MHC class I Dk expression in hematopoietic and nonhematopoietic cells confers natural killer cell resistance to murine cytomegalovirus

NK cell-mediated murine cytomegalovirus (MCMV) resistance (Cmv^r) is under H-2^k control in MA/My mice, but the underlying gene(s) is unclear. Prior genetic analysis mapped Cmv^r to the MHC class I (MHC-I) D^k gene interval. Because NK cell receptors are licensed by and responsive to MHC class I molecules, D^k itself is a candidate gene. A 10-kb genomic D^k fragment was subcloned and microinjected into MCMV-susceptible (Cmv^s) (MA/My.L-H2^b × C57L)F₁ or (B6 × DBA/2)F₂ embryos. Transgenic founders, which are competent for D^k expression and germline transgene transmission, were identified and further backcrossed to MA/My.L-H2^b or C57L mice. Remarkably, D^k expression delivered NK-mediated resistance in either genetic background. Further, NK cells with cognate inhibitory Ly49G receptors for self-MHC-I D^k were licensed and critical in protection against MCMV infection. In radiation bone marrow chimeras, NK resistance was significantly diminished when MHC-I D^k expression was restricted to only hematopoietic or nonhematopoietic cells. Thus, MHC-I D^k is the H-2^k-linked Cmv^r locus; these findings suggest a role for NK cell interaction with D^k-bearing hematopoietic and nonhematopoietic cells to shape NK-mediated virus immunity.     

Chronic ethanol consumption decreases murine Langerhans cell numbers and delays migration of Langerhans cells as well as dermal dendritic cells

Background: Chronic alcoholics experience increased incidence and severity of infections, the mechanism of which is incompletely understood. Dendritic cells (DC) migrate from peripheral locations to lymph nodes (LN) to initiate adaptive immunity against infection. Little is known about how chronic alcohol exposure affects skin DC numbers or migration. Methods: Mice received 20% EtOH in the drinking water for up to 35 weeks. Baseline Langerhans cell (LC) and dermal DC (dDC) numbers were enumerated by immunofluorescence (IF). LC repopulation after inflammation was determined following congenic bone marrow (BM) transplant and ultraviolet (UV) irradiation. Net LC loss from epidermis was determined by IF following TNF‐α or CpG stimulation. LC and dDC migration into LN was assessed by flow cytometry following epicutaneous FITC administration. Results: Chronic EtOH consumption caused a baseline reduction in LC but not dDC numbers. The deficit was not corrected following transplantation with non‐EtOH‐exposed BM and UV irradiation, supporting the hypothesis that the defect is intrinsic to the skin environment rather than LC precursors. Net loss of LC from epidermis following inflammation was greatly reduced in EtOH‐fed mice versus controls. Ethanol consumption for at least 4 weeks led to delayed LC migration into LN, and consumption for at least 8 weeks led to delayed dDC migration into LN following epicutaneous FITC application. Conclusions: Chronic EtOH consumption causes decreased density of epidermal LC, which likely results in decreased epidermal immunosurveillance. It also results in altered migratory responsiveness and delayed LC and dDC migration into LN, which likely delays activation of adaptive immunity. Decreased LC density at baseline appears to be the result of an alteration in the skin environment rather than an intrinsic LC defect. These findings provide novel mechanisms to at least partially explain why chronic alcoholics are more susceptible to infections, especially those following skin penetration.     

HBsAg stimulates NKG2D receptor expression on natural killer cells and inhibits hepatitis C virus replication

