Angel Zaballos

Tumour biology: Senescence in premalignant tumours

Oncogene-induced senescence is a cellular response that may be crucial for protection against cancer development, but its investigation has so far been restricted to cultured cells that have been manipulated to overexpress an oncogene. Here we analyse tumours initiated by an endogenous oncogene, ras, and show that senescent cells exist in premalignant tumours but not in malignant ones. Senescence is therefore a defining feature of premalignant tumours that could prove valuable in the diagnosis and prognosis of cancer.

CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses

CCR6 expression in dendritic, T, and B cells suggests that this beta-chemokine receptor may regulate the migration and recruitment of antigen-presenting and immunocompetent cells during inflammatory and immunological responses. Here we demonstrate that CCR6-/- mice have underdeveloped Peyers patches, in which the myeloid CD11b+ CD11c+ dendritic-cell subset is not present in the subepithelial dome. CCR6-/- mice also have increased numbers in T-cell subpopulations within the intestinal mucosa. In 2,4-dinitrofluorobenzene-induced contact hypersensitivity (CHS) studies, CCR6-/- mice developed more severe and more persistent inflammation than wild-type (WT) animals. Conversely, in a delayed-type hypersensitivity (DTH) model induced with allogeneic splenocytes, CCR6-/- mice developed no inflammatory response. The altered responses seen in the CHS and DTH assays suggest the existence of a defect in the activation and/or migration of the CD4(+) T-cell subsets that downregulate or elicit the inflammation response, respectively. These findings underscore the role of CCR6 in cutaneous and intestinal immunity and the utility of CCR6-/- mice as a model to study pathologies in these tissues. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Bcl-3 Expression Promotes Cell Survival following Interleukin-4 Deprivation and Is Controlled by AP1 and AP1-Like Transcription Factors

ABSTRACT We have analyzed the interleukin-4 (IL-4)-triggered mechanisms implicated in cell survival and show here that IL-4 deprivation induces apoptotic cell death but does not modulate Bcl-2 or Bcl-x expression. Since Bcl-x expression is insufficient to ensure cell survival in the absence of IL-4, we speculate that additional molecules replace the antiapoptotic role of Bcl-2 and Bcl-x in an alternative IL-4-triggered pathway. Cell death is associated with Bcl-3 downregulation and Bcl-3 expression blocks IL-4 deprivation-induced apoptosis, suggesting that Bcl-3 acts as a survival factor in the absence of growth factor. To characterize the IL-4-induced regulation of murine Bcl-3 expression, we cloned the promoter of this gene. Sequencing of the promoter showed no TATA box element but did reveal binding sites for AP1, AP1-like, and SP1 transcription factors. Retardation gels showed that IL-4 specifically induces AP1 and AP1-like binding activity and that mutation of these binding sites abolishes the IL-4-induced Bcl-3 promoter activity, suggesting that these transcription factors are important in Bcl-3 promoter transactivation. IL-4 deprivation induces downregulation of Jun expression and upregulation of Fos expression, both of which are proteins involved in the formation of AP1 and AP1-like transcription factors. Overexpression of Jun family proteins transactivates the promoter and restores Bcl-3 expression in the absence of IL-4 stimulation. Taken together, these data describe a new biological role for Bcl-3 and define the regulatory pathway implicated in Bcl-3 expression.

CCR6 regulates EAE pathogenesis by controlling regulatory CD4+ T-cell recruitment to target tissues.

The T‐cell subsets, characterized by their cytokine production profiles and immune regulatory functions, depend on correct in vivo location to interact with accessory or target cells for effective immune responses. Differentiation of naive CD4+ T cells into effectors is accompanied by sequentially regulated expression of the chemokine receptors responsible for cell recruitment to specific tissues. We studied CCR6 function in EAE, a CD4+ T‐cell‐mediated CNS disease characterized by mononuclear infiltration and demyelination. CCR6−/− mice showed an altered time course of EAE development, with delayed onset, a higher clinical score, and more persistent symptoms than in controls. An imbalanced cytokine profile and reduced Foxp3+ cell frequency characterized CNS tissues from CCR6−/− compared with CCR6+/+ mice during the disease effector phase. Transfer of CCR6+/+ Treg to CCR6−/− mice the day before EAE induction reduced the clinical score associated with an increased in infiltrating Foxp3+ cells and recovery of the cytokine balance in CCR6−/− mouse CNS. Competitive assays between CCR6+/+ and CCR6−/− Treg adoptively transferred to CCR6−/− mice showed impaired ability of CCR6−/− Treg to infiltrate CNS tissues in EAE‐affected mice. Our data indicate a CCR6 requirement by CD4+ Treg to downregulate the CNS inflammatory process and neurological signs associated with EAE.

