A NEW METHOD FOR THE RAPID SEPARATION OF HUMAN BLOOD MONOCYTE SUBSETS BY COUNTER - CURRENT CENTRIFUGAL ELUTRIATION

Abstract

The existence of two human peripheral blood monocyte subpopulations, the regular monocytes (RN) and the intermediate monocytes (IM), was previously reported (Akiyama et al. 1983). The RN and the IM monocytes were characterized by their differential ability to mediate antibody-dependent cellular cytotoxicity (ADCC), secrete alpha interferon in response to polyI:C, and express surface antigens reactive with the OKM1 monoclonal antibody. To date RN and IM monocytes have been isolated by a combination of counter-current centrifugal elutriation (CCE) followed by purification over discontinuous human serum albumin gradients. Although this method produced enriched IM monocytes, the yield was low. In addition, the procedure was cumbersome, and still failed to produce pure lymphocytes. This thesis describes a improved method of isolating IM monocytes and lymphocyte subsets to greater than 90% purity with increased yield. The new gradient elutriation method purified IM monocyte were assayed and found to retain three characteristics of IM monocytes. The three characteristics that differentiate IM monocytes from RN are 1) decreased capability for ADCC, 2) increased polyI:C induced alpha interferon secretion, and 3) low OKM1 antigen expression. In addition to these three properties the RN and IM monocytes ability to secret tumor necrosis factor, produce hydrogen peroxide and the surface expression of the human leukocyte antigen (HLA) HLA-Dr and leu-M3 antigens were also examined. No differences in the tumor necrosis factor or hydrogen peroxide production were found; there was no difference in the surface expression of HLA-Dr, but there was a difference in the expression of the surface antigen for Leu-M3.