Background: Higher hepatitis B surface antigen(HBs Ag) facilitates hepatitis C virus(HCV) clearance in patients with hepatitis B virus(HBV)/HCV co-infection. We investigated the effect of exogenous HBs Ag on the inhibition of HCV replication mediated by natural killer(NK) cells.Methods: After isolated from peripheral blood of 42 chronic hepatitis B(CHB) patients and 16 healthy individuals, NK cells were co-cultured with HCV-infected Huh7 cells, respectively, with or without HBs Ag.Three days later, the co-cultured supernatants were collected and HCV RNA levels were measured by realtime quantitative PCR. NKG2 D, NKp46 and NKG2 A expression levels were measured by flow cytometry.NKG2 D on NK cells from CHB responsive subgroup was blocked and HCV RNA levels were examined again.Results: HCV RNA levels in the co-cultured system were significantly reduced by NK cells isolated from healthy donors(P 0.01) but not from CHB patients. However, HCV RNA levels in CHB cultures were significantly decreased following HBs Ag addition(P 0.05), whereas no such effect was seen in control cultures. No significant difference was observed in basic NKG2 D expression between the CHB patients and healthy donors. On NK cells from CHB patients, the expression of NKG2 D was increased significantly by HBs Ag stimulation(P 0.01), and higher than that from healthy controls(P 0.05). HCV RNA levels were increased significantly after the blockage of NKG2 D on NK cells from responsive CHB patients in the co-cultured system(P 0.05).Conclusion: Exogenous HBs Ag stimulated NKG2 D expression on NK cells from CHB patients which inhibit HCV replication, suggesting that HBs Ag may facilitate the clearance of HCV in patients with HBV/HCV co-infection.     

Chronic hepatitis C: Effect of alcohol on hepatic activity and viral titre

Background/Aims: Alcohol and the hepatitis C virus have been postulated to interact to adversely affect the natural history of patients with chronic liver disease. The aim of this study was to examine the effect of alcohol on hepatitic activity and serum HCV RNA levels in patients with chronic hepatitis C.Methods: Forty-five consecutive patients with chronic hepatitis C were classified according to alcohol intake over the 3-month period preceding study entry: group 1 (n=23), >10 g alcohol/day; group 2 (n=22), ≤10 g alcohol/day. Hepatitic activity and alcohol intake were assessed at study entry and, following moderation of alcohol intake, after a mean follow-up period of 4.4±0.2 months.Results: Hepatitic activity was significantly greater in the patients who consumed >10 g of alcohol/day. Moderation of alcohol consumption in patients consuming >10 g/day resulted in a significant decrease in both disease activity (p=0.0002) and viral RNA titre (p=0.018); there was no change over the study period in patients with a consistently low alcohol intake.Conclusion: The results support the hyptheses that, in patients with chronic hepatitis C, alcohol aggravates hepatic injury, increases viral load and adversely affects the natural history of the associated liver disease.     

Enhanced natural killer cell activity is found in exposed uninfected recipients of hepatitis C‐contaminated blood

A minority of injecting drug users, termed exposed uninfected, are resistant to hepatitis C (HCV) infection despite repeated low‐dose exposures. We identify for the first time a cohort of blood recipients who remained uninfected despite large‐dose exposure to HCV‐contaminated blood and characterize immune factors that may confer protection. Of 1340 blood recipients from the English Look Back database who were transfused HCV‐contaminated blood, we identified 8 who remained uninfected. In these 8 exposed uninfecteds, we characterized their natural killer (NK) cell populations and HCV‐specific T‐cell responses. Findings were compared with 10 spontaneous resolvers of HCV infection, 10 patients with chronic HCV infection and 10 healthy controls. Exposed uninfecteds had significantly greater numbers of NK cells with the activating receptor NKp30+ on CD56^bright and CD56^dim subsets compared with other groups (P < .05). Following interleukin‐2 activation, NK cells of exposed uninfecteds had enhanced cytotoxicity that positively correlated with NKp30 expression (P = .02). Differences in NKp80 and KIR2DL3 expression were also observed. HCV‐specific T‐cell responses were observed in some exposed uninfecteds but of low amplitude. Exposure without infection following transfusion of HCV‐contaminated blood is a very rare phenomenon and suggests a high level of resistance to infection. Enhanced NK cell activation and killing, with weak HCV‐specific T‐cell responses, were observed many years after exposure in uninfected recipients and may contribute to protection from HCV acquisition, although additional protective factors are being sought in this important cohort.     

