肝脏GJIC对大鼠体内肝卵圆细胞增殖的影响

目的:研究肝脏缝隙连接细胞间通讯(GJIC)对大鼠体内肝卵圆细胞(HOC)增殖的影响.方法:健康♂Wistar大鼠,随机分成对照组(n=6)、模型组、苯巴比妥(Phenobarbital,PB)组.模型组大鼠按每天20 mg/kg剂量灌喂2-AFF,连续4 d,第5天不灌喂行2/3肝切除,术后次日按每天20 mg/kg剂量继续灌喂5 d (2-AFF/PH);PB组予以0.8 g/L PB饮水7 d,第8天按模型组处理,0.8 g/L PB饮水持续至实验结束.模型组、PB组在术后4 h,4,8,12和16 d随机取6只大鼠检测.采用组织病理技术观察肝组织的形态学变化;免疫组化和细胞形态学方法计数HOC;切开标记/染料示踪技术(incision loading/dye transfer,IL/DT)技术确定GJIC:免疫组化及RT-PCR技术检测CX32蛋白及mRNA表达:免疫组化、Western blot及RT-PCR技术分析CX43蛋白及mRNA水平.结果:对照组及模型组和PB组4 h未见HOC增殖.模型组4 d汇管区有HOC增殖反应,8 d HOC增殖达峰值,12 d HOC从汇管区向肝实质内浸润,16 d HOC增殖较12 d减少.与模型组比较PB组4-16 d各时点HOC明显增加(P<0.01);IL/DT显示各时点模型组大鼠肝脏GJIC均低于对照组(P<0.01),与模型组比较PB组各时点GJIC进一步降低(P>0.01):模型组、PB组各时点CX32的表达低于对照组(P<0.05),与模型组比较PB组CX32表达在4 h,12,16 d时点减少(P<0.05),在4,8 d时点表达增多(P<0.05):模型组4 h-16 d时点CX32 mRNA水平分别为对照组的0.82±0.13,0.33±0.11,0.51±0.13,0.68±0.14,1.12±0.18倍,与模型组比较PB组4 h时点无显著差异(P>0.05),4-16 d时点明显升高(P<0.05).模型组4 h-16 d各时点CX43蛋白表达分别为对照组的1.14±0.17,3.87±0.35,5.28±0.48,2.96±0.33,2.12±0.19倍,与模型组比较PB组CX43蛋白4 h上调(P>0.05),4-16 d明显减少(P<0.05).模型组4 h-16 d各时点CX43 mRNA水平分别为对照组的1.09±0.16,2.82±0.23,5.46±0.58,3.34±0.64.0.91±0.11倍.与模型组比较PB组各时点CX43 mRNA表达上调(P<0.05).结论:改变大鼠2-AAF/PH模型肝脏CX32、CX43的时空表达模式,降低肝脏肝细胞及HOC与其偶联细胞的GJIC,可解除HOC生长抑制,促进HOC的增殖.     

大鼠肝卵圆细胞增殖模型肝脏的GJIC功能和意义

目的:研究大鼠肝卵圆细胞(HOC)增殖模型肝脏缝隙连接蛋白32,43(CX)的表达及缝隙连接细胞间通讯(GJIC)的功能,探讨HOC增殖的可能机制.方法:健康(?)Wistar大鼠,随机分成正常对照组(n=6),模型组.模型组大鼠按每天20 mg/kg剂量灌喂2-AFF,连续4 d,第5天不灌喂行2/3肝切除,术后次日按每天20 mg/kg剂量继续灌喂5 d(2-AFF/PH).模型组在术后4h,4,8,12和16d随机取6只大鼠检测.采用组织病理技术观察肝组织的形态学变化;免疫组化和细胞形态学方法计数HOC;切开标记/染料示踪技术(incision loading/dye transfer,IL/DT)技术确定GJIC;免疫组化及RT-PCR技术检测CX32蛋白及mRNA表达;免疫组化、Western blot及RT-PCR技术分析CX43蛋白及mRNA水平.结果:对照组及模型组4 h未见HOC增殖.模型组4d汇管区有HOC增殖反应,8d HOC增殖达峰值,12d HOC从汇管区向肝实质内浸润,16d HOC增殖减少较12 d减少;IL/DT检测结果显示,模型组各时点(4h,4,8,12和16d)染料扩散距离低于对照组(84.5±3.4,60.6±3.3,108.6±4.2,150.6±2.6,199.6±3.7μm vs 250.0±5.0 um,P<0.01),GJIC功能降低.模型组各时点CX32表达均低于对照组(P<0.05),在4 h下调,4d达低峰(2.85±0.39),8d后逐渐恢复;RT-PCR显示模型组CX32 mRNA在4h开始下降,4d达低峰(0.33±0.11),8 d后逐渐恢复,16 d高于对照组,但无显著差异(P>0.05).Western blot结果显示模型组CX43蛋白表达在4 h上调(P>0.05)、4-16 d明显升高;RT-PCR显示模型组4h CX43 mRNA上调(P>0.05),4 d表达明显升高,12 d达高峰(5.46±0.58),16 d低于对照组,但无显著差异(P>0.05).结论:采用Solt-Farber方法成功建立了HOC增殖动物模型;大鼠2-AAF/PH肝脏CX表达呈时空特异性变化,使肝脏GJIC功能抑制.肝脏的GJIC抑制可能启动了HOC增殖.     

