Oligomerization of CXCL10 Is Necessary for Endothelial Cell Presentation and In Vivo Activity

Harvard University, Cambridge, Massachusetts, United States
The Journal of Immunology (Impact Factor: 4.92). 12/2006; 177(10):6991-8. DOI: 10.4049/jimmunol.177.10.6991
Source: PubMed


The chemokine IFN-gamma-inducible protein of 10 kDa (IP-10; CXCL10) plays an important role in the recruitment of activated T lymphocytes into sites of inflammation by interacting with the G protein-coupled receptor CXCR3. IP-10, like other chemokines, forms oligomers, the role of which has not yet been explored. In this study, we used a monomeric IP-10 mutant to elucidate the functional significance of oligomerization. Although monomeric IP-10 had reduced binding affinity for CXCR3 and heparin, it was able to induce in vitro chemotaxis of activated T cells with the same efficacy as wild-type IP-10. However, monomeric IP-10 was unable to induce recruitment of activated CD8+ T cells into the airways of mice after intratracheal instillation. Use of a different IP-10 mutant demonstrated that this inability was due to lack of oligomerization rather than reduced CXCR3 or heparin binding. Molecular imaging demonstrated that both wild-type and monomeric IP-10 were retained in the lung after intratracheal instillation. However, in vitro binding assays indicated that wild-type, but not monomeric, IP-10 was retained on endothelial cells and could induce transendothelial chemotaxis of activated T cells. We therefore propose that oligomerization of IP-10 is required for presentation on endothelial cells and subsequent transendothelial migration, an essential step for lymphocyte recruitment in vivo.

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Available from: Jan Grimm, Sep 26, 2014
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    • "We next investigated the requirement of CXCR3 binding for CXCL10's anti-proliferative effect on HUVEC utilizing a series of CXCL10 mutants we previously described [28]. In particular, mutant R8A has greatly decreased CXCR3 binding affinity and does not induce chemotaxis or calcium-flux through CXCR3 in vitro, or recruitment of T cells in vivo [29], whereas it binds normally to glycosaminoglycans (GAGs). Mutant R8A was however fully able to inhibit HUVEC proliferation to the same extent as wildtype CXCL10 (Fig. 8), again suggesting that the anti-proliferative effect of CXCL10 on endothelial cells in this assay is not dependent on CXCR3 binding and signaling. "
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    ABSTRACT: CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10) is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.
    Preview · Article · Sep 2010 · PLoS ONE
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    • "In contrast to our observation, a disulfide-linked CXCL12 was shown not capable of chemotaxis [42]. CXC chemokine IP-10/CXCL10 forms dimers and oligomers, and a trapped CXCL10 monomer was shown to be inactive in an in vivo mouse lung model [43]. Monomeric and GAG-binding-deficient mutants of CC chemokines RANTES/CCL5, MIP-1β/CCL4, and MCP-1/CCL2 are inactive in the mouse peritoneum model [44], and a trapped CC MIP-1β dimer has been shown incapable of binding its receptor [45]. "
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    Full-text · Article · Jul 2010 · PLoS ONE
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    • "This makes them capable of migrating from the blood through the vascular endothelium into the site of inflammation [39,40]. Previous studies on IP-10 and transendothelial migration indicated that IP-10 retained on endothelial cells could induce transendothelial chemotaxis of activated T cells [41]. Another investigation on the biological activity of human recombinant IP-10 investigation further verified its chemotactic properties towards human peripheral blood monocytes and stimulated human peripheral blood T lymphocytes, but not neutrophils [42,43]. "
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