Effects of chronic ethanol feeding on murine dendritic cell numbers, turnover rate, and dendropoiesis

Background: Chronic alcoholics have increased susceptibility to and severity of infection, which are likely to be a result of impaired immune defense mechanisms. The contribution of dendritic cells (DC) to these immune defense changes is not well understood. Alterations in DC numbers, dendropoiesis, and lifespan have not been specifically studied in vivo in chronic ethanol (EtOH) exposure models. As DC play an essential role in initiating immune responses, alterations in these DC characteristics would help explain changes observed in adaptive immune responses. Methods: Mice received 20% EtOH (w/v) in the drinking water for up to 28 weeks, with mouse chow ad libitum. In EtOH‐fed and water control mice, DC were enumerated by flow cytometry. The effect of EtOH on DC precursor numbers was determined by differentiation in vitro in the presence of granulocyte‐macrophage colony‐stimulating factor and interleukin‐4, and the effect of an EtOH environment on untreated DC differentiation was measured following bone marrow transfer to irradiated hosts. DC turnover rate was also examined by bromodeoxyuridine incorporation and loss. Results: The percentage and absolute numbers of DC were decreased in spleen and increased in thymus beginning as early as 4 weeks of EtOH feeding. In addition, the overall cellularity of spleen and thymus were altered by this regimen. However, chronic EtOH consumption did not adversely affect DC precursor numbers, differentiation abilities, or turnover rates. Conclusions: Decreased splenic DC numbers observed following chronic murine EtOH consumption are not because of altered DC precursor numbers or differentiation, nor increased DC turnover rate. Similarly, increased thymic DC numbers are not the result of alterations in DC precursor differentiation or turnover rate. Compartment size plays a role in determining splenic and thymic DC numbers following chronic EtOH feeding. EtOH‐induced alterations in total DC numbers provide several mechanisms to partially explain why chronic alcoholics have increased susceptibility to infections.     

Studies on natural killer cell activity and the influence ofCorynebacterium parvum on murine T-cell leukemogenesis induced by butylnitrosourea

Summary Butylnitrosourea (BNU) was used to induce thymic lymphomas in BDF₁ mice. During and after the 12-week BNU exposure the spontaneous NK cell activity against YAC-1 cells and that arising 4 days after stimulation withCorynebacterium parvum (CP) were measured, as were the mitogen responses of splenic T and B cells. In addition to BNU, groups of mice received multiple injections of the interferon inducer CP during or after the BNU exposure period. The results show a slight impairment of the NK cell activity by BNU and also after the injections of CP depending heavily on the treatment protocol. After the multiple injections of CP, either into BNU-treated mice or into controls, no further stimulation by CP was possible. The mitogen responses, reduced after BNU, were further reduced after the additional treatment. Both effects can be explained by the known induction of suppressor cells by CP. Although these treatments had pronounced effects on the parameters tested in vitro there was no significant influence on the development of thymic lymphomas in vivo.Abbreviations BNU butylnitrosourea - CP Corynebacterium parvum - PEC peritoneal exudate cells - NK natural killer - ConA concanavalin A - LPS lipopolysaccharide B - MLC mixed lymphocyte culture With technical assistance by E. Barthel and K. Steinhoff. Supported by Deutsche Forschungsgemeinschaft (SFB 112) and the Bundesministerium des Inneren     

Impairment of natural killer cell activity in Chlamydia trachomatis infected individuals

Natural killer (NK) cell activity is impaired in Chlamydia trachomatis-infected patients. The mechanisms behind the altered NK functions are not clear, but data concerning NK and antibody-dependent cellular cytotoxicity (ADCC) activity have been reported. To investigate whether this impairment is related to a defect at the target cell binding and/or the postbinding level, we evaluated highly purified NK cells obtained from 125 C. trachomatis-infected patients and compared them with 101 normal controls for their ability to kill K-562 and U-937 cell lines using a 51Cr release assay; release tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma); and kill anti-IgM preincubated P-815 cell line (ADCC activity). We found a decrease in the lytic capability of NK cells from C. trachomatis-infected patients against target cell lines; decreased ability to kill bound target cells; and low levels of released TNF-alpha and INF-gamma after incubation with U-937 cells. Taken together, these findings suggest that the impaired NK cell reaction during chlamydial infection is related to defects both at the target and postbinding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long-term culture in the presence of high levels of recombinant IL-2 support the hypothesis of an anergic process during chlamydial infection.     

