Positive controls yielded a CI (mean + standard error of the mean [SEM]) of 3
Positive controls yielded a CI (mean + standard error of the mean [SEM]) of 3.7 + 0.81. Treatment of MW-150 supernatants with anti-IL-8 antibodies. the peripheral blood, through the mammary tissue, and into the mammary secretions is called chemotaxis (24). Briefly, chemotaxis is a highly regulated process in which selectins, integrins, and chemoattractants interact to generate cell migration (32). Selectins are adhesion molecules on leukocyte membranes that have an N-terminal domain homologous to that of Ca2+-dependent lectins, and they are responsible for attachment of leukocytes to vessel walls (4). Integrins are responsible for leukocyte-endothelial cell interactions preceding migration into tissue (13, 14). Lastly, chemoattractants are soluble mediators released at or near the site of chemotaxis. They function to regulate integrins, bind leukocytes, and modulate migration (24, 32). The cytokine interleukin-8 (IL-8) is one such chemotactic factor. IL-8 is a chemokine that is produced by numerous cell types, including lymphocytes (10), neutrophils (35), monocytes/macrophages (29, 34), and epithelial cells (8, 9), including human mammary gland epithelial cells (2, 17, 20). IL-8 has several biological roles, including the following: recruiting and activating neutrophils (11), inducing neutrophil degranulation (29), stimulating phagocytosis of opsonized particles (7), and recruiting T lymphocytes (17, 36). In addition, IL-8 has been detected in human mammary secretions, and human maternal cells in breast milk express mRNA for IL-8 (33). IL-8 has also been detected in mammary secretions from glands challenged with (30, 31) and in mastitic mammary Spp1 secretions (1). In this study we examined whether caprine mammary epithelial cells (CMEC) and caprine mammary myoepithelial cells (CMMyoEC) were able to produce chemoattractants for caprine neutrophils, whether the chemokine IL-8 was present, and whether chemoattractant production by these cells was inducible by a variety of agents. The cell lines used have been previously described (21C23). Briefly, the CMEC show functional differentiation when grown on a plastic substratum by expressing lactation-specific proteins preferentially in cells which form dome-like structures. Morphologic differentiation is observed with the formation of duct-like and acinus-like structures when cells are grown within a collagen matrix. CMEC proliferate in response to insulin, insulin-like growth factor 1, transforming growth factor alpha, hydrocortisone, and the ovarian steroid estradiol, when estradiol is combined with triiodothyronine. MW-150 The complementing syngeneic CMMyoEC line (21) was derived from the same primary mixed MW-150 mammary cell culture as CMEC. CMMyoEC have been shown to be alpha-smooth muscle actin positive and to have a contractile response to exogenous oxytocin. Coculture and culture supernatant bioassay experiments with epithelial and myoepithelial cells suggest the presence of paracrine-cell-mediated epithelial modulation of mammary myoepithelial cells. CMEC culture supernatants are able to augment myoepithelial-cell proliferation and are chemotactic for myoepithelial cells. However, myoepithelial-cell culture supernatants are not chemotactic for epithelial or myoepithelial cells. Our previous studies have shown that epithelial and myoepithelial cell lines are a relevant in vitro model in which to study mammary gland function. In this study, we found that CMEC but not CMMyoEC culture supernatants were chemotactic for neutrophils. In confluent cultures, the chemotactic activity was inhibited by anti-IL-8 antibodies. Also, chemotactic activity of CMEC cultures was induced by the proinflammatory cytokine IL-1, by plus alpha-toxin was inhibited by anti-IL-8 antibodies. These studies indicate that epithelial cells, but not myoepithelial cells, produce IL-8 in the mammary gland. MATERIALS AND METHODS Reagents. All reagents were obtained from Sigma Chemical Co., St. Louis, Mo., unless otherwise noted. Anti-human IL-8 antiserum that was produced in chickens and that was found to cross-react with (ruminant) bovine IL-8 (25) was kindly provided by Donald L. Kreutzer (Departments of Pathology and Surgery, School of Medicine, University of Connecticut, Farmington). Cells and culture conditions. The CMEC and CMMyoEC established by our laboratory were used in all experiments. The cell lines were originally derived from a biopsy specimen of a mammary gland from a lactating (114 days postparturition) Anglo-Nubian (bacteria per ml of new culture media. Bacteria were enumerated by using the BBL Prompt Inoculation System (Becton Dickinson, Cockeysville, Md.). The cells were incubated for 18 h and the supernatants were collected. Isolation of responder cells. Caprine blood was collected via venipuncture into an EDTA Vacutainer (Fisher). Whole blood was centrifuged at 400 for 20 min. The plasma and buffy coat layers were aspirated and the erythrocyte pellet,.