Concomitant expression of the chemokines RANTES and MCP-1 in human breast cancer: a basis for tumor-promoting interactions.

The chemokines RANTES (CCL5) and MCP-1 (CCL2) were suggested to contribute, independently, to breast malignancy. In the present study, we asked if the two chemokines are jointly expressed in clinical samples of breast cancer patients, and do they interact in breast tumor cells. We found that RANTES and MCP-1 were expressed by breast tumor cells in primary tumors of Ductal Carcinoma In Situ and of Invasive Ductal Carcinoma, but minimally in normal breast epithelial duct cells. The chemokines were also detected in metastases and pleural effusions. Novel findings showed that co-expression of RANTES and MCP-1 in the same tumor was associated with more advanced stages of disease, suggesting that breast tumors "benefit" from interactions between the two chemokines. Accordingly, MCP-1 significantly promoted the release of RANTES from endogenous pre-made vesicles, in an active process that depended on calcium from intracellular and extracellular sources, and on intracellular transport of RANTES towards exocytosis. Our findings show a chemokine-triggered release of stored pro-malignancy chemokine from breast tumor cells. These observations support a major tumor-promoting role for co-expression of the chemokines in breast malignancy, and agree with the significant association of joint RANTES and MCP-1 expression with advanced stages of breast cancer.

Interleukin 8 and cell migration to inflammatory sites: the regulation of focal adhesion kinase under conditions of migratory desensitization.

BACKGROUND: Interleukin 8 is a prototypical inflammatory chemokine that induces leukocyte migration to inflammatory sites. Leukocyte recruitment in response to gradients of this chemokine is attenuated at advanced stages of inflammation to prevent damage to surrounding healthy tissues. Our published studies suggest that over-phosphorylation of focal adhesion kinase in migration-desensitizing conditions is involved in cessation of cell motility. This over-phosphorylation of FAK was induced by IL-8 only when the receptor transmitting the chemokine signals was CXCR2 and not CXCR1, indicating that the two IL-8 receptors diverge in their signaling properties. OBJECTIVES: To analyze the regulation of FAK in CXCR2-expressing hematopoietic cells under conditions of migratory desensitization, focusing on the roles played by adhesion-related components in this process. METHODS: Under conditions of migratory desensitization, we determined IL-8-induced cell spreading and FAK localization following disruption of actin filaments, and evaluated the role of integrins in FAK phosphorylation. RESULTS: The disturbance of intact activity of actin filaments resulted in inhibition of cell spreading and modification of FAK intracellular localization upon IL-8 stimulation. Also, adhesion-dependent pre-stimulation of integrins was required for IL-8-induced FAK phosphorylation. CONCLUSIONS: Intact actin filaments and integrins are required for optimal IL-8-induced FAK phosphorylation in conditions of migratory desensitization. These observations suggest that lack of adequate activity/regulation of adhesion-related components may give rise to FAK activities that are not appropriately controlled, possibly leading to pathological conditions that are associated with perturbed leukocyte migration phenotypes.

Reduced expression of chemoattractant receptors by polymorphonuclear leukocytes in Hyper IgE Syndrome patients.