Reconstitution of natural killer cell receptors influences natural killer activity and relapse rate after haploidentical transplantation of T- and B-cell depleted grafts in children

Background

Natural killer cells have been demonstrated to exert remarkable graft-versus-leukemia effects after haploidentical transplantation. Acquisition of both, inhibiting and activating, receptors on developing natural killer cells is an important step in their functional maturation. Here, we report on the reconstitution of natural killer receptors after haploidentical transplantation of T-and B-cell (CD3/CD19) depleted grafts with co-transfusion of natural killer cells in children and its influence on natural killer cell activity and clinical outcome.

Design and Methods

We analyzed reconstitution patterns of natural killer receptors at different time intervals after haploidentical transplantation by multi-color flow cytometry. Natural killer cell activity and antibody-dependent cellular cytotoxicity was tested against cell lines and leukemic blasts in vitro. Survival was analyzed using Kaplan-Meier estimates.

Results

Recovery of CD56⁺/CD16⁺ cells was fast with high cytolytic activity against K562 and strong antibody-dependent cellular cytotoxicity activity against neuroblastoma and leukemic blasts as early as day 14 posttransplant. KIR reconstitution showed a predominance of KIR negative natural killer cells early after transplantation and an early reconstitution of CD158b compared to CD158a and CD158e. These differences were independent of presence or absence of the corresponding KIR ligands in donors or recipients. This reconstitution pattern was associated with a higher relapse probability of patients homozygous for HLA-C1-alleles compared to patients homozygous or even heterozygous for HLA-C2-alleles.

Conclusions

Our results indicate a fast recovery of functional and alloreactive natural killer cells with a constant KIR pattern after haploidentical transplantation with T- and B-cell depleted grafts. Moreover, these natural killer cells can mediate antibody-dependent cellular cytotoxicity and therefore may allow for an early use of antibodies against residual malignant cells.     

Modulation of natural killer cell antitumor activity by the aryl hydrocarbon receptor

The aryl hydrocarbon receptor (AhR) has become increasingly recognized for its role in the differentiation and activity of immune cell subsets; however, its role in regulating the activity of natural killer (NK) cells has not been described. Here, we show that AhR expression is induced in murine NK cells upon cytokine stimulation. We show that in the absence of AhR, NK cells have reduced cytolytic activity and reduced capacity to control RMA-S tumor formation in vivo, despite having normal development and maturation markers. Although AhR was first identified to bind the xenobiotic compound dioxin, AhR is now known to bind a variety of natural exogenous (e.g., dietary) and endogenous ligands. We show that activation of AhR with an endogenous tryptophan derivative, 6-formylindolo[3,2-b]carbazole, potentiates NK cell IFN-γ production and cytolytic activity. Further, administration of 6-formylindolo[3,2-b]carbazole in vivo enhances NK cell control of tumors in an NK cell- and AhR-dependent manner. Finally, similar effects on NK cell potency occur with AhR dietary ligands, potentially explaining the numerous associations that have been observed in the past between diet and NK cell function. Our studies introduce AhR as another regulator of NK cell activity in vivo.     

Toll-like receptor-stimulated non-parenchymal liver cells can regulate hepatitis C virus replication

BACKGROUND/AIMS: The aim of this study was to further elucidate the role of the IFN and the Toll-like receptor (TLR) system in the control of HCV replication by non-parenchymal liver cells (NPC). METHODS: Murine HCV replicon bearing MH1 cells were incubated with supernatants from TLR1-9-stimulated murine NPC (Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC)) and bone marrow-derived myeloid dendritic cells (mDC). HCV replication and expression of IFN-stimulated genes (ISGs) as well as TLR1-9 mRNA were determined by real-time rtPCR. RESULTS: IFNs (-alpha, -beta, -gamma) and TLR3 ligands only (despite the expression of TLR1-7 and TLR9 mRNA) achieved direct suppression of HCV replication by about 80-90% or 60%, respectively. Supernatants from TLR3- and 4-stimulated NPC only, however, led to potent suppression of HCV replication through IFN-beta and induction of ISGs. By contrast, mDCs could be stimulated by TLR2, -3, -4, -7 and -9 to produce antiviral cytokines. CONCLUSIONS: TLR3- and TLR4-stimulated NPC are able to regulate HCV replication through production of IFN-beta. This can also, at least partly explain the high level of ISG expression in HCV infected livers. These novel findings are of particular relevance for the control of HCV replication by the innate immune system of the liver.     

