EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

Meteorin-like/Meteorin-ß Is a Novel Immunoregulatory Cytokine Associated with Inflammation.

We have described a novel cytokine encoded by a gene called Meteorin-like (Metrnl). Metrnl is a small (∼28 kDa) secreted protein expressed by activated macrophages and barrier tissues (mucosa and skin). Metrnl production by bone marrow macrophages is induced by several cytokines including TNF-α, IL-17α, IL-12, and IL-4 and inhibited by IFN-γ and TGF-ß. Metrnl expression in macrophages is also induced by LPS, and its levels in circulation are associated with inflammatory responses in vivo. Furthermore, Metrnl regulates the production of several cytokines and chemokines in macrophages. We have produced a Metrnl-/- mouse, which is viable and shows normal development. However, it exhibits dysregulated cytokine production, alterations in IgG production, and is highly susceptible to LPS in a sepsis model. Furthermore, older Metrnl-/- mice develop inflammatory lesions, suggesting that Metrnl participates in the control of inflammatory responses. Taken together, these observations indicate that Metrnl encodes a novel immunoregulatory cytokine associated with inflammatory responses that we have designated Meteorin-ß.

Identification of IL-40, a Novel B Cell-Associated Cytokine.

We describe a novel B cell-associated cytokine, encoded by an uncharacterized gene (C17orf99; chromosome 17 open reading frame 99), that is expressed in bone marrow and fetal liver and whose expression is also induced in peripheral B cells upon activation. C17orf99 is only present in mammalian genomes, and it encodes a small (∼27-kDa) secreted protein unrelated to other cytokine families, suggesting a function in mammalian immune responses. Accordingly, C17orf99 expression is induced in the mammary gland upon the onset of lactation, and a C17orf99-/- mouse exhibits reduced levels of IgA in the serum, gut, feces, and lactating mammary gland. C17orf99-/- mice have smaller and fewer Peyer's patches and lower numbers of IgA-secreting cells. The microbiome of C17orf99-/- mice exhibits altered composition, likely a consequence of the reduced levels of IgA in the gut. Although naive B cells can express C17orf99 upon activation, their production increases following culture with various cytokines, including IL-4 and TGF-ß1, suggesting that differentiation can result in the expansion of C17orf99-producing B cells during some immune responses. Taken together, these observations indicate that C17orf99 encodes a novel B cell-associated cytokine, which we have called IL-40, that plays an important role in humoral immune responses and may also play a role in B cell development. Importantly, IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observations suggest that it may play a role in the pathogenesis of certain human diseases.

Omics techniques and biobanks to find new biomarkers for the early detection of acute lymphoblastic leukemia in middle-income countries: a perspective from Mexico / Técnicas ómicas y biobancos para el hallazgo de nuevos biomarcadores para la detección temprana de leucemia linfoblástica aguda en países de ingresos medios: una perspectiva mexicana

Abstract: Acute lymphoblastic leukemia (ALL) affects the quality of life of many children in the world and particularly in Mexico, where a high incidence has been reported. With a proper financial investment and with well-organized institutions caring for those patients, together with solid platforms to perform high-throughput analyses, we propose the creation of a Mexican repository system of serum and cells from bone marrow and blood samples derived from tissues of pediatric patients with ALL diagnosis. This resource, in combination with omics technologies, particularly proteomics and metabolomics, would allow longitudinal studies, offering an opportunity to design and apply personalized ALL treatments. Importantly, it would accelerate the development of translational science and will lead us to further discoveries, including the identification of new biomarkers for the early detection of leukemia.

