Early myeloid cells are high producers of nitric oxide upon CD40 plus IFN‐γ stimulation through a mechanism dependent on endogenous TNF‐α and IL‐1α

Bone marrow contains nonadherent low‐density wheat germ agglutinin‐positive (Fr3‐WGA+) cells that release large amounts of NO and show natural suppressor activity if stimulated with activated T cells. We have assessed the involvement of CD40‐derived signals in NO production and their cytokine requirements. Production of NO by Fr3‐WGA+ cells in co‐culture 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 CD40 plus IFN‐γ activation. Production of NO through CD40 is strictly dependent on endogenous TNF‐α and / or IL‐1α, since it is inhibited by neutralizing these cytokines or blocking the TNF receptor (p55). Both cytokines are transcribed when Fr3‐WGA+ cells are stimulated by CD40 signaling plus IFN‐γ, although TNF‐α remains below detection limits in stimulated Fr3‐WGA+ cell cultures. Phenotypic studies combined with data on intracellular 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 activated T cells through CD40 and IFN‐γ‐derived signals, in a mechanism involving the production of TNF‐α and / or IL‐1α.