(Received for publication, April 20, 1994; and in revised form, December 15, 1994)
From the
Cell adhesion between circulating monocytes and the endothelium is a critical component of vascular thromboregulation and atherogenesis. The biochemical and genetic consequences of adhesion are poorly understood. We have found that monocyte surface expression of CD36, an integral membrane receptor for thrombospondin, collagen, and oxidized low density lipoprotein, increased dramatically upon adhesion to tumor necrosis factor-activated human umbilical vein endothelial cells (HUVEC). Expression was assessed by indirect immunofluorescence microscopy and immunoblotting using monoclonal antibodies to CD36. Steady-state CD36 mRNA levels, detected by RNase protection assay, also showed a similar pattern of up-regulation. To verify the adhesion dependence of the observed phenomenon, monocytes were co-cultured with tumor necrosis factor-activated HUVEC in a transwell apparatus that physically separated monocytes from the endothelial cells. Under these conditions, no increase in CD36 expression was detected, demonstrating that the enhanced monocyte CD36 expression observed is not due to soluble factors released by HUVEC. To characterize the specific adhesion molecules involved in the process, co-culture assays were performed on murine L cells transfected with either human E-selectin or intercellular adhesion molecule-1 cDNAs. A dramatic increase in CD36 mRNA was seen upon monocyte adhesion to E-selectin-transfected L cells compared with adhesion to intercellular adhesion molecule-1 or control transfectants. Furthermore, monoclonal antibodies to E-selectin inhibited the adhesion-dependent up-regulation of CD36 mRNA induced by transfected L cells or cytokine-activated endothelial cells. These findings demonstrate adhesiondependent gene regulation of monocyte CD36 and suggest the possible involvement of E-selectin in initiating this process.
Monocyte adhesion to cytokine-activated endothelium is mediated
by specific adhesion molecules expressed on monocyte and endothelial
cell surfaces (Bevilacqua, 1993; Butcher, 1991; Shimazu et
al., 1992; Springer, 1990). The biochemical and genetic
consequences that follow these adhesive events remain poorly
characterized. Previous studies suggest that engagement of one adhesion
molecule may sequentially activate another in an adhesion cascade (Lo et al., 1991). For example, E-selectin-mediated adhesion of
neutrophils and T cell receptor ligation on lymphocytes have been shown
to enhance the functional activity of the -integrins
CD11b/CD18 (Lo et al., 1991) and CD11a/CD18 (Dustin and
Springer, 1989), respectively. Furthermore, we have recently shown that
monocyte adhesion to cytokine-activated endothelial cells resulted in
monocyte tissue factor expression (Lo et al., 1995). In
contrast to integrin activation, this induction was at the level of
mRNA transcription, suggesting that specific adhesion events may be
capable of initiating a genetic program in the adherent cells.
Similarly, Eierman et al.(1989) have shown that monocyte
adhesion to immobilized substrates induces c-fos and TNF-
(
)mRNAs.
CD36 is an 88-kDa transmembrane glycoprotein expressed on monocytes, platelets, and microvascular endothelium. It functions as an adhesive receptor for thrombospondin (Asch et al., 1987; Silverstein et al., 1992) and collagen (Tandon et al., 1989) and mediates cytoadherence of Plasmodium falciparum-infected erythrocytes to the endothelium (Barnwell et al., 1985). The CD36-thrombospondin (TSP) interaction participates in platelet-tumor cell adhesion (Silverstein et al., 1992) and macrophage uptake of aged neutrophils (Savill et al., 1992). Furthermore, CD36 has also been shown to function as a scavenger receptor on macrophages for oxidized low density lipoprotein (Endemann et al., 1993; Nicholson et al., 1995). Cellular regulation of this multifunctional receptor has not yet been well studied. Asch et al.(1993) have recently shown that ligand specificity may be regulated by phosphorylation of an extracellular domain, and we have shown that cytokines may regulate monocyte mRNA levels (Yesner et al., 1993).
