Diepoxybutane induces the expression of a novel p53-target gene XCL1 that mediates apoptosis in exposed human lymphoblasts.

Diepoxybutane (DEB) is the most potent active metabolite of the environmental chemical 1,3-butadiene (BD). BD is a human carcinogen that exhibits multiorgan systems toxicity. Our previous studies demonstrated that the X-C motif chemokine ligand 1 (XCL1) gene expression was upregulated 3.3-fold in a p53-dependent manner in TK6 lymphoblasts undergoing DEB-induced apoptosis. The tumor-suppressor p53 protein is a transcription factor that regulates a wide variety of cellular processes, including apoptosis, through its various target genes. Thus, the objective of this study was to determine whether XCL1 is a novel direct p53 transcriptional target gene and deduce its role in DEB-induced toxicity in human lymphoblasts. We utilized the bioinformatics tool p53scan to search for known p53 consensus sequences within the XCL1 promoter region. The XCL1 gene promoter region was found to contain the p53 consensus sequences 5'-AGACATGCCTAGACATGCCT-3' at three positions relative to the transcription start site (TSS). Furthermore, the XCL1 promoter region was found, through reporter gene assays, to be transactivated at least threefold by wild-type p53 promoter in DEB-exposed human lymphoblasts. Inactivation of the XCL1 promoter p53-binding motif located at -2.579 kb relative to TSS reduced the transactivation function of p53 on this promoter in DEB-exposed cells by 97%. Finally, knockdown of XCL1 messenger RNA with specific small interfering RNA inhibited DEB-induced apoptosis in human lymphoblasts by 50%. These observations demonstrate, for the first time, that XCL1 is a novel DEB-induced direct p53 transcriptional target gene that mediates apoptosis in DEB-exposed human lymphoblasts.

A functional variant near XCL1 gene improves breast cancer survival via promoting cancer immunity.

Most genome-wide association studies (GWASs) identify genetic variants for breast cancer occurrence. In contrast, few are for recurrence and mortality. We conducted a GWAS on breast cancer survival after diagnosis in estrogen receptor-positive patients, including 953 Taiwanese patients with 159 events. Through Cox proportional hazard models estimation, we identified 24 risk SNPs with p < 1 × 10-5 . Based on imputation and integrated analysis, one SNP, rs1024176 (located in 1q24.2, p = 2.43 × 10-5 ) was found to be a functional variant associated with breast cancer survival and XCL1 gene expression. A series of experimental approaches, including cell-based analyses and CRISPR/Cas9 genome-editing system, were then used and identified the transcription factor MYBL2 was able to discriminately bind to the A allele of rs1024176, the protective variant for breast cancer survival, which promoted XCL1 expression, but not to the G allele of rs1024176. The chemokine XCL1 attracts type 1 dendritic cells (DC1s) to the tumor microenvironment. In breast cancer tissues, we applied a two-step Mendelian randomization analysis, using expression quantitative trait loci as instrumental variables, to confirm higher XCL1 expression was correlated with higher DC1 signatures and favorable disease progression, through the causal effect of rs1024176-A allele. Our study supports the genetic effect on preventing breast cancer survival through XCL1-induced DC1 recruitment in tumor microenvironment.

Expression of Chemokine XCL2 and CX3CL1 in Lung Cancer.

BACKGROUND Chemokines are a family of small proteins secreted by cells with chemotactic activity, and they play important roles in cell adhesion. However, the expression of chemokine XCL2 and CX3CL1 in lung cancers in different pathological stages remains unclear. MATERIAL AND METHODS XCL2 and CX3CL1 expression in lung cancers and adjacent non-cancerous tissues was detected by quantitative PCR and ELISA. The relative expression of both chemokines in lung cancers in different pathological stages was compared by immunohistochemical assay. RESULTS The relative expression level of XCL2 and CX3CL1 in lung cancer was significantly higher compared with adjacent normal tissues (P<0.001). The expression level of both chemokines was significantly increased with higher pathological stages, as indicated by immunohistochemical assay (P<0.05 or P <0.001). Their expression level in cancers with higher numbers of metastatic lymph nodes was also significantly increased compared with cancers with lower numbers of metastatic lymph nodes (P<0.05 or P<0.001). CONCLUSIONS The expression of XCL2 and CX3CL1 increases with increasing degree of malignancy, indicating that both chemokines might be important targets in gene therapy for lung cancer.

Induction of chemokine (C-C motif) ligand 2 by sphingosine-1-phosphate signaling in neuroblastoma.

