Chemokine regulation of human megakaryocytopoiesis

Abstract

We have previously shown that platelet factor 4 (PF4), a platelet-specific CXC chemokine, can directly and specifically inhibit human megakaryocyte colony formation. We therefore hypothesized that PF4 might function as a negative autocrine regulator of megakaryocytopoiesis. Herein we present additional studies characterizing the inhibitory effect of CXC chemokines on human megakaryocyte development. We first corroborated our initial studies by showing that recombinant human (rH) PF4, like the native protein, inhibited megakaryocytopoiesis. We then examined the inhibitory properties of other CXC family members. Neutrophil activating peptide-2 (NAP-2), a naturally occurring N-terminally cleaved beta TG peptide, was found to inhibit megakaryocytopoiesis with two to three orders of magnitude greater potency than PF4. Structure function studies showed that an N-terminal mutation, which eliminated NAP-22s neutrophil activating properties (NAP-2E2-->A), also abrogated its ability to inhibit megakaryocyte development. Further investigations of this type demonstrated that a chimeric PF4 protein (AELR/PF4) in which PF42s 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 inhibitory protein-1 alpha [MIP-1 alpha], MIP-1 beta, and C10) also specifically inhibited megakaryocyte colony formation at NAP-2 equivalent doses. CXC and CC chemokine inhibition was additive suggesting that the effects might be mediated through a common pathway. The inhibitory effects of NAP-2 and MIP-1 alpha could not be overcome by adding physiologically relevant amounts of recombinant human megakaryocyte growth and development factor (MGDR) (50 ng/mL) to the cultures. Using Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) based analyses, we documented mRNA expression of IL-8 receptor isoforms alpha and beta in total platelet RNA and in normal human megakaryocytes, respectively. Based on these results, we hypothesize that chemokines play a physiologic role in regulating megakaryocytopoiesis. Because chemokines are elaborated by ancillary marrow cells, both autocrine and paracrine growth control is suggested, the effects of which might be exerted, in part, through alpha and beta IL-8 receptors.

Platelet factor 4 platelet levels are inversely correlated with steady‐state platelet counts and with platelet transfusion needs in pediatric leukemia patients

“…Another candidate to ameliorate thrombocytopenia, especially after marrow injury [5,6], is platelet factor 4 (PF4, CXCL4), a platelet-specific and megakaryocyte-specific chemokine stored in a-granules and released from activated platelets. In vitro studies have shown that PF4 is a negative regulator of human and murine megakaryopoiesis via LDL-receptor related protein-1 [6][7][8]. In mice, we have shown that steady-state platelet counts are inversely related to platelet PF4 (pPF4) content [6].…”

“…Prescreening pPF4 levels in these patients may permit more intensive dosing of chemotherapy or shorter intervals between cycles, which have been associated with better event-free survival in some cancer types [10,11]. Additionally, strategies to block PF4 have been effective in altering the duration and severity of thrombocytopenia in animal models of CIT and RIT [6,7]. Therefore, prospective validation of pPF4 linkage to CIT in a variety of cancers is important as a first step in the eventual development of strategies that block PF4Õs negative paracrine effect on megakaryocytes as a therapeutic strategy for CIT.…”

Thrombopoietin‐induced CXC chemokines, NAP‐2 and PF4, suppress polyploidization and proplatelet formation during megakaryocyte maturation

“…Chemokines are divided into two major subfamilies, CXC chemokines and CC chemokines, based on whether the first two cysteines are separated by one amino acid (CXC) or are adjacent (CC). Chemokines are known to act not only as immune and inflammatory regulators but also as regulators of haematopoiesis (Gewirtz et al . 1995;Butcher & Picker 1996;Rollins 1997;Brandt et al .…”

“…1995). However, CXC chemokines including PF-4, NAP-2 and interlukin-8, as well as CC chemokines, macrophage inflammatory protein-1 α (MIP-1 α ), MIP-1 β and C10, were found to act negatively on megakaryocyte colony formation (Gewirtz et al . 1995).…”

Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

“…To facilitate these studies we have also generated mutants of MIP-la which have a reduced tendency for self aggregation (Graham et al, 1994). Other members of the wider chemokine family also display proliferation regulatory functions ranging from stimulation of keratinocyte (Tuschil et al, 1992) or haemopoietic progenitor cell proliferation (Broxmeyer et al, 1990), to inhibition of endothelial cell (Gupta and Singh, 1994;Luster et al, 1995) or megakaryocyte proliferation (Gewirtz et al, 1995).…”