In this paper, we report changes in monocyte CD36 levels upon adhesion to activated endothelial cells. The data presented demonstrate significant enhancement of CD36 expression at both the mRNA and protein levels upon monocyte adhesion to TNF-activated endothelium. Furthermore, our data suggest that adhesion of monocytes to HUVEC via E-selectin is likely to be involved in the induction of CD36 on monocytes.
Figure 1:
Monocyte CD36 levels increase upon
co-culture with TNF-stimulated HUVEC. Confluent HUVEC in 12-well
culture plates were pretreated with TNF (200 units/ml, 4 h), thrombin
(2 units/ml, 30 min), or buffer; washed thoroughly; and then
co-cultured with Ficoll/Percoll-purified human peripheral blood
monocytes (5 10
cells) for 4 h. Well contents were
then lysed, resolved by SDS-polyacrylamide gel electrophoresis, and
immunoprobed for CD36 with monoclonal anti-CD36 IgG (FA6). LaneA shows monocytes co-cultured with TNF-stimulated HUVEC, laneC with resting HUVEC, and laneD with thrombin-stimulated HUVEC. LaneB shows
TNF-stimulated HUVEC in the absence of added
monocytes.
To localize the induced CD36 protein, indirect immunofluorescence was performed. As shown in Fig. 2, monocytes co-cultured with control HUVEC demonstrated faint rim fluorescence around the cell membrane (Fig. 2A), representing the basal cell-surface CD36 protein level on the monocytes. In comparison, significantly enhanced cell-surface fluorescence was observed on monocytes upon adhesion to TNF-activated HUVEC (Fig. 2B). These results verify changes in CD36 protein upon adhesion to TNF-activated HUVEC at the single cell level while demonstrating that the increased CD36 level was localized to the monocyte plasma membrane. In these experiments, >90% of the mononuclear cells stained with anti-CD36 IgG. Staining intensity on the adherent cells was uniform.
Figure 2: Increased surface expression of CD36 on monocytes adherent to TNF-activated HUVEC. HUVEC-monocyte co-cultures were prepared as described for Fig. 1on glass coverslips and fixed with methanol after a 4-h coincubation. Immunolocalization of CD36 was performed using indirect immunofluorescence microscopy with monoclonal anti-CD36 antibody FA6. A shows monocytes co-cultured with unstimulated HUVEC, and B shows cells co-cultured with TNF-stimulated HUVEC (200 units/ml, 4 h).
To determine if the observed increases in CD36 expression were associated with concomitant changes in CD36 mRNA levels, we measured steady-state CD36 mRNA by RNase protection assay. As shown in Fig. 3, monocytes adherent to TNF-activated HUVEC demonstrated a >10-fold increase in steady-state CD36 mRNA levels, detected as a 760-bp protected fragment (laneA), relative to monocytes adherent to thrombin-activated HUVEC (laneB) or to cells co-cultured with control HUVEC (laneC). No CD36 mRNA was detected in TNF-stimulated HUVEC lysates alone (laneD). The lack of visible bands in lanesB and C is not due to lack of CD36 expression on nonadherent monocytes. Rather, it is a photographic artifact reflecting the dramatic up-regulation of CD36 in laneA. The basal level of monocyte CD36 mRNA expression is seen better in Fig. 6. Time course studies (data not shown) revealed that the induction of CD36 mRNA was detected at 4 h and persisted for at least 24 h.
Figure 3:
Monocyte
CD36 mRNA levels increase upon co-culture with TNF-stimulated HUVEC.
Confluent HUVEC were pretreated as described for Fig. 1prior to
the addition of purified human peripheral blood monocytes. After a 4-h
co-culture, well contents were lysed in 5 M guanidine
thiocyanate, 0.1 M EDTA and hybridized with P-labeled CD36 and glyceraldehyde-phosphate dehydrogenase
antisense riboprobe at 37 °C for 20 h. The samples were then
digested with RNase A and RNase T1 and electrophoresed on 5%
polyacrylamide gels, and autoradiograms were obtained. LaneA shows a 759-bp protected fragment from monocytes
incubated with TNF-stimulated HUVEC, laneB with
thrombin-stimulated HUVEC, and laneC with
unstimulated HUVEC. LaneD shows the TNF-stimulated
HUVEC lysate in the absence of added monocytes. The bargraph for each lane shows densitometric scanning results
represented as -fold change in densitometric intensity. Total RNA per
lane was normalized by comparison with total RNA hybridized with a
glyceraldehyde-phosphate dehydrogenase (GAPDH)
riboprobe.
