Cutting edge: CD91-independent cross-presentation of GRP94(gp96)-associated peptides

GRP94(gp96) elicits CD8(+) T cell responses against its bound peptides, a process requiring access of its associated peptides into the MHC class I cross-presentation pathway of APCs. Entry into this pathway requires receptor-mediated endocytosis, and CD91 (low-density lipoprotein receptor-related protein) has been reported to be the receptor mediating GRP94 uptake into APC. However, a direct role for CD91 in chaperone-based peptide Ag re-presentation has not been demonstrated. We investigated the contribution of CD91 to GRP94 cell surface binding, internalization, and trafficking in APCs. Whereas a clear role for CD91 in alpha(2)-macroglobulin binding and uptake was readily obtained, the addition of excess CD91 ligand, activated alpha(2)-macroglobulin, or receptor-associated protein, an antagonist of all known CD91 ligands, did not affect GRP94 cell surface binding, receptor-mediated endocytosis, or peptide re-presentation. These data identify a CD91-independent, GRP94 internalization pathway that functions in peptide Ag re-presentation.     

SREC-I, a type F scavenger receptor, is an endocytic receptor for calreticulin

Calreticulin and gp96 (GRP94) traffic associated peptides into the major histocompatibility complex class-I cross-presentation pathway of antigen-presenting cells (APCs). Efficient accession of the cross-presentation pathway requires APC receptor-mediated endocytosis of the chaperone/peptide complexes. Previously, scavenger receptor class-A (SRA) was shown to play a substantial role in trafficking gp96 and calreticulin into macrophages, accounting for half of total receptor-mediated uptake. However, the scavenger receptor ligand fucoidin competed the chaperone uptake beyond that accounted for by SRA, indicating that another scavenger receptor(s) may also contribute. Consistent with this hypothesis, we showed that the residual calreticulin uptake into SRA(-/-) macrophages is competed by the scavenger receptor ligand acetylated low density lipoprotein (LDL). We now report that an additional scavenger receptor, SREC-I (scavenger receptor expressed by endothelial cell-I), mediates the endocytosis of calreticulin and gp96. Ectopic expression of SREC-I in Chinese hamster ovary cells yielded chaperone recognition and uptake, and these processes were competed by the inhibitory ligands fucoidin and acetylated (Ac)LDL. Although AcLDL competes for the chaperone interactions with SRA and SREC, we showed that not all of the scavenger receptors, which bind AcLDL, bind calreticulin or gp96. The overexpression of SREC-I in macrophages increased chaperone endocytosis, indicating that SREC-I functions in APCs and that the cytosolic components necessary for the endocytosis of SREC-I and its cargo are present and not limiting in APCs. These data identify a novel class of ligands for SREC-I and provide insight into the mechanisms by which APCs and potentially endothelial cells traffic chaperone/antigen complexes.     

Adjuvanticity of alpha 2-macroglobulin, an independent ligand for the heat shock protein receptor CD91

We recently have identified CD91 as a receptor for the heat shock protein gp96. CD91 was identified initially as a receptor for alpha(2)-macroglobulin (alpha(2)M). Gp96 and alpha(2)M are both ligands for CD91. Because gp96-chaperoned peptides can prime CD8(+) T cell responses and are re-presented by APCs, we tested alpha(2)M for similar properties. Our studies show that alpha(2)M binds peptides in vitro and that the peptides, chaperoned by alpha(2)M, efficiently prime peptide-specific CD8(+) T cell responses in mice immunized with alpha(2)M-peptide complexes. Furthermore, peptides chaperoned by alpha(2)M, like those chaperoned by gp96, can be re-presented by CD91(+) APCs on their MHC I molecules. These studies demonstrate that alpha(2)M molecules, like the heat shock protein molecules, are T cell adjuvants that can channel exogenous Ags into the endogenous pathway of Ag presentaion. The remarkable similarities between an intracellular chaperone and an extracellular serum chaperone may have interesting physiological ramifications.     

