Heterogeneous expression of apolipoprotein-E by human macrophages

Abstract

Apolipoprotein-E (apoE) is expressed at high levels by macrophages. In addition to its role in lipid transport, macrophage-derived apoE plays an important role in immunoregulation. Previous studies have identified macrophage subpopulations that differ substantially in their ability to synthesize specific cytokines and enzymes, however, potential heterogeneous macrophage apoE expression has not been studied. Here we examined apoE expression in human THP-1 macrophages and monocyte-derived macrophages (MDM). Using immunocytochemistry and flow cytometry methods we reveal a striking heterogeneity in macrophage apoE expression in both cell types. In phorbol-ester-differentiated THP-1 macrophages, 5% of the cells over-expressed apoE at levels more than 50-fold higher than the rest of the population. ApoE over-expressing THP-1 macrophages contained condensed/fragmented nuclei and increased levels of activated caspase-3 indicating induction of apoptosis. In MDM, 3-5% of the cells also highly over-expressed apoE, up to 50-fold higher than the rest of the population; however, this was not associated with obvious nuclear alterations. The apoE over-expressing MDM were larger, more granular, and more autofluorescent than the majority of cells and they contained numerous vesicle-like structures that appeared to be coated by apoE. Flow cytometry experiments indicated that the apoE over-expressing subpopulation of MDM were positive for CD14, CD11b/Mac-1 and CD68. These observations suggest that specific macrophage subpopulations may be important for apoE-mediated immunoregulation and clearly indicate that subpopulation heterogeneity should be taken into account when investigating macrophage apoE expression.

Figure 1

Analysis of apoE expression in THP-1 macrophages. THP-1 cells were treated with PMA 50 ng/ml for 72 hr then assessed for apoE expression by immunocytochemical staining and nuclear morphology by DAPI staining. (a–f) ApoE expression. The nuclear morphology for the series is shown directly below each panel. (a) Typical apoE expression in most cells is compared to high expression in the arrowed cell which contains distinct micronuclei in addition to the major nucleus. (b) ApoE expression is high in multinucleated giant cells. The arrowed cell contains six intact nuclei in addition to several clustered micronuclei. (c) In the arrowed cell, apoE is highly over-expressed and the nucleus shows a typical apoptotic morphology as it is condensed and fragmented. (d) Higher magnification of apoE over-expressing cell with condensed/fragmented nucleus. The apoE appears to encapsulate the fragmented nuclei. (e) Binuclear cell over-expressing apoE. (f) Staurosporine treated THP-1 macrophages displaying cytoplasmic shrinkage and relocalization of intracellular apoE to the plasma membrane. Note that in (f), the nuclear morphology panel is overlayed with a phase contrast image in order to illustrate plasma membrane alterations. Bar = 25 µm: (a, b, c and f) same magnification; (d and e) higher magnification all croped and resized.

Figure 2

ApoE over-expression is colocalized with active caspase-3 in THP-1 macrophages. THP-1 cells were treated with PMA 50 ng/ml for 72 hr then assessed for apoE and active caspase-3 expression by immunocytochemical staining and laser scanning confocal microscopy. (a) ApoE expression detected with an FITC-conjugated secondary antibody. (b) Active caspase-3 expression detected with an Alexa Fluor 633-conjugated secondary antibody. In the arrowed cell, both apoE and active caspase-3 are over-expressed. Bar = 50 µm

Figure 3

ApoE expression and caspase-3 activation in staurosporine-treated THP-1 cells. (a) THP-1 macrophages and MDM were treated with 0·1 µm staurosporine for the times indicated and caspase-3 activation was assessed by hydrolysis of the caspase-3 substrate Ac-DEVD-AMC to yield the fluorophore AMC in cell lysates (containing 12 µg protein per sample). Both THP-1 macrophages (○) and MDM (•) contained caspase-3 activity and this was either moderately increased (THP-1) or unchanged (MDM) with staurosporine treatment. In contrast, fibroblasts (□) contained very low caspase-3 activity initially but dramatically increased their activity after staurosporine treatment. The data for human foreskin fibroblasts are based on our previously published data generated using an identical experimental protocol and are included here for the purposes of comparison only. Data are means ± SE, n = 3. (b) THP-1 macrophages were treated with 0·1 µm staurosporine for 8 h and cell lysates (34 µg protein per sample) were analysed for apoE by Western blot. Recombinant human apoE3 was included as a positive control and the positions of molecular weight markers are indicated. Analysis of integrated optical densities of the apoE bands shown indicated that staurosporine (Stsp) treatment increased apoE levels by 64%.

Figure 4

Analysis of apoE expression in human MDM. MDM were cultured for either 7 or 14 days and assessed for apoE expression by immunocytochemical staining and nuclear morphology by DAPI staining. (a, d, g and j) Nuclear morphology. The corresponding apoE expression for each panel is shown directly to the right (b, e, h and k) and the arrowed cells are also shown at higher magnification (c, f, i and l). (a, b, c) Typical 7-day-old MDM are compared to an apoE over-expressing (arrowed) MDM. (d, e, f) Fourteen-day-old MDM. Clear Golgi and plasma membrane staining are demonstrated in the arrowed cell. (g, h, i) An apoE over-expressing 14-day-old MDM is compared with typical neighbouring cells. At higher magnification and using shorter exposure time, apoE is detected as numerous ring-like structures, which are presumably coating vesicles. (j, k, l) Treatment of 14-day-old MDM with 0·1 µm staurosporine for 16 hr resulted in disruption of the typical staining pattern (Golgi and plasma membrane) and the formation of numerous large apoE-containing intracellular deposits (l). Bar = 25 µm. Note: (c, f, i and l) at higher magnification.

Figure 5

Demonstration of predominant MDM plasma membrane apoE immunostaining. MDM were grown under standard culture conditions for 2 weeks, immunostained using an anti-apoE monoclonal antibody and examined under an Olympus LSM-GB 200 laser scanning confocal microscope. Cells were treated with propidium iodide after immunostaining and mounted in Vectashield medium. Optical serial sections of 2 µm are shown from the basal to apical cell surface (a–f).

Figure 6

Flow cytometric analysis of apoE in 14-day-old MDM. MDM were cultured for 14 days and analysed for size, granularity and for expression of apoE, CD14 and CD11b/Mac-1 by immunocytochemical staining and flow cytometric analysis. (a) Forward and side scatter analysis revealed a subpopulation of large granular MDM ‘R2’. (b) Analysis of MDM autofluorescence. (c) Three obvious levels of apoE immunostaining were present with a minor fraction ‘M3’ highly over-expressing apoE. (d) IgG control for apoE immunostaining did not reveal significant staining. (e) The M3 MDM population over-expressing apoE were clustered within the larger granular R2 MDM subpopulation. (f) The M2 MDM coincided with the major R1 population. (g, h and i) The R2 subpopulation were positive for apoE, CD11b and CD14, respectively. Fluorescence resulting from the relevant IgG isotype control conditions are also shown. Except for cell counts, data are log scale.