Similarly, addition of one clone of CD11c antibodies had only modest effects on IL-12 production (Fig.1,AandB). adhesion molecules (15). CR3 is expressed mainly on polymorphonuclear leukocytes, monocytes and macrophages, and natural killer cells (1,2), and RGFP966 interacts with a variety of ligands including complement fragment iC3b, intercellular adhesion molecule 1 (ICAM1)1, fibrinogen, and -glucan (2,4). Moreover, it mediates the binding of opsonized or unopsonized infectious agents such asHistoplasma capsulatum, Leishmania major,group B streptococci, Bordetella pertussis, Candida albicans, and several mycobacteria (2,57). Previous studies have shown that CR3 is involved in several monocyte and macrophage functions including transmigrational adhesion (2,4,8), phagocytosis, nitric oxide production and the generation of a respiratory burst (2,3,5,8,10). In addition, CR3 signaling may indirectly affect T cell function RGFP966 as the administration of antibodies to CR3 to animals suppresses delayed type hypersensitivity (DTH) reactions (11,12), and fatally potentiates infections withListeria monocytogenesorToxoplasma gondii(13,14).Since these phenomena are dependent on an intact cell-mediated immunity (CMI) and Th1 responses, we explored the role of CR3 signaling in regulating cytokines that mediate such responses. == Materials and Methods == == Cell Isolation, Cell Culture Conditions and Assessment of Cytokine Production. == Human monocytes were obtained from healthy donors (totaln= 14) by standard leukapheresis and were highly purified (9599%) by counterflow centrifugal centrifugation (15). Cell purity was checked by flow cytometry analysis using monoclonal antibodies to CD14 and CD11b (Becton Dickinson, San Jose, CA). Monocytes were cultured at 2 106cells/ml for 24 h RGFP966 in 1 ml of RPMI 1640 medium (Whittaker, Walkersville, MD) supplemented with 10% fetal calf serum (Whittaker), 100 g/ml penicillin, 100 g/ml streptomycin and 0.03% glutamine and stimulated at the beginning of the culture with the following substances as indicated: heat-killed, formalin-fixedStaphylococcus aureus, Cowan I strain (SAC; Calbiochem, Cambridge, MA), LPS (Escherichia coliserotype 0127:B8;SigmaChem. Co., St. Louis, MO) (16,17), recombinant IFN- (Genzyme, Cambridge, MA), CD40L trimer (kindly provided by Immunex Corporation, Seattle, WA), IFN- (Endogen, Cambridge, MA), or antiIFN- (Endogen). One experiment was typically performed with the monocytes from one donor, and there was individual variability in IL-12 production (e.g., range for SAC plus IFN- stimulation 3803,300 pg/ml IL-12 p70, mean 1,750 pg/ml). Monocytes were incubated with heat-killedHistoplasma capsulatum(HC), (strain GS-57, kindly provided by Dr. R. Seder, Lymphokine Regulation Unit, LCI, NIAID, NIH) or iC3b-SRBC (2 107/ml) 2 h before stimulation as indicated. iC3b-SRBC were prepared by sequential addition of anti-sheep erythrocyte antibodies (IgM;PharMingen, San Diego, CA) and C5-deficient human serum (Sigma) to SRBC as described (18,19). The presence of iC3b on the cell surface was verified by flow cytometry showing a >100fold increase in fluorescence intensity with a monoclonal antibody to the iC3b neoantigen (Quidel, San Diego, CA). Human PBMC were isolated by Ficoll-Hypaque density gradient centrifugation from leukocyte concentrates prepared by automated leukapheresis of healthy donors (totaln= 5) as described (15). PBMC were cultured similar to monocytes, except that they were cultured at 1 106cells/ml for 48h and stimulated with PHA (Sigma). In some experiments, recombinant human IL-12, or recombinant IL-2 (PharMingen) were RGFP966 added as indicated. Culture supernatants were assessed in duplicate by ELISA using kits or antibody pairs for IL-12 p70, TNF- (both from R&D Systems), IL-12 p40, IL-10 (both fromPharMingen), IL-6, IL-1, IFN-, IFN- (all from Endogen, Cambridge, MA), and TGF-1 (Genzyme). Unstimulated cultures (with or without added integrin antibodies) contained cytokines below the detection limit (IL-12 p70 >5 pg/ml, IL-12 p40 >20 pg/ml, IFN- >10 pg/ml, IL-10 >100 pg/ml, TGF- >50 pg/ml, IL-6 >50 pg/ml, IL-1 >25 pg/ml). For TNF-, the detecting limit was >25 pg/ml and the background was below 200 pg/ml. Integrin antibodies were added at the beginning of the culture at the indicated concentrations and were obtained in a preservative-free preparation or dialyzed overnight before use. RGFP966 The antibodies and monocyte stimulants (e.g., CD40L trimer) Mouse monoclonal to ERBB2 contained low endotoxin levels as.