异型缝隙连接通道和磷酸化对心脏缝隙连接的调变

目的 检测由缝隙连接蛋白(connexin，Cx)43和Cx45组成的多种异型缝隙连接通道(her—eromultimeric gap junction channels，HGJC)和磷酸化对缝隙连接(gap junction，GJ)的调变作用。方法 将转染了编码为Cx43或Cx45的DNA后的Hela细胞放置在一起共同培养组成双侧和单侧异型GJ通道。显微注射若丹明123(rhodamine123，Rh)检测经200nmol／L十四(烷)酰佛波醇乙酸酯(12-0-tetrade—canoylphorbol-13-acetae，TPA)处理前后，在紫外光显示下由Cx43和Cx45所组成的不同GJ通道对荧光染料的偶联率(coupling ratio)。结果 在不同的GJ中，同型GJ通道Cx43(homotypie Cx43，HoCx43)偶联率最高。从Cx45侧注入荧光染料的单侧异型GJ通道45(mono-heteromeric Cx45-Cx43／45，MH45)偶联率较之从Cx43／45侧注入荧光染料的MH45、双侧异型GJ通道Cx43／45(bi-heteromeric Cx43／45，BH43／45)及同型GJ通道Cx45(homotypic Cx45，HoCx45)等的偶联率是最低的。根据HoCx43或HoCx45通道的偶联率对各型通道偶联率进行标准化处理。BH43／45和MH43通道的偶联率均较HoCx43降低。对MH45通道来说，从Cx43／45侧注射的通道偶联率大于从Cx45侧注射的偶联率。TPA处理后HoCx43的偶联率降低，而当Cx43和Cx45组合成BH43／45和MH43通道后其偶联率下降更显著。结论 Cx43和Cx45共同表达可构成BH43／45、MH43和MH45等异型通道，而这些通道可降低细胞间的通讯并对磷酸化的作用不敏感。单侧异型GJ通道的偶联率取决于染料注射的方向。     

Modulation of steroidogenic activities in testis Leydig cells

Recent developments in the regulation of testicular Leydig-cell function support the following conclusions. (1) Enzymic activities involved in steroid production in the testis are mainly localized in Leydig cells. The aromatase enzyme complex for oestrogen production appears to be localized in Leydig cells as well as in Sertoli cells. (2) LH- (or hCG-) induced alterations of Leydig cells depend on dose and duration of exposure of the cell to the hormone. Locally produced oestradiol is probably involved in the inhibition of steroidogenesis. (3) The stimulatory action of LH on Leydig cells involves different proteins in concert with the activation of the cleavage activity of mitochondrial cholesterol side-chain. However, most of the functional properties of these proteins are yet unknown. (4) Different populations of Leydig cells are present in the testis. These different cell populations can be characterized by quantitatively and qualitatively different responses to hormones.     