Absence of CCR8 Does Not Impair the Response to Ovalbumin-Induced Allergic Airway Disease

Interaction of chemokines with their specific receptors results in tight control of leukocyte migration and positioning. CCR8 is a chemokine receptor expressed mainly in CD4+ single-positive thymocytes and Th2 cells. We generated CCR8-deficient mice (CCR8−/−) to study the in vivo role of this receptor, and describe in this study the CCR8−/− mouse response in OVA-induced allergic airway disease using several models, including an adoptive transfer model and receptor-blocking experiments. All CCR8−/− mice developed a pathological response similar to that of wild-type animals with respect to bronchoalveolar lavage cell composition, peripheral blood and bone marrow eosinophilia, lung infiltrates, and Th2 cytokine levels in lung and serum. The results contrast with a recent report using one of the OVA-induced asthma models studied here. Similar immune responses were also observed in CCR8−/− and wild-type animals in a different model of ragweed allergen-induced peritoneal eosinophilic inflammation, with an equivalent number of eosinophils and analogous increased levels of Th2 cytokines in peritoneum and peripheral blood. Our results show that allergic diseases course without critical CCR8 participation, and suggest that further work is needed to unravel the in vivo role of CCR8 in Th2-mediated pathologies.

Molecular cloning, functional characterization and mRNA expression analysis of the murine chemokine receptor CCR6 and its specific ligand MIP‐3α1

We have cloned the murine CCR6 receptor and its ligand, the β‐chemokine mMIP‐3α. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP‐3α. Murine MIP‐3α RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT‐PCR analysis of FACS‐sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8− splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP‐3α will allow the study of the role of these proteins in mouse models of inflammation and immunity.

Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice

Lymphocyte infiltration to pancreatic islets is associated to chemoattraction, as are other inflammatory autoimmune processes. We examined whether development of insulitis and diabetes dependson chemoattraction of lymphocytes via the CCR5 chemokine receptor. In non‐obese diabetic (NOD) mice, a substantial fraction of peripheral T cells and virtually all B cells expressed high CCR5 levels. CCR5 expression characterized the effector T cell phenotype, suggesting their potential involvement in disease development. In view of these findings and the CCL5 (RANTES, the CCR5 ligand) expression by pancreatic islets, we treated NOD mice with a neutralizing anti‐CCR5 antibody. This did not influence peri‐insulitis advancement, but inhibited β‐cell destruction and diabetes. These data demonstrate a role of CCR5‐dependent chemoattraction in insulitis progression to islet destruction, suggesting the potential value of therapeutic intervention by CCR5 targeting.

Functional inactivation of CXC chemokine receptor 4-mediated responses through SOCS3 up-regulation

Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca2+ flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Gαi pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system.

Statins Induce Regulatory T Cell Recruitment via a CCL1 Dependent Pathway

The statins, a group of inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, are reported to influence a variety of immune system activities through 3-hydroxy-3-methylglutaryl coenzyme A reductase-dependent and -independent mechanisms. How statin treatment regulates immune system function in vivo nonetheless remains to be fully defined. We analyzed the immunomodulatory effects of lovastatin in a Candida albicans-induced delayed-type hypersensitivity reaction in mice. In this model, lovastatin administration reduced the acute inflammatory response elicited by C. albicans challenge. This anti-inflammatory activity of lovastatin was associated with a shift from a Th1 to a Th2 immune response, as well as an increase in the percentage of regulatory T cells at the inflammation site and in the regional draining lymph node. The lovastatin-induced increase in regulatory T cells in the inflamed skin was dependent on expression of CCL1, a chemokine that is locally up-regulated by statin administration. The anti-inflammatory effect of lovastatin was abrogated in CCL1-deficient mice. These results suggest that local regulation of chemokine expression may be an important process in statin-induced modulation of the immune system.

Down-regulation of the beta-chemokine receptor CCR6 in dendritic cells mediated by TNF-alpha and IL-4.

Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response. We have generated DC from human umbilical cord blood CD34+ progenitors cultured with granulocyte‐macrophage colony‐stimulating factor, tumor necrosis factor α (TNF‐α), and stem cell factor. Using an anti‐CCR6 monoclonal antibody, we observed that these cells showed maximum expression of this β‐chemokine receptor when they were immature, as determined by their relatively low expression of several DC maturation markers such as CD1a, CD11c, CD14, CD40, CD80, and CD83. Immature DC responded strongly to macrophage inflammatory protein‐3α (MIP‐3α), the CCR6 ligand, in migration and calcium mobilization assays. CCR6 expression decreased in parallel with the DC maturation induced by prolonged TNF‐α treatments. Interleukin‐4 was also able to decrease CCR6 protein levels. Our findings suggest that the MIP‐3α/CCR6 interaction plays an important role in the trafficking of immature DC to chemokine production sites such as injured or inflamed peripheral tissues, where DC undergo maturation on contact with antigens. J. Leukoc. Biol. 66: 837–844; 1999.