Alessandra Saccani

Regulation of the Chemokine Receptor CXCR4 by Hypoxia

Cell adaptation to hypoxia (Hyp) requires activation of transcriptional programs that coordinate expression of genes involved in oxygen delivery (via angiogenesis) and metabolic adaptation (via glycolysis). Here, we describe that oxygen availability is a determinant parameter in the setting of chemotactic responsiveness to stromal-derived factor 1 (CXCL12). Low oxygen concentration induces high expression of the CXCL12 receptor, CXC receptor 4 (CXCR4), in different cell types (monocytes, monocyte-derived macrophages, tumor-associated macrophages, endothelial cells, and cancer cells), which is paralleled by increased chemotactic responsiveness to its specific ligand. CXCR4 induction by Hyp is dependent on both activation of the Hyp-inducible factor 1 α and transcript stabilization. In a relay multistep navigation process, the Hyp–Hyp-inducible factor 1 α–CXCR4 pathway may regulate trafficking in and out of hypoxic tissue microenvironments.

Autocrine Production of IL-10 Mediates Defective IL-12 Production and NF-κB Activation in Tumor-Associated Macrophages

IL-12 is a central cytokine in the activation of inflammation and immunity and in the generation of Th1-type responses. Tumor-associated macrophages (TAM) from mouse and human tumors showed defective production of IL-12. Defective IL-12 production was associated with lack of p50/p65 NF-κB activation. TAM produced increased amounts of the immunosuppressive cytokine IL-10. Abs against IL-10 restored the defective capacity of TAM to produce IL-12. Our data suggest that during tumor growth an IL-10-dependent pathway of diversion of macrophage function can be activated into the tumor microenvironment and results in the promotion of the IL-10+ IL-12− phenotype of TAM. Blocking IL-10, as well as other immunosuppressive cytokines present in the tumor microenvironment, such as TGF-β, may complement therapeutic strategies aimed at activating type I antitumor immune responses.

p50 nuclear factor-κB overexpression in tumor-associated macrophages inhibits M1 inflammatory responses and antitumor resistance

Tumor-associated macrophages (TAM) are a major inflammatory infiltrate in tumors and a major component of the protumor function of inflammation. TAM in established tumors generally have an M2 phenotype with defective production of interleukin-12 (IL-12) and high IL-10. Here, we report that defective responsiveness of TAM from a murine fibrosarcoma and human ovarian carcinoma to M1 activation signals was associated with a massive nuclear localization of the p50 nuclear factor-kappaB (NF-kappaB) inhibitory homodimer. p50 overexpression inhibited IL-12 expression in normal macrophages. TAM isolated from p50(-/-) mice showed normal production of M1 cytokines, associated with reduced growth of transplanted tumors. Bone marrow chimeras showed that p50 inactivation in hematopoietic cells was sufficient to result in reduced tumor growth. Thus, p50 NF-kappaB overexpression accounts for the inability of TAM to mount an effective M1 antitumor response capable of inhibiting tumor growth.

Defective Expression of the Monocyte Chemotactic Protein-1 Receptor CCR2 in Macrophages Associated with Human Ovarian Carcinoma

Monocyte chemotactic protein-1 (MCP-1, CCL2) is an important determinant of macrophage infiltration in tumors, ovarian carcinoma in particular. MCP-1 binds the chemokine receptor CCR2. Recent results indicate that proinflammatory and anti-inflammatory signals regulate chemokine receptor expression in monocytes. The present study was designed to investigate the expression of CCR2 in tumor-associated macrophages (TAM) from ovarian cancer patients. TAM isolated from ascitic or solid ovarian carcinoma displayed defective CCR2 mRNA (Northern blot and PCR) and surface expression and did not migrate in response to MCP-1. The defect was selective for CCR2 in that CCR1 and CCR5 were expressed normally in TAM. CCR2 gene expression and chemotactic response to MCP-1 were decreased to a lesser extent in blood monocytes from cancer patients. CCR2 mRNA levels and the chemotactic response to MCP-1 were drastically reduced in fresh monocytes cultured in the presence of tumor ascites from cancer patients. Ab against TNF-α restored the CCR2 mRNA level in monocytes cultured in the presence of ascitic fluid. The finding of defective CCR2 expression in TAM, largely dependent on local TNF production, is consistent with previous in vitro data on down-regulation of chemokine receptors by proinflammatory molecules. Receptor inhibition may serve as a mechanism to arrest and retain recruited macrophages and to prevent chemokine scavenging by mononuclear phagocytes at sites of inflammation and tumor growth. In the presence of advanced tumors or chronic inflammation, systemic down-regulation of receptor expression by proinflammatory molecules leaking in the systemic circulation may account for defective chemotaxis and a defective capacity to mount inflammatory responses associated with advanced neoplasia.

Role of the autocrine chemokines MIP-1α and MIP-1β in the metastatic behavior of murine T cell lymphoma

The ESb‐MP T‐cell line is a highly malignant murine lymphoma, which preferentially metastasizes toward the kidney. This could be a result of the local production of monocyte chemoattractant protein‐1 (MCP‐1) and regulated on activation, normal T expressed and secreted (RANTES), which are chemotactic for ESb‐MP cells. Here, we demonstrate that ESb‐MP cells are already responsive to the chemotactic activity of macrophage inflammatory protein‐1α (MIP‐1α) and MIP‐1β from 1 ng/ml onward. Moreover, upon stimulation with lipopolysaccharide (LPS) or virus, ESb‐MP cells themselves produce significant amounts of MIP‐1 (∼200 ng/ml). Indeed, the major autocrine chemoattractants, isolated from ESb‐MP cells, were intact MIP‐1α and MIP‐1β. Pretreatment with LPS or addition of MIP‐1 inhibited the in vitro migration of ESb‐MP cells toward various chemokines. Moreover, compared with untreated lymphoma cells, LPS‐treated cells produced significantly less metastasis in mice. The results represented here suggest that the role of chemokines in attracting tumor cells at secondary sites depends on a balance between autocrine‐produced and tissue‐derived chemokines. This delicate balance should be considered in the design of antichemokine strategies in different tumor types.

Divergent effects of dithiocarbamates on AP-1-containing and AP-1-less NFAT sites.

Changes in the redox status of cells affect NF‐κB and activator protein (AP)‐1 nuclear expression and activity. In particular, antioxidants decrease NF‐κB and increase AP‐1 transcriptional activity, thereby regulating gene expression. In T cells, low concentrations of antioxidants enhance IL‐2 and inhibit IL‐4 expression. Since NFAT binding sites play an essential role in regulating IL‐2 and IL‐4 gene transcription, we studied the effects of dithiocarbamates, using the pyrrolidine derivative of dithiocarbamate (PDTC), on the activity of the distinct AP‐1‐containing IL‐2 NFAT and AP‐1‐less IL‐4 NFAT enhancers elements. Consistent with the presence of AP‐1 proteins within the IL‐2 NFAT complex, PDTC strongly enhanced phorbol 12‐myristate 13‐acetate/phytohemagglutinin‐induced NFAT binding to the IL‐2 NFAT enhancer and transcriptional activity of a reporter plasmid driven by this NFAT enhancer. In contrast, the activity of the IL‐4 NFp enhancer, which does not bind AP‐1, was abolished by PDTC treatment. In the Jurkat T cell line treated with PDTC, co‐expression of the Ca2+/calmodulin‐dependent phosphatase, calcineurin, completely restored the IL‐4 NFp enhancer activity. Our data indicate that calcineurin‐mediated NFAT activity is a target for antioxidants and provides new insights into the molecular mechanisms controlling differential cytokine gene expression.