Determination of Serum BRAK/CXCL14 Levels in Healthy Volunteers

BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., K(d), approximately 2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.

In obese individuals, white adipose tissue (WAT) is infiltrated by large numbers of macrophages, resulting in enhanced inflammatory responses that contribute to insulin resistance. Here we show that expression of the CXC motif chemokine ligand-14 (CXCL14), which targets tissue macrophages, is elevated in WAT of obese mice fed a high fat diet (HFD) compared with lean mice fed a regular diet. We found that HFD-fed CXCL14-deficient mice have impaired WAT macrophage mobilization and improved insulin responsiveness. Insulin-stimulated phosphorylation of Akt kinase in skeletal muscle was severely attenuated in HFD-fed CXCL14+/- mice but not in HFD-fed CXCL14-/- mice. The insulin-sensitive phenotype of CXCL14-/- mice after HFD feeding was prominent in female mice but not in male mice. HFD-fed CXCL14-/- mice were protected from hyperglycemia, hyperinsulinemia, and hypoadiponectinemia and did not exhibit increased levels of circulating retinol-binding protein-4 and increased expression of interleukin-6 in WAT. Transgenic overexpression of CXCL14 in skeletal muscle restored obesity-induced insulin resistance in CXCL14-/- mice. CXCL14 attenuated insulin-stimulated glucose uptake in cultured myocytes and to a lesser extent in cultured adipocytes. These results demonstrate that CXCL14 is a critical chemoattractant of WAT macrophages and a novel regulator of glucose metabolism that functions mainly in skeletal muscle.

Although numerous chemokines act on monocytes, none of them is specific for these cells. Here, we show that breast and kidney–expressed chemokine (BRAK) is a highly selective monocyte chemoattractant. Migration efficacy and Bordetella pertussis toxin–sensitive Ca2+ mobilization responses to BRAK were strongly enhanced after treatment of monocytes with the cyclic AMP–elevating agents prostaglandin E2 and forskolin. BRAK is the first monocyte-selective chemokine, as other types of blood leukocytes or monocyte-derived dendritic cells and macrophages did not respond. Expression in normal skin keratinocytes and dermal fibroblasts as well as lamina propria cells in normal intestinal tissues suggests a homeostatic rather than an inflammatory function for this chemokine. In addition, macrophages were frequently found to colocalize with BRAK-producing fibroblasts. We propose that BRAK is involved in the generation of tissue macrophages by recruiting extravasated precursors to fibroblasts, which are known to secrete essential cytokines for macrophage development.