17beta-estradiol reduces the effect of metabolic inhibition on gap junction intercellular communication in rat cardiomyocytes via the estrogen receptor

The effects of 17beta-estradiol (E2) on gap junction intercellular communication (GJIC) were assessed by Lucifer yellow dye coupling in cultured neonatal rat cardiomyocytes after metabolic inhibition (MI) using potassium cyanide and sodium iodoacetate. MI significantly reduced dye coupling of cardiomyocytes to 8.5% +/- 0.6% of control levels, and pretreatment with E2, but not its inactive isomer 17alpha-estradiol, dose-dependently (EC(50) = 0.41 microM) increased the dye coupling up to 76% +/- 15% of control levels. The effect of E2 on MI-induced dye uncoupling was abolished by tamoxifen, a potent estrogen receptor (ER) antagonist. The ligand, E2-BSA-FITC, labeled the cardiomyocyte surface, whereas BSA-FITC did not, suggesting the presence of membrane-associated E2 receptors. Double immunofluorescence microscopy showed that MI-induced the accumulation of non-phosphorylated Cx43 at the gap junction and that this was prevented by E2 pretreatment. Labeling of Lucifer yellow-microinjected cardiomyocytes with antibodies specific for Ser368-phosphorylated Cx43 (Ser368Cx43) or non-phosphorylated Cx43 confirmed that E2 reduced the MI-induced inhibition of dye coupling and accumulation of non-phosphorylated Cx43 concomitant with the reappearance of Ser368Cx43 at the gap junction. MI caused a decrease in Ser368Cx43 protein levels, and pretreatment with E2 significantly increased the levels of Ser368Cx43. Inhibition of protein kinase C (PKC) with chelerythrine blocked the E2-induced increase of Ser368Cx43 levels in MI-treated cardiomyocytes. These results suggest that E2 attenuates the inhibitory effect of MI on GJIC in cardiomyocytes by affecting the phosphorylation of Cx43, possibly mediated by activation of PKC via a membrane-associated signaling mechanism.     

Modulation of cardiac gap junction expression and arrhythmic susceptibility

Connexin43 (Cx43), the predominant ventricular gap junction protein, is critical for maintaining normal cardiac electrical conduction, and its absence in the mouse heart results in sudden arrhythmic death. The mechanisms linking reduced Cx43 abundance in the heart and inducibility of malignant ventricular arrhythmias have yet to be established. In this report, we investigate arrhythmic susceptibility in a murine model genetically engineered to express progressively decreasing levels of Cx43. Progressively older cardiac-restricted Cx43 conditional knockout (CKO) mice were selectively bred to produce a heart-specific Cx43-deficient subline ("O-CKO" mice) in which the loss of Cx43 in the heart occurs more gradually. O-CKO mice lived significantly longer than the initial series of CKO mice but still died suddenly and prematurely. At 25 days of age, cardiac Cx43 protein levels decreased to 59% of control values (P<0.01), but conduction velocity was not significantly decreased and no O-CKO mice were inducible into sustained ventricular tachyarrhythmias. By 45 days of age, cardiac Cx43 abundance had decreased in a heterogeneous fashion to 18% of control levels, conduction velocity had slowed to half of that observed in control hearts, and 80% of O-CKO mice were inducible into lethal tachyarrhythmias. Enhanced susceptibility to induced arrhythmias was not associated with altered invasive hemodynamic measurements or changes in ventricular effective refractory period. Thus, moderately severe reductions in Cx43 abundance are associated with slowing of impulse propagation and a dramatic increase in the susceptibility to inducible ventricular arrhythmias.     

前列腺癌细胞连接蛋白43表达和细胞间隙连接交流状况

目的　观察前列腺癌 (PCa)细胞连接蛋白 (Connexin ,Cx)表达和细胞间隙连接交流(GJIC)状况 ,探讨胸苷激酶 (TK)自杀基因治疗前列腺癌时旁观者效应不够强大的原因以及GJIC与前列腺癌发生的关系。　方法　分别采用逆转录 聚合酶链式反应 (RT PCR)、链霉亲和素免疫组化法 (SABC法 )和划痕标记染料示踪技术 (SLDT)检测前列腺癌细胞系PC 3m的连接蛋白 4 3(Cx4 3)mRNA、蛋白表达和GJIC功能状况 ,并观察Cx4 3蛋白在正常前列腺和前列腺癌组织中的表达。　结果　PC 3m有Cx4 3mRNA和蛋白表达 ,但其表达较弱 ,且Cx4 3蛋白大多异常定位于细胞浆中而非细胞膜上 ;组织切片显示前列腺癌组织Cx4 3蛋白异常定位且表达水平较正常前列腺组织明显减弱 ,与病理分级呈负相关关系 (χ2 =4 0 2 5 ,P <0 0 5 ) ,高分化和中等分化、低分化腺癌的表达率分别为5 2 9%及 7 1%。此外 ,PC 3m细胞GJIC功能低下 ,半定量为 ( )或 (- )。　结论　前列腺癌细胞GJIC功能低下 ,Cx4 3基因下调表达和蛋白异常定位均可能为导致这一现象的原因。GJIC功能缺陷可能是引起单纯疱疹病毒 胸苷激酶 /更昔洛韦系统杀伤前列腺癌细胞时旁观者效应不够强大的原因 ,亦可能是前列腺癌发生、发展中的分子事件。     

