Styles Biotechnol. was Rabbit polyclonal to AMDHD2 more potent in inhibiting parasite growth, although it was much less efficient than gamma interferon (IFN-). Nevertheless, JE potentiates parasite killing by macrophages incubated with low doses of IFN-. Together, these results suggest that in addition to their chemotactic activity, murine -chemokines may also contribute to enhancing parasite uptake and promoting control GW 7647 of parasite replication in macrophages and may play a role in resistance to infection. The infection of inbred mice with has been associated with the production of the proinflammatory cytokine interleukin-12 (IL-12), which triggers the production of gamma interferon (IFN-) by NK and T cells (1, 7). The IFN- produced in change activates macrophages to GW 7647 release nitric oxide and kill the obligate intracellular amastigote forms of the parasite (15, 28). Tumor necrosis factor alpha (TNF-), another cytokine associated with macrophage activation, provides a second transmission to induce microbicidal activity in IFN–activated macrophages by stimulating NO production (12). Since (29), we hypothesized that chemokines may be secreted by infected GW 7647 cells and may mediate resistance to contamination. These chemokine-mediated effects may depend not only on the ability of chemokines to induce the attraction of leukocytes but also on their ability to induce NO synthase (NOS) activation. For this purpose, we examined whether trypomastigotes brought on -chemokine (MIP-1, MIP-1, RANTES, and JE/MCP-1) mRNA expression and protein production and whether these chemokines were involved in the regulation of NO production by infected murine macrophages. The choice of chemokines to be investigated was based on our preliminary studies, which exhibited the expression of MIP-1, MIP-1, RANTES, and JE/MCP-1 mRNAs in the hearts of was used in this study. Trypomastigote forms were produced and purified from a monkey fibroblast cell collection (LLC-MK2). Macrophage cultures for RNA extraction. C3H/HeJ mouse inflammatory macrophages were harvested from peritoneal cavities three days after the injection of 1 1 ml of 3% sodium thioglycolate (Sigma Chemical Co., St. Louis, Mo.). The cells were washed in Hanks medium and resuspended to 106/ml in RPMI-C (RPMI 1640 [Flow Laboratories, Inc., MCLean, Va.] supplemented with 5% fetal bovine serum [HyClone, Logan, Utah], 5 10?5 M 2-mercaptoethanol, 2 mM l-glutamine, and antibiotics [all from Sigma Chemical Co.]). The adherent cells were obtained after a 2- to 4-h incubation of single cell suspensions in 24-well tissue culture plates at 37C. Nonadherent cells were removed by exhaustive washing with Hanks medium. Parasites were added in a 1:1 parasite/cell ratio and incubated for 6 h at 37C in a humidified chamber made up of 5% CO2. The cells were then washed three times with Hanks medium. One milliliter of Trizol LS reagent (Life Technologies, Grand Island, N.Y.) was added to each 107 cells, incubated at room heat for 5 min, and stored at ?70C until RNA was extracted. RNA was also purified from peritoneal cavity cells harvested from your mice 6 h after intraperitoneal injection of 5 105 trypomastigote forms in 200 l of phosphate-buffered saline (PBS). As a control, we used cells from mice inoculated with PBS only. Total RNA extraction and cDNA preparation by reverse transcription (RT). The extraction of total RNA was performed with the Trizol LS reagent according to the instructions of the manufacturer. Briefly, the samples were homogenized and 0.2 ml of chloroform (Sigma) was added to each 1.