Assay for quantitation of mature monocytes in murine haemopoietic tissues

We propose a new assay which allows the extent of production of mature monocytes in murine haemopoietic tissues to be monitored with precision. To develop such a quantitative assay, we chose a T-cell function easily detectable in vivo, namely the ability of T lymphocytes to locally transfer a delayed-type hypersensitivity (DTH) reaction. T cells co-transferred with antigen into footpads of syngeneic mice are mediators of DTH through their ability to recruit phagocytes recently produced in haemopoietic tissues. Therefore, if recipients of DTH-mediating T cells (DTH-T cells) are depleted of phagocytes by lethal irradiation given 36 to 48 h before local transfer of these DTH-T cells mixed with antigen, they no longer respond by a DTH reaction, measured as an increase in footpad thickness, unless they receive i.v. bone marrow cells as a source of recruitable phagocytes. In such conditions, the footpad thickness increase is linearly related to the number of cells injected i.v. Pretreatment of bone marrow cells with the rat IgG2b F4/80 monoclonal antibody abolishes their ability to restore expression of DTH, indicating that monocytes (F4/80+ cells) are the phagocytes to be measured. This assay system thus provides a means of measuring levels of recruitable mature monocytes present in haemopoietic tissues. One illustration of the assay is given by the study of the recovery of recruitable phagocytes in bone marrow following a single treatment with cyclophosphamide.