Natural killer cell promotes the HBV clearance via secreting IFN-γ / 安徽医科大学学报

Objective To investigate the mechanism of NK cell in eliminating the Hepatitis B virus during HBV in-fection . Methods Acute HBV infection model was established by injecting adult mice hydrodynamically with 20μg of pGEM4Z/HBV1. 2 plasmid. This model was evaluated by detecting serum level of HBsAg and HBcAg in liver tis-sue at the indicated time points by radioimmunoassay and immunohistochemistry respectively. The variation of fre-quency and absolute number of NK cell was analyzed between wide type ( WT ) mice and HBV plasmid-injected mice. Furthermore, the activation and the IFN-γproduction of NK cell were investigated in these mice by flow cy-tometry. HE staining and alanine transaminase( ALT) dectection were used to observe liver injury. To test whether NK cell and IFN-γwere involved in HBV elimination, we used PK136 antibody to clear NK cell and IFN-γneutral-ization antibody toblock IFN-γeffect. Results After the hydrodynamic injection with 20 μg of pGEM4Z/HBV1.2, the serum level of HBsAg and expression of HBcAg in liver tissue were very high at 1 week, but then decreased gradually. However, these antigens almost became negative at 4 to 5 weeks, which mimic acute HBV infection pa-tients. Compared with NK cell from WT mice, the frequency and absolute number of NK cell increased significantly from HBV mice. Also, the NK cells express higher level of CD69 and produce more IFN-γ. Meanwhile, there was no liver injury in HBV mice. Depletion of NK cell or blocking IFN-γ effect in HBV mice could significantly in-crease the level of HBV related antigens. Conclusion In the mouse model of acute HBV infection, NK cell could promote the HBV elimination through secreting IFN-γ.

Effect of simvastatin on production of reactive oxygen species and secretion of IL-1βin macrophages induced by oxLDL / 中国药理学通报

Aim To study the effect of simvastatin on the production of reactive oxygen species ( ROS ) and the secretion of interleukin-1 beta ( IL-1β) in oxidized low density lipoprotein ( oxLDL )-induced macropha-ges. Methods After the murine macrophage J774A. 1 was treated with 0,50,100,200 mg·L-1 oxLDL, the contents of aggregated lipid in macrophages were ob-served and determined by oil red O staining. Then, the oxLDL-primed macrophages were treated with 0 . 5 ,1 . 0μmol·L-1 simvastatin, the production of ROS was de-termined by flow cytometry and the expressions of pro-caspase-1 , cleaved caspase-1 and mature IL-1βon pro-tein level were determined by Western blot. Results The oil red O staining results showed that oxLDL could induce obvious lipid aggregation in macrophages, and reached the saturation point with 100 mg·L-1 concen-tration. Flow cytometry results indicated that oxLDL could induce the production of ROS in macrophages, up to 167% ± 0. 47%, and ROS level decreased to 139% ± 0. 97% in a dose-dependent manner after treatment with simvastatin. Western blot indicated that simvastatin could inhibit the expression of cleaved caspase-1 and mature IL-1β in macrophages triggered by oxLDL;compared with oxLDL group, the expression of cleaved caspase-1 and mature IL-1β decreased in simvastatin treated group, and all results had statistical significance ( P<0. 05 ) . Conclusion In the lipid ag-gregation model of macrophages induced by oxLDL, simvastatin can inhibit the production of ROS, caspase-1 activation, and secretion of IL-1β in macrophages.

CXC chemokine ligand 13 and CC chemokine ligand 19 cooperatively render resistance to apoptosis in B cell lineage acute and chronic lymphocytic leukemia CD23+CD5+ B cells.

CXCL13/CXCR5 and CCL19/CCR7 play a quite important role in normal physiological conditions, but the functions of both chemokine/receptor pairs in pathophysiological events are not well-investigated. We have investigated expression and functions of CXCL13/CXCR5 and CCL19/CCR7 in CD23+CD5+ and CD23+CD5- B cells from cord blood (CB) and patients with B cell lineage acute or chronic lymphocytic leukemia (B-ALL or B-CLL). CXCR5 and CCR7 are selectively expressed on B-ALL, B-CLL, and CB CD23+CD5+ B cells at high frequency, but not on CD23+CD5- B cells. Although no significant chemotactic responsiveness was observed, CXCL13 and CCL19 cooperatively induce significant resistance to TNF-alpha-mediated apoptosis in B-ALL and B-CLL CD23+CD5+ B cells, but not in the cells from CB. B-ALL and B-CLL CD23+CD5+ B cells express elevated levels of paternally expressed gene 10 (PEG10). CXCL13 and CCL19 together significantly up-regulate PEG10 expression in the same cells. We have found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulate PEG10 expression and function, subsequently stabilize caspase-3 and caspase-8 in B-ALL and B-CLL CD23+CD5+ B cells, and further rescue the cells from TNF-alpha-mediated apoptosis. Therefore, we suggest that normal lymphocytes, especially naive B and T cells, use CXCL13/CXCR5 and CCL19/CCR7 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoid tissues organogenesis. In addition, certain malignant cells take advantages of CXCL13/CXCR5 and CCL19/CCR7 for infiltration, resistance to apoptosis, and inappropriate proliferation.