The class A scavenger receptor binds to proteoglycans and mediates adhesion of macrophages to the extracellular matrix

The class A scavenger receptor (SR-A) binds modified lipoproteins and has been implicated in cholesterol ester deposition in macrophages. The SR-A also contributes to cellular adhesion. Using SR-A(+/+) and SR-A(-)/- murine macrophages, we found SR-A expression important for both divalent cation-dependent and -independent adhesion of macrophages to the human smooth muscle cell extracellular matrix. The SR-A mediated 65 and 85% of macrophage adhesion to the extracellular matrix in the presence and absence of serum, respectively. When EDTA was added to chelate divalent cations, the SR-A mediated 90 and 95% of the macrophage adhesion without and with serum, respectively. SR-A-mediated adhesion to the extracellular matrix was prevented by fucoidin, an SR-A antagonist. Biglycan and decorin, proteoglycans of the extracellular matrix, were identified as SR-A ligands. Compared with control cells, Chinese hamster ovary cells expressing the SR-A showed 5- and 6-fold greater cell association (binding and internalization) of (125)I-decorin and -biglycan, respectively. In competition studies, unlabeled proteoglycan or fucoidin competed for binding of (125)I-labeled decorin and -biglycan, and biglycan and decorin competed for the SR-A-mediated cell association and degradation of (125)I-labeled acetylated LDL, a well characterized ligand for the SR-A. These results suggest that the SR-A could contribute to the adhesion of macrophages to the extracellular matrix of atherosclerotic plaques.     

Differential CD91 dependence for calreticulin and Pseudomonas exotoxin-A endocytosis

Calreticulin (CRT) interaction with cell-surface receptors is integral to its function in escorting associated peptides into the antigen-presenting cell (APC) antigen presentation pathway. Additionally, extracellular CRT is proposed to be required for lung APC interaction with collectins. In both cases, CD91 has been proposed to act as the APC cell-surface receptor requisite for mediating these processes. However, the evidence for a CRT interaction with CD91 is indirect, predicated on partial competition of cellular binding by gp96, of which CD91 has been proposed as the unique endocytic receptor, and by the CD91 ligand alpha2-macroglobulin. Here, we directly investigate the function of CD91 in binding and trafficking CRT. We find that the ability of CRT to interact with APC does not correlate with cellular CD91 expression or function. Additionally, in the first genetic test of CD91 function regarding CRT, CD91 expression neither conferred CRT association nor did CD91-deficient (CD91-/-) and CD91-expressing cells differ in their ability to traffic CRT. Finally, cellular CRT trafficking did not parallel that of Pseudomonas exotoxin-A, an obligate CD91 ligand, by the criteria of CD91 dependence, cell-type specificity and endocytic itinerary. These data identify that CRT trafficking is not, as previously hypothesized, CD91 dependent and indicate usage of alternative cellular trafficking pathways.     

A CD91-positive subset of CD11c+ blood dendritic cells: characterization of the APC that functions to enhance adaptive immune responses against CD91-targeted antigens

Dendritic cells (DC) and other APCs rely on a number of specialized receptors to facilitate the uptake and intracellular accumulation of Ags. In this capacity, APCs use receptor-mediated endocytosis to enhance Ag presentation and the stimulation of Ag-specific T cells. Studies have demonstrated that the targeted delivery of Ags in vivo to CD91/the low-density lipoprotein receptor-related protein (CD91/LRP) induces enhanced activation of the adaptive immune system. However, the APC that mediates these augmented, Ag-specific responses remains to be characterized. In this study, we show that a subset of CD11c(+) lineage-negative (lin(-)) DC expresses the scavenger receptor CD91/LRP and that these rare APC are primarily responsible for the T cell activation that occurs following CD91/LRP-mediated Ag uptake in whole blood. The targeting of Ags to CD91/LRP results in enhanced receptor-mediated uptake within both lin(-) DCs and monocytes, and this uptake results in markedly increased T cell activation. Finally, purified cellular populations were used to demonstrate that CD11c(+) lin(-) DC, but not monocytes, are capable of stimulating T cell activation following CD91/LRP-mediated Ag uptake. Therefore, CD11c(+) lin(-) DC use CD91/LRP to facilitate the uptake and subsequent presentation of an array of Ags complexed within the CD91/LRP ligand, the activated form of alpha2-macroglobulin (alpha2M*).     