聚肌胞刺激后慢性乙型肝炎患者树突状细胞内Toll样受体3的表达变化

目的 研究慢性乙型肝炎(CHB)患者树突状细胞(DC)内Toll样受体3(TLR3)的表达变化,探讨HBV持续感染与TLR3表达之间的关系.方法 选取CHB患者60例(CHB组)及健康对照者20名(对照组)为研究对象,应用免疫磁珠细胞分选法获得CD14~+单核细胞,体外诱导培养成未成熟的髓样树突状细胞(imDC),并以聚肌胞(poly I;C)刺激以获取成熟的树突状细胞(mDC).刺激48 h后测定细胞内TLR3蛋白及mRNA的表达,表面标志CD80和人白细胞抗原(HLA)-DR的蛋白表达.计量资料采用t检验,计数贤料采用卡方检验.结果 poly I:C刺激前,对照组和CHB组imDC内TLR3的流式平均荧光密度(MFI)分别为1192.95±301.40和1212.05±250.80,组间差异无统计学意义(t=0.280,P>0.05);刺激后MFI均明显升高,对照组与CHB组分别为1593.00±349.65和1352.98±313.67,前者比后者升高更显著(t=2.880,P<0.05).polyI:C刺激后,对照组与CHB组mDC胞内TLR3 mRNA的表达水平均明显升高,分别为0.1780±0.0664和0.1204±0.0267,前者升高更显著(t=3.909,P<0.05).CHB组内再分为HBeAg(+)和HBeAg(-)组,刺激后两组mDC内TLR3的MFI和mRNA水平均较imDC有明显升高,但组间比较差异均无统计学意义(t=0.366,P>0.05).结论 poly I:C刺激后,对照组和CHB组中mDC胞内TLR3的表达均明显升高,但CHB患者升高的程度显著低于健康者,提示CHB患者的TLR3合成不足可能与HBV持续感染相关.     

Modulation of hypothalamic beta-endorphin-regulated expression of natural killer cell cytolytic activity regulatory factors by ethanol in male Fischer-344 rats

BACKGROUND: We have previously shown that ethanol administration suppresses natural killer (NK) cell cytolytic activity, partly by decreasing the action of hypothalamic beta-endorphin (beta-EP) on the spleens of male Fischer-344 rats. This study was conducted to examine the effects of ethanol and central administration of beta-EP on perforin, granzyme B, and the cytokine interferon (IFN)-gamma--factors that modulate NK cell cytolytic activity--to understand the mechanism involved in ethanol's suppression of NK cell activity. METHODS: A group of male Fischer-344 rats were fed an ethanol-containing diet (8.7% v/v), and a control group was pair-fed an isocaloric diet. At the end of 2 weeks, both groups were infused with beta-EP 100 ng/hr into the paraventricular nucleus of the hypothalamus for 18 hr, and spleen tissues were immediately removed for analysis of perforin, granzyme B, and IFN-gamma messenger RNA (mRNA) and protein levels. The mRNA levels of perforin, granzyme B, and IFN-gamma were evaluated by quantitative real-time polymerase chain reaction, and the protein levels of perforin and granzyme B were analyzed by Western blot. RESULTS: Paraventricular nucleus administration of beta-EP increased the mRNA and protein expression of granzyme B and mRNA expression of IFN-gamma in pair-fed animals. Ethanol significantly reduced both basal and beta-EP-induced levels of granzyme B and IFN-gamma. CONCLUSIONS: These data suggest that chronic ethanol consumption suppresses beta-EP-induced NK cytolytic activity, granzyme B, and IFN-gamma in male Fischer-344 rats.     

