©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CD36 Induction on Human Monocytes upon Adhesion to Tumor Necrosis Factor-activated Endothelial Cells (*)

(Received for publication, April 20, 1994; and in revised form, December 15, 1994)

Ho Young Huh (1) Siu Kong Lo Lewis M. Yesner Roy L. Silverstein (§)

From the Program in Cell Biology and Genetics and the Department of Medicine, Division of Hematology/Oncology and the National Institutes of Health Specialized Center of Research in Thrombosis, Cornell University Medical College, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 beta(2)-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-alpha (^1)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.


EXPERIMENTAL PROCEDURES

Materials and Probes

Murine monoclonal anti-CD36 IgG (FA6) was obtained from the Fifth International Workshop on Human Leukocyte Antigens. Monoclonal anti-ICAM-1 (RR1/1) was from Dr. Robert Rothlein (Boehringer Mannheim), and anti-E-selectin (H18/7) was from Dr. Michael Bevilacqua (University of California at San Diego, La Jolla, CA). A full-length CD36 cDNA was obtained from Brian Seed. CD36 antisense riboprobes were generated by subcloning a 792-bp BamHI fragment from the 5`-end of the CD36 cDNA into pBluescript KS (Stratagene, San Diego, CA) and linearized with EcoRI and incubated with T7 RNA polymerase. Glyceraldehyde-phosphate dehydrogenase antisense riboprobe was obtained from Ambion Inc. (Austin, Texas). Human recombinant TNF was graciously provided by Genentech (South San Francisco, CA), and human thrombin by Dr. J. Fenton. Actinomycin D was obtained from Boehringer Mannheim. RPMI 1640 medium, Medium 199, Versene, Dulbecco's phosphate-buffered saline, fetal bovine serum, and the antibiotics G418, gentamicin, penicillin, and streptomycin were obtained from Life Technologies, Inc. Gelatin, human type AB serum, and paraformaldehyde were purchased from Sigma. Transwell apparatuses and tissue culture 12-well plates were obtained from Costar (Cambridge, MA). Ficoll and Percoll for monocyte preparation were obtained from Pharmacia Biotech Inc. The colorogenic phosphatase substrates 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium for immunoblotting were from Promega. All other reagents were of analytical grade.

Monocyte Isolation and Culture

Monocytes were isolated from heparinized whole blood (1.5 units/ml) drawn from healthy adults upon informed consent. Peripheral blood mononuclear cells were isolated from leukocyte-rich fractions diluted 1:1 with Versene on Ficoll gradients. The collected mononuclear fraction was further separated by Percoll gradient centrifugation to obtain monocytes (Wright and Silverstein, 1982). Monocytes were washed in phosphate-buffered saline and resuspended in RPMI 1640 medium containing gentamicin and supplemented with 5% heat-inactivated human type AB serum. The purity of the monocytes was >80% as assessed by CD14 immunostaining, while their viability was >98% as assessed by trypan blue exclusion.

HUVEC Isolation and Culture

HUVEC were isolated by collagenase treatment of human umbilical veins as described (Jaffe et al., 1973). HUVEC were cultured in Medium 199 plus 20% heat-inactivated human serum and were verified as endothelial cells based on their typical cobblestone morphology and by immunostaining with anti-von Willebrand factor. The cultures derived from pooled primary cultures were passaged with collagenase onto fibronectin-treated 12-well tissue culture plates and were grown to confluence. Confluent cultures were treated with either human recombinant TNF (200 units/ml, 4 h) or thrombin (2 units/ml, 30 min). Preliminary studies verified that these treatments were optimal for up-regulating HUVEC adhesion molecules and promoting monocyte adhesion. Cells were washed extensively with medium prior to the addition of monocytes to eliminate any TNF or thrombin remaining in the medium or associated with the HUVEC surface.

