Neutrophil products that inhibit cell proliferation: relation to granulocytic "chalone"

Various investigators have proposed the existence of a “chalone” or product of mature granulocytes which inhibits the replication of granulocytic progenitors in a tissue-specific, species-nonspecific regulatory system. We tested this system utilizing purified populations of human leukocytes and a variety of proliferating target cell populations. Granulocyte-conditioned medium potently inhibited 3Hthymidine incorporation in normal mouse bone marrow, in mouse erythroid precursors from spleens of phenylhydrazinetreated animals, and in human leukemic myeloblasts. This effect occurred without demonstrable cytotoxicity. Human marrow was only slightly inhibited and mitogen-stimulated proliferating lymphocytes were unaffected. Granulocyte-conditioned medium was also inhibitory in colony-forming assays for erythroid precursors but not for granulocyte precursors. It also reduced the mitotic index of proliferating erythroid progenitors in mouse spleen. Inhibitory activity was separable into at least two components on the basis of stability during heating and ultrafiltration, and in susceptibility to digestion by proteolytic enzymes. On the basis of these observations, we concluded that human granulocytes contain potent inhibitors of growth and 3H-thymidine uptake of some proliferating hematopoietic cells, but these are not true granulocyte “chalones.” I N 1960, Bullough and Lawrence provided experimental evidence for the control of epidermal cell mitosis by products of mature cells in culture in vitro.’ They evolved the concept of a tissue-specific product of differentiated cells that served as a mitotic inhibitor of early cells of the same line. They termed the inhibitor a “chalone” (from the Greek, “to loosen” or “make slack”). Since then, a number of experimenters have suggested the existence of chalones specific for various tissues.2 More recently, various investigators have emphasized theoretical reasons why granulopoiesis should be under negative feedback control and have adduced evidence for the existence of granulocyte chalones.38 However, the diversity of model systems, methodologic limitations, and frequently inadequate experimental design have made interpretations difficult. RytOmaa and Kiviniemi,7’8 Paukovitz,5’6 and Bateman3 have presented evidence suggesting that rat and calf serum solutions “conditioned” by granulocyte-rich cell suspensions and spleen homogenates inhibit DNA synthesis in developing myeloid precursors in short-term cultures of rat bone marrow cells. This “chalone” material has been partially purified by gel filtration, ultrafiltration, and electrophoresis.57 It is reported to be a polypeptide of a molecular weight of approximately 4000-5000 daltons, which is unstable at 4#{176}C,8 with