Figure 6: Monocyte CD36 mRNA up-regulation on E-selectin-transfected L cells is inhibited by monoclonal antibodies to E-selectin or by the transcriptional inhibitor actinomycin D. Monocytes were co-incubated with E-selectin-transfected L cells for 4 h as described for Fig. 5, and lysates were probed with CD36 and glyceraldehyde-phosphate dehydrogenase (GAPDH) riboprobes as described for Fig. 3. LaneA shows monocytes co-cultured with E-selectin transfectants in the absence of antibody, laneB with murine monoclonal anti-E-selectin H18/7, and laneC with control antibody against ICAM-1. LaneD shows monocytes co-cultured with E-selectin transfectants in the presence of actinomycin D, and laneE represents E-selectin transfectants alone.
Figure 5: Monocyte CD36 mRNA increases upon co-culture with E-selectin-transfected L cells. Monocytes were co-incubated with confluent transfectants for 4 h, and total lysates were probed for CD36 and glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNAs as described for Fig. 3. LaneA shows monocytes co-cultured with mock-transfected L cells, laneB with ICAM-1-transfected L cells, and laneC with E-selectin-transfected L cells. Each lane was normalized as described for Fig. 3with glyceraldehyde-phosphate dehydrogenase.
Figure 4: Contact dependence of increased monocyte CD36 protein expression by TNF-stimulated HUVEC. HUVEC were treated as described for Fig. 1, and monocytes were added either directly to the wells or in transwell devices. After a 4-h co-incubation, total cell lysates were analyzed by immunoblotting as described for Fig. 1. Lane A shows total cell lysates of monocytes and TNF-stimulated HUVEC separated by transwell devices. Lane B shows the total cell lysate of monocytes in direct contact with TNF-stimulated HUVEC. Lane C shows monocytes cultured alone on tissue culture wells, and lane D on transwells. Lane E shows TNF-stimulated HUVEC in the absence of monocytes. The bargraph for each lane shows densitometric scanning results represented as -fold change in densitometric intensity.
To verify the role of E-selectin in eliciting CD36 induction, we showed that inhibitory antibodies to E-selectin blocked >70% of the increase in CD36 mRNA induced by co-culture with E-selectin transfectants (Fig. 6, laneB). Antibodies to ICAM-1 (laneC) had no effect. Parallel antibody blocking experiments were also performed on monocytes co-cultured with TNF-stimulated HUVEC (Fig. 7). There was a >50% reduction in the steady-state level of CD36 mRNA in cultures incubated with anti-E-selectin (laneA) compared with cultures incubated with control antibody (laneB). The antibody inhibition was not complete as mRNA levels were still increased compared with co-cultures with nontreated HUVEC (laneC). These results suggest that E-selectin may be a critical component of the adhesion-dependent signal transduction machinery involved in the up-regulation of monocyte CD36 upon adhesion to TNF-activated HUVEC, possibly acting at the level of RNA synthesis.
Figure 7: Induction of monocyte CD36 mRNA by TNF-stimulated HUVEC is partially inhibited by monoclonal anti-E-selectin antibody. Monocytes were co-cultured with TNF-stimulated HUVEC for 4 h, and then lysates were probed with CD36 and glyceraldehyde-phosphate dehydrogenase riboprobes as described for Fig. 3. LaneA shows the effect of including murine monoclonal anti-E-selectin H18/7, and laneB control antibody in the co-culture. LaneC shows monocytes co-cultured with resting HUVEC, and laneD shows lysates from TNF-activated HUVEC without monocytes. Total RNA per lane was normalized using glyceraldehyde-phosphate dehydrogenase as described for Fig. 3.