Hyper IgE Syndrome (HIES) is a rare genetic disorder, characterized by elevated serum IgE levels and reduced inflammatory responses to bacterial infections. This leads to dermatitis, recurrent lung infections and "cold abscesses". Recently, progress was made in HIES research, when mutations in STAT3 were found in the autosomal dominant form of HIES, and impaired responses of T helper 17 cells were reported. However, the causes for reduced inflammatory responses in these patients were not fully elucidated. In view of studies that indicated that polymorphonuclear leukocytes (PMN) of HIES patients are defective in their chemotactic properties, we asked if the PMN of these patients have reduced expression of receptors for chemoattractants. To analyze this possibility, we focused on fMLP and ELR(+)-CXC chemokines - which are essential for mounting acute inflammatory responses - and determined the coding sequences and expression levels of their corresponding receptors: FPR (for fMLP) as well as CXCR1 and CXCR2 (the receptors for ELR(+)-CXC chemokines). The analyses of these receptors in HIES patients indicated that their coding sequences were intact and normal. However, the percentages of PMN that expressed FPR, CXCR1 and CXCR2 were significantly lower in HIES patients. In addition, lower expression levels per cell were denoted for CXCR1 in PMN of the patients. A cumulative score that was calculated for the three chemoattractant receptors together indicated that in some of the patients there were prominent reductions, of up to approximately 50% in the overall expression of the receptors (indicated by % positive cells and mean expression levels per cell). In addition, we asked whether deregulation of PMN activities in HIES may result from binding of IgE to corresponding receptors on HIES PMN. Our findings indicate that this is probably not the case, because similarly to normal PMN, the cells of HIES patients did not express notable levels of the IgE receptors FcvarepsilonRI and FcvarepsilonRII. Together, these results provide novel information on the expression of key determinants in PMN migration in HIES, suggesting that a defect in the expression of chemoattractant receptors may lead to impaired chemotaxis found in HIES patients, and to decreased inflammatory responses.

Intracellular cross-talk between the GPCR CXCR1 and CXCR2: role of carboxyl terminus phosphorylation sites.

In the present study, we used the human chemokine receptors CXCR1 and CXCR2 as a model system for the study of intracellular cross-talk between two closely related G protein-coupled receptors (GPCR). In cells expressing either CXCR1 or CXCR2, exposure to the CXCL8 ligand resulted in prominent reduction in cell surface expression of the receptors. We have shown previously that the reduction in cell surface expression of CXCR1 and CXCR2, to be termed herein "down-regulation", is significantly lower in cells expressing both receptors together. Now we show that reduced receptor down-regulation was specific to the CXCR1+CXCR2 pair. Also, CXCR2 carboxyl terminus phosphorylation sites were required for inducing inhibition of CXCR1 down-regulation, and vice versa. Accordingly, phosphorylation of CXCR2 carboxyl terminus domain was intact when expressed together with CXCR1. Moreover, specific carboxyl terminus phosphorylation sites on each of the wild type receptors protected them from more severe inhibition of down-regulation, induced by joint expression with the other receptor. When concomitantly expressed, CXCR1 and CXCR2 were impaired in recycling to the plasma membrane, despite their undergoing intact dephosphorylation. Overall, we show that cross-talk between two GPCR is manifested by impairment of their intracellular trafficking, primarily of ligand-induced down-regulation, via carboxyl terminus phosphorylation sites.

CXCL8-induced FAK phosphorylation via CXCR1 and CXCR2: cytoskeleton- and integrin-related mechanisms converge with FAK regulatory pathways in a receptor-specific manner.

CXCL8 is a potent chemokine, inducing focal adhesion kinase (FAK) phosphorylation, and migration via a FAK-mediated pathway. Since, unlike growth factors, chemokines directly control integrins and cytoskeleton rearrangements, we determined whether these elements regulate CXCL8-induced FAK phosphorylation. The analysis intentionally dissociated between the CXCL8 receptors CXCR1 and CXCR2. In both CXCR1- and CXCR2-expressing cells, actin and microtubules were required for CXCL8-induced FAK phosphorylation, and CXCL8-induced cell spreading was accompanied by concordant re-localization of FAK with actin and beta-tubulin. The phosphorylation of five FAK sites depended on intact actin filaments and microtubules. While in CXCR2-expressing cells FAK phosphorylation was adhesion-dependent and was stimulated by fibronectin, in CXCR1-expressing cells FAK phosphorylation was adhesion-independent. Of note, even in the absence of integrin stimulation, the CXCL8-induced phosphorylation of FAK in CXCR1-expressing cells required cytoskeletal elements. CXCL8-induced migration in both cell types was highly reliant on actin filaments, but only the migration of CXCR1-expressing cells was fully dependent on microtubules. Overall, several aspects of CXCL8-induced FAK phosphorylation and migration are regulated in a receptor-specific manner. These observations lay the basis for future investigation of the equilibrium between CXCR1 and CXCR2 in cells expressing both receptors together, such as neutrophils, endothelial cells and tumor cells.