A potential immune escape mechanism by melanoma cells through the activation of chemokine-induced T cell death.

The immune system attempts to prevent or limit tumor growth, yet efforts to induce responses to tumors yield minimal results, rendering tumors virtually invisible to the immune system [1]. Several mechanisms may account for this subversion, including the triggering of tolerance to tumor antigens [2, 3], TGF-alpha or IL-10 production, downregulation of MHC molecules, or upregulation of FasL expression [4, 5]. Melanoma cells may in some instances use FasL expression to protect themselves against tumor-infiltrating lymphocytes (TIL) [4, 5]. Here, we show another, chemokine-dependent mechanism by which melanoma tumor cells shield themselves from immune reactions. Melanoma-inducible CCL5 (RANTES) production by infiltrating CD8 cells activates an apoptotic pathway in TIL involving cytochrome c release into the cytosol and activation of caspase-9 and -3. This process, triggered by CCL5 binding to CCR5, is not mediated by TNFalpha, Fas, or caspase-8. The effect is not unique to CCL5, as other CCR5 ligands such as CCL3 (MIP-1alpha) and CCL4 (MIP-1beta) also trigger TIL cell death, nor is it limited to melanoma cells, as it also operates in activated primary T lymphocytes. The model assigns a role to the CXC chemokine CXCL12 (SDF-1alpha) in this process, as this melanoma cell-produced chemokine upregulates CCL5 production by TIL, initiating TIL cell death.

Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.

The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2.

Cytokines interact with hematopoietin superfamily receptors and stimulate receptor dimerization. We demonstrate that chemoattractant cytokines (chemokines) also trigger biological responses through receptor dimerization. Functional responses are induced after pairwise crosslinking of chemokine receptors by bivalent agonistic antichemokine receptor mAb, but not by their Fab fragments. Monocyte chemoattractant protein (MCP)-1-triggered receptor dimerization was studied in human embryonic kidney (HEK)-293 cells cotransfected with genes coding for the CCR2b receptor tagged with YSK or Myc sequences. After MCP-1 stimulation, immunoprecipitation with Myc-specific antibodies revealed YSK-tagged receptors in immunoblotting. Receptor dimerization also was validated by chemical crosslinking in both HEK-293 cells and the human monocytic cell line Mono Mac 1. Finally, we constructed a loss-of-function CCR2bY139F mutant that acted as a dominant negative, blocking signaling through the CCR2 wild-type receptor. This study provides functional support for a model in which the MCP-1 receptor is activated by ligand-induced homodimerization, allowing discussion of the similarities between bacterial and leukocyte chemotaxis.

Conformational changes required in the human growth hormone receptor for growth hormone signaling.

Growth hormone (GH) plays a significant role in normal growth and development. Signaling to the cell is believed to require growth hormone receptor (GHR) dimerization, which occurs following binding of a single growth hormone molecule to each of two receptors. We have developed human growth hormone receptor-specific monoclonal antibodies, one of which was used here to characterize hormone/receptor interactions. This antibody, GHR05, is directed against the hinge spanning subdomains I and II of the receptor's extracellular region. Antibody binding to the cell surface receptor increases upon receptor binding to growth hormone, but not when it binds a mutant form, hGHG120R, which does not trigger receptor activation. Growth hormone binding thus appears to lead to a conformational change in the receptor epitope recognized by GHR05, giving rise to the active dimer configuration, necessary for signal transduction. Using a chimeric receptor-expressing, growth hormone-dependent murine cell line, we find that GHR05 binds to the receptor in the absence of human GH and delivers a signal leading to cell proliferation. Finally, GHR05 treatment of IM-9 cells, a human cell line expressing a functional human GHR, leads to cell proliferation mediated by the generation of GH-specific signals, including phosphorylation of the JAK2 tyrosine kinase and activation of STAT5.