大蒜素对胃上皮细胞Cx37、Cx43 mRNA表达及细胞间隙连接通讯功能的影响

目的观察体外实验中大蒜素对胃上皮细胞系MGC-803Cx37 mRNA、Cx43 mRNA表达及细胞间隙连接通讯功能的影响。方法终浓度为3μg／ml、6μg／ml、9μg／ml、12μg／ml大蒜素分别加入胃上皮细胞MGC-803培养24h、48h、72h，同时设立不加药物的阴性对照，用RT，PCR法检测细胞cx37mRNA、Cx43 mRNA表达，LY染料传输方法检测细胞间隙连接通讯功能。结果MGC-803细胞cx37mRNA有表达，cx43mRNA无表达，GJIC功能弱，加入不同浓度大蒜素后，cx37mRNA表达和细胞间隙连接通讯功能增强，cx37mRNA表达随大蒜素浓度和培养时间的增加而增强（均P〈0．05），Cx43 mRNA仍无表达。结论大蒜素在转录水平上调胃上皮细胞MGC-803 Cx37基因的表达，改善细胞间隙连接通讯功能。     

Luteinizing and human chorionic gonadotropin hormones increase intercellular communication and gap junctions in cultured mouse leydig cells

The effect of luteinizing (LH) and human chorionic gonadotropin (hCG) hormones on gap junctions (Gjs) and intercellular communication (ic) was evaluated in Leydig (interstitial) cells from mouse testes. Cell cultures enriched in Leydig cells were studied under control conditions and when maintained in the presence of 100 ng/mL LH, 10 ng/mL hCG, or 1 mM dibutiryl-cAMP (db-cAMP), for 8, 24, and 36 h. To monitor the extent of ic, Lucifer yellow (LY) was injected through a patch pipet into one cell of-small cell aggregates (6–10), and its transfer was evaluated using fluorescent microscopy. The expression of GJs was monitored using immunofluorescent (IF) labeling of connexin 43 (Cx43) with a specific antibody. Testosterone secretion was determined by radioimmunoassay. At all culture times, testosterone levels in the medium were higher in treated than in control cell cultures. In cell cultures of 8 h, LY transferred to most of the neighboring cells (93%) and cell membrane appositions showed abundant Cx43; no difference was found between control and treated cells. In contrast, in control cell cultures of 24 and 36 h, LY transferred to a reduced fraction of neighboring cells (46 and 21%, respectively) and Cx43 labeling was markedly decreased. Addition of LH, hCG, or db-cAMP, to cell cultures for 24 and 36 h completely prevented the decrease in ic and Cx43 expression. Immunoblot studies, from total protein homogenates of cell cultures of 36 h, showed that relative levels of 40- and 43-kDa bands, characteristic of Cx43, were higher in treated than in control cells. These results demonstrate that the expression of Cx43 and ic in Leydig cells is modulated by LH and hCG, and suggest that their effect is mediated by the second messenger of these hormones, cAMP.     

Modulation of intercellular communication mediated at the cell surface and on extracellular,plasma membrane-derived vesicles by ionizing radiation