BACKGROUND/PURPOSE: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Preliminary data derived from a human angiogenesis array in NB showed that the bioactive lipid sphingosine-1-phosphate (S1P) induced the secretion of several angiogenesis-related proteins including the important inflammatory factor chemokine (C-C motif) ligand 2 (CCL2). In the present study, we investigated the mechanism of S1P-induced CCL2 expression in NB. METHODS: Quantitative real-time PCR and CCL2 ELISA were conducted to detect the mRNA expression and protein secretion of CCL2 in NB cells. Gain and loss of function studies were performed by using specific S1PR antagonists, adenoviral transduction and siRNA transfection. Macrophage F4/80 receptor in NB xenografts was detected by quantitative real-time PCR and immunohistochemistry staining. RESULTS: S1P induced CCL2 mRNA expression and protein secretion in a time- and concentration-dependent manner in NB cells. Blockade of S1P2 signaling using the selective S1P2 antagonist JTE-013 inhibited S1P-induced CCL2 expression. Overexpression of S1P2 by adenoviral transduction increased CCL2 secretion while knockdown of S1P2 by siRNA transfection decreased S1P-induced CCL2 secretion in NB cells. Macrophage infiltration, as detected by F4/80 staining, was significantly decreased in JTE-013-treated NB xenografts. CONCLUSIONS: Taken together, our data for the first time demonstrate that S1P induced the macrophage-recruiting factor CCL2 expression in NB cells via S1P2, providing new insights into the complicated functions of S1P2 in cancer.

The lymphotactin receptor is expressed in epithelial ovarian carcinoma and contributes to cell migration and proliferation.

Chemokine receptor-ligand interactions are important to support functioning of both normal and pathologic cells. The expression and function of chemokine receptors in epithelial ovarian carcinoma (EOC) is largely unknown. Here, we report that the lymphotactin receptor (XCR1) was expressed in primary and metastatic human epithelial ovarian carcinoma (EOC) specimens and cell lines. In contrast, expression of XCR1 was not detected in the normal ovary or in human normal ovarian surface epithelial cells. Our data indicate that XCL1 and XCL2 are either present in the malignant ascites or expressed by the ovarian carcinoma cells. The addition of lymphotactin (XCL1 and XCL2) stimulated migration and proliferation of XCR1-positive cells. Reduction of XCR1 expression in ovarian carcinoma cell line SKOV-3 resulted in abrogated diaphragm and peritoneal wall tumor formation and in reduced frequency of colonic, splenetic, and liver nodules in an in vivo xenograft mouse model. Taken together, our data suggest that XCR1 is expressed early during the course of tumorigenic transformation and contributes towards increased cell migration and proliferation, which can facilitate the prometastatic behavior of EOC cells.

T inflammatory memory CD8 T cells participate to antiviral response and generate secondary memory cells with an advantage in XCL1 production.

Besides the classically described subsets of memory CD8 T cells generated under infectious conditions, are T inflammatory memory cells generated under sterile priming conditions, such as sensitization to allergens. Although not fully differentiated as pathogen-induced memory cells, they display memory properties that distinguish them from naive CD8 T cells. Given these memory cells are generated in an antigen-specific context that is devoid of pathogen-derived danger signals and CD4 T cell help, we herein questioned whether they maintained their activation and differentiation potential, could be recruited in an immune response directed against a pathogen expressing their cognate antigen and further differentiate in fully competent secondary memory cells. We show that T inflammatory memory cells can indeed take part to the immune response triggered by a viral infection, differentiate into secondary effectors and further generate typical central memory CD8 T cells and effector memory CD8 T cells. Furthermore, the secondary memory cells they generate display a functional advantage over primary memory cells in their capacity to produce TNF-α and the XCL1 chemokine. These results suggest that cross-reactive stimulations and differentiation of cells directed against allergens or self into fully competent pathogen-induced memory cells might have incidences in inflammatory immuno-pathologies.

Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development.

Dendritic cells (DCs) in the thymus (tDCs) are predominantly accumulated in the medulla and contribute to the establishment of self-tolerance. However, how the medullary accumulation of tDCs is regulated and involved in self-tolerance is unclear. We show that the chemokine receptor XCR1 is expressed by tDCs, whereas medullary thymic epithelial cells (mTECs) express the ligand XCL1. XCL1-deficient mice are defective in the medullary accumulation of tDCs and the thymic generation of naturally occurring regulatory T cells (nT reg cells). Thymocytes from XCL1-deficient mice elicit dacryoadenitis in nude mice. mTEC expression of XCL1, tDC medullary accumulation, and nT reg cell generation are diminished in Aire-deficient mice. These results indicate that the XCL1-mediated medullary accumulation of tDCs contributes to nT reg cell development and is regulated by Aire.

Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice.

Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vaccine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine XCR1 was the only chemokine receptor selectively expressed in CD8alpha(+) conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8(+) T cells increased XCL1 production upon activation. These expression patterns were conserved in human blood cells, including the BDCA3(+) DC subset. Thus, in human and mice, certain DC subsets should be chemotactic towards NK or activated CD8(+) T cells through XCR1.