Resumen: La leucemia linfoblástica aguda (LLA) afecta la calidad de vida de una gran cantidad de individuos en edad pediátrica en todo el mundo; particularmente en México, donde se ha reportado una alta incidencia. Con un apropiado fondo de inversión financiera, así como instituciones adecuadamente organizadas al cuidado de los pacientes con LLA, en conjunto con plataformas sólidas para llevar a cabo análisis globales y de alto rendimiento, se propone la creación de un repositorio para la conservación de suero y células provenientes de médula ósea y sangre derivadas de pacientes pediátricos con LLA al diagnóstico. Estos recursos, en combinación con las tecnologías ómicas, en particular la proteómica y la metabolómica, podrían permitir el establecimiento de estudios longitudinales y ofrecer una oportunidad para el diseño y aplicación de tratamientos personalizados para la LLA. Esta estrategia permitiría acelerar el desarrollo de la ciencia traslacional, favoreciendo el hallazgo de importantes descubrimientos, incluyendo la identificación de nuevos biomarcadores para la detección temprana de la leucemia.

Transient Cannabinoid Receptor 2 Blockade during Immunization Heightens Intensity and Breadth of Antigen-specific Antibody Responses in Young and Aged mice.

The hallmark of vaccines is their ability to prevent the spread of infectious pathogens and thereby serve as invaluable public health tool. Despite their medical relevance, there is a gap in our understanding of the physiological factors that mediate innate and adaptive immune response to vaccines. The endocannabinoid (eCB) system is a critical modulator of homeostasis in vertebrates. Our results indicate that macrophages and dendritic cells produce the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG) upon antigen activation. We have also established that 2-AG levels are upregulated in the serum and in the lymph node of mice during vaccination. We hypothesized that the intrinsic release of eCBs from immune cells during activation by pathogenic antigens mitigate inflammation, but also suppress overall innate and adaptive immune response. Here we demonstrate, for the first time, that transient administration of the cannabinoid receptor 2 antagonist AM630 (10 mg/kg) or inverse agonist JTE907 (3 mg/kg) during immunization heightens the intensity and breadth of antigen-specific immune responses in young and aged mice through the upregulation of immunomodulatory genes in secondary lymphoid tissues.

Omics techniques and biobanks to find new biomarkers for the early detection of acute lymphoblastic leukemia in middle-income countries: a perspective from Mexico.

Acute lymphoblastic leukemia (ALL) affects the quality of life of many children in the world and particularly in Mexico, where a high incidence has been reported. With a proper financial investment and with well-organized institutions caring for those patients, together with solid platforms to perform high-throughput analyses, we propose the creation of a Mexican repository system of serum and cells from bone marrow and blood samples derived from tissues of pediatric patients with ALL diagnosis. This resource, in combination with omics technologies, particularly proteomics and metabolomics, would allow longitudinal studies, offering an opportunity to design and apply personalized ALL treatments. Importantly, it would accelerate the development of translational science and will lead us to further discoveries, including the identification of new biomarkers for the early detection of leukemia.

Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage.

M-CSF and GM-CSF are 2 important cytokines that regulate macrophage numbers and function. Here, we review their known effects on cells of the macrophage-monocyte lineage. Important clues to their function come from their expression patterns. M-CSF exhibits a mostly homeostatic expression pattern, whereas GM-CSF is a product of cells activated during inflammatory or pathologic conditions. Accordingly, M-CSF regulates the numbers of various tissue macrophage and monocyte populations without altering their "activation" status. Conversely, GM-CSF induces activation of monocytes/macrophages and also mediates differentiation to other states that participate in immune responses [i.e., dendritic cells (DCs)]. Further insights into their function have come from analyses of mice deficient in either cytokine. M-CSF signals through its receptor (CSF-1R). Interestingly, mice deficient in CSF-1R expression exhibit a more significant phenotype than mice deficient in M-CSF. This observation was explained by the discovery of a novel cytokine (IL-34) that represents a second ligand of CSF-1R. Information about the function of these ligands/receptor system is still developing, but its complexity is intriguing and strongly suggests that more interesting biology remains to be elucidated. Based on our current knowledge, several therapeutic molecules targeting either the M-CSF or the GM-CSF pathways have been developed and are currently being tested in clinical trials targeting either autoimmune diseases or cancer. It is intriguing to consider how evolution has directed these pathways to develop; their complexity likely mirrors the multiple functions in which cells of the monocyte/macrophage system are involved.

