Macrophage Scavenger Receptor CD36 Research Articles

Assessment of the Macrophage Scavenger Receptor CD163 in Mediating Glaesserella parasuis Infection of Host Cells.

The macrophage CD163 surface glycoprotein is a member of the SRCR family class B, which has been identified as the key trigger in host-pathogen interactions, but its specific roles in sensing Glaesserella parasuis (G. parasuis) infection are largely unknown. Here, we investigated porcine CD163 in mediating the adhesion and immune response of G. parasuis using in vitro host-bacteria interaction models. CD163-overexpressing Chinese hamster ovary K1 cells (CHO-K1) showed obvious subcellular localization in the cytoplasm, especially in the cytomembrane. Although detection using scanning electron microscopy (SEM) confirmed the bacterial adhesion, there was no significant difference in the adhesion of G. parasuis to CHO-K1 cells between the presence and absence of CD163. In addition, similar results were observed in 3D4/21 cells. Meanwhile, bindings of G. parasuis to nine synthetic peptides, the bacterial binding motifs within SRCR domains of CD163, were weak based on a solid-phase adhesion assay and agglutination assay. Moreover, CD163 had no effect on the expression of G. parasuis-induced inflammatory cytokines (IL-6, INF-γ, IL-10, IL-4 and TGF-β) in CHO-K1 cells. In conclusion, these findings indicate that porcine CD163 plays a minor role in sensing G. parasuis infection.

Assessment of Haptoglobin 2-2 Genotype in Type 2 Diabetes and Cardiovascular Patients in North Indian Population: A Case-control Study

Introduction: Haptoglobin (Hp), an acute phase protein, is a plasma inflammatory marker. It is important in both infectious and non infectious illnesses. When intravascular haemolysis occurs, the dead red blood cells are discharged into the circulation and link to Hp to form the haemoglobin (Hb)-haptoglobin (Hb-Hp) complex that is recognised by the macrophage scavenger receptor CD163. The Hp1 and Hp2 are its two alleles. The Hp2 is formed by a partial duplication of the Hp1 gene. The Hp1 has five exons, but Hp2 has seven exons owing to the duplication of Hp1 third and fourth exon. There are three genotypes of Hp: Hp1-1, Hp2-1, and Hp2-2. The encoded products of genotypes are attached to haemoglobin with varying degrees of affinity: Hp1-1 has the most affinity, Hp2-1 has medium affinity, and Hp2-2 has the lowest affinity. Hp allelic and genotypic variation differs amongst populations and different geographic regions of the world. Aim: To study the haptoglobin 2-2 genotype in type 2 diabetes and cardiovascular patients in north Indian population. Materials and Methods: It was a case-control study and a total of 200 participants were recruited randomly including 50 participants of type 2 Diabetes Mellitus (DM), 50 participants of Cardiovascular Disorders (CVD), 50 participants with Cardiovascular Disorders With type 2 Diabetes Mellitus (CVDWDM) and 50 normal healthy controls from Punjab Institute of Medical Sciences (PIMS) Hospital and clinics and Aashirwad laboratory of Jalandhar, Punjab, India in February 2021 to January 2022. Anthropometric variables were determined by using standard methods and biochemical parameters were measured by commercial available kits. Allele specific Polymerase Chain Reaction (PCR) or Amplification Refractory Mutation System (ARMS) PCR was used to detect Hp genotypes. Data were represented as mean±Standard Deviation and the statistical difference i.e. p<0.05 between all parameters was determined by using the Mann-Whitney U test. Results: Anthropometric parameters i.e. Body Mass Index (BMI) was found to be statistically significant in CVDWDM as compared to CVD, N and T2DM i.e. p=0.023, p=0.01 and p=0.014. Waist Circumference (WC) was found to be statistically significant in CVDWDM as compared to CVD, N and T2DM i.e. p=0.02, p=0.013 and p=0.012 and Hip Circumference (HC) was found to be statistically significant in CVDWDM as compared to CVD, N and T2DM i.e. p=0.02, p=0.017 and p=0.014. However, Apolipoprotein A1 mean value was found to be higher and statistically significant i.e. p=0.03 in Type 2 Diabetes Mellitus (T2DM) as compared to healthy participants. Triglycerides mean value was increased in T2DM as compared to CVD, CVD as compared to N and CVDWDM as compared to healthy controls (p-value=0.012,0.04,0.015, respectively). The findings had shown that a 349 bp band was detected in patients with T2DM, CVD and CVDWDM, implying that the Hp2-2 genotypes were observed but not in healthy controls. Conclusion: The study finds that people with DM, CVD, or CVDWDM all have a 349bp band. It means that the genotypes of the Hp2-2 allele were found in patients of diabetes, CVD and diabetics with CVD but not in the healthy individuals.