Different neurotropic pathogens elicit neurotoxic CCR9- or neurosupportive CXCR3-expressing microglia.

What mechanism that determines microglia accomplishing destructive or constructive role in CNS remains nebulous. We report here that intracranial priming and rechallenging with Toxoplasma gondii in mice elicit neurotoxic CCR9+ Irg1+ (immunoresponsive gene 1) microglia, which render resistance to apoptosis and produce a high level of TNF-alpha; priming and rechallenging with lymphocytic choriomeningitis virus elicit neurosupportive CXCR3+ Irg1- microglia, which are sensitive to apoptosis and produce a high level of IL-10 and TGF-beta. Administration of CCR9 and/or Irg1 small interfering RNA alters the frequency and functional profiles of neurotoxic CCR9+ Irg1+ and neurosupportive CXCR3+ Irg1- microglia in vivo. Moreover, by using a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury to intracranially prime and subsequent rechallenge mice, the bi-directional elicitation of microglia has been confirmed as neurotoxic CCR9+ Irg1+ and neurosupportive CXCR3+ Irg1- cells in these mouse models. These data suggest that there exist two different types of microglia, providing with a novel insight into microglial involvement in neurodegenerative and neuroinflammatory pathogenesis such as Alzheimer's disease and AIDS dementia.

V alpha 24-invariant NKT cells from patients with allergic asthma express CCR9 at high frequency and induce Th2 bias of CD3+ T cells upon CD226 engagement.

We have demonstrated that Valpha24(+)Vbeta11(+) invariant (Valpha24(+)i) NKT cells from patients with allergic asthma express CCR9 at high frequency. CCR9 ligand CCL25 induces chemotaxis of asthmatic Valpha24(+)i NKT cells but not the normal cells. A large number of CCR9-positive Valpha24(+)i NKT cells are found in asthmatic bronchi mucosa, where high levels of Th2 cytokines are detected. Asthmatic Valpha24(+)i NKT cells, themselves Th1 biased, induce CD3(+) T cells into an expression of Th2 cytokines (IL-4 and IL-13) in cell-cell contact manner in vitro. CD226 are overexpressed on asthmatic Valpha24(+)i NKT cells. CCL25/CCR9 ligation causes directly phosphorylation of CD226, indicating that CCL25/CCR9 signals can cross-talk with CD226 signals to activate Valpha24(+)i NKT cells. Prestimulation with immobilized CD226 mAb does not change ability of asthmatic Valpha24(+)i NKT cells to induce Th2-cytokine production, whereas soluble CD226 mAb or short hairpin RNA of CD226 inhibits Valpha24(+)i NKT cells to induce Th2-cytokine production by CD3(+) T cells, indicating that CD226 engagement is necessary for Valpha24(+)i NKT cells to induce Th2 bias of CD3(+) T cells. Our results are providing with direct evidence that aberration of CCR9 expression on asthmatic Valpha24(+)i NKT cells. CCL25 is first time shown promoting the recruitment of CCR9-expressing Valpha24(+)i NKT cells into the lung to promote other T cells to produce Th2 cytokines to establish and develop allergic asthma. Our findings provide evidence that abnormal asthmatic Valpha24(+)i NKT cells induce systemically and locally a Th2 bias in T cells that is at least partially critical for the pathogenesis of allergic asthma.

Selectively frequent expression of CXCR5 enhances resistance to apoptosis in CD8(+)CD34(+) T cells from patients with T-cell-lineage acute lymphocytic leukemia.

We investigated CD4(+)CD34(+), CD8(+)CD34(+), CD4(+)CD34(-), and CD8(+)CD34(-) T cells from cord blood and from typical patients with T-cell-lineage acute lymphocytic leukemia and T-cell-lineage chronic lymphocytic leukemia in terms of expression and functions of CXCR5/CXCL13. We found that CXCR5 was selectively frequently expressed on T-cell-lineage acute (chronic) lymphocytic leukemia (T-ALL) CD8(+)CD34(+) T cells, but not on T-ALL CD4(+)CD34(+), CD4(+)CD34(-), and CD8(+)CD34(-) T cells. CXCR5 was rarely expressed on all types of CD34(+) and CD34(-) CB or T-CLL T cells. CXCL13/B cells attracting chemokine 1 induced significant resistance to TNF-alpha-mediated apoptosis in T-ALL CD8(+)CD34(+) T cells, instead of induction of chemotactic and adhesive responsiveness. A proliferation-inducing ligand expression in T-ALL CD8(+)CD34(+) T cells was upregulated by CXCL13/BCA-1 (B-cell attracting chemokine 1). The CXCR5/CXCL13 pair by means of activation of APRIL (A proliferation-inducing ligand) induced resistance to apoptosis in T-ALL CD8(+)CD34(+) T cells in livin-dependent manner. In this process, cell-cell contact in culture was necessary. Based on our findings, we suggested that there were differential functions of CXCR5/CXCL13 in distinct types of cells. Normal lymphocytes, especially naive B and T cells, utilized CXCR5/CXCL13 for migration, homing, maturation, and cell homeostasis, as well as secondary lymphoid tissue organogenesis. Meanwhile, certain malignant cells took advantages of CXCR5/CXCL13 for infiltration, resistance to apoptosis, and inappropriate proliferation.

