B cells control the migration of a subset of dendritic cells into B cell follicles via CXC chemokine ligand 13 in a lymphotoxin-dependent fashion

Certain classes of dendritic cells (DCs) meet rare cognate Ag-specific T and B cells inside primary B cell follicles for the development of germinal centers. However, the mechanisms underlying this coordination are still undefined. Cysteine-rich (CR) domain of the mannose receptor (CR-Fc)(+) DCs are a newly discovered subset of DCs that migrate rapidly into the primary lymphoid follicles from marginal zone after immunization. In this work, we uncover the key role of B cells in the establishment of a microenvironment that allows these DCs to be in the B cell area in a lymphotoxin (LT)-dependent fashion. CR-Fc(+) DCs are absent from the spleens of both LTbetaR- and LTalpha-deficient mice, suggesting that signaling by membrane LT is required for the presence of CR-Fc(+) DCs in the spleen. Interestingly, analysis of mutant mice that lack T, B, or NK cells demonstrates that B cell-derived membrane LT is essential for the unique localization of CR-Fc(+) DCs in the spleen. Using bone marrow transfer and ligand-blocking approaches, we provide evidence that B cell-derived LT acts indirectly on CR-Fc(+) DCs through LTbetaR(+) stromal cells. In analogous fashion to certain Ag-activated T and B cells, CR-Fc(+) DCs, expressing CXCR5, localize to primary lymphoid follicles in response to CXC ligand 13 (B lymphocyte chemoattractant). Together, we propose that B cells play a central role in establishing the chemotactic gradient that attracts not only Ag-activated T and B cells but also Ag-carrying CR-Fc(+) DCs. In turn, CR-Fc(+) DCs and T cells home to B cell follicles to interact with B cells in the developing germinal center.     

The CXC chemokine murine monokine induced by IFN-gamma (CXC chemokine ligand 9) is made by APCs,targets lymphocytes including activated B cells,and supports antibody responses to a bacterial pathogen in vivo

Monokine induced by IFN-gamma (Mig; CXC chemokine ligand 9) is an IFN-gamma-inducible CXC chemokine that signals through the receptor CXCR3 and is known to function as a chemotactic factor for human T cells, particularly following T cell activation. The mig gene can be induced in multiple cell types and organs, and Mig has been shown to contribute to T cell infiltration into immune/inflammatory reactions in peripheral tissues in mice. We have investigated the expression and activities of Mig and CXCR3 in mouse cells and the role of Mig in models of host defense in mice. Murine (Mu)Mig functioned as a chemotactic factor for resting memory and activated T cells, both CD4(+) and CD8(+), and responsiveness to MuMig correlated with surface expression of MuCXCR3. Using mig(-/-) mice, we found that MuMig was not necessary for survival after infections with a number of intracellular pathogens. Surprisingly, however, we found that mig(-/-) mice showed reductions of 50-75% in Abs produced against the intracellular bacterium Francisella tularensis live vaccine strain. Furthermore, we found that MuMig induced both calcium signals and chemotaxis in activated B cells, and that B cell activation induced expression of MuCXCR3. In addition, IFN-gamma induced the expression of mumig in APCs, including CD8 alpha(+) and CD8 alpha(-) dendritic cells. Together, our data suggest that Mig and CXCR3 may be important not only to recruit T cells to peripheral inflammatory sites, but also in some cases to maximize interactions among activated T cells, B cells, and dendritic cells within lymphoid organs to provide optimal humoral responses to pathogens.     

Distinct roles for the NK cell-activating receptors in mediating interactions with dendritic cells and tumor cells

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)-mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.     

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.     

Upregulated monocytic expression of CXC chemokine ligand 10 (CXCL-10) and its relationship with serum interleukin-6 levels in the syndrome of frailty

