The uptake of cholesterol-rich LDL by macrophages is thought to be critical in mediating the development of atherosclerotic plaques. Therefore, understanding and targeting the pathways mediating LDL uptake by macrophages is an important goal. In this study, by using inhibitors of LDL uptake by M-CSF–differentiated human macrophages, we quantitatively and morphologically demonstrate that native LDL uptake by macrophages occurs by both macropinocytosis and micropinocytosis. Targeting both pinocytotic pathways is necessary for efficient inhibition of LDL uptake by M-CSF–differentiated macrophages because inhibition of either macropinocytosis or micropinocytosis only results in an approximate 40% decrease in LDL uptake, whereas inhibiting both pathways results in an 80% decrease in LDL uptake.
Fluid-phase pinocytosis is a receptor-independent mechanism of solute uptake by cells that occurs by macropinocytosis and/or micropinocytosis.35
Because it was previously noted that fluid-phase pinocytosis of native LDL by M-CSF–differentiated macrophages accounts for all native LDL uptake by these macrophages,7
fluid-phase macropinocytosis and fluid-phase micropinocytosis must account for all macrophage uptake of LDL in this study.
We found that the kinase inhibitor SU6656 inhibited macropinocytosis. SU6656 was identified as a specific small-molecule inhibitor of Src family kinases.22
showed that overexpression of Src or other nonpalmitoylated Src family kinases promotes macropinocytosis in certain cell lines, suggesting that Src family kinases may mediate macropinocytosis by M-CSF–differentiated macrophages. However, when M-CSF–differentiated macrophages were treated with other Src family kinase inhibitors, PP1 or PP2, there was no effect on LDL uptake, suggesting that macropinocytosis may be mediated by a non-Src family kinase. Although SU6656 was initially identified as a “specific” Src family kinase inhibitor,22
non-Src family kinases can be efficiently inhibited by this drug.39
showed that minimally modified LDL stimulates murine macrophage macropinocytosis by activating the non-Src family kinase Syk. We observed no role for Syk-mediating macrophage macropinocytosis. Because we did not stimulate macrophages with minimally modified LDL, it is not surprising that Syk inhibition did not affect macrophage macropinocytosis in this study. Although beyond the scope of this study, future studies will determine the kinase mediating macropinocytosis in M-CSF–differentiated macrophages.
We also found that the Rho GTPase inhibitor toxin B inhibited macropinocytosis, in agreement with previous studies28–32
showing that Rho GTPases mediate macropinocytosis. However, similar to SU6656, although toxin B substantially inhibited macropinocytosis, this agent only partially inhibited macrophage LDL uptake and cholesterol accumulation, further supporting that micropinocytosis contributes substantially to LDL uptake and cholesterol accumulation.
The inhibition of both macropinocytosis with SU6656 and micropinocytosis with bafilomycin A1 resulted in an 80% decrease in LDL uptake by macrophages, showing that inhibition of at least 1 pathway is incomplete. In the presence of bafilomycin A1, we found a nearly complete absence of micropinosomes containing the fluid-phase tracer HRP by electron microscopy, suggesting that inhibition of macropinocytosis is incomplete. Also, electron microscopic analysis showed occasional macropinosomes present in SU6656-treated macrophages; and SU6656-treated macrophages showed residual macropinocytosis, as observed by time-lapse microscopy (supplemental Figure II
). Therefore, the remaining 20% of macrophage LDL uptake in the presence of SU6656 and bafilomycin A1 is most likely the result of incomplete inhibition of macropinocytosis by SU6656. Insufficient dosage of SU6656 was not the reason for incomplete inhibition of macrophage macropinocytosis because the doses used in this study maximally inhibited macropinocytosis and LDL uptake (data not shown), suggesting that at least 1 SU6656-insensitive macropinocytotic pathway exists. Attributing 20% of macrophage LDL uptake to SU6656-insensitive macropinocytosis and 40% to SU6656-sensitive macropinocytosis results in 60% of LDL uptake mediated by macropinocytosis. Consequently, 40% of LDL uptake can be attributed to micropinocytosis.