CC chemokine ligand 25 enhances resistance to apoptosis in CD4+ T cells from patients with T-cell lineage acute and chronic lymphocytic leukemia by means of livin activation.

We investigated CD4 and CD8 double-positive thymocytes, CD4(+) T cells from typical patients with T-cell lineage acute lymphocytic leukemia (T-ALL) and T cell lineage chronic lymphocytic leukemia (T-CLL), and MOLT4 T cells in terms of CC chemokine ligand 25 (CCL25) functions of induction of resistance to tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis. We found that CCL25 selectively enhanced resistance to TNF-alpha-mediated apoptosis in T-ALL and T-CLL CD4(+) T cells as well as in MOLT4 T cells, but CD4 and CD8 double-positive thymocytes did not. One member protein of the inhibitor of apoptosis protein (IAP) family, Livin, was selectively expressed in the malignant cells at higher levels, particularly in T-ALL CD4(+) T cells, in comparison with the expression in CD4 and CD8 double-positive thymocytes. After stimulation with CCL25 and apoptotic induction with TNF-alpha, the expression levels of Livin in these malignant cells were significantly increased. CCL25/thymus-expressed chemokine (TECK), by means of CC chemokine receptor 9 (CCR9) ligation, selectively activated Livin to enhance resistance to TNF-alpha-mediated apoptosis in c-jun-NH(2)-kinase 1 (JNK1) kinase-dependent manner. These findings suggested differential functions of CCR9/CCL25 in distinct types of cells. CD4 and CD8 double-positive thymocytes used CCR9/CCL25 for migration, homing, development, maturation, selection, cell homeostasis, whereas malignant cells, particularly T-ALL CD4(+) T cells, used CCR9/CCL25 for infiltration, resistance to apoptosis, and inappropriate proliferation.

Aberration of CCR7 CD8 memory T cells from patients with systemic lupus erythematosus: an inducer of T helper type 2 bias of CD4 T cells.

Chemokine receptors are important in the entry of leucocytes into the inflammatory sites of systemic lupus erythematosus (SLE). CCR7(+) and CCR7(-) memory T cells exert different functions in homing, cytokine production and cytotoxicity. To determine whether differential expression and functions of the CCR7 occur in SLE patients, we examined CCR3, CCR4, CCR5, CCR7 and CCR9 on CD4(+) and CD8(+) T cells from normal and SLE subjects. Flow cytometry, real-time quantitative reverse transcription polymerase chain reactions and Northern blotting were used to detect the expression of chemokine receptors and cytokines; a chemotaxis assay was used to detect their functions. CD4(+) T-cell stimulation with syngeneic CCR7(+) CD8(+) CD45RO(+) T cells and dendritic cells (including transwell chambers) was used to induce cytokine expression. We demonstrated that CCR7 was selectively, frequently and functionally expressed on CD8(+) (94.8%) but not on CD4(+) (16.1%) T cells from patients with active SLE, whereas this phenomenon was not seen in normal subjects and in those whose SLE was inactive. CCR7(+) CD8(+) CD45RO(+) memory T cells from patients with active SLE, themselves T helper type 2 (Th2) biased, were inducers of Th2 bias in CD4(+) T cells in a cell-cell contact manner in vitro, meanwhile, the cells from both normal subjects and those whose SLE was inactive drove CD4(+) T cells into a regulatory T-cell-derived cytokine pattern. Our findings might provide new clues to understanding the functions of CCR7(+) CD8(+) CD45RO(+)'central' memory T cells in autoimmune diseases (such as SLE). We suggest that in the case of active SLE, CCR7(+) central memory T cells were able to enter peripheral blood and inflammatory sites from secondary lymphoid organs, were continuously expressing CCR7, and interacted with dendritic cells and functioned as CCR7(-)'effector' memory T cells, which were described in normal humans.

Highly up-regulated CXCR3 expression on eosinophils in mice infected with Schistosoma japonicum.

