CHEMOKINE REGULATION OF HUMAN MEGAKARYOCYTOPOIESIS

We have previously shown that platelet factor 4 (PF4), a platelet-spec ific CXC chemokine, can directly and specifically inhibit human megaka ryocyte colony formation. We therefore hypothesized that PF4 might fun ction as a negative autocrine regulator of megakaryocytopoiesis. Herei n we present additional studies characterizing the inhibitory effect o f CXC chemokines on human megakaryocyte development. We first corrobor ated our initial studies by showing that recombinant human (rH) PF4, l ike the native protein, inhibited megakaryocytopoiesis. We then examin ed the inhibitory properties of other CXC family members. Neutrophil a ctivating peptide-2 (NAP-2), a naturally occurring N-terminally cleave d beta TG peptide, was found to inhibit megakaryocytopoiesis with two to three orders of magnitude greater potency than PF4. Structure funct ion studies showed that an N-terminal mutation, which eliminated NAP-2 's neutrophil activating properties (NAP-2(E2-->A)), also abrogated it s ability to inhibit megakaryocyte development. Further investigations of this type demonstrated that a chimeric PF4 protein (AELR/PF4) in w hich PF4's N-terminus was replaced with the first four amino acids of NAP-2 was also a potent inhibitor of megakaryocytopoiesis. Interleukin (IL)-8, another CXC chemokine, and three CC chemokines (macrophage in hibitory protein-1 alpha [MIP-1 alpha], MIP-1 beta, and C10) also spec ifically inhibited megakaryocyte colony formation at NAP-2 equivalent doses. CXC and CC chemokine inhibition was additive suggesting that th e effects might be mediated through a common pathway. The inhibitory e ffects of NAP-2 and MIP-1 alpha could not be overcome by adding physio logically relevant amounts of recombinant human megakaryocyte growth a nd development factor (MGDR) (50 ng/mL) to the cultures. Using Norther n blot and reverse transcriptase-polymerase chain reaction (RT-PCR) ba sed analyses, we documented mRNA expression of IL-8 receptor isoforms alpha and beta in total platelet RNA and in normal human megakaryocyte s, respectively. Based on these results, we hypothesize that chemokine s play a physiologic role in regulating megakaryocytopoiesis. Because chemokines are elaborated by ancillary marrow cells, both autocrine an d paracrine growth control is suggested, the effects of which might be exerted, in part, through alpha and beta IL-8 receptors. (C) 1995 by The American Society of Hematology.