Transfected L Cells

Murine L cells stably transfected with human ICAM-1 or E-selectin cDNA were a generous gift of Dr. Thomas Tedder (Duke University). L cells stably transfected with vector alone were a gift of Dr. William Muller (The Rockefeller University). These cells were transfected by electroporation with full-length cDNAs inserted into a plasmid vector (pZIP/neo) containing the neomycin resistance gene and were maintained in G418 (250 µg/ml)-containing medium. Protein expression of ICAM-1 and E-selectin was confirmed prior to use by immunofluorescence cytofluorography.

Co-culture Assays

HUVEC cultures were treated with TNF, thrombin, or buffer control as described above and washed thoroughly, and then peripheral blood monocytes (10^6 cells) were added to each well. After 4 h of HUVEC-monocyte co-culture at 37 °C, total cell lysates were prepared in radioimmune precipitation buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 0.1% SDS, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 20 µg/ml phenylmethylsulfonyl fluoride, pH 8.0) for immunoblot analysis or in 5 M guanidine thiocyanate, 0.1 M EDTA for mRNA analysis. In some experiments, transfected L cells were substituted for cytokine-treated HUVEC. In other experiments, monocytes were added in transwell devices (0.4-µm pore size) and placed into HUVEC-confluent wells for 4 h, preventing physical cell-cell contact. For some studies, inhibitory antibodies to E-selectin or ICAM-1 (10 µg/ml) or the transcriptional inhibitor actinomycin D (5 µg/ml) was added to the co-culture assay at the time of monocyte addition.

Immunoblot Analysis

HUVEC-monocyte cultures were lysed in radioimmune precipitation buffer, and proteins (25 µg/lane) were resolved by 10% SDS-polyacrylamide gel electrophoresis and then electrophoretically transferred to nitrocellulose paper. The nitrocellulose was then blocked for 30 min in 20 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4 (TBST), with 1% bovine serum albumin. The blocking solution was replaced with monoclonal anti-CD36 IgG (FA6) at 2 µg/ml in TBST/bovine serum albumin for 1 h and washed with the same buffer three times for 10 min each. Immunoreactive bands were detected after incubation with alkaline phosphatase-conjugated goat anti-mouse F(ab`)(2) for 30 min, followed by the addition of the colorogenic phosphatase substrates 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.

Indirect Immunofluorescence

HUVEC-monocyte adhesion was performed on gelatinized glass coverslips for indirect immunofluorescence microscopy. The cells were fixed at -20 °C in 100% methanol for 10 min and then washed thoroughly in phosphate-buffered saline, incubated with monoclonal anti-CD36 IgG for 30 min, and washed three times for 10 min each in phosphate-buffered saline, followed by incubation in fluorescein-labeled goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) for 30 min. The coverslips were mounted on slides using 15% polyvinyl alcohol, 65% glycerol in phosphate-buffered saline, dried overnight in the dark, and photographed with a Nikon epifluorescence microscope using Kodak Ektachrome ASA 400 slide film.

RNase Protection Assay

HUVEC-monocyte co-cultures were lysed in 5 M guanidine thiocyanate, 0.1 M EDTA. Hybridization was performed on 20-µl aliquots of total cell lysate with P-labeled RNA probe (specific activity of 2 times 10^9 cpm/µg [P]UTP) at 37 °C for 20 h (Haines and Gillespie, 1992; Yesner et al., 1993). After overnight incubation, the samples were digested with RNase A and RNase T1 and electrophoresed on 5% polyacrylamide gels. Autoradiograms of the dry gels were assessed by densitometric scanning using a UMAX UC630 flat-bed scanner. Total mRNA levels were normalized using a control transcript (glyceraldehyde-phosphate dehydrogenase). The CD36 riboprobe generates a protected fragment of 760 bp when hybridized to CD36 mRNA, while the glyceraldehyde-phosphate dehydrogenase riboprobes generate fragments of 480 or 983 bp.


