The gender‐related differences in the incidence of development of respiratory complications, and the role of estrogen

Sir, In patients aged between 20 and 45 years (female patients presumed to be pre-menopausal), who were admitted to our hospital between 2002 and 2004, the number who required mechanical ventilation for respiratory complications and were admitted to the intensive care unit was significantly lower for women than for men (number of patients aged between 20 and 45 years admitted to our hospital between 2002 and 2004: men, 74 474; women, 75 518; number of patients requiring mechanical ventilation and admitted to the intensive care unit: men, 583; women, 340; P < 0.0001, w-test). In order to elucidate the molecular basis of the gender-related difference in the incidence of development of respiratory complications, we sought to clarify the role of 17-b-estradiol on the inflammatory responses induced by bacterial infection. RAW 264.7 cells, a murine macrophage-lineage cell line, cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (GibcoBRL, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS) and antibiotics, were transiently transfected with cisreporting nuclear factor-kB (NF-kB)-dependent reporter plasmid (Stratagene, La Jolla, CA) as described previously (1), and stimulated with CpG oligodeoxynucleotide (ODN), a constitution of bacterial DNA which has been shown to stimulate strong immune responses in the mammalian immune system (2, 3), in the absence or presence of various concentrations of 17-b-estradiol (SigmaAldrich, St. Louis, MO). Unmethylated CpG ODN has the following nucleotide sequence and was synthesized by Sawady, Tokyo, Japan: 50-TCCATGACGTTCCTGACGTT-30. One day before transfection, 1 10 cells/well were seeded into six-well plates. On the following day, the cells were washed twice with phosphatebuffered saline, and 1 mg of reporter plasmid was transfected according to the manufacturer’s instructions (TransFast Transfection Reagent; Promega, Madison, WI), and incubated overnight. In order to ensure equivalent uptake of plasmid DNA, eukaryotic expression plasmid carrying b-galactosidase cDNA (2) was cotransfected simultaneously. Cells were then treated with 1 mM of CpG ODN in the absence or presence of 17-b-estradiol (Table 1), and the luciferase activities were measured according to the manufacturer’s instructions (Dual Luciferase Reporter Assay System; Promega) using a Mini Lumat LB9506 (Berthold, Tokyo, Japan). As shown in Table 1, the presence of a physiological concentration of 17-b-estradiol (10 M) suppressed NF-kB activation following stimulation with CpG ODN. In addition to the classic effects of estrogen on the female reproductive system, it has been shown to exert a diverse array of biological effects on bone density, plasma lipid profiles and the cardiovascular system (4). Here, we have shown that estrogen also exerts anti-inflammatory properties by preventing inflammatory gene transcription via inhibition of NF-kB activation. Cellular events associated with inflammation are mediated through NFkB, a redox-sensitive transcription factor that regulates a multitude of inflammatory genes, including cytokines, adhesion molecules and acute phase proteins (5, 6). Although the precise mechanism by which estrogen interferes with the activation of NF-kB and the subsequent nuclear localization at which it binds to DNA elements in the promoter of a number of pro-inflammatory gene families remain to be elucidated, the results presented herein provide novel insights into the therapeutic approach to potentially life-threatening complications such as acute respiratory distress syndrome.