© 2000 American Heart Association, Inc.
Letters to the Editor |
Oxidized LDL Can Promote Human Monocyte Survival
Arthritis and Inflammation Research Centre, University of Melbourne, Department of Medicine, The Royal Melbourne Hospital, Parkville, Victoria, Australia, 3050
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, 
-modification (-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
Table
). A dose response for the
ox-LDL effect is provided in the online Figure
(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 I; please see
http://atvb.ahajournals.org).
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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 II and III; 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 II and III); 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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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.
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