RESULTS

Monocyte CD36 Expression Increased upon Co-culture with TNF-activated Endothelial Cells

Human peripheral blood monocytes co-cultured for 4 h with TNF-treated HUVEC, thrombin-treated HUVEC, or control HUVEC were assayed for CD36 expression by immunoblot assay with a specific murine monoclonal anti-CD36 IgG. As shown in Fig. 1, monocytes adherent to TNF-activated endothelium (laneA) had a 3-5-fold increase in CD36 protein levels relative to cells co-cultured with control HUVEC (laneC) or thrombin-stimulated HUVEC (laneD). The increased CD36 expression was likely monocyte-derived since TNF-treated HUVEC alone (laneB) did not express CD36.


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 times 10^6 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.



Adhesion Dependence of CD36 Up-regulation

To determine if direct cell-cell contact between monocytes and TNF-stimulated HUVEC was necessary for the increased CD36 expression, monocytes were cultured in transwell apparatuses that physically separated them from the underlying endothelial cell layer while allowing free diffusion of soluble mediators. As shown in Fig. 4, monocytes in physical contact with TNF-treated HUVEC (laneB) demonstrated a 5-7-fold enhancement of CD36 expression relative to monocytes in transwells co-cultured with TNF-treated HUVEC (laneA). In fact, monocytes co-incubated in transwells for 4 h with TNF-treated HUVEC (laneA) showed no apparent differences in CD36 expression as compared with monocytes cultured alone in transwells (laneC) or in tissue culture wells (laneD). LanesC and D account for the effects of filter and plastic substrates and thus represent the basal level of CD36 expression upon nonspecific substrate adhesion, in contrast to the negative control of TNF-stimulated HUVEC alone (laneE). These results suggest that monocyte adhesion to TNF-stimulated HUVEC is necessary for the induction of CD36 expression.


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.



E-selectin May Be Involved in the Adhesion-dependent Up-regulation of CD36

TNF activation of HUVEC has been shown to induce cell-surface expression of a number of molecules that are involved in monocyte cell adhesion. These include E-selectin, ICAM-1, and VCAM-1 (Bevilacqua, 1993). To test for their possible involvement in the up-regulation of CD36 upon adhesion to TNF-activated HUVEC, monocytes were co-cultured with mouse L cells stably transfected with either E-selectin or ICAM-1 cDNA. RNase protection assays were performed on the cell lysates to determine CD36 mRNA changes (Fig. 5). Monocytes adherent to E-selectin-transfected L cells (laneC) demonstrated a dramatic enhancement (6-8-fold) of CD36 mRNA expression relative to ICAM-1-transfected L cells (laneB) or to L cells transfected with vector alone (laneA). The RNA synthesis inhibitor actinomycin D also blocked the adhesion-induced increase in CD36 mRNA in these studies (Fig. 6, laneD), suggesting that the increase in CD36 mRNA is due to increased transcription.

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.




DISCUSSION

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, alpha(4)beta(1), 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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Specialized Center of Research in Thrombosis Grants HL 42540 and HL 18828 and Program Project in Vascular Cell Signalling Grant HL 46403 (to R. L. S.) and by National Institutes of Health Grant HL49883, the Dorothy Rodbell Cohen Foundation for Sarcoma Research, and a grant-in-aid from the American Heart Association (to S. K. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Div. of Hematology and Oncology, Rm. C606, Cornell University Medical College, New York, NY 10021. Tel.: 212-746-2060; Fax: 212-746-8866.

(^1)
The abbreviations used are: TNF, tumor necrosis factor; TSP, thrombospondin; HUVEC, human umbilical vein endothelial cell(s); ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular adhesion molecule-1; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Drs. Eric Jaffe and Rosemary Kraemer for providing cultured human umbilical vein endothelial cells; Dr. Thomas Tedder for the transfected L cells; and Dr. Frieda Pearce, Qinghu Zheng, and George Lam for technical assistance.


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