Obese Patients With Nonalcoholic Fatty Liver Disease Have an Increase in Soluble Plasma CD163 and a Concurrent Decrease in Hepatic Expression of CD163.

Macrophages play an important role in the development of nonalcoholic fatty liver disease (NAFLD) and its progression to nonalcoholic steatohepatitis (NASH). In this study, we investigated the hepatic expression of the macrophage scavenger receptor CD163 and the plasma level of its shed soluble form (sCD163) in patients with obesity and NASH, non-NASH NAFLD (NAFL), or healthy livers (no NAFLD). Paired liver biopsies and plasma samples were collected from 61 patients with obesity (body mass index ≥35). Hepatic expression of CD163 was analyzed by immunohistochemistry and data-independent acquisition mass spectrometry, whilst plasma levels of sCD163 were determined by enzyme-linked immunosorbent assay and data-independent acquisition mass spectrometry. NAFLD stage and activity were assessed using the Kleiner fibrosis and NASH Clinical Research Network (NAS-CRN) scoring system. sCD163 turned out as a promising predictor of NASH with an area under the receiver-operating characteristic curve of 0.78 [0.65;0.92] (P= .0008). sCD163 increased with more severe NAFLD both in univariate (odds ratio [OR]= 3.31[1.80;6.11], P < .001) and multivariable ordinal logistic regression adjusting for NAFLD risk factors (OR= 2.02 [1.03;3.97], P= .042). On the other hand, hepatic expression of CD163 was negatively associated with more severe NAFLD in univariate ordinal logistic regression determined by immunohistochemistry (OR= 0.91[0.84;0.98], P= .015) and proteomics (OR= 0.13[0.02;0.80], P= .028). Taking NAFLD risk factors into account, hepatic expression of CD163 was only associated with the fibrosis stage (OR= 0.01 [0.0003;0.21], P= .004). Accordingly, hepatic CD163 surface expression and sCD163 were negatively correlated (rho=-0.478, P= .0001). An increased plasma sCD163 and a concurrent decreased hepatic expression of CD163 are strongly associated with NAFLD in obese patients.

Hyperglycemia Induces Inflammatory Response of Human Macrophages to CD163-Mediated Scavenging of Hemoglobin-Haptoglobin Complexes.

Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 internalizes and degrades hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our aim was to identify whether hyperglycemia is able to amplify inflammation via Hb-Hp complex interactions with the immune system. M(IFNγ), M(IL-4), and control M0 macrophages were differentiated out of primary human monocytes in normo- (5 mM) and hyperglycemic (25 mM) conditions. CD163 gene expression was decreased 5.53 times in M(IFNγ) with a further decrease of 1.99 times in hyperglycemia. Hyperglycemia suppressed CD163 surface expression in M(IFNγ) (1.43 times). Flow cytometry demonstrated no impairment of Hb-Hp uptake in hyperglycemia. However, hyperglycemia induced an inflammatory response of M(IFNγ) to Hb-Hp1-1 and Hb-Hp2-2 uptake with different dynamics. Hb-Hp1-1 uptake stimulated IL-6 release (3.03 times) after 6 h but suppressed secretion (5.78 times) after 24 h. Contrarily, Hb-Hp2-2 uptake did not affect IL-6 release after 6h but increased secretion after 24 h (3.06 times). Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.