Phylogenetic conservation of glycoprotein 96 ability to interact with CD91 and facilitate antigen cross-presentation

Although the ability of gp96 to activate APCs and generate CD8 CTLs against peptides they chaperone through interaction with the endocytic receptors CD91 is supported by solid evidence, its biological relevance in immune surveillance is debated. We have used an evolutionary approach to determine whether gp96 interacts with receptors expressed on APCs and promotes MHC class I cross-presentation of minor histocompatibility Ags (H-Ags) to CTLs in the frog Xenopus. We show that in Xenopus gp96 binds the CD91 homolog at the surface of peritoneal leukocytes, and that this binding is inhibited by molar excess of unlabeled gp96 or the CD91 ligand alpha2-macroglobulin, by anti-CD91 Ab and by the specific CD91 antagonist receptor-associated protein. Surface binding followed by internalization of gp96 was confirmed by fluorescent microscopy. Furthermore, adoptive transfer of peritoneal leukocytes pulsed with as little as 800 ng of gp96 chaperoning minor H-Ags, but not minor H-Ag-free gp96, induces potent CD8 T cell infiltration and Ag-specific accelerated rejection of minor H-locus disparate skin grafts. Inhibition of gp96-CD91 interaction by pretreatment with anti-CD91 Ab and receptor-associated protein impairs both CD8 T cell infiltration and acute skin graft rejection. These data provide evidence of the conserved ability of gp96 to facilitate cross-presentation of chaperoned Ags by interacting with CD91. The persistence of this biological process for >350 million years that separate mammals and amphibians from a common ancestor strongly supports the proposition that gp96 and CD91 are critically involved in immune surveillance.     

The receptor for heat shock protein 60 on macrophages is saturable, specific, and distinct from receptors for other heat shock proteins.

Previous studies have shown that human heat shock protein (hsp) 60 elicits a strong proinflammatory response in cells of the innate immune system with CD14, Toll-like receptor (TLR) 2, and TLR4 as mediators of signaling, but probably not of binding. In the present study, we directly demonstrate binding of hsp60 to the macrophage surface and find the binding receptor for hsp60 different from the previously described common receptor for several other heat shock proteins, including hsp70, hsp90, and gp96. Fluorescence-labeled human hsp60 bound to cell surfaces of the murine macrophage lines J774 A.1 and RAW264.7 and to mouse bone marrow-derived macrophages. By flow cytometry, we could demonstrate for the first time that hsp60 binding to macrophages occurred at submicromolar concentrations, is saturable, and can be competed by unlabeled hsp60, but not by unrelated proteins, thus confirming the classic characteristics of specific ligand-receptor interactions. Binding of hsp60 at 4 degrees C was followed by endocytosis at 37 degrees C. Hsp60 binding to macrophages could not be competed by excess hsp70, hsp90, or gp96, all of which share the alpha(2)-macroglobulin receptor as binding site. Hsp60 binding occurred in the absence of surface TLR4. However, no cytokine response was induced by hsp60 in TLR4-deficient macrophages. We conclude that hsp60 binds to a stereo-specific receptor on macrophages, and that different surface molecules are engaged in binding and signal transduction. Furthermore, the binding site for hsp60 is separate from the common receptor for hsp70, hsp90, and gp96, which suggests an independent role of hsp60 as danger Ag and in immunoregulation.     