Frailty is an important geriatric syndrome that predicts disability and mortality. Substantial evidence suggests inflammation marked by elevated IL-6 levels as a key pathophysiologic factor that contributes to frailty. CXCL-10, a potent pro-inflammatory chemokine, has increased levels with age and is implicated in several inflammatory conditions. To better understand molecular mechanisms of inflammation activation in frailty, we evaluated monocytic expression of CXCL-10 and other inflammatory pathway genes by pathway-specific gene array analysis and quantitative RT-PCR. Frailty status was determined by the validated criteria. Sixteen pairs of community-dwelling frail and age-, race-, and sex-matched non-frail participants (mean age 83 years, range 72–94) completed the study. Here we report that frail participants had higher CXCL-10 expression levels than matched non-frail controls (1.05 ± 0.88 versus 0.53 ± 0.39, p = 0.04). CXCL-10 expression correlated with IL-6 levels only in frail participants (Spearman correlation coefficient r = 0.52, p = 0.03). Furthermore, frailty-associated CXCL-10 upregulation was highly correlated with IL-6 elevation, both measured by frail-over-non-frail ratios (r = 0.93, p < 0.0001). These findings suggest upregulated monocytic expression of CXCL-10 as an important molecular mechanism that contributes to inflammation activation in frail older adults. Therapeutic implications include potential development of CXCL-10-based interventional strategies for the prevention and treatment of frailty in older adults.     

Recruitment of uterine NK cells: induction of CXC chemokine ligands 10 and 11 in human endometrium by estradiol and progesterone

Uterine NK (uNK) cells express a unique set of markers compared with blood NK cells. However, recent studies suggest that uNK cells may be derived from the recruitment of blood NK cells into the endometrium. In this study, we used an in vitro organ culture system to demonstrate that estradiol induces expression of chemokines CXCL10 and/or CXCL11 within human endometrium in 85% of patient samples tested. The average increase in gene expression after 10(-9) M estradiol treatment was 8.5-fold for CXCL10 and 7.7-fold for CXCL11 compared with medium alone. We observed that a specific estrogen receptor antagonist (ICI182780) was able to prevent chemokine gene induction, indicating that the effect of estradiol was receptor mediated. Moreover, our study showed that progesterone induced CXCL10 and CXCL11 expression in 83% of endometrial samples tested. We have also found that uNK cells and blood NK cells express the receptor for CXCL10 and CXCL11, CXCR3, with the highest expression found on uNK cells and CD56(bright) blood NK cells. These data indicate that sex hormones induce specific chemokines in nonpregnant human endometrium that can activate NK cell migration, and suggest that this mechanism may account for the increased NK cell numbers in endometrium during the menstrual cycle.     

Role of CXC chemokine ligand 13, CC chemokine ligand (CCL) 19, and CCL21 in the organization and function of nasal-associated lymphoid tissue

Nasal-associated lymphoid tissue (NALT) orchestrates immune responses to Ags in the upper respiratory tract. Unlike other lymphoid organs, NALT develops independently of lymphotoxin-alpha (LTalpha). However, the structure and function of NALT are impaired in Ltalpha(-/-) mice, suggesting a link between LTalpha and chemokine expression. In this study we show that the expression of CXCL13, CCL19, CCL21, and CCL20 is impaired in the NALT of Ltalpha(-/-) mice. We also show that the NALT of Cxcl13(-/-) and plt/plt mice exhibits some, but not all, of the structural and functional defects observed in the NALT of Ltalpha(-/-) mice. Like the NALT of Ltalpha(-/-) mice, the NALT in Cxcl13(-/-) mice lacks follicular dendritic cells, BP3(+) stromal cells, and ERTR7(+) lymphoreticular cells. However, unlike the NALT of Ltalpha(-/-) mice, the NALT of Cxcl13(-/-) mice has peripheral node addressin(+) high endothelial venules (HEVs). In contrast, the NALT of plt/plt mice is nearly normal, with follicular dendritic cells, BP3(+) stromal cells, ERTR7(+) lymphoreticular cells, and peripheral node addressin(+) HEVs. Functionally, germinal center formation and switching to IgA are defective in the NALT of Ltalpha(-/-) and Cxcl13(-/-) mice. In contrast, CD8 T cell responses to influenza are impaired in Ltalpha(-/-) mice and plt/plt mice. Finally, the B and T cell defects in the NALT of Ltalpha(-/-) mice lead to delayed clearance of influenza from the nasal mucosa. Thus, the B and T cell defects in the NALT of Ltalpha(-/-) mice can be attributed to the impaired expression of CXCL13 and CCL19/CCL21, respectively, whereas impaired HEV development is directly due to the loss of LTalpha.     