Anti-atherogenic effects of CD36-targeted epigallocatechin gallate-loaded nanoparticles

Intimal macrophages play a critical role in atherosclerotic lesion initiation and progression by taking up oxidized low-density lipoprotein (oxLDL) and promoting inflammatory process. 1-(Palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), a major type of oxidized phosphatidylcholines (PC) found on oxLDL, has a high binding affinity to the macrophage scavenger receptor CD36 and participates in CD36-mediated recognition and uptake of oxLDL by intimal macrophages. We successfully synthesized epigallocatechin gallate (EGCG)-loaded nanoparticles (Enano), which were composed of EGCG, PC, (+) alpha-tocopherol acetate, and surfactant. We also incorporated KOdiA-PC on the surface of Enano to make ligand-coated Enano (L-Enano) to target intimal macrophages. The objectives of this study were to determine the anti-atherogenic effects of Enano and L-Enano in LDL receptor null (LDLr−/−) mice.Our in vitro data demonstrated that L-Enano had a higher binding affinity to mouse peritoneal macrophages than Enano. This high binding affinity was diminished by CD36 antibodies or knockdown of the CD36 receptor in mouse peritoneal macrophages, confirming the specific binding of L-Enano to the macrophage CD36 receptor. LDLr−/− mice were randomly divided to six groups and received weekly tail vein injection with PBS, EGCG, void nanoparticles (Vnano), Enano, ligand-coated Vnano (L-Vnano), or L-Enano once per week for 22 weeks. The dose of EGCG was 25 mg per kg body weight. L-Enano at 20 μg/mL significantly decreased production of monocyte chemoattractant protein-1, tumor necrosis factor alpha, and interleukin-6 from mouse macrophages, while having no effect on their plasma levels compared to the PBS control. There were no significant differences in blood lipid profiles among six treatment groups. Mice treated with L-Enano also had significantly smaller lesion surface areas on aortic arches compared to the PBS control. Liver EGCG content was decreased by treatments in the order of EGCG>Enano>L-Enano. Native EGCG had inhibitory effects on liver and body fat accumulation. This molecular target approach signals an important step towards inhibiting atherosclerosis development via targeted delivery of bioactive compounds to intimal macrophages.

Haptoglobin.

Haptoglobin (Hp) is an abundant human plasma protein that tightly captures hemoglobin (Hb) during hemolysis. The Hb-Hp complex formation reduces the oxidative properties of heme/Hb and promotes recognition by the macrophage scavenger receptor CD163. This leads to Hb-Hp breakdown and heme catabolism by heme oxygenase and biliverdin reductase. Gene duplications of a part of or the entire Hp gene in the primate evolution have led to variant Hp gene products that collectively may be designated "the haptoglobins (Hps)" as they all bind Hb. These variant products include the human-specific multimeric Hp phenotypes in individuals, which are hetero- or homozygous for an Hp2 gene allele. The Hp-related protein (Hpr) is another Hp duplication product in humans and other primates. Alternative functions of the variant Hps are indicated by numerous reports on association between Hp phenotypes and disease as well as the elucidation of a specific role of Hpr in the innate immune defense. Recent Advances: Recent functional and structural information on Hp and receptor systems for Hb removal now provides insight on how Hp carries out essential functions such as the Hb detoxification/removal, and how Hpr, by acting as an Hp-lookalike, can sneak a lethal toxin into trypanosome parasites that cause mammalian sleeping sickness. Critical Issues and Future Directions: The new structural insight may facilitate ongoing attempts of developing Hp derivatives for prevention of Hb toxicity in hemolytic diseases such as sickle cell disease and other hemoglobinopathies. Furthermore, the new structural knowledge may help identifying yet unknown functions based on other disease-relevant biological interactions involving Hps. Antioxid. Redox Signal. 26, 814-831.

Neopterin and 7,8-dihydroneopterin are generated within atherosclerotic plaques

Abstract Plasma neopterin correlates with the level of cardiovascular disease. Neopterin is the oxidation product of 7,8-dihydroneopterin, which is released by γ-interferon-stimulated macrophages. 7,8-Dihydroneopterin is a potent antioxidant, which inhibits lipid oxidation, macrophage cell death and scavenger receptor CD36 expression. The concentration of neopterin within atherosclerotic plaques was measured in tissue removed from carotid and femoral arteries. The excised plaques were cut into 3-mm-thick sections, and each section was analysed for neopterin, total neopterin, cholesterol, lipid peroxides, α-tocopherol and protein-bound 3,4-dihydroxyphenylalanine. Selected plaques were placed in tissue culture, and the media was analysed for 7,8-dihydroneopterin and neopterin release. Total neopterin levels ranged from 14 to 18.8 nmol/g of tissue. Large ranges of values were seen both within the same plaque and between plaques. No correlation between neopterin and any of the other analytes was observed, nor was there any significant trend in levels along the length of the plaques. γ-Interferon stimulation of cultured plaque generated total neopterin concentrations from 1 to 4 nmol/(g 24 h). The level of 7,8-dihydroneopterin generated within the plaque was within the range that inhibits lipid oxidation. The data show that atherosclerotic plaques are extremely dynamic in biochemistry and are the likely source of the plasma 7,8-dihydroneopterin and neopterin.