show that murine macrophages take up fluid by micropinocytosis and macropinocytosis. We extend this finding, relating it to macrophage pinocytosis of LDL. M-CSF–differentiated human macrophages were generally found to contain macropinosomes (>0.5 µm) and micropinosomes (<0.2 µm). These pinosome sizes are consistent with previous observations for murine M-CSF–differentiated macrophages.42
In murine M-CSF–differentiated macrophages, most macropinosomes are 0.5 to 1.0 µm,42
similar to what we observed with human M-CSF–differentiated macrophages in this study. Macropinosomes, because of their size, contain substantially more fluid than micropinosomes. For example, assuming a spherical macropinosome, we calculate that 524 aL of fluid is contained within a single 1 µm-diameter macropinosome. In contrast, a spherical 0.1 µm-diameter micropinosome (the approximate diameter of a clathrin-coated vesicle) only contains 0.5 aL of fluid. Because the fluid volume within macropinosomes is much greater than within micropinosomes, the amount of LDL taken up by an individual macropinosome must be substantially greater (> 1000-fold) than that of a micropinosome. Therefore, extensive micropinocytosis by macrophages must occur for 40% of LDL uptake to be attributed to micropinocytosis. Assessment of HRP-treated macrophages by electron microscopy showed more micropinosomes than macropinosomes containing HRP reaction product (supplemental Figure IX
), albeit much less than the greater than 1000-fold difference calculated. However, static microscopy does not provide an indication of rates of formation and turnover of vesicles and vacuoles. Thus, the relative numbers of pinocytotic vesicles and vacuoles observed in electron micrographs does not provide an accurate evaluation of the flux of solute carried by micropinosomes and macropinosomes.
Murine macrophages cultured without M-CSF deliver 0.43% of the cell volume per minute by fluid-phase pinocytosis mediated by micropinocytosis.33
We calculate that human macrophages take up 0.89% of the cell volume per minute by fluid-phase pinocytosis, of which 0.36% can be attributed to micropinocytosis, roughly the amount of fluid pinocytosed through micropinocytosis in murine macrophages cultured without M-CSF. Because it was later found that M-CSF stimulation of murine macrophages induces macropinocytosis and increases fluid-phase pinocytosis approximately 2-fold,41
it would be expected that 0.86% of the cell volume per minute would be pinocytosed for murine macrophages in the presence of M-CSF, similar to the 0.89% we calculate for human macrophages. Thus, these calculations show that the relative amount of fluid taken up through micropinocytosis and macropinocytosis in murine and human macrophages is similar. In contrast, an important difference is that M-CSF must be present for macropinocytosis in M-CSF–differentiated murine macrophages41
but is not required for macropinocytosis in M-CSF–differentiated human macrophages, which show constitutive macropinocytosis.7
Because of multiple micropinocytotic subpathways, targeting 1 subpathway may not result in substantial inhibition of solute uptake by cells. Furthermore, inhibition of certain pinocytotic pathways may upregulate other pinocytotic pathways. For example, the molecule dynamin is essential for clathrin-dependent and some clathrin-independent endocytic processes. The expression of nonfunctional mutant dynamin in human cell line cells results in upregulation of other fluid-phase pinocytotic pathways, completely compensating for the loss of fluid uptake by dynamin-dependent endocytosis.43
We found that inhibition of dynamin using the inhibitor dynasore increased rather than decreased LDL uptake by macrophages (JJA, HSK, unpublished observation, 2008), showing that upregulation of other endocytic pathways can also occur in macrophages. Because of the potential for upregulation of pinocytotic pathways, we cannot exclude the possibility that the macropinocytosis inhibitors used in this study upregulate micropinocytosis. Therefore, the percentage of LDL macropinocytosed by untreated macrophages may be greater than macrophages treated with macropinocytosis inhibitors. This possibility of secondary upregulation of micropinocytosis pathways may preclude substantial inhibition of macrophage LDL uptake and cholesterol accumulation with macropinocytosis inhibitors alone.
Clathrin- and caveolin-dependent micropinocytotic pathways can be inhibited by depletion of cellular cholesterol.44–47
Previously, it was reported that cholesterol depletion of M-CSF–differentiated macrophages with the cholesterol sequesterant methyl-β-cyclodextrin had no effect on macrophage uptake of the fluid-phase pinocytosis tracer 125
showing that micropinocytotic pathways of M-CSF–differentiated macrophages do not depend on cellular cholesterol. These results suggest that clathrin- and caveolin-dependent micropinocytotic pathways do not contribute substantially to fluid uptake by M-CSF–differentiated macrophages. In a separate study, it was also determined that greater than 95% of LDL uptake by M-CSF–differentiated macrophages is dependent on actin polymerization.7
Because micropinocytosis has previously been reported to be actin independent,49,50
these data suggested that nearly all macrophage uptake of LDL occurs by macropinocytosis, a process requiring actin polymerization. In contrast, we clearly demonstrate in this study that micropinocytosis contributes substantially to LDL uptake by macrophages, showing that almost all micropinocytotic uptake of LDL occurs in an actin-dependent manner. Although micropinocytosis is generally actin independent, certain cell types show actin-dependent micropinocytosis.51,52
Therefore, our results show that almost all micropinocytosis by M-CSF–differentiated macrophages is actin dependent and cholesterol independent. These data emphasize that the properties of endocytic processes can vary between cell types and warrant caution in generalizing findings to all cell types.