Secreted forms of the amyloid-beta precursor protein are ligands for the class A scavenger receptor

Upon activation, platelets secrete a 120-kDa protein that competes for the binding and internalization of acetyl low density lipoproteins (AcLDL) by macrophages. From the amino-terminal amino acid sequence, amino acid composition, and immunoblot analysis, we identified the active factor in platelet secretion products as sAPP, an alpha-secretase cleavage product of the beta-amyloid precursor protein (APP), that contains a Kunitz-type protease inhibitor (KPI) domain. We showed that both sAPP751 (also called Nexin II) and sAPP695, which does not contain a KPI domain, are ligands for the class A scavenger receptor (SR-A). Chinese hamster ovary cells stably transfected to express the SR-A bound and internalized 4-fold more human platelet-derived sAPP than control cells. The binding and internalization of sAPP were inhibited by the SR-A antagonist fucoidin. In addition, sAPP competed as effectively as fucoidin for SR-A-mediated cell association and degradation of (125)I-AcLDL. To determine if the KPI domain is required for the binding of sAPP to the SR-A, APP751 and APP695 were expressed in Chinese hamster ovary cells, and sAPP751 and sAPP695 purified from the medium were tested for their binding to the SR-A. sAPP751 and sAPP695 were equally effective in competing for the cell association of (125)I-AcLDL by SR-A-expressing cells, demonstrating that the KPI domain is not essential for binding. We also found that sAPP751 is present in extracts of atherosclerotic lesions and that sAPP competes for the SR-A-mediated cell association of oxidized low density lipoprotein. Deletion mutagenesis indicated that a negatively charged region of APP (residues 191-264) contributes to binding to the SR-A. These results suggest that the SR-A contributes to the clearance of sAPP and that sAPP competes for the cell association of other SR-A ligands.     

Class B scavenger receptors CD36 and SR-BI are receptors for hypochlorite-modified low density lipoprotein

The presence of HOCl-modified epitopes inside and outside monocytes/macrophages and the presence of HOCl-modified apolipoprotein B in atherosclerotic lesions has initiated the present study to identify scavenger receptors that bind and internalize HOCl-low density lipoprotein (LDL). The uptake of HOCl-LDL by THP-1 macrophages was not saturable and led to cholesterol/cholesteryl ester accumulation. HOCl-LDL is not aggregated in culture medium, as measured by dynamic light scattering experiments, but internalization of HOCl-LDL could be inhibited in part by cytochalasin D, a microfilament disrupting agent. This indicates that HOCl-LDL is partially internalized by a pathway resembling phagocytosis-like internalization (in part by fluid-phase endocytosis) as measured with [14C]sucrose uptake. In contrast to uptake studies, binding of HOCl-LDL to THP-1 cells at 4 degrees C was specific and saturable, indicating that binding proteins and/or receptors are involved. Competition studies on THP-1 macrophages showed that HOCl-LDL does not compete for the uptake of acetylated LDL (a ligand to scavenger receptor class A) but strongly inhibits the uptake of copper-oxidized LDL (a ligand to CD36 and SR-BI). The binding specificity of HOCl-LDL to class B scavenger receptors could be demonstrated by Chinese hamster ovary cells overexpressing CD36 and SR-BI and specific blocking antibodies. The lipid moiety isolated from the HOCl-LDL particle did not compete for cell association of labeled HOCl-LDL to CD36 or SR-BI, suggesting that the protein moiety of HOCl-LDL is responsible for receptor recognition. Experiments with Chinese hamster ovary cells overexpressing scavenger receptor class A, type I, confirmed that LDL modified at physiologically relevant HOCl concentrations is not recognized by this receptor.     

Re-examination of CD91 function in GRP94 (glycoprotein 96) surface binding, uptake, and peptide cross-presentation

