Di-(2-ethylhexyl) phthalate induces production of inflammatory molecules in human macrophages.

OBJECTIVE AND DESIGN: To investigate whether di-(2-ethylhexyl) phthalate (DEHP) affects the production of inflammatory cytokines by human macrophages. MATERIALS AND METHODS: Differentiated macrophage-like THP-1 cells were exposed to 200 µM DEHP for 3 h, followed by incubation in the presence or absence of opsonized zymosan A, and the concentrations of TNF-α, IL-1ß, IL-8, and IL-6 in the culture media were determined by ELISA. DNA microarray and quantitative real-time RT-PCR analyses were performed to identify genes that showed changes in expression in response to DEHP. RESULTS: DEHP treatment increased the concentrations of TNF-α, IL-1ß, IL-8, and IL-6 in the media, regardless of whether the cells phagocytosed zymosan. DNA microarray analysis showed that DEHP increased the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP3, MMP10, MMP14, and CSF2 mRNA, and real-time RT-PCR showed that DEHP significantly enhanced the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP10, CSF2, TNF-α, IL-1ß, and IL-6 mRNA in THP-1 cells. DEHP significantly induced translocation of p65 NF-κB into the nucleus. CONCLUSION: DEHP enhances the production of inflammatory cytokines and chemokines by macrophages, and exacerbates their inflammatory response.

Proteomic analysis of plasma membrane lipid rafts of HL-60 cells.

Neutrophils acquire phagocytic activity as they differentiate. Recently, plasma membrane lipid rafts have been shown to play important roles in the process of phagocytosis in neutrophils. To characterize the proteins involved in phagocytosis and to elucidate the process by which they acquire phagocytic activity, we investigated by nano-LC-MS/MS analysis the changes in protein composition of plasma membrane lipid rafts during DMSO-induced differentiation of the human leukemia cell line HL-60 cells into neutrophilic lineage. Based on the spectrum counts of 147 proteins identified, 25 proteins were upregulated and 49 were downregulated by DMSO treatment. CD11b/CD18 subunits of beta2-integrin Mac-1, CD35, and GPI-80, which are known to be upregulated during differentiation, were dominantly detected in the lipid rafts of DMSO-treated cells. Many known membrane proteins, G proteins, and cytoskeletal proteins were also detected and they showed characteristic distributions. Absolute quantification of nine proteins in the lipid rafts using internal standard peptides labeled with stable isotopes showed that the amount of protein almost corresponded to the results obtained by spectrum count. Identified proteins, expression of which was altered by DMSO treatment, are expected to be candidate proteins involved in differentiation and functions of neutrophils.