CXCR3, predominately expressed on memory/activated T cells, is a receptor for both interferon-gamma inducible protein-10/CXC ligand 10 (CXCL10) and monokine induced by interferon-gamma/CXCL9. We reported here that CXCR3 was highly up-regulated on infiltrating eosinophils in Schistosoma japonicum egg-induced granuloma in the mouse liver. It was also highly and functionally up-regulated on peritoneal exudate eosinophils in mice infected with S. japonicum. The phenomena were demonstrated at protein and mRNA levels using immunohisto- and immunocytochemistry evaluation of biopsy, flow cytometry and real-time quantitative reverse transcriptase-polymerase chain reaction technique, and verified by Northern blotting and chemotaxis assay in vitro. We also found that CCR3 expression on the infiltrating and peritoneal exudate cells was significantly decreased, CXCR4 expression was unchanged during the 42-day period of infection. We screened mRNA expression levels of the all known chemokine receptors in purified peritoneal exudate eosinophils and liver granuloma dominated by eosinophils. CXCR3 was highly and functionally up-regulated on peritoneal exudate eosinophils in mice infected with S. japonicum, meanwhile CCR3 was significantly and functionally down-regulated in these cells. The findings could lead to a better understanding of the chemokine receptor expression pattern of eosinophils at inflamed tissue sites caused by parasites. These could be also crucial for establishing a therapeutic strategy for eosinophilic inflammation via intervention in chemokine actions.

Selectively increased expression and functions of chemokine receptor CCR9 on CD4+ T cells from patients with T-cell lineage acute lymphocytic leukemia.

In a total of 38 typical T-cell lineage acute lymphocytic leukemia (T-ALL) and T-cell lineage chronic lymphocytic leukemia (T-CLL) cases investigated, we found that CC chemokine receptor CCR9 was selectively and frequently expressed on T-ALL CD4+ T cells, was moderately expressed on T-CLL CD4+ T cells, and was rarely expressed on normal CD4+ T cells. These findings were demonstrated at protein and mRNA levels using flow cytometry and real-time quantitative reverse transcription-PCR technique and were verified by digital confocal microscopy and Northern blotting. Thymus-expressed chemokine, a ligand for CCR9, selectively induced T-ALL CD4+ T-cell chemotaxis and adhesion. Interleukin (IL)-2 and IL-4, together, down-regulated the expression and functions of CCR9 in T-ALL CD4+ T cells including chemotaxis and adhesion. It was also demonstrated that IL-2 and IL-4, together, internalized CCR9 on T-ALL CD4+ T cells and subsequently inhibited functions of CCR9 in these cells. Thymus-expressed chemokine mRNA was highly expressed in CD4+ T cells, involving lymph node and skin in T-ALL patients, and was expressed at moderate levels in lymph node and skin tissues in T-CLL patients. Our findings may provide new clues to understanding various aspects of T-ALL CD4+ T cells, such as functional expression of CCR9-thymus-expressed chemokine receptor-ligand pairs as well as the effects of IL-2 and IL-4, which may be especially important in cytokine/chemokine environment for the pathophysiological events of T-ALL CD4+ T-cell trafficking.

Peptide nucleic acid antisense prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into graft.

CXCR3, predominantly expressed on memory/activated T cells, is a receptor for both IFN-gamma-inducible protein 10/CXC chemokine ligand (CXCL)10 and monokine induced by IFN-gamma/CXCL9. It was reported that CXC chemokines IFN-gamma-inducible protein 10/CXCL10 and monokine induced by IFN-gamma/CXCL9 play a critical role in the allograft rejection. We report that CXCR3 is a dominant factor directing T cells into mouse skin allograft, and that peptide nucleic acid (PNA) CXCR3 antisense significantly prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into allografts in mice. We found that CXCR3 is highly up-regulated in spleen T cells and allografts from BALB/c recipients by day 7 of receiving transplantation, whereas CCR5 expression is moderately increased. We designed PNA CCR5 and PNA CXCR3 antisenses, and i.v. treated mice that received skin allograft transplantations. The PNA CXCR3 at a dosage of 10 mg/kg/day significantly prolonged mouse skin allograft survival (17.1 +/- 2.4 days) compared with physiological saline treatment (7.5 +/- 0.7 days), whereas PNA CCR5 (10 mg/kg/day) marginally prolonged skin allograft survival (10.7 +/- 1.1 days). The mechanism of prolongation of skin allograft survival is that PNA CXCR3 directly blocks the CXCR3 expression in T cells, which is responsible for directing T cells into skin allograft to induce acute rejection, without interfering with other functions of the T cells. These results were obtained at mRNA and protein levels by flow cytometry and real-time quantitative RT-PCR technique, and confirmed by chemotaxis, Northern and Western blot assays, and histological evaluation of skin grafts. The present study indicates the therapeutic potential of PNA CXCR3 to prevent acute transplantation rejection.