Neurocognitive and neuroinflammatory correlates of PDYN and OPRK1 mRNA expression in the anterior cingulate in postmortem brain of HIV-infected subjects

Chronic inflammation may contribute to neuropsychological impairments in individuals with HIV, and modulation of this inflammatory response by opiate receptor ligands is important in light of the prevalence of drug use in HIV populations. Exogenous MOR and KOR agonists have differential effects on central nervous system (CNS) immunity and, while some data suggest KOR agonists are immunosuppressive, the KOR agonist dynorphin has been shown to stimulate human monocyte chemotaxis. In this study, we examined mRNA levels of endogenous opioid receptors OPRK1 and OPRM1, prodynorphin (PDYN), macrophage scavenger receptor CD163, and microglia/macrophage marker CD68 in the caudate and anterior cingulate of postmortem brains from HIV-positive and HIV-negative subjects. Brain tissues of HIV-infected (n = 24) and control subjects (n = 15) were obtained from the Manhattan HIV Brain Bank. Quantification of the gene mRNA was performed using SYBR Green RT-PCR. CD68 and CD163 were increased in HIV-positive (HIV+) compared to HIV-negative (HIV-) individuals in both brain regions. There were higher OPRK1 (P <0.005), and lower PDYN mRNA (P <0.005) levels in the anterior cingulate of HIV+ compared to HIV- subjects. This difference between the clinical groups was not found in the caudate. There was no difference in the levels of OPRM1 mRNA between HIV+ and HIV- subjects. Using linear regression analysis, we examined the relationship of OPRK1 and PDYN mRNA levels in the HIV+ subjects with seven cognitive domain T scores of a neuropsychological test battery. Within the HIV+ subjects, there was a positive correlation between anterior cingulate PDYN mRNA levels and better T-scores in the motor domain. Within the HIV+ subjects there were also positive correlations of both OPRK1 and PDYN mRNA levels with the anti-inflammatory marker CD163, but not with proinflammatory CD68 levels. In this setting, decreased PDYN mRNA may reflect a homeostatic mechanism to reduce monocyte migration, accompanied by compensatory increases in the cognate receptor (KOR) to dampen pro-inflammatory responses. It is possible that enhanced neuroprotection and better motor performance are associated with higher levels of dynorphin and the recruitment of neuroprotective CD163-positive macrophages. Further studies are needed to test this hypothesis.

Thrombospondin-1 triggers macrophage IL-10 production and promotes resolution of experimental lung injury.

Mononuclear phagocyte recognition of apoptotic cells triggering suppressive cytokine signaling is a key event in inflammation resolution from injury. Mice deficient in thrombospondin-1 (thbs1−/−), an extracellular matrix glycoprotein that bridges cell-cell interactions, are prone to LPS-induced lung injury and show defective macrophage IL-10 production during the resolution phase of inflammation. Reconstitution of IL-10 rescues thbs1−/− mice from persistent neutrophilic lung inflammation and injury and thbs1−/− alveolar macrophages show defective IL-10 production following intratracheal instillation of apoptotic neutrophils despite intact efferocytosis. Following co-culture with apoptotic neutrophils, thbs1−/− macrophages show a selective defect in IL-10 production whereas PGE2 and TGF-β1 responses remain intact. Full macrophage IL-10 responses require the engagement of thrombospondin-1 structural repeat 2 domain and the macrophage scavenger receptor CD36 LIMP-II Emp sequence homology (CLESH) domain in vitro. Although TSP-1 is not essential for macrophage engulfment of apoptotic neutrophils in vivo, TSP-1 aids in the curtailment of inflammatory responses during the resolution phase of injury in the lungs by providing a means by which apoptotic cells are recognized and trigger optimal IL-10 production by macrophages.

Structure of the haptoglobin–haemoglobin complex

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected β-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).