Murine malaria: dissociation of natural killer (NK) cell activity and resistance to Plasmodium chabaudi

Striking differences in the resistance to P. chabaudi infection among different inbred mouse strains have previously been correlated with the level of both the spontaneous and the infection-induced enhanced level of NK cell activity. We have examined this putative correlation in individual animals of backcross progeny derived from A/J (malaria-susceptible, low NK cell activity) and B10.A (malaria-resistant, high NK cell activity) progenitors. We have found that NK cell activity and resistance to malaria segregated independently. Furthermore, C57BL/6-bg/bg mice which are deficient in NK cell activity were found to be as resistant to malaria as their heterozygous C57BL/6-bg/+siblings. We conclude that low NK cell activity, characteristic of A/J strain mice, is not a sufficient determinant of the exquisite susceptibility of these animals to infection with Plasmodium chabaudi.     

Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases

Cytotoxicity of liver natural killer cells against regenerating hepatocytes has been reported as a possible mechanism of regeneration failure in fulminant hepatitis. An augmenter of liver regeneration (ALR) inhibits liver natural killer cell activity in rats. In this study, we measured hepatic expression of ALR mRNA, blood levels of ALR, and peripheral blood natural killer cell activity in patients with various types of acute liver disease to investigate the relationship between failure of liver regeneration and hepatic natural killer cells. Hepatic ALR mRNA expression was higher in liver disease patients than in non-liver disease controls, and a correlation was found between serum ALR values and hepatic levels of ALR mRNA. In acute liver injury, the serum ALR level also showed a negative correlation with NK activity. ALR was produced by and released from the liver at the time of hepatic injury. Our findings suggest that ALR may protect against failure of regeneration by inhibition of hepatic natural killer cell activity in acute liver injury. Received: December 7, 1998 / Accepted: August 27, 1999     

Role of beta-endorphin, corticotropin-releasing hormone, and autonomic nervous system in mediation of the effect of chronic ethanol on natural killer cell cytolytic activity

BACKGROUND: We have recently shown that alcohol feeding suppresses natural killer (NK) cell cytolytic activity partly by decreasing the function of hypothalamic beta-endorphin (beta-EP) neurons. The neuronal mechanism by which hypothalamic beta-EP communicates with the spleen to regulate the action of ethanol on NK cells is not known. In the present study, we evaluated the roles of beta-EP neurons, corticotropin releasing hormone (CRH) neurons, and the autonomic nervous system (ANS) in regulation of the ethanol effect on splenic NK cell cytolytic function. METHODS: Male rats were fed an ethanol-containing liquid diet or control diets. These rats were used to determine the hormone release from the paraventricular nuclei (PVN) of the hypothalamus or used to determine the splenic NK cell cytolytic function after PVN administration of CRH or intraperitoneal (i.p.) administration of a ganglionic blocker chlorisondamine. The release of hormones from the PVN was measured using the push-pull perfusion method. Splenic cytolytic activity was determined using the 4-hour (51)Cr release assay against YAC-1 lymphoma target cells. RESULTS: Alcohol feeding decreased the amount of beta-EP but increased the amount of CRH in the push-pull perfusate (PPP) samples collected from the PVN. When exogenous beta-EP was perfused into the PVN, it suppressed the release of endogenous CRH found in PPP samples of the PVN. Conversely, perfusion of an opiate antagonist naltrexone into the PVN increased the levels of endogenous CRH in PPP samples of the PVN. In addition, administration of exogenous beta-EP in the PVN stimulated the cytolytic function of NK cells, an action that was antagonized by CRH as well as by ethanol. Corticotropin-releasing hormone and ethanol alone also had an inhibitory action on NK cells. Finally, the ganglionic blocker used prevented the effect that ethanol, beta-EP, and CRH had on NK cells. These data suggest that ethanol inhibits the function of NK cells partly by suppressing the influence of the beta-EP-CRH-ANS signal to the spleen.