Torres-Morales, L. with induces the secretion of inflammatory cytokines, including tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-8. IL-8, first isolated from monocytes as a neutrophil attractant (1), is usually a member of the CXC MSI-1436 lactate () chemokine family that is also chemotactic for T lymphocytes (19). IL-8 has been found during the course of the immune response to contamination, resulting in tissue inflammation, granuloma formation, and a major chemotactic factor for neutrophils (11, 12, 35). Neutrophils chemoattracted into the alveolus by IL-8 during pulmonary tuberculosis may have a direct role in mycobacterial killing (14). Alveolar macrophages and monocytes, recruited early in the course of pulmonary contamination with has not been shown previously. The aim of the present work was to determine the effects of IL-10 and TGF- on BCG. MATERIALS AND METHODS Reagents. Recombinant IL-10 (19110V) was purchased from PharMingen (San Diego, Calif.). Recombinant TGF- (240-B) was purchased from R&D Systems (Minneapolis, Minn.). The following antibodies were obtained: anti-human IL-10 (AB217-NA), anti-TGF- (AB-101-NA), and isotype-matched control antibody from R&D Systems and fluorescein isothiocyanate (FITC)-conjugated anti-CD40 (5C3) from PharMingen. Live Danish strain 1331 was kindly supplied by J. Ruiz (Birmex, Mxico, D.F., Mxico). Cell culture. Peripheral blood mononuclear cells were obtained from normal healthy volunteers and separated out by density gradient centrifugation on Ficoll Histopaque 1077 (Sigma, St. Louis, Mo.). The peripheral blood mononuclear cells were enriched for monocytes by adherence on petri dishes at 37C for 1 h. Then, the plates were vortexed, and nonadherent cells were removed by vigorous washing with RPMI 1640 (GIBCO BRL, Rockville, MSI-1436 lactate Md.). Adherent cells were then cultured overnight in RPMI 1640 supplemented with 2 mM l-glutamine, 100 U of penicillin/ml, 100 g of streptomycin/ml, and 10% fetal calf serum, and the monolayers were washed again before contamination with For activation studies, human peripheral blood monocytes at 105 per well were infected with 105 organisms or exposed Mouse monoclonal to WNT5A to medium alone at 37C in a 5% CO2-95% humidified air flow incubator. Neutralizing antibody to IL-10 was added to some cultures. Culture supernatants for the detection of IL-8 and IL-10 were harvested, centrifuged to remove any debris, and then stored in frozen aliquots at ?20C. Cytokine protein measurement. Concentrations of IL-8 and IL-10 in the supernatant were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) system purchased from Amersham (Aylesbury, United Kingdom). The assays were carried out MSI-1436 lactate according to the methodology suggested by the manufacturer. Concentrations of cytokines are expressed as picograms per 105 cells. IL-8 mRNA expression. IL-8 mRNA expression in organisms overnight. Total RNA from cells was prepared according to the method of Chomczynski and Sacchi (3). Briefly, the cells were lysed in Trizol (Life Technologies), and total RNA was quantified on a spectrophotometer, followed by a reverse-transcription reaction using random hexamer primers (GIBCO BRL) and Superscript II reverse transcriptase (GIBCO BRL). The producing cDNAs were amplified by PCR in the standard reaction combination using DNA polymerase and sense and antisense primers for IL-8 (5 TTG GCA GCC TTC CTG ATT TC 3 and 5 AAC TTC TCC ACA ACC CTC TG 3). The PCR product was subjected to electrophoresis and visualized by staining it with ethidium bromide. Bands were quantified by densitometry. IL-8 levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to account for loading differences between lanes. Circulation cytometric analysis. Cells were cultured for 4 h in RPMI 1640 medium in the presence or absence of TGF- and then infected with 105 organisms at 37C overnight. The cells were washed twice with phosphate-buffered saline (PBS) and then reacted with FITC-conjugated anti-CD40 antibody for 30 min, washed twice with 0.2% bovine serum albumin-PBS, and fixed with 1% paraformaldehyde in PBS (pH 7.2) for 10 min. After being washed with PBS, the producing cells were subjected to.