GRP94 (gp96)-peptide complexes can be internalized by APCs and their associated peptides cross-presented to yield activation of CD8(+) T cells. Investigations into the identity (or identities) of GRP94 surface receptors have yielded conflicting results, particularly with respect to CD91 (LRP1), which has been proposed to be essential for GRP94 recognition and uptake. To assess CD91 function in GRP94 surface binding and endocytosis, these parameters were examined in mouse embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via RNA interference or eliminated by genetic disruption of the CD91 locus. Reduction or loss of CD91 expression abrogated the binding and uptake of receptor-associated protein, an established CD91 ligand. Surface binding and uptake of an N-terminal domain of GRP94 (GRP94.NTD) was unaffected. GRP94.NTD surface binding was markedly suppressed after treatment of MEF cell lines with heparin, sodium chlorate, or heparinase II, demonstrating that heparin sulfate proteoglycans can function in GRP94.NTD surface binding. The role of CD91 in the cross-presentation of GRP94-associated peptides was examined in the DC2.4 dendritic cell line. In DC2.4 cells, which express CD91, GRP94.NTD-peptide cross-presentation was insensitive to the CD91 ligands receptor-associated protein or activated α(2)-macroglobulin and occurred primarily via a fluid-phase, rather than receptor-mediated, uptake pathway. These data clarify conflicting data on CD91 function in GRP94 surface binding, endocytosis, and peptide cross-presentation and identify a role for heparin sulfate proteoglycans in GRP94 surface binding.     

A role for class A scavenger receptor in dendritic cell nibbling from live cells

Monocyte-derived dendritic cells (DC) possess the unique capacity to capture Ag from live cells through intimate cell contact, a process referred to as nibbling. We sought to define the receptor(s) mediating DC nibbling. Uptake of fluorescently labeled plasma membrane from live cells by DC was inhibited by protease treatment and by a panel of polyanionic ligands, implicating scavenger receptors (SR) in this process. Differential expression of SR on DC and macrophages correlated with the capacity to acquire membrane from live cells. Internalized membrane colocalized with SR ligand and entered the endosomal pathway. DC very efficiently acquired and internalized gp100 tumor Ag expressed at the surface of viable adenocarcinoma cells via recombinant adenoviral infection. Cross-presentation of gp100 by DC to MHC class I-restricted T cells was inhibited by polyanionic SR ligand and an Ab to type A SR (SR-A), whereas Ab to the class B SR CD36, which mediates uptake of apoptotic cells, induced no inhibition. DC capture of fluorescently labeled membrane from live cells was partially blocked by SR-A-specific Ab, suggesting that other SR may also be contributing to nibbling. DC maturation resulted in a switch in expression from type II SR-A (SR-AII) to the SR-AI splice variant. Finally, SR-A was identified on interdigitating DC isolated from monkey lymph nodes. These findings define a novel role for SR-A, and suggest that Ag uptake from live cells by DC may be important in the generation of immunity and in the maintenance of peripheral tolerance in vivo.     

Caveolae-dependent endocytosis is required for class A macrophage scavenger receptor-mediated apoptosis in macrophages

SR-A (class A macrophage scavenger receptor) is a transmembrane receptor that can bind many different ligands, including modified lipoproteins that are relevant to the development of vascular diseases. However, the precise endocytic pathways of SR-A/mediated ligands internalization are not fully characterized. In this study, we show that the SR-A/ligand complex can be endocytosed by both clathrin- and caveolae-dependent pathways. Internalizations of SR-A-lipoprotein (such as acLDL) complexes primarily go through clathrin-dependent endocytosis. In contrast, macrophage apoptosis triggered by SR-A-fucoidan internalization requires caveolae-dependent endocytosis. The caveolae-dependent process activates p38 kinase and JNK signaling, whereas the clathrin-mediated endocytosis elicits ERK signaling. Our results suggest that different SR-A endocytic pathways have distinct functional consequences due to the activation of different signaling cascades in macrophages.     

Scavenger receptor A (SR-A) is required for LPS-induced TLR4 mediated NF-κB activation in macrophages

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15 min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A(-/-)), TLR4(-/-) and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4(-/-) or SR-A(-/-) macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.     

Surfactant protein A (SP-A)-mediated clearance of Staphylococcus aureus involves binding of SP-A to the staphylococcal adhesin eap and the macrophage receptors SP-A receptor 210 and scavenger receptor class A

Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap(+)) but not Eap-deficient (Eap(-)) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap(+) S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap(+) but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap(+) S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap(-) S. aureus was impaired. Macrophages express two isoforms: SP-R210(L) and SP-R210(S). The results show that WT alveolar macrophages are distinguished by expression of SP-R210(L), whereas SR-A(-/-) alveolar macrophages are deficient in SP-R210(L) expressing only SP-R210(S). Accordingly, SR-A(-/-) mice were highly susceptible to both Eap(+) and Eap(-) S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210(L) mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A.     

Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide

Scavenger receptor expressed by endothelial cells I (SREC-I) is a novel endocytic receptor for acetylated low density lipoprotein (LDL). Here we show that SREC-I is expressed in a wide variety of tissues, including macrophages and aortas. Lipopolysaccharide (LPS) robustly stimulated the expression of SREC-I in macrophages. In an initial attempt to clarify the role of SREC-I in the uptake of modified lipoproteins as well as in the development of atherosclerosis, we generated mice with a targeted disruption of the SREC-I gene by homologous recombination in embryonic stem cells. To exclude the overwhelming effect of the type A scavenger receptor (SR-A) on the uptake of Ac-LDL, we further generated mice lacking both SR-A and SREC-I (SR-A(-/-);SREC-I(-/-)) by cross-breeding and compared the uptake and degradation of Ac-LDL in the isolated macrophages. The contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 85 and 5%, respectively, in a non-stimulated condition. LPS increased the uptake and degradation of Ac-LDL by 1.8-fold. In this condition, the contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 90 and 6%, respectively. LPS increased the absolute contribution of SR-A and SREC-I by 1.9- and 2.3-fold, respectively. On the other hand, LPS decreased the absolute contribution of other pathways by 31%. Consistently, LPS did not increase the expression of other members of the scavenger receptor family such as CD36. In conclusion, SREC-I serves as a major endocytic receptor for Ac-LDL in LPS-stimulated macrophages lacking SR-A, suggesting that it has a key role in the development of atherosclerosis in concert with SR-A.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

Heat shock proteins gp96 and hsp70 activate the release of nitric oxide by APCs

NO is a cytotoxic and immunomodulatory cytokine produced by macrophages and dendritic cells. We show that stimulation of murine and human macrophages with the heat shock proteins gp96 and hsp70 results in induction of inducible NO synthase and the production of NO. The release of NO by monocytes exposed to hsp60 has been documented previously. Immature, but not mature, dendritic cells respond in the same manner. The activity of heat shock proteins is relatively unaffected by an antagonist of LPS, and is abrogated by heat denaturation. Macrophages have been shown previously to produce NO in response to stimulation with IFN-gamma; stimulation of macrophages with mixtures of IFN-gamma and gp96 or hsp70 leads to a synergistic production of NO. The present observations extend the roles of these heat shock proteins in innate immune responses to another potent and highly conserved function of APC.     

CD36 is not involved in scavenger receptor-mediated endocytic uptake of glycolaldehyde- and methylglyoxal-modified proteins by liver endothelial cells

Circulating proteins modified by advanced glycation end-products (AGE) are mainly taken up by liver endothelial cells (LECs) via scavenger receptor-mediated endocytosis. Endocytic uptake of chemically modified proteins by macrophages and macrophage-derived cells is mediated by class A scavenger receptor (SR-A) and CD36. In a previous study using SR-A knockout mice, we demonstrated that SR-A is not involved in endocytic uptake of AGE proteins by LECs [Matsumoto et al. (2000) Biochem. J. 352, 233-240]. The present study was conducted to determine the contribution of CD36 to this process. Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were used as AGE proteins. 125I-GA-BSA and 125I-MG-BSA underwent endocytic degradation by these cells at 37 degrees C, and this process was inhibited by several ligands for the scavenger receptors. However, this endocytic uptake of 125I-GA-BSA by LECs was not inhibited by a neutralizing anti-CD36 antibody. Similarly, hepatic uptake of (111)In-GA-BSA after its intravenous injection was not significantly attenuated by co-administration of the anti-CD36 antibody. These results clarify that CD36 does not play a significant role in elimination of GA-BSA and MG-BSA from the circulation, suggesting that the receptor involved in endocytic uptake of circulating AGE proteins by LEC is not SR-A or CD36.