Purification and further characterization of a non-tumor necrosis factor alpha or beta differentiation-inducing cytokine, P48

Abstract

In this report, we present the further characterization and purification of a cytokine differentiation factor, termed P48, which unlike previously described differentiation factors is antigenically unrelated to tumor necrosis factor alpha (TNF-alpha), tumor necrosis factor beta (TNF-beta), and gamma interferon. HL-60 cells and phorbol diester-resistant HL-60-1E3 cells exposed to conditioned medium from Reh cells mature along the monocyte/macrophage pathway, as assessed by several assays (express nonspecific esterase, produce superoxide anion, morphologically resemble monocytes, mediate phorbol diester-triggered extracellular cytolytic activity). Reh cell conditioned medium is antiproliferative toward a panel of cell lines, is not nonspecifically cytotoxic, has no antiviral or colony-stimulating factor activities, and is not affected by exposure to insolubilized anti-gamma interferon. A 48-kDa glycoprotein (P48) which mediates this differentiation factor activity has been purified to homogeneity from Reh cell conditioned medium, and a polyclonal neutralizing antiserum has been produced. P48 activity is not blocked by either anti-TNF-alpha and anti-TNF-beta and on Western blot analysis is antigenically distinct from TNF-alpha and TNF-beta. In addition, polyclonal anti-P48 does not block either TNF-alpha or TNF-beta activities or recognize either on Western blots. Unlike gamma interferon, colony-stimulating factor, TNF-alpha, or TNF-beta, P48 reverses phorbol diester resistance of HL-60-1E3 cells. These studies present strong evidence for the existence of a previously unrecognized cytokine which, unlike other reported differentiation factors, is antigenically unrelated to TNF-alpha or TNF-beta. P48 may play an important role in growth and development of normal and abnormal (leukemic) hematopoietic and nonhematopoietic cells.