Cutting edge: GPR35/CXCR8 is the receptor of the mucosal chemokine CXCL17.

Chemokines are chemotactic cytokines that direct the traffic of leukocytes and other cells in the body. Chemokines bind to G protein-coupled receptors expressed on target cells to initiate signaling cascades and induce chemotaxis. Although the cognate receptors of most chemokines have been identified, the receptor for the mucosal chemokine CXCL17 is undefined. In this article, we show that GPR35 is the receptor of CXCL17. GPR35 is expressed in mucosal tissues, in CXCL17-responsive monocytes, and in the THP-1 monocytoid cell line. Transfection of GPR35 into Ba/F3 cells rendered them responsive to CXCL17, as measured by calcium-mobilization assays. Furthermore, GPR35 expression is downregulated in the lungs of Cxcl17(-/-) mice, which exhibit defects in macrophage recruitment to the lungs. We conclude that GPR35 is a novel chemokine receptor and suggest that it should be named CXCR8.

METEORIN-LIKE is a cytokine associated with barrier tissues and alternatively activated macrophages.

Cytokines are involved in many functions of the immune system including initiating, amplifying and resolving immune responses. Through bioinformatics analyses of a comprehensive database of gene expression (BIGE: Body Index of Gene Expression) we observed that a small secreted protein encoded by a poorly characterized gene called meteorin-like (METRNL), is highly expressed in mucosal tissues, skin and activated macrophages. Further studies indicate that Metrnl is produced by Alternatively Activated Macrophages (AAM) and M-CSF cultured bone marrow macrophages (M2-like macrophages). In the skin, METRNL is expressed by resting fibroblasts and IFNγ-treated keratinocytes. A screen of human skin-associated diseases showed significant over-expression of METRNL in psoriasis, prurigo nodularis, actinic keratosis and atopic dermatitis. METRNL is also up-regulated in synovial membranes of human rheumatoid arthritis. Taken together, these results indicate that Metrnl represents a novel cytokine, which is likely involved in both innate and acquired immune responses.

B cells participate in tolerance and autoimmunity through cytokine production.

In the 1950s, the discovery of autoantibodies produced by B cells seemed to provide a compelling mechanism underlying autoimmune diseases. The discovery of T regulatory cells and other T helper cell subsets shifted the field back towards a T cell central view. The success of rituxan, a chimeric mAb targeting CD20 on B cells, in the treatment of rheumatoid arthritis forced a review of the role of B cells in autoimmunity. Rituxan was first developed to treat lymphomas, and it also proved effective in treating rheumatoid arthritis, a disease not previously associated with B cells. One of the side effects of rituxan is a pronounced depletion of peripheral blood B cells, an effect that seemed to correlate with effectiveness in preclinical and clinical models of autoimmune diseases. B cell depletion was also shown to affect T cell populations, suggesting an antibody-independent mechanism through which B cells influenced rheumatic disease. Most recently, the identification of cytokine producing B cells (B regulatory and B effector cells) that modulate tolerance has added to our understanding of human health and disease and the mechanisms that break tolerance, as the B cell cytokine network produced by B cell subsets were shown to influence T cell numbers, as well as the polarization of T cell subsets (Tregs/Th1/Th2). Therefore, B cells have once again taken the center stage in tolerance and autoimmunity. Here, we review the role of B cells in autoimmunity, mainly through their ability to produce cytokines.

International Union of Basic and Clinical Pharmacology. [corrected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors.