We have demonstrated that monocyte expression of CD36 was
dramatically increased following adhesion to cytokine-activated HUVEC.
This induction was likely due to increased gene transcription since
steady-state mRNA levels were up-regulated in parallel and since the
RNA synthesis inhibitor actinomycin D effectively prevented the
observed induction. CD36 up-regulation required direct cell-cell
contact and was the result of engagement of E-selectin expressed on the
surface of cytokine-treated endothelial cells with specific
counter-receptors on the monocyte cell surface. This conclusion is
based on a number of observations. 1) Co-culture of cells separated by
transwell devices did not lead to increased CD36 expression; 2)
induction of CD36 could be duplicated by co-culture with E-selectin
transfectants, while neither thrombin-stimulated HUVEC nor ICAM-1
transfectants had any effect; and 3) inhibitory antibodies to
E-selectin blocked the adhesion-dependent up-regulation of CD36.
Antibodies to E-selectin resulted in a partial inhibition of monocyte
CD36 induction on TNF-stimulated HUVEC, suggesting the possibility of
multiple and independent adhesion receptor-mediated signaling pathways
for the regulation of CD36 expression. While these studies cannot rule
out a role for other endothelial adhesion molecules, such as VCAM-1,
the data from transfected L cells, which do not express VCAM-1, suggest
that VCAM-1 and its counter-receptor,
, are not necessary for
adhesion-dependent induction of CD36. Taken together, these data
suggest that specific endothelial cell-monocyte adhesion mediated by
engagement of E-selectin ligands on the monocyte surface may initiate a
unique signaling sequence resulting in increased CD36 expression.
Regulation of CD36 expression on monocytes in vivo may be
complex, involving a coordinated interplay between soluble mediators
and cell-surface adhesion molecules. While our present data suggest
that soluble mediators derived from cytokine-stimulated endothelial
cells do not account for the observed changes in CD36 expression, we
have previously found that a number of soluble factors including
macrophage colony-stimulating factor, interleukin-4, and
lipopolysaccharide modulated the expression of monocyte CD36 mRNA
levels (Yesner et al., 1993). Interestingly, macrophage
colony-stimulating factor and interleukin-4 treatment resulted in
increased CD36 mRNA levels, with only minimal changes in surface
protein expression. In contrast, lipopolysaccharide and
interferon- treatment dramatically down-regulated both mRNA and
protein expression. Thus, a cooperative interplay of adhesion receptors
and soluble mediators may regulate the appropriate functional
expression of CD36 on monocyte surfaces. It is also possible that CD36
production may be regulated by its ligands, such as TSP or oxidized low
density lipoprotein. Although endothelial secretion of TSP has been
reported to be influenced by cytokines (Majack et al., 1987;
Donoviel et al., 1990), it is not likely that altered
secretion of TSP was responsible for the increase in monocyte CD36 seen
in our system as monocytes grown in 0.4-µm pore transwells should
allow TSP exchange, but did not elicit a similar up-regulation of CD36.
Increased expression of monocyte CD36 may have important implications in monocyte/macrophage biology. Rapid up-regulation of CD36 as monocytes pass through the endothelial barrier and begin the process of further differentiation into macrophages may serve to enhance several macrophage functions important in inflammation and vascular biology. For example, CD36 has been shown to be an adhesive glycoprotein functioning as a receptor for thrombospondin and collagen. An increase in cell-surface CD36 expression may thus serve to enhance monocyte-matrix interactions. The TSP-CD36 interaction has also been shown to mediate macrophage binding and internalization of apoptotic neutrophils. Thus, this critical function that serves to limit tissue damage from infiltrating neutrophils is up-regulated concomitantly with leukocyte infiltration into tissues. CD36 has also recently been shown to serve as a scavenger receptor for oxidized low density lipoprotein. Experimental data from a number of laboratories, including our own, suggest that CD36 may be responsible for >50% of macrophage uptake of oxidized low density lipoprotein (Endemann et al., 1993; Nicholson et al., 1995). Adhesion-mediated modulation of CD36 surface expression may therefore serve as a critical component of foam cell formation and atherogenesis.