Macropinocytosis only occurs in some cell types, whereas micropinocytosis is common to all cells, suggesting that other cell types may accumulate cholesterol by this pathway. Fibroblasts have been shown to take up LDL by fluid-phase pinocytosis.53,54
Because macropinocytosis is not known to occur in fibroblasts, the uptake of LDL by fibroblasts most likely occurs by micropinocytosis; however, cholesterol accumulation in these cells does not occur because all the LDL cholesterol taken up is excreted by the fibroblasts.54
In contrast, micropinocytosis of LDL by M-CSF–differentiated macrophages leads to cholesterol accumulation because LDL uptake and cholesterol accumulation by macrophages occurred in the presence of macropinocytosis inhibitors.
Previously, it was shown that human serum-differentiated macrophages stimulated with phorbol 12-myristate 13-acetate (PMA) take up LDL and accumulate cholesterol by macropinocytosis.6
Nearly all LDL uptake and cholesterol accumulation by PMA-stimulated human serum–differentiated macrophages were attributed to macropinocytosis because inhibitors of macropinocytosis almost completely inhibited LDL uptake and cholesterol accumulation. Although not directly investigated in that study, a small amount of LDL uptake by human serum–differentiated macrophages can be attributed to micropinocytosis because, in the absence of macropinocytosis (ie, without PMA stimulation), LDL uptake occurred. The amount of LDL micropinocytosed by unstimulated human serum–differentiated macrophages was approximately 20% to 30% of the total uptake of PMA-stimulated human serum–differentiated macrophages and only led to a small increase in cellular cholesterol (approximately 10% of PMA-stimulated cellular cholesterol). In contrast, micropinocytosis of LDL by M-CSF–differentiated macrophages accounts for at least 40% of LDL and cholesterol accumulation. It remains to be determined if micropinocytosis of LDL contributes substantially to cholesterol accumulation in other cell types or is a unique feature of M-CSF–differentiated macrophages.
One commonly known inhibitory function of bafilomycin A1 is disruption of lysosomal function.55
This occurs by inhibition of the vacuolar type H(+)-ATPase, causing an increase in lysosomal pH, thereby preventing enzymatic degradation of endocytosed cargo. Consistent with this, we found that bafilomycin A1 substantially inhibited macrophage degradation of LDL compared with untreated cells (26% and 76% of pinocytosed LDL was degraded, respectively). Bafilomycin A1 can also inhibit other cellular functions. For example, bafilomycin A1 slows endosomal recycling to the plasma membrane and prevents the formation of multivesicular bodies.56
Furthermore, bafilomycin A1 may have other undiscovered inhibitory properties. Although it has not been previously described that bafilomycin A1 inhibits pinocytosis, we found that bafilomycin A1 inhibited micropinocytosis in M-CSF–differentiated human macrophages. Previously, bafilomycin A1 was used to determine whether phorbol ester–stimulated human serum–differentiated macrophages degrade LDL using lysosomes.6
Although not investigated in that study, bafilomycin A1 was also found to inhibit fluid phase–mediated LDL uptake by phorbol ester–stimulated human serum–differentiated macrophages, supporting the inhibitory effect on pinocytosis by bafilomycin A1. Further supporting our finding that bafilomycin A1 can inhibit pinocytosis, a previous study57
showed that bafilomycin A1 inhibits receptor-mediated endocytosis of albumin by mouse tubular cells. In that study, the vacuolar type H(+)-ATPase was reported to act as a sensor of endosomal pH, with increases in endosomal pH inhibiting endocytosis. Additional studies have shown that the vacuolar type H(+)-ATPase is found in the plasma membrane of kidney epithelial cells58
and can form a vesicle coat, suggesting a potential role for the vacuolar type H(+)-ATPase in mediating vesicle trafficking. Taken together, these studies substantiate that the vacuolar type H(+)-ATPase inhibitor bafilomycin A1 can inhibit micropinocytosis.
In summary, we find that fluid-phase macropinocytosis and fluid-phase micropinocytosis each contribute substantially to LDL uptake by human M-CSF–differentiated macrophages, with both pathways promoting macrophage cholesterol accumulation. This study shows that targeting macrophage uptake of LDL should not only consider macropinocytosis as a mechanism of macrophage LDL uptake but also micropinocytosis and that both uptake pathways can facilitate substantial cholesterol accumulation in macrophages.