Sixteen years ago, the Nomenclature Committee of the International Union of Pharmacology approved a system for naming human seven-transmembrane (7TM) G protein-coupled chemokine receptors, the large family of leukocyte chemoattractant receptors that regulates immune system development and function, in large part by mediating leukocyte trafficking. This was announced in Pharmacological Reviews in a major overview of the first decade of research in this field [Murphy PM, Baggiolini M, Charo IF, Hébert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, and Power CA (2000) Pharmacol Rev 52:145-176]. Since then, several new receptors have been discovered, and major advances have been made for the others in many areas, including structural biology, signal transduction mechanisms, biology, and pharmacology. New and diverse roles have been identified in infection, immunity, inflammation, development, cancer, and other areas. The first two drugs acting at chemokine receptors have been approved by the U.S. Food and Drug Administration (FDA), maraviroc targeting CCR5 in human immunodeficiency virus (HIV)/AIDS, and plerixafor targeting CXCR4 for stem cell mobilization for transplantation in cancer, and other candidates are now undergoing pivotal clinical trials for diverse disease indications. In addition, a subfamily of atypical chemokine receptors has emerged that may signal through arrestins instead of G proteins to act as chemokine scavengers, and many microbial and invertebrate G protein-coupled chemokine receptors and soluble chemokine-binding proteins have been described. Here, we review this extended family of chemokine receptors and chemokine-binding proteins at the basic, translational, and clinical levels, including an update on drug development. We also introduce a new nomenclature for atypical chemokine receptors with the stem ACKR (atypical chemokine receptor) approved by the Nomenclature Committee of the International Union of Pharmacology and the Human Genome Nomenclature Committee.

TSPAN33 is a novel marker of activated and malignant B cells.

We have identified Tspan33 as a gene encoding a transmembrane protein exhibiting a restricted expression pattern including expression in activated B cells. TSPAN33 is a member of the tetraspanin family. TSPAN33 is not expressed in resting B cells, but is strongly induced in primary human B cells following activation. Human 2E2 cells, a Burkitt's lymphoma-derived B cell model of activation and differentiation, also upregulate TSPAN33 upon activation. TSPAN33 is expressed in several lymphomas including Hodgkin's and Diffuse large B cell lymphoma. TSPAN33 is also expressed in some autoimmune diseases where B cells participate in the pathology, including rheumatoid arthritis patients, systemic lupus erythematosus (SLE), and in spleen B cells from MRL/Fas(lpr/lpr) mice (a mouse model of SLE). We conclude that TSPAN33 may be used as a diagnostic biomarker or as a target for therapeutic antibodies for treatment of certain B cell lymphomas or autoimmune diseases.

Systematic identification and characterization of novel human skin-associated genes encoding membrane and secreted proteins.

Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics.

Microarray analysis of port wine stains before and after pulsed dye laser treatment.

BACKGROUND AND OBJECTIVES: Neither the pathogenesis of port wine stain (PWS) birthmarks nor tissue effects of pulsed dye laser (PDL) treatment of these lesions is fully understood. There are few published reports utilizing gene expression analysis in human PWS skin. We aim to compare gene expression in PWS before and after PDL, using DNA microarrays that represent most, if not all, human genes to obtain comprehensive molecular profiles of PWS lesions and PDL-associated tissue effects. MATERIALS AND METHODS: Five human subjects had PDL treatment of their PWS. One week later, three biopsies were taken from each subject: normal skin (N); untreated PWS (PWS); PWS post-PDL (PWS + PDL). Samples included two lower extremity lesions, two facial lesions, and one facial nodule. High-quality total RNA isolated from skin biopsies was processed and applied to Affymetrix Human gene 1.0ST microarrays for gene expression analysis. We performed a 16 pair-wise comparison identifying either up- or down-regulated genes between N versus PWS and PWS versus PWS + PDL for four of the donor samples. The PWS nodule (nPWS) was analyzed separately. RESULTS: There was significant variation in gene expression profiles between individuals. By doing pair-wise comparisons between samples taken from the same donor, we were able to identify genes that may participate in the formation of PWS lesions and PDL tissue effects. Genes associated with immune, epidermal, and lipid metabolism were up-regulated in PWS skin. The nPWS exhibited more profound differences in gene expression than the rest of the samples, with significant differential expression of genes associated with angiogenesis, tumorigenesis, and inflammation. CONCLUSION: In summary, gene expression profiles from N, PWS, and PWS + PDL demonstrated significant variation within samples from the same donor and between donors. By doing pair-wise comparisons between samples taken from the same donor and comparing these results between donors, we were able to identify genes that may participate in formation of PWS and PDL effects. Our preliminary results indicate changes in gene expression of angiogenesis-related genes, suggesting that dysregulation of angiogenic signals and/or components may contribute to PWS pathology.