Selection of reliable reference genes during THP-1 monocyte differentiation into macrophages

BackgroundReliable reference genes are a vital prerequisite for any functional study employing quantitative real-time RT-PCR (RT-qPCR) for analyzing gene expression. Yet a proper selection and assessment of the chosen reference genes is only rarely included into a study. To date, no reference genes have been validated for differentiation of THP-1 monocytes. Here we report on the selection of validated reference genes during differentiation of THP-1 monocytes into macrophages induced by phorbol 12-myristate 13-acetate (PMA).ResultsThe mRNA expression of 21 preselected potential reference genes was measured by RT-qPCR at several time-points over six days of PMA-induced THP-1 monocyte-to-macrophage differentiation. A ranking according to expression stability was calculated. Calculations were performed using Microsoft Excel-based applets GeNorm, NormFinder and BestKeeper. Our results indicated ACTB (β-actin) (Cq ± SD, 14.1 ± 0.3) and RPL37A (ribosomal protein L37a) (14.5 ± 0.3) as the most stable genes. While other frequently used reference genes such as GAPDH (glycereraldehyde-3-phosphate dehydrogenase) (20.8 ± 0.8) or G6PD (glucose-6-phophate dehydrogenase) (16.1 ± 1.0) were found to be not as reliable and were therefore unsuited for use as reference genes. These findings were validated by investigating mRNA expression of macrophage scavenger receptor CD36, known to be regulated during monocyte-to-macrophage differentiation. Using ACTB and RPL37A as reference genes a profound and significant regulation of CD36 could be demonstrated, while use of G6PD resulted in a much less pronounced apparent regulation of CD36.ConclusionConsequently, it is recommended to normalize any real-time PCR-based expression data obtained during THP-1 monocyte differentiation using ACTB and RPL37A.

Deletion of the cytoplasmic domain of CD163 enhances porcine reproductive and respiratory syndrome virus replication

The macrophage scavenger receptor CD163 is a major determinant for the entry of porcine reproductive and respiratory syndrome virus (PRRSV). The present study was undertaken to assess the functional significance of the intracellular region of CD163 for PRRSV infection. We stably transfected non-susceptible BHK cells with plasmids encoding full-length and cytoplasmic-domain-deleted (tailless) CD163 and evaluated their permissiveness to PRRSV. The experimental data revealed that the tailless CD163 was not only completely sufficient for converting non-target cells to PRRSV susceptibility, but it also notably enhanced virus replication, with significant increases in viral protein synthesis and progeny release. Taken together, our results suggest that the intracellular domain of CD163 may be associated with an important yet-unknown function during PRRSV infection.

Abstract 1354: Expression of macrophage scavenger receptor (CD163) by tumor-infiltrating macrophages and malignant cells in bladder cancer: association with disease severity and poor patient survival

Abstract Introduction: Infiltrating cells of the tumor microenvironment are important for the growth and spreading of cancer. CD163 is a macrophage-specific hemoglobin scavenger receptor. It is predominantly expressed by alternatively activated macrophages (M2), which is a cell subtype thought to have important tumor-supporting functions. We hypothesized that CD163 mRNA and protein expression may be significant markers of disease severity in bladder cancer. Furthermore, recent findings have indicated that malignant cells express molecular pathways normally only expressed in macrophages, which led us to investigate the possible tumor cell CD163 expression. Materials and Methods: CD163, IL-6, and IL-10 mRNA expression levels were measured using quantitative real-time polymerase chain reaction (RT-qPCR) in bladder cancer biopsy samples obtained from 87 patients followed for a median of 5.1 years (range, 0.07 to 12.9 years). The subcellular distribution of CD163-positive cells was investigated by immunohistochemistry in a subset of 46 bladder cancer biopsy samples. After co-culturing human monocyte-derived macrophages with T24 bladder cancer cells for 48 hours, the expression of the macrophage scavenger receptor CD163 was examined using flow cytometry. Results: CD163 mRNA was expressed in all 87 biopsies, and its expression was strongly correlated with overall (p=0.0028) and relapse-free survival (p=0.0003). Moreover, it was significantly increased in invasive (T2-T4) (p=0.017) and aggressive (grade III/IV) cancers (p=0.015). The expression strongly correlated with local expression of IL-6 (r=0.72, p&amp;lt;0.0001) and IL-10 (r=0.75, p&amp;lt;0.0001), mediators known to induce CD163 expression in vitro. An association between the density of CD163 immunohistochemical positive infiltrating cells and histologically advanced bladder cancer, confirmed the mRNA expression findings. Surprisingly, a large fraction of biopsies (39%) showed definite expression of CD163 by the tumor cells, which indicated the possibility of horizontal mRNA transfer or heterotypic cell fusion. RT-qPCR and flow cytometric analysis showed that human bladder cancer cell lines did not express CD163; however, co-culturing bladder cancer cells with macrophages induced significant tumor cell CD163-expression. Conclusion: In conclusion, we show a strong association between CD163 mRNA expression in bladder cancer biopsies and the severity of the disease, on the one hand, and poor survival on the other hand. CD163 expression was not confined to the infiltrating macrophages. It was also observed in a significant portion of the tumor cells. Our findings identify CD163 as a possible target for novel cancer therapy and underline the importance of the cellular interaction between tumor cells and immune cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1354.

