Gregory R. Johnson

Long-term exposure to retrovirally expressed granulocyte-colony-stimulating factor induces a nonneoplastic granulocytic and progenitor cell hyperplasia without tissue damage in mice.

Murine marrow cells infected with a retroviral vector (MPZen) bearing a granulocyte-colony-stimulating factor (G-CSF) cDNA insert were transplanted into lethally irradiated recipients to study the effects of autocrine production of G-CSF in normal hemopoietic cells. Most animals remained healthy with no evidence of tissue damage throughout the observation period (4-30 wk) despite high circulating G-CSF levels (range 2,000-26,000,000 U/ml). A dramatic neutrophilic granulocytosis was observed in all hemopoietic tissues with neutrophilic infiltration occurring in the lung and liver. Spleen, peritoneal, and peripheral blood cellularity increased approximately three-, two-, and eightfold, respectively, but total bone marrow cell counts remained unchanged. Progenitor cell numbers granulocyte-macrophage colony-forming cell (GM-CFC), granulocyte colony-forming cell (G-CFC), burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E) and mixed colony-forming cells (Mix-CFC) were elevated between 10-100-fold in the spleen, peritoneal cavity, and peripheral blood, but were unaffected or slightly depressed in the marrow. No tumors developed in syngeneic recipients transplanted with bone marrow or spleen cells from such mice, confirming the nonneoplastic nature of the hyperplasia induced by chronic G-CSF stimulation. These experiments also indicated the stable integration of MPZen vectors in infected cells, as evident from the continuous expression of the inserted gene for at least 6 mo, and from the ability of infected stem cells from the primary recipients to express the gene in lethally irradiated secondary recipients.

Preparation of colony stimulating factors from human placental conditioned medium

Abstract Human placental conditioned medium is able to stimulate human bone marrow cells to form neutrophilic granulocyte-monocyte colonies and eosinophil colonies in agar cultures. Methods are described for preparing highly active preparations (capable of supramaximal stimulation of colony formation) by partial purification of the conditioned medium using calcium phosphate absorption and gel filtration chromatography. Partially purified calcium phosphate gel eluates appear to increase in specific activity after concentration by ultrafiltration. This phenomenon was not observed with the active fractions after further purification on Sephadex G-100. Fractionation of the conditioned medium using gel filtration and hydrophobic chromatography (Cibacron Blue-Sepharose) showed that distinct and partially separable factors were responsible for stimulating granulocyte-macrophage and eosinophil colony formation. From the gel filtration data the apparent molecular weight of the eosinophil colony stimulating factor was higher than the molecular weight of the granulocyte-macrophage colony stimulating factor. Cibacron Blue-Sepharose chromatography led to the separation of one molecular species of granulocyte-macrophage colony stimulating factor from eosinophil colony stimulating factor so that a specific stimulus for human neutrophilic granulocyte and/or monocyte-macrophage progenitors is now available.

Peritoneal Cell Population of Mice Infected with Mesocestoides corti as a Source of Eosinophils

A prominent feature of the inflammatory cellular response in the peritoneal cavity of Mesocestoides corti-infected mice is a marked and sustained increase in the number of eosinophils. In intact mice, the total number of nucleated cells in the peritoneal cavity rises from less than 5 X 10(6) to more than 50 X 10(6) and, at certain time points, in excess of 50% of these cells are eosinophils. Peritoneal eosinophils are absent in infected hypothymic nude (nu/nu) mice of three genotypes, and eosinophils counts can be elevated in infected nude mice by injection of peripheral lymphoid cells or thymocytes. The peritoneal cells of M. corti-infected mice are a convenient starting cell population for eosinophil purification.