Production of biologically active human lymphotactin (XCL1) by Lactococcus lactis.

Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8+ T-cells and directs migration of CD4+ and CD8+ lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level.

XCL1 enhances regulatory activities of CD4+ CD25(high) CD127(low/-) T cells in human allergic asthma.

Chemokine-mediated recruitment of regulatory cell subsets to the airway during inflammation and enhancement of their activities are potential strategies for therapeutic development in allergic asthma (AA). In this study, we aim to explore the role of XCL1, a chemokine associated with immune suppression and allergy, on CD4(+)CD25(high)CD127(low/-) regulatory T cell (Treg) function in AA. Flow cytometry and PCR analysis showed a reduction in XCL1 and XCR1 expression in AA Treg compared with healthy control and nonallergic asthmatic counterparts. This reduction in XCL1 expression was associated with the suboptimal regulatory function of Treg in AA. Interestingly, incubation with recombinant human XCL1 significantly increased Treg-mediated suppression and cytotoxicity by up-regulating expression of XCL1 and chief effector molecules of Treg function. Altogether, these results suggest an association between dysregulated XCL1 expression and reduced Treg activities in AA, as well as a potential role of XCL1 in reversing defective Treg function in the disease.

Mycobacterium tuberculosis antigen Wag31 induces expression of C-chemokine XCL2 in macrophages.

Tuberculosis is still a major threat to human health. To date, only approximately half of the proteins encoded by Mycobacterium tuberculosis H37Rv have been assigned specific functions. Wag31 (Rv2145c) is one of the bacterial proteins whose function is mostly unknown. Using a modified split-ubiquitin membrane yeast two-hybrid system, we screened a macrophage cDNA library with Wag31 as bait and identified XCL2, a C-subfamily chemokine, as a binding partner for Wag31. More importantly, Wag31 was found to specifically stimulate XCL2 expression in macrophages. The results from this study demonstrate that expression of C-chemokine is not restricted to certain types of T cells and natural killer cells. Because C-chemokine is chemotactic for CD8+ and CD4+ T cells, our novel findings could provide a new mechanism by which the bacteria induce cell-mediated immunity and by which Wag31 could be a potential target for controlling M. tuberculosis infection.

Protein expression pattern in experimental pneumococcal meningitis.

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.

Induction of C chemokine XCL1 (lymphotactin/single C motif-1 alpha/activation-induced, T cell-derived and chemokine-related cytokine) expression by HIV-1 Tat protein.

HIV-1 Tat has been proposed as a key agent in many AIDS-related disorders, including HIV-1-associated neurological diseases. We have recently shown that Tat expression induces a significant increase in T lymphocytes in the brains of Tat transgenic mice. The CNS infiltration of T lymphocytes has been noted in AIDS patients. In the present study using this unique genetic system we attempted to understand the underlying mechanisms of Tat expression-induced infiltration of T lymphocytes by examining chemokine expression. RNase protection assay revealed that in addition to CCL2 (monocyte chemoattractant protein-1), CCL3 (macrophage inflammatory protein-1alpha (MIP-1alpha)), CCL4 (MIP-1beta), CCL5 (RANTES), CXCL2 (MIP-2), and CXCL10 (inducing protein-10), XCL1 (lymphotactin/single C motif-1alpha/activation-induced, T cell-derived and chemokine-related cytokine) was identified to be up-regulated by Tat expression. XCL1 is a C chemokine and plays a specific and important role in tissue-specific recruitment of T lymphocytes. Thus, we further determined the relationship between Tat and XCL1 expression. Tat-induced XCL1 expression was further confirmed by XCL1-specific RT-PCR and ELISA. Combined in situ hybridization and immunohistochemical staining identified astrocytes, monocytes, and macrophages/microglia as XCL1-producing cells in vivo. Using human astrocytes, U87.MG cells, as an in vitro model, activation of XCL1 expression was positively correlated with Tat expression. Moreover, the XCL1 promoter-driven reporter gene assay showed that Tat-induced XCL1 expression occurred at the transcriptional level. Taken together, these results demonstrate that Tat directly trans-activated XCL1 expression and suggest potential roles of Tat-induced XCL1 expression in the CNS infiltration of T lymphocytes during HIV-1 infection and subsequent HIV-1-induced neurological diseases.

CD8+ alpha beta+ T cells that lack surface CD5 antigen expression are a major lymphotactin (XCL1) source in peripheral blood lymphocytes.