The macrophage scavenger receptor CD163 functions as an innate immune sensor for bacteria

AbstractThe plasma membrane glycoprotein receptor CD163 is a member of the scavenger receptor cystein-rich (SRCR) superfamily class B that is highly expressed on resident tissue macrophages in vivo. Previously, the molecule has been shown to act as a receptor for hemoglobin-haptoglobin complexes and to mediate cell-cell interactions between macrophages and developing erythroblasts in erythroblastic islands. Here, we provide evidence for a potential role for CD163 in host defense. In particular, we demonstrate that CD163 can function as a macrophage receptor for bacteria. CD163 was shown to bind both Gram-positive and -negative bacteria, and a previously identified cell-binding motif in the second scavenger domain of CD163 was sufficient to mediate this binding. Expression of CD163 in monocytic cells promoted bacteria-induced proinflammatory cytokine production. Finally, newly generated antagonistic antibodies against CD163 were able to potently inhibit cytokine production elicited by bacteria in freshly isolated human monocytes. These findings identify CD163 as a macrophage receptor for bacteria and suggest that, during bacterial infection, CD163 on resident tissue macrophages acts as an innate immune sensor and inducer of local inflammation.

The Lipid Whisker Model of the Structure of Oxidized Cell Membranes

An essential feature of the innate immune system is maintaining cellular homeostasis by identifying and removing senescent and apoptotic cells and modified lipoproteins. Identification is achieved through the recognition of molecular patterns, including structurally distinct oxidized phospholipids, on target cells by macrophage receptors. Both the structural nature of the molecular patterns recognized and their orientation within membranes has remained elusive. We recently described the membrane conformation of an endogenous oxidized phospholipid ligand for macrophage scavenger receptor CD36, where the truncated oxidized sn-2 fatty acid moiety protrudes into the aqueous phase, rendering it accessible for recognition. Herein we examine the generality of this conformational motif for peroxidized glycerophospholipids within membranes. Our data reveal that the addition of a polar oxygen atom on numerous peroxidized fatty acids reorients the acyl chain, whereby it no longer remains buried within the membrane interior but rather protrudes into the aqueous compartment. Moreover, we show that neither a conformational change in the head group relative to the membrane surface nor the presence of a polar head group is essential for CD36 recognition of free oxidized phospholipid ligands within membranes. Rather, our results suggest the following global phenomenon. As cellular membranes undergo lipid peroxidation, such as during senescence or apoptosis, previously hydrophobic portions of fatty acids will move from the interior of the lipid bilayer to the aqueous exterior. This enables physical contact between pattern recognition receptor and molecular pattern ligand. Cell membranes thus "grow whiskers" as phospholipids undergo peroxidation, and many of their oxidized fatty acids protrude at the surface.

Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages

We used a panel of monoclonal antibodies to characterize DCs in the dermis of normal human skin. Staining for the CD11c integrin, which is abundant on many kinds of DCs, revealed cells in the upper dermis. These cells were positive for blood DC antigen-1 (BDCA-1; also known as CD1c), HLA-DR, and CD45, markers that are also expressed by circulating myeloid DCs. A small subset of CD11c+ dermal cells expressed DEC-205/CD205 and DC-lysosomal-associated membrane glycoprotein/CD208 (DC-LAMP/CD208), suggesting some differentiation or maturation. When BDCA-1+ cells were selected from collagenase digests of normal dermis, they proved to be strong stimulators for T cells in a mixed leukocyte reaction. A second major population of cells located throughout the dermis was positive for factor XIIIA (FXIIIA