Leukocyte infiltration,but not neurodegeneration,in the CNS of transgenic mice with astrocyte production of the CXC chemokine ligand 10

The CXC chemokine ligand (CXCL)10 is induced locally in the CNS in diverse pathologic states. The impact of CXCL10 production in the CNS was examined in transgenic mice with astrocyte-directed production of this chemokine. These glial fibrillary acidic protein (GF)-CXCL10 transgenic mice spontaneously developed transgene dose- and age-related leukocyte infiltrates in perivascular, meningeal, and ventricular regions of the brain that were composed of, surprisingly, mainly neutrophils and, to a lesser extent, T cells. No other overt pathologic or physical changes were evident. In addition, the cerebral expression of a number of inflammation-related genes (e.g., cytokines) was not significantly altered in the transgenic mice. The extent of leukocyte recruitment to the brain could be enhanced markedly by peripheral immunization of GF-CXCL10 mice with CFA and pertussis toxin. This was paralleled by a modest, transient increase in the expression of some cytokine and chemokine genes. Analysis of the expression of the CXCL10 receptor, CXCR3, by the brain-infiltrating leukocytes from immunized GF-CXCL10 transgenic mice revealed a significant enrichment for CXCR3-positive cells in the CNS compared with spleen. The majority of cells positive for CXCR3 coexpressed CD3, whereas Gr1-positive granulocytes were negative for CXCR3 expression. Thus, while astrocyte production of CXCL10 can promote spontaneous and potentiate immune-induced recruitment of leukocytes to the CNS, this is not associated with activation of a degenerative immune pathology. Finally, the accumulation of neutrophils in the brain of GF-CXCL10 transgenic mice is apparently independent of CXCR3 and involves an unknown mechanism.     

CXC chemokine ligand 10 neutralization suppresses the occurrence of diabetes in nonobese diabetic mice through enhanced beta cell proliferation without affecting insulitis

We have shown that neutralization of IFN-inducible protein 10/CXCL10, a chemokine for Th1 cells, breaks Th1 retention in the draining lymph nodes, resulting in exacerbation in Th1-dominant autoimmune disease models induced by immunization with external Ags. However, there have been no studies on the role of CXCL10 neutralization in Th1-dominant disease models induced by constitutive intrinsic self Ags. So, we have examined the effect of CXCL10 neutralization using a type 1 diabetes model initiated by developmentally regulated presentation of beta cell Ags. CXCL10 neutralization suppressed the occurrence of diabetes after administration with cyclophosphamide in NOD mice, although CXCL10 neutralization did not significantly inhibit insulitis and gave no influence on the trafficking of effector T cells into the islets. Because both CXCL10 and CXCR3 were, unexpectedly, coexpressed on insulin-producing cells, CXCL10 was considered to affect mature and premature beta cells in an autocrine and/or paracrine fashion. In fact, CXCL10 neutralization enhanced proliferative response of beta cells and resultantly increased beta cell mass without inhibiting insulitis. Thus, CXCL10 neutralization can be a new therapeutic target for beta cell survival, not only during the early stage of type 1 diabetes, but also after islet transplantation.     

CXC chemokine ligand 12/Stromal cell-derived factor-1 regulates cell adhesion in human colon cancer cells by induction of intercellular adhesion molecule-1

Background

Gene therapy and viral therapy are used for cancer therapy for many years, but the results are less than satisfactory. Our aim was to construct a new recombinant adenovirus which is more efficient to kill hepatocarcinoma cells but more safe to normal cells.

Methods

By using the Cancer Targeting Gene-Viro-Therapy strategy, Apoptin, a promising cancer therapeutic gene was inserted into the double-regulated oncolytic adenovirus AD55 in which E1A gene was driven by alpha fetoprotein promoter along with a 55 kDa deletion in E1B gene to form AD55-Apoptin. The anti-tumor effects and safety were examined by western blotting, virus yield assay, real time polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, Hoechst33342 staining, Fluorescence-activated cell sorting, xenograft tumor model, Immunohistochemical assay, liver function analysis and Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling assay.

Results

The recombinant virus AD55-Apoptin has more significant antitumor effect for hepatocelluar carcinoma cell lines (in vitro) than that of AD55 and even ONYX-015 but no or little impair on normal cell lines. Furthermore, it also shows an obvious in vivo antitumor effect on the Huh-7 liver carcinoma xenograft in nude mice with bigger beginning tumor volume till about 425 mm3 but has no any damage on the function of liver. The induction of apoptosis is involved in AD55-Apoptin induced antitumor effects.

