP1 expression by day 2, whereas MMP9, 13, and 14 were unchanged compared with sham controls. Despite substantial cellular and molecular changes during repair, collagen resorption was the same in both groups CONCLUSION: Dexamethasone has clear effects on both the hepatic macrophage populations and infiltrating neutrophils following biliary decompression. Altered MMP and TIMP gene expression might suggest that steroids have the potential to modify matrix metabolism during repair. Nevertheless, successful resorption of collagen fibrosis proceeded presumably through other MMP activating mechanisms. We conclude that steroids do not impede the rapid intrinsic repair mechanisms of matrix degradation required for successful repair.
Assuntos
Anti-Inflamatórios/farmacologia , Atresia Biliar/terapia , Dexametasona/farmacologia , Células de Kupffer/efeitos dos fármacos , Fígado/efeitos dos fármacos , Animais , Ductos Biliares/cirurgia , Atresia Biliar/fisiopatologia , Colestase/fisiopatologia , Colestase/terapia , Colágeno/biossíntese , Citocinas/biossíntese , Descompressão Cirúrgica , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/biossíntese , Fígado/fisiopatologia , Cirrose Hepática/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Masculino , Neutrófilos/efeitos dos fármacos , RatosRESUMO
BACKGROUND: Cholestatic liver injury from extrahepatic biliary obstruction is well characterized by inflammatory and fibrogenic mechanisms. Little is known, however, about mechanisms required to reverse injury and effect liver repair. We sought to determine the cellular and molecular requirements for repair after biliary decompression, focusing on the role of hepatic macrophages in regulating inflammation and matrix resolution. METHODS: Male Sprague-Dawley rats underwent bile duct obstruction for 7 days followed by ductular decompression. Rats were treated with gadolinium chloride (GdCl(3)) to deplete the macrophage populations 24 or 48 hours before decompression. Liver tissue obtained at the time of decompression or after 2 days of repair was processed for morphometric analysis, immunohistochemistry, quantitative RT-PCR and in situ hybridization. RESULTS: GdCl(3) treatment for either 24 or 48 hours before decompression reduced the numbers of ED2(+) Kupffer cells and ED1(+) inflammatory macrophages in obstructed livers; only 48 hours of pretreatment, however, reduced the neutrophil counts. Furthermore, 48-hour GdCl(3) pretreatment blocked matrix degradation. Quantitative polymerase chain reaction demonstrated decreased cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and intercellular adhesion molecule-1 mRNA expression after GdCl(3) treatment and the elimination of hepatic macrophages. Immunohistochemistry and in situ hybridization revealed that neutrophils and CINC-1 mRNA localize within regions of fibrotic activity during both injury and repair. CONCLUSION: We conclude that the macrophage population is not directly involved in fibrotic liver repair. Rather, hepatic macrophages regulate the influx of neutrophils, which may play a direct role in matrix degradation.
Assuntos
Colestase Extra-Hepática/imunologia , Cirrose Hepática/imunologia , Regeneração Hepática/imunologia , Macrófagos/fisiologia , Neutrófilos/fisiologia , Animais , Quimiocina CXCL1/metabolismo , Colestase Extra-Hepática/complicações , Expressão Gênica , Fígado/imunologia , Fígado/metabolismo , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Regeneração Hepática/fisiologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND & AIMS: Biliary obstruction and cholestasis can cause hepatocellular apoptosis and necrosis. Ligation of the common bile duct in mice provides an excellent model in which to study the underlying mechanisms. Kupffer cells play a key role in modulating the inflammatory response observed in most animal models of liver injury. This study was performed to determine the role of Kupffer cells in the injury attending cholestasis. METHODS: Mice were not treated or were rendered Kupffer cell-depleted by intravenous inoculation of multilamellar liposome-encapsulated dichloromethylene diphosphonate, the common bile duct was ligated and divided; sham-operated animals served as controls. Similarly, interleukin-6 (IL-6)-deficient and tumor necrosis factor-receptor-deficient mice underwent bile duct ligation (BDL) or sham operations. RESULTS: Serum alanine transaminase levels were increased in all BDL mice at 3 days after surgery, but were significantly higher in IL-6-deficient mice or mice rendered Kupffer cell-depleted before ligation. Histologic examination of BDL livers showed portal inflammation, neutrophil infiltration, bile duct proliferation, and hepatocellular necrosis. Photoimage analyses confirmed more necrosis in the livers of Kupffer cell-depleted and IL-6-deficient animals. Purified Kupffer cells derived from BDL animals produced more IL-6 in culture. Similarly, Kupffer cells obtained by laser capture microdissection from the livers of BDL mice expressed increased levels of IL-6 messenger RNA. Recombinant mouse IL-6 administered 1 hour before BDL completely reversed the increased liver damage assessed otherwise in Kupffer cell-depleted mice. CONCLUSIONS: These findings indicate that Kupffer cells abrogate cholestatic liver injury by cytokine-dependent mechanisms that include the production of IL-6.
Assuntos
Colestase/patologia , Células de Kupffer/fisiologia , Fígado/patologia , Animais , Ácido Clodrônico/administração & dosagem , Feminino , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Interleucina-6/genética , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Microdissecção , Necrose , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/biossínteseRESUMO
BACKGROUND: Although timely surgical treatment of liver disease can interrupt inflammation and reduce fibrosis, the mechanisms of repair are unknown. We questioned whether these mechanisms of repair include changes in the inflammatory infiltrate and associated biological activity of matrix metalloproteinases (MMPs) 8 and 2. METHODS: Rats (n >or= 3) underwent biliary ductal suspension for 7 days followed by decompression. Livers were collected after 7 days of obstruction (d0) and after 2, 5, and 7 days of repair (d2, d5, d7, respectively), and assessed morphometrically for collagen, polymorphonuclear cells (PMNs), Kupffer cells (KCs), and inflammatory mononuclear phagocytes (MNPs). In situ zymography was performed by using fluorogenic substrates for MMP-8 and MMP-2 to spatially localize enzymatic activity. RESULTS: Cholestatic injury resulted in significantly elevated (P Assuntos
Colestase/imunologia
, Colestase/metabolismo
, Fígado/imunologia
, Metaloproteinase 8 da Matriz/metabolismo
, Neutrófilos/imunologia
, Animais
, Bilirrubina/sangue
, Colestase/cirurgia
, Colágeno/metabolismo
, Descompressão Cirúrgica
, Fígado/metabolismo
, Cirrose Hepática/imunologia
, Cirrose Hepática/metabolismo
, Cirrose Hepática/cirurgia
, Hepatopatias/imunologia
, Hepatopatias/metabolismo
, Hepatopatias/cirurgia
, Masculino
, Metaloproteinase 2 da Matriz/metabolismo
, Neutrófilos/enzimologia
, Ratos
, Ratos Endogâmicos