The plasma membrane is a dynamic organelle whose function includes receptor-mediated signal transduction into the cell. Conversely, the plasma membrane is the origin of inter-cellular signaling. In addition to expressing and releasing growth factors in a soluble form(through exocytosis) and via proteolysis of cell surface components, membrane ligands may signal nearby cells through juxtacrine stimulation or by the exfoliation or shedding of plasma membrane-derived vesicles. Ionizing radiation (IR) has a profound effect on plasma membrane structure and function. IR-induced ultrastructural alterations are mediated via lipid interaction with water radiolysis products (e.g., hydroxyl radicals, hydrogen radicals, and hydrated electrons). Ionizing radicals act directly on lipid molecules to promote lipid hydro-peroxides and lipid hydroperoxide breakdown products (e.g., alpha, beta unsaturated aldehydes) that contribute to altered plasma membrane lipid composition. A change in lipid composition increases membrane lipid microviscosity and results in membrane fenestrations that enhance permeability to small molecules and ions. Reactive ionizing species also stimulate sphingomyelinase activity, leading to sphingomyelin hydrolysis and ceramide generation that further contributes to altered membrane lipid composition and cellular apoptosis. In addition, exposure to IR results in impaired rate of and cumulative shedding of plasma membrane-associated growth factors. Mechanisms of exfoliation are reviewed for normal cells and the impact of radiation on modulating signal transduction mediated by exfoliation is summarized.     

Taurine preserves gap junctional intercellular communication in rat hepatocytes under oxidative stress

Gap junctional intercellular communication (GJIC) between hepatocytes is important for the maintenance of differentiated liver functions. Taurine is known to be cytoprotective, and is used clinically to improve liver functions. We evaluated the effect of taurine on GJIC in hepatocyte doublets under oxidative stress. Hepatocyte doublets were isolated from female Wistar rats, using a collagenase perfusion technique, and cultured in Leibovitz-15 medium containing fetal bovine serum (10%). H₂O₂ (2 mM) and/or taurine (0.1–1 mM) were added 2 h after inoculation, and the culture was incubated for 3 h. Fluorescent dye (Lucifer Yellow CH) coupling between adjacent cells was evaluated by microinjection. The distribution and quantity of connexin 32 (Cx32) in hepatocytes were detected using indirect immunofluorescence analysis and Western blotting. Steady state mRNA levels of Cx32 were detected by Northern blotting. The percentage of dye coupling 5 h after inoculation was 88 ± 6.3% in the control, however, this was decreased to almost half the control value by H₂O₂. Taurine prevented the decrease caused by H₂O₂ in a dose-dependent manner. Immunofluorescence analysis for Cx32 demonstrated numerous punctate fluorescent spots along the intercellular plasma membrane in controls, which were significantly decreased by H₂O₂. Taurine prevented the decrease of Cx32. Western blot analysis also showed the decrease of Cx32 protein levels by H₂O₂ treatment, which decrease was prevented by taurine. Interestingly, H₂O₂ and/or taurine treatments did not affect Cx32 mRNA levels. Our findings indicated that H₂O₂ treatment decreased GJIC between hepatocytes, most likely due to augmenting the degradation of Cx32 proteins, whereas taurine prevented this process. This effect of taurine is beneficial for the preservation of differentiated functions in the liver under oxidative stress. Received: August 12, 1999 / Accepted: November 26, 1999     

Up-regulation of gap junctional intercellular communication and connexin43 expression by retinoic acid in human endometrial stromal cells

CONTEXT: Gap junctions, made up of connexins (Cxs), play fundamental roles in coordinating a number of cellular processes through their ability to directly regulate cell-cell communication. Cx43 is the most widely expressed Cx in the endometrium and is known to be important in a variety of physiological and pathological processes in this tissue. OBJECTIVE: In this study, we investigated the ability of the retinoid, all-trans-retinoic acid (RA), to regulate Cx43 expression in human endometrial stromal cells. DESIGN: Primary endometrial stromal cells obtained from patients undegoing surgery for infertility workup were treated in vitro with RA and control compounds for different time periods, up to 48 h. Cx43 mRNA and protein levels, protein phosphorylation, and gap junctional intercellular communication (GJIC) were analyzed. RESULTS: Treatment of the cells with RA showed a dose-dependent increase in Cx43 expression at both the mRNA and protein levels. In addition, RA induced a relative decrease in the phosphorylated species of Cx43 while causing a corresponding increase in the nonphosphorylated form. Concomitant with these changes, RA-treated cells demonstrated up to a 250% enhancement of GJIC as assessed by dye transfer experiments. Augmentation of GJIC and alterations of Cx43 expression were observed over the same range of RA concentrations. Treatment of cells with the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate increased the phosphorylated species of Cx43 and correspondingly inhibited GJIC. CONCLUSIONS: Phosphorylation of Cx43 is inversely related to GJIC in endometrial stromal cells. Retinoids increase GJIC in endomentrial stromal cells through upregulation of Cx43 expression while inducing a decrease in the phosphorylated species of the protein. The data suggest a novel mechanism by which retinoids can influence endometrial cell biology.     