Conclusion

The AD55-Apoptin can be a potential anti-hepatoma agent with remarkable antitumor efficacy as well as higher safety in cancer targeting gene-viro-therapy system.     

Excessive production of IFN-gamma in patients with systemic lupus erythematosus and its contribution to induction of B lymphocyte stimulator/B cell-activating factor/TNF ligand superfamily-13B

Expression and immunological significance of IFN-gamma, a pivotal cytokine in murine lupus, have not been clearly demonstrated in human systemic lupus erythematosus (SLE). In the present study we investigated the expression of IFN-gamma in peripheral blood T cells from patients with SLE and its role in the production of the soluble B lymphocyte stimulator (sBLyS). Peripheral blood T cells from patients with SLE expressed significantly larger amounts of IFN-gamma in response to stimulation with anti-CD3 mAb plus anti-CD28 mAb than those from normal controls as shown by three analytical methods, including ELISA, flow cytometry, and quantitative RT-PCR. The ratio of IFN-gamma-producing T cells to effector memory T cells in CD3(+)CD4(+) and CD3(+)CD8(+) populations in patients with SLE was significantly higher than that of normal controls. The T-box expressed in T cells (T-bet) mRNA/GATA-binding protein-3 (GATA-3) mRNA ratio was significantly higher in patients with SLE than in normal controls. T cell culture supernatants from patients with SLE contained significantly higher sBLyS-inducing activity than normal controls; this was almost completely inhibited by the addition of anti-human IFN-gamma mAb. Percentages of BLyS-expressing peripheral blood monocytes in patients with SLE were significantly higher than those of normal controls. Monocytes from patients with SLE produced significantly larger amounts of sBLyS in response to IFN-gamma than those from normal controls. Taken together, these data strongly indicate that the overexpression of IFN-gamma in peripheral blood T cells contributes to the immunopathogenesis of SLE via the induction of sBLyS by monocytes/macrophages, which would promote B cell activation and maturation.     

Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer's patches and affects B cell trafficking across high endothelial venules

While CCR7 ligands direct T cell trafficking into lymph nodes (LNs) and Peyer's patches (PPs), chemokines that regulate B cell trafficking across high endothelial venules (HEVs) remain to be fully elucidated. Here we report that CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant) is detected immunohistologically in the majority of HEVs in LNs and PPs of nonimmunized mice. Systemically administered anti-CXCL13 Ab bound to the surface of approximately 50% of HEVs in LNs and PPs, but not to other types of blood vessels, indicating that CXCL13 is expressed in the HEV lumen. In CXCL13-null mice, B cells rarely adhered to PP HEVs, whereas T cells did efficiently. Superfusion of CXCL13-null PPs with CXCL13 restored the luminal presentation of CXCL13 and also B cell arrest in PP HEVs at least partially. Collectively, these results indicate that CXCL13 expressed in the HEV lumen plays a crucial role in B cell trafficking into secondary lymphoid tissues such as PPs.     

Th1-biased tertiary lymphoid tissue supported by CXC chemokine ligand 13-producing stromal network in chronic lesions of autoimmune gastritis

Secondary lymphoid tissue is developmentally programmed and characterized by well-ordered compartmentalization of lymphocyte subsets and specialized stromal cells supporting the tissue architecture. By contrast, tertiary lymphoid tissue is defined as that induced in ectopic sites by inflammation, although its immunological role is largely unknown. In this study, we characterize the lymphoid tissue induced in the chronic lesion of murine autoimmune gastritis (AIG). Within the lymphoid cluster in the gastric mucosa, there is a clear segregation of T and B cells. Follicle-like B cell areas are always located on the luminal side of the mucosa, while T cells are located in the basal part. A typical lymphoid reticular network and follicular dendritic cells support the structure. Importantly, complement receptor 1(+) follicular dendritic cells within the follicle express a B cell homing chemokine, CXC chemokine ligand 13. The number and size of the clusters correlate with the age of the mice and the serum autoantibody titer, suggesting the functional importance of the clusters in local Ab production, although involvement of the autoantibody in the disease progression is still unclear. AIG gastric lesions are known to constitute a Th1-biased, memory T cell-dependent immunomicroenvironment. The expression pattern of cytokines, including lymphotoxin-beta, and chemokines in the AIG stomach is consistent with this observation. Taken together, these facts suggest that, during the chronic phase of autoimmunity, long-lasting lymphocyte infiltration probably induces a unique tertiary lymphoid tissue that has a function distinct from that of regional lymph nodes. These neolymphoid tissues may maintain the local self reactivity supporting the vicious cycle of Th1-type reaction as well as autoantibody production.     

