Early myeloid cells are high producers of nitric oxide upon CD40 plus IFN-gamma stimulation through a mechanism dependent on endogenous TNF-alpha andIL-1 alpha

Bone marrow contains nonadherent low-density wheat germ agglutinin-positive (Fr3-WGA(+)) cells that release large amounts of NO and show natural suppr essor activity if stimulated with activated T cells. We have assessed the i nvolvement of CD40-derived signals in NO production and their cytokine requ irements. Production of NO by Fr3-WGA(+) cells in coculture with activated T cells is inhibited by a competing CD40 soluble fusion protein. Fr3-WGA(+) cells express the inducible NO synthase (iNOS) and release NO following CD 40 plus IFN-gamma activation. Production of NO through CD40 is strictly dep endent on endogenous TNF-alpha and/or IL-1 alpha, since it is inhibited by neutralizing these cytokines or blocking the TNF receptor (p55), Both cytok ines are transcribed when Fr3-WGA(+) cells are stimulated by CD40 signaling plus IFN-gamma, although TNF-cr remains below detection limits in stimulat ed Fr3-WGA(+) cell cultures. Phenotypic studies combined with data on intra cellular iNOS expression and cell sorting indicate that NO-producing cells are CD40, CD31 (ER-MP12), CD11b (Mac-1)low, ER-MP20 (Ly-6C) and Gr-1 (Ly-6G ) positive, consistent with myeloid progenitors. The results point to early myeloid cells as an important cell source of NO once triggered by activate d T cells through CD40 and IFN-gamma-derived signals, in a mechanism involv ing the production of TNF-alpha and/or IL-1 alpha.