Abstract
Improved techniques of cell isolation resulted in 90 to 100 million monocytes from a single donor. Addition of low density lipoprotein (LDL) to to cultures of these cells resulted in the down regulation of LDL receptor activity. Addition of malondialdehyde-altered LDL. which enters the cell through a receptor for negatively charged proteins (the scavenger receptor), produced an even greater down regulation of the LDL receptor, indicating that both receptors are present on the same cell. Within hours of adherence of the cells, there was a dramatic decrease in the activity of both receptors. LDL receptor activity was highest during the first week in culture and then declined, despite the maintenance of a constant LDL concentration in the medium. Scavenger receptor activity surpassed LDL receptor activity by the 6th day and was maximally expressed during the second week. Increasing cell density resulted in a slight increase in the activity of the LDL receptor and a dramatic increase in scavenger receptor activity. Insulin had no significant effect on either receptor. Removing serum from the culture medium for 48 hr resulted in a 3.5-fold increase in LDL receptor activity and a 2-fold decrease in scavenger receptor activity. Twenty-four hr after the cells were re-exposed to serum, the activities of both receptors essentially returned to base line. Heat-inactivation of serum was associated with an increased cholesteryl ester content of the cells and depressed receptor activities. Scavenger receptor activity appears related to the maturation of monocytes into macrophages and is promoted by increasing cell density and serum factors that are heat labile.
Highlights
Improved techniques of cell isolation resulted in 90 to 100million monocytes froma single donor
In order to carry out these experiments, we often required greater numbers of cells than could be obtained by the methods that we developed earlier(5).we devised variations of these methods that produce[90] to 100 million monocytes from a single donor
We found thata factor such as insulin that has been shown to affect the activity of the low density lipoprotein (LDL) receptor in cultured human fibroblasts (6) has no effect on either the LDL receptor or the scavenger receptor of human monocyte-macrophages
Summary
(sp act 15 mCi lZ5I/pgof iodine) was purchased from Amersham,ArlingtonHeights,IL. Crystallized Human Albumin T h e tubes from which the cells had been taken were washed successively with an additional 10 ml of buffer Awhich was added to the two tubes containing the cells. The supernatant was again discarded and the cells were resuspended in buffer A containing penicillin The supernatant was discarded and thceells were resuspended in 10 ml of buffer B by sequentially transferring the contentosf each tube. This method differed from method aBs described previously (2,5)in that 300 ml of blood was processed for cells andthe Plasmagel supernatant was centrifuged once at 150g at 5°C for 10 min in order to remove the platelets. One tubewas kept on ice whilethe contentsof the otherwas injected into the Elutriator system and processed as described previously (2,5),except thatan additional 40 ml of monocyte fraction was collected at aflow rate of 18.2ml/min. The addition of the Fungizone had no effect on protein content
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