To better characterize the cellular source of lymphotactin (XCL1), we compared XCL1 expression in different lymphocyte subsets by real-time PCR. XCL1 was constitutively expressed in both PBMC and CD4(+) cells, but its expression was almost 2 log higher in CD8(+) cells. In vitro activation was associated with a substantial increase in XCL1 expression in both PBMC and CD8(+) cells, but not in CD4(+) lymphocytes. The preferential expression of XCL1 in CD8(+) cells was confirmed by measuring XCL1 production in culture supernatants, and a good correlation was found between figures obtained by real-time PCR and XCL1 contents. XCL1 expression was mostly confined to a CD3(+)CD8(+) subset not expressing CD5, where XCL1 expression equaled that shown by gammadelta(+) T cells. Compared with the CD5(+) counterpart, CD3(+)CD8(+)CD5(-) cells, which did not express CD5 following in vitro activation, showed preferential expression of the alphaalpha form of CD8 and a lower expression of molecules associated with a noncommitted/naive phenotype, such as CD62L. CD3(+)CD8(+)CD5(-) cells also expressed higher levels of the XCL1 receptor; in addition, although not differing from CD3(+)CD8(+)CD5(+) cells in terms of the expression of most alpha- and beta-chemokines, they showed higher expression of CCL3/macrophage inflammatory protein-1alpha. These data show that TCR alphabeta-expressing lymphocytes that lack CD5 expression are a major XCL1 source, and that the contribution to its synthesis by different TCR alphabeta-expressing T cell subsets, namely CD4(+) lymphocytes, is negligible. In addition, they point to the CD3(+)CD8(+)CD5(-) population as a particular T cell subset within the CD8(+) compartment, whose functional properties deserve further attention.

Evidence of CXC, CC and C chemokine production by lymphatic endothelial cells.

Although production of chemokines by vascular endothelial cells has been documented, there is only limited information regarding the expression of chemokines by the lymphatic endothelium. Here we used lymphatic endothelial cells (LEC) derived from experimentally induced murine lymphangiomas to investigate the pattern of chemokine expression by these cells. Histological analysis of the lymphatic hyperplasia revealed the presence of leucocytes in the tissues surrounding the lesions, suggesting the presence of chemoattractant activity. A functional chemotactic assay on human polymorphonuclear cells and on purified subpopulations of murine leucocytes using culture supernatants from LEC primary cultures confirmed the presence of chemoattractant activity. The identity of different cytokines of the CXC, CC and C subfamilies was investigated by reverse trancriptase-polymerase chain reaction on total endothelial cell RNA. Amplified fragments corresponding to KC, IP10, Mig-1, BCL, MIP-2, SLC, RANTES, MCP-1, C10, and Lptn were obtained, and confirmed by Southern blot and sequencing. In contrast, MIP-1alpha, MIP-1beta, and MIP-1gamma were not detected. Higher levels of expression were revealed by Northern blot analysis for Mig-1, MCP-1 and C10. The lymphatic endothelium-restricted production of these chemokines was also confirmed by in situ hybridization. The presence of high C10 mRNA expression levels in LEC was particularly unexpected, because the production of this molecule has been previously identified only in cells of the haematopoietic lineage. These observations represent the first detailed analysis of chemokine production by lymphatic endothelial cells and may account, in part, for the mechanism of leucocyte recruitment into the lymphatics, and of lymphocyte recirculation within the lymphatic system.

Monomeric solution structure of the prototypical 'C' chemokine lymphotactin.

Lymphotactin, the sole identified member of the C class of chemokines, specifically attracts T lymphocytes and natural killer cells. This 93-residue protein lacks 2 of the 4 conserved cysteine residues characteristic of the other 3 classes of chemokines and possesses an extended carboxyl terminus, which is required for chemotactic activity. We have determined the three-dimensional solution structure of recombinant human lymphotactin by NMR spectroscopy. Under the conditions used for the structure determination, lymphotactin was predominantly monomeric; however, pulsed field gradient NMR self-diffusion measurements and analytical ultracentrifugation revealed evidence of dimer formation. Sequence-specific chemical shift assignments were determined through analysis of two- and three-dimensional NMR spectra of (15)N- and (13)C/(15)N-enriched protein samples. Input for the torsion angle dynamics calculations used in determining the structure included 1258 unique NOE-derived distance constraints and 60 dihedral angle constraints obtained from chemical-shift-based searching of a protein conformational database. The ensemble of 20 structures chosen to represent the structure had backbone and heavy atom rms deviations of 0.46 +/- 0.11 and 1.02 +/- 0.14 A, respectively. The results revealed that human lymphotactin adopts the conserved chemokine fold, which is characterized by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. Two regions are dynamically disordered as evidenced by (1)H and (13)C chemical shifts and [(15)N]-(1)H NOEs: residues 1-9 of the amino terminus and residues 69-93 of the C-terminal extension. A functional role for the C-terminal extension, which is unique to lymphotactin, remains to be elucidated.