人脐动脉内皮细胞和平滑肌细胞体外培养鉴定及缝隙连接通讯功能测定

目的:应用双光子激光扫描共聚焦显微镜鉴定体外培养的脐动脉内皮细胞(ECs)和平滑肌细胞(SMCs),应用荧光光漂白恢复技术(FRAP)测定血管ECs、SMCs之间的缝隙连接通讯(GJIC)功能。方法:人脐动脉ECs、SMCs分离培养,Ⅷ因子和SMα-actin相关抗原鉴定ECs和SMCs,应用FRAP技术测定血管内皮细胞、平滑肌细胞之间的GJIC功能,记录实时成像结果,应用动态比(M)计算漂白区域内标记荧光的分子中动态分子的比例。结果:第一组ECs和SMCs单独培养,选择漂白细胞与周围至少3个同种细胞相连接,SMCs被漂白后平均M值为31.79±5.69;ECs被漂白后平均M值为23.43±2.11;第二组ECs和SMCs混合培养,选择ECs和SMCs独立相连的2个细胞,SMCs被漂白后平均M值为14.47±3.28,ECs被漂白后平均M值为6.41±0.80。结论:FRAP实时动态恢复曲线可直接观察荧光恢复强度及速度,参照FRAP恢复曲线,M值可做为组间GJIC比较相对定量的可靠指标,通过检测证实ECs和SMCs之间存在GJIC,且荧光由ECs向SMCs方向的传递大于由SMCs向ECs方向的传递。     

Development of gap junctional channels and intercellular communication in rat liver during ontogenesis

BACKGROUND/AIMS: We investigated the expression of connexin (Cx) 32 and 26 subunit proteins of the gap junction (GJ) in the rat liver during ontogenesis to clarify their roles in control of growth and differentiation, and observed their channels in association with development of gap junctional intercellular communication (GJIC). METHODS: The expression of Cx32 and 26 in prenatal and postnatal livers was examined by Western blot and immunofluorescence. GJ channels were investigated not only by double immunofluorescence study but also by immunogold electron microscopy. The spread of lucifer yellow 5 min after its microinjection was examined in the cultured liver tissues. RESULTS: 1) Western blot showed the expression of both Cx from the late stage of gestation and their peak a week after birth. 2) Cx32- or 26-positive plaques were scattered on hepatocytes of the fetal liver and some of them were colocalized; both were increased just after birth. On day 7 after birth, Cx32-positive plaques were present on all hepatocytes within a lobule, and Cx26-positive plaques were distributed in the periportal area. 3) Double-immunogold electron microscopy just after birth showed that most GJ channels were homotypic type of Cx32 or 26, and that few were heterotypic. On day 7 after birth, most channels had the homotypic type of type of Cx32 in the middle and pericentral areas, and there was a heterotypic type of Cx32 and 26 in the periportal area. 4) The dye transfer of lucifer yellow showed a wider spread in the liver tissues on day 7 after birth than on day 1. CONCLUSION: Increased GJ formation and compatibility or incompatibility of GJ channels are closely associated with development of GJIC, and GJIC may develop at cytodifferentiation during ontogenesis.     

DNA-triggered innate immune responses are propagated by gap junction communication

Cells respond to infection by sensing pathogens and communicating danger signals to noninfected neighbors; however, little is known about this complex spatiotemporal process. Here we show that activation of the innate immune system by double-stranded DNA (dsDNA) triggers intercellular communication through a gap junction-dependent signaling pathway, recruiting colonies of cells to collectively secrete antiviral and inflammatory cytokines for the propagation of danger signals across the tissue at large. By using live-cell imaging of a stable IRF3-sensitive GFP reporter, we demonstrate that dsDNA sensing leads to multicellular colonies of IRF3-activated cells that express the majority of secreted cytokines, including IFNβ and TNFα. Inhibiting gap junctions decreases dsDNA-induced IRF3 activation, cytokine production, and the resulting tissue-wide antiviral state, indicating that this immune response propagation pathway lies upstream of the paracrine action of secreted cytokines and may represent a host-derived mechanism for evading viral antiinterferon strategies.