Flow Cytometric Analysis of I‐J Expression on Murine Bone Marrow‐Derived Macrophages

Attempts to analyze bone marrow‐derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I‐A, I‐J, and Mac‐1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50–60% Mac 1‐positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1‐positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I‐J until these cells have been in culture 3 to 4 days, and the number of cells expressing I‐J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I‐A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I‐A, and only 20% of peritoneal cells had I‐J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I‐J positive (up to 70%), and only about 30% of these cells expressed I‐A cell surface antigens. The binding of anti‐I‐J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 μg of an IgG 2a myeloma protein did not block anti‐I‐J antibody binding. The addition of 25‐200 μg of monoclonal anti‐Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti‐I‐J k antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti‐I‐J IgM antibody. BMDM provide a pure population of macrophages that express a significant level of cell surface I‐J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I‐J‐positive cells parallels the increase in macrophages in these cultures. Thus, the further study of BMDM may permit resolution of questions raised by molecular biologists and immunologists regarding the nature of the I‐J protein, the location of I‐J genes, and the role of I‐J in regulating immune responses.