PRODUCTION AND CONSUMPTION OF THE TETRAPEPTIDE ACSDKP, A NEGATIVE REGULATOR OF HEMATOPOIETIC STEM-CELLS, BY HEMATOPOIETIC MICROENVIRONMENTAL CELLS

This study was performed to evaluate the role of human microenvironmen tal cells in the metabolism of AcSDKP, a physiological inhibitor of he matopoietic stem cells. Using long-term marrow cultures (LTMCs), whose medium already contained a baseline value of AcSDKP, we found after 2 weeks a net output in the culture supernatant indicating that release by cells from the adherent layer was superior to consumption of the p eptide. Since human microenvironmental cells consist of macrophages an d vascular smooth-muscle-like stromal cells we generated pure populati ons of macrophages (by culturing cord blood cells in the presence of g ranulomonocytic colony-stimulating factor) and of stromal cells (gener ated by stromal colonies). We found in supernatants of macrophage cult ures a significantly (p < 0.01) increased level of AcSDKP (compared wi th value in medium) while in supernatants of stromal cell cultures the level was decreased. Cell content of angiotensin-converting enzyme (A CE) in stromal cells was higher than in macrophages, which suggests a degradation of AcSDKP by stromal cells because of their higher amount of ACE. Finally, we analyzed the content of AcSDKP in adherent layers of LTMCs (with or without extracellular matrix [ECM] components), macr ophages, and stromal cells. We found levels of AcSDKP of 1.5 pMol per 10(6) cells in extracts from macrophages or from stromal cells. On the contrary, extracts from primary layers of LTMCs contained 3 times mor e AcSDKP; however, after treatment of primary layers by collagenase, A cSDKP level fell to 1 pMol per 10(6) cells. Immunofluorescence using a n anti-AcSDKP monoclonal antibody showed an extracellular network in c ertain areas of LTMCs. This study shows that 1) macrophages synthesize and release in the supernatant AcSDKP, 2) stromal cells probably degr ade the peptide via ACE, and 3) components of the ECM from LTMCs serve as a reservoir for the peptide. These results are reminiscent of what has been described for growth factors, produced by microenvironmental cells, and stored in the ECM in close vicinity to hematopoietic precu rsors.