The transcriptional response of human macrophages to murabutide reflects a spectrum of biological effects for the synthetic immunomodulator

Abstract

The synthetic immunomodulator murabutide (MB) presents multiple biological activities with minimal toxicity in animals and in man. Although MB is known to target cells of the reticuloendothelial system and to regulate cytokine synthesis, the molecular mechanisms underlying several of its biological effects are still largely unknown. In an effort to define cellular factors implicated in the immunomodulatory and HIV-suppressive activities of MB, we have undertaken profiling the regulated expression of genes in human monocyte-derived macrophages (MDM) following a 6-h stimulation with this synthetic glycopeptide. Oligonucleotide microarray analysis was performed on RNA samples of differentiated MDM from four separate donors, using probe sets corresponding to 1081 genes. We have identified, in a reproducible fashion, the enhanced expression of 40 genes and the inhibition of 16 others in MB-treated MDM. These regulated genes belonged to different families of immune mediators or their receptors, transcription factors and kinases, matrix proteins and their inhibitors, ion channels and transporters, and proteins involved in cell metabolic pathways. Additional verification of the regulated expression of selected genes was carried out using Northern blots or the quantification of released proteins in MDM cultures. The profile of MB-regulated genes in MDM provides a molecular basis for some of its previously reported biological activities, and reveals new set of genes targeted by the immunomodulator suggesting potential application in novel therapeutic indications.

Fig. 1

Kinetics of gene induction in MDM following stimulation with MB. (a) RNA samples (20 μg) prepared from MDM that were either left untreated (0 h) or stimulated with MB (2, 4 and 6 h) have been analysed by Northern blot using ³²P-labelled oligonucleotide probes specific for NINJ-1, HSP70 or GLUT-1. The same membrane was hybridized with a GAPDH cDNA probe. (b) graphical representation of the changes in NINJ-1, HSP70 and GLUT-1 gene expresssion following MB stimulation. Autoradiographs were analysed by densitometry and the relative mRNA levels of each gene were normalized to those of GAPDH (results shown are of one representative experiment of three independent ones). ✦, HSP70; □, NINJ-1; ▴, GLUT-1.

Fig. 2

Profile of cytokines released in culture supernatants of MDM left untreated (c) or stimulated with 10 μg/ml MB for a 24-h period. The levels of cytokines and chemokines in the culture supernatants of MDM derived from six different donors were determined using commercially avalaible ELISA kits.