CXCL17 is a mucosal chemokine elevated in idiopathic pulmonary fibrosis that exhibits broad antimicrobial activity.

The mucosal immune network is a crucial barrier preventing pathogens from entering the body. The network of immune cells that mediates the defensive mechanisms in the mucosa is likely shaped by chemokines, which attract a wide range of immune cells to specific sites of the body. Chemokines have been divided into homeostatic or inflammatory depending upon their expression patterns. Additionally, several chemokines mediate direct killing of invading pathogens, as exemplified by CCL28, a mucosa-associated chemokine that exhibits antimicrobial activity against a range of pathogens. CXCL17 was the last chemokine ligand to be described and is the 17th member of the CXC chemokine family. Its expression pattern in 105 human tissues and cells indicates that CXCL17 is a homeostatic, mucosa-associated chemokine. Its strategic expression in mucosal tissues suggests that it is involved in innate immunity and/or sterility of the mucosa. To test the latter hypothesis, we tested CXCL17 for possible antibacterial activity against a panel of pathogenic and opportunistic bacteria. Our results indicate that CXCL17 has potent antimicrobial activities and that its mechanism of antimicrobial action involves peptide-mediated bacterial membrane disruption. Because CXCL17 is strongly expressed in bronchi, we measured it in bronchoalveolar lavage fluids and observed that it is strongly upregulated in idiopathic pulmonary fibrosis. We conclude that CXCL17 is an antimicrobial mucosal chemokine that may play a role in the pathogenesis of interstitial lung diseases.

Homeostatic chemokine receptors and organ-specific metastasis.

It has been 10 years since the role of a chemokine receptor, CXCR4, in breast cancer metastasis was first documented. Since then, the field of chemokines and cancer has grown significantly, so it is timely to review the progress, analyse the studies to date and identify future challenges facing this field. Metastasis is the major factor that limits survival in most patients with cancer. Therefore, understanding the molecular mechanisms that control the metastatic behaviour of tumour cells is pivotal for treating cancer successfully. Substantial experimental and clinical evidence supports the conclusion that molecular mechanisms control organ-specific metastasis. One of the most important mechanisms operating in metastasis involves homeostatic chemokines and their receptors. Here, we review this field and propose a model of 'cellular highways' to explain the effects of homeostatic chemokines on cancer cells and how they influence metastasis.

Gene expression patterns in livers of Hispanic patients infected with hepatitis C virus.

We report a gene expression study aimed at the identification of genes differentially expressed in the livers of Hispanic patients infected with hepatitis C virus (HCV). Six uninfected controls were compared with 14 HCV(+) patients in which the liver biopsies were obtained at the time of diagnosis. Among the latter, five patients were also analyzed 4 weeks after the onset of standard anti-HCV therapy (pegylated interferon-α + ribavirin). We identified many genes up- or down-regulated by the infection with HCV in the human livers. When these genes were subjected to pathway analysis, several prominent pathways were revealed including many interferon (IFN)-inducible pathways as well as immune cell trafficking, inflammation, anti-microbial responses, and even cancer. We detected expression of many genes that have previously been associated with HCV infection, as well as several novel genes including CD47. The genes induced by HCV infection showed large expression changes, whereas the genes induced by the IFN-α combination therapy were relatively few (including MX2, ORMDL3, GPAM, KOPX18, TMEM56, and HBP1) and they reflected relatively small expression changes. This is the first study to identify changes in gene expression in livers of HCV(+) Hispanic patients and the first to identify genes induced by anti-HCV combination therapy in the human liver.

Expression of Siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology.

Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor ß1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential roles for Siglec-11 in ovarian physiology and human evolution.