Different diabetogenic potential of autoaggressive CD8+ clones associated with IFN-gamma-inducible protein 10 (CXC chemokine ligand 10) production but not cytokine expression, cytolytic activity, or homing characteristics

Type 1 diabetes mellitus is an autoimmune disease characterized by T cell-mediated destruction of the insulin-producing beta cells in the islets of Langerhans. From studies in animal models, CD8(+) T cells recognizing autoantigens such as islet-specific glucose-6-phosphatase catalytic subunit-related protein, insulin, or glutamic acid decarboxylase (GAD) are believed to play important roles in both the early and late phases of beta cell destruction. In this study, we investigated the factors governing the diabetogenic potential of autoreactive CD8(+) clones isolated from spleens of NOD mice that had been immunized with GAD65(515-524) or insulin B-chain(15-23) peptides. Although these two clones were identical in most phenotypic and functional aspects, for example cytokine production and killing of autologous beta cells, they differed in the expression of IFN-gamma-inducible protein-10, which was only produced at high levels by the insulin-specific clone, but not by the GAD65-specific clone, and other autoantigen-specific nonpathogenic CD8 T cell clones. Interestingly, upon i.p. injection into neonatal mice, only the insulin B-chain(15-23)-reactive CD8(+) T clone accelerated diabetes in all recipients after 4 wk, although both insulin- and GAD-reactive clones homed to pancreas and pancreatic lymph nodes with similar kinetics. Diabetes was associated with increased pancreatic T cell infiltration and, in particular, recruitment of macrophages. Thus, secretion of IFN-gamma-inducible protein-10 by autoaggressive CD8(+) lymphocytes might determine their diabetogenic capacity by affecting recruitment of cells to the insulitic lesion.     

趋化因子CXCL14在SLE患者外周血单个核细胞中的表达及启动子甲基化分析

目的：分析趋化因子CXCL14在系统性红斑狼疮（SLE）患者外周血单个核细胞（PBMC）中的表达及其启动子甲基化特征。方法：收集28例SLE患者和20名健康人外周血单个核细胞（PBMC）标本,抽提细胞中RNA,逆转录后,以GAPDH为内参,通过定量PCR检测PBMC中CXCL14的表达,统计分析CXCL14表达水平与SLE各种临床资料的相关性,探讨PBMC中CXCL14表达与SLE的关系,通过重亚硫酸盐修饰的全基因组DNA用以BSP测序来明确不同标本中CXCL14启动子中甲基化位点的甲基化率。结果：CXCL14在SLE患者和正常人PBMC中的表达量存在显著差异（P < 0.05）。与健康人PBMC中CXCL14的含量相比较,CXCL14在SLE患者PBMC中表达量显著降低。与进一步CXCL14表达水平与SLE各种临床资料的相关性分析显示,CXCL14表达水平与SSB抗体（干燥综合征B抗体）、蛋白尿及血小板计数相关。与SSB抗体阴性SLE患者比较,SSB抗体阳性患者CXCL14表达水平更低（P <0.05）;与蛋白尿阴性SLE患者相比,蛋白尿阳性患者CXCL14表达水平更低（P < 0.05）;而血小板升高患者的CX-CL14表达水平更高（P < 0.05）。CXCL14表达水平与SLE活动、肾损害指标、抗ds-DNA、C反应蛋白（CRP）、补体C3水平等指标未见显著相关性。CXCL14启动子区甲基化分析显示,SLE患者CpG岛甲基化明显高于正常对照。结论：PBMC中低表达的CXCL14与SLE发生发展相关,其启动子区CpG岛过度甲基化是SLE患者CXCL14低表达的重要机制。