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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2329-2331

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2329-a.)
© 2000 American Heart Association, Inc.

Letters to the Editor

Oxidized LDL Can Promote Human Monocyte Survival

John A. Hamilton; Genevieve Whitty

Arthritis and Inflammation Research Centre, University of Melbourne, Department of Medicine, The Royal Melbourne Hospital, Parkville, Victoria, Australia, 3050

Wendy Jessup

Heart Research Institute, Camperdown, New South Wales, Australia

To the Editor:

It is likely that in the early stages of atherosclerosis, circulating monocytes migrate into the subendothelial space, where they can mature into foam cells.1 2 3 4 5 There is in vivo and in vitro evidence for both foam cell death but also enhanced survival and growth.6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Human peripheral blood monocytes (>=95% pure) were obtained by countercurrent elutriation and usually cultured in minimal essential medium, {alpha}-modification ({alpha}-MEM)/1% pooled normal human serum (HS).23 24 The number of viable cells was measured by scraping the tissue culture surface and counting them in a hemocytometer with trypan blue exclusion or by propidium iodide staining (flow cytometry). Oxidized LDL (ox-LDL) was prepared as before.14

The number of viable monocytes declined when they were left untreated or treated with native LDL; this loss was reduced by both ox-LDL and acetylated LDL (ac-LDL; see the TableDown). A dose response for the ox-LDL effect is provided in the online FigureDown (please see http://atvb.ahajournals.org) and, as we found before with murine macrophages,14 doses of ox-LDL <=50 µg/mL generally promoted survival; at these survival-inducing doses, the cells spread on the tissue culture surface and remained attached. In contrast, at higher concentrations, viable cell numbers again declined. With different ox-LDL preparations, the effective survival dose response varied to some extent. The ability of ox-LDL to enhance human monocyte survival was confirmed with monocytes from 30 donors. We previously found that prior adherence of the monocytes for a short period under serum-free conditions, followed by culture in 1% HS, improved the subsequent viability of the cells.24 Under these conditions, ox-LDL was able to maintain the original cell number (online Table IDown; please see http://atvb.ahajournals.org).


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  		Table 1. Effect of Ox-LDL and Ac-LDL on Human Monocyte Survival



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  		Figure 1. Effect of ox-LDL dose on human monocyte survival. Elutriation-purified human monocytes were plated at 2x105 monocytes (t=0) in {alpha}-MEM/1% HS and were either left untreated or treated with increasing concentrations of ox-LDL. After 5 days, viable cell number was determined (hemocytometer, trypan blue exclusion). Data are from a representative experiment, which was repeated 12 lines with monocytes from different donors, and are mean values|Pd+SEM from triplicate cultures.

It is possible that the enhanced human monocyte survival by ox-LDL described above is due to endogenous granulocyte macrophage–colony stimulating factor (GM-CSF) and/or CSF-1.25 26 27 For ox-LDL–treated cultures, no evidence could be found for a requirement for either CSF by using blocking monoclonal antibodies to the ligands and to the CSF-1 receptor (online Tables DownII and IIIDown; please see http://atvb.ahajournals.org). For most experiments, the antibodies reduced the number of viable cells in the untreated cultures, suggesting that endogenous GM-CSF and CSF-1 play a role in monocyte survival in 1% HS (online Tables IIDown and IIIDown); this inhibitory effect on basal survival led, in some experiments, to an apparent reduction in the number of viable cells in the ox-LDL–treated cultures, which could, however, be accounted for by an effect on the survival of the non–ox-LDL–treated cells (data not shown).


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  		Table 2. Effect of Antibody to GM-CSF on Ox-LDL–Induced Human Monocyte Survival


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  		Table 3. Effect of Antibodies to CSF-1 and to Its Receptor on Ox-LDL–Induced Human Monocyte Survival

Prior studies have found that ox-LDL caused apoptosis in adherence-prepared human monocyte cultures.12 However, in that study, only ox-LDL concentrations >=50 µg/mL were examined, and the toxic response increased as the concentration of the lipoprotein was raised to 200 µg/mL; the effects of lower concentrations were not reported. From our studies, it is important to titrate the concentration of each ox-LDL batch on human monocytes. Our findings on the reversal of cell death by ox-LDL are similar to what we have published previously with murine macrophages.14 Others have found that human macrophages, derived after maturation from 9-day cultures of monocytes, subsequently showed a proliferative response when treated with 10 to 50 µg/mL ox-LDL.5 We found no evidence of increased DNA synthesis (tritiated thymidine incorporation) over the 5-day period in our ox-LDL–treated human monocytes (data not shown).

The few studies that have measured the amounts of oxidation products, eg, oxysterols, present in foam cells from human lesions have found them to be small28 ; also during the early stages of atherosclerosis, the amount of ox-LDL is likely to be low. It could therefore be argued that lower ox-LDL loadings could more likely better represent the in vivo situation than the high (toxic) levels, although it could be imagined that at more advanced stages of the disease, increased accumulation of ox-LDL may generate a toxic effect.29 Our data could help explain both the increased numbers of foam cells, as well as the presence of apoptotic cells, in atheroma (see also Reference 14 ).

We have demonstrated above that ac-LDL was quite potent in promoting human monocyte survival. Uptake of ox-LDL by macrophages occurs in part through the ac-LDL receptor,30 31 but several lines of evidence point to the existence of a number of receptors for ox-LDL.32 The contribution of different receptor usage to the effects on human monocyte survival remains to be elucidated. Our result with ac-LDL and human monocytes is consistent with our findings in murine macrophages;14 in contrast, others have distinguished ac-LDL from ox-LDL by the inability of the former to induce murine macrophage growth.33

In summary, foam cells in atherosclerotic plaques are widely believed to result from the uptake by monocytes/macrophages of LDL after its modification, eg, by oxidation. Human monocytes slowly die in vitro, an apoptotic process that has been reported to be enhanced after addition of ox-LDL.12 We report here that the effect of ox-LDL on the survival of elutriation-purified human monocytes in vitro is dose dependent, with high concentrations being toxic but lower concentrations in fact promoting survival. Ac-LDL, but not native LDL, was also active in enhancing monocyte survival. Addition of blocking monoclonal antibodies to either GM-CSF or CSF-1 failed to provide evidence for an essential role for these CSFs in ox-LDL–promoted monocyte survival. The data could help explain both the increased numbers of foam cells, as well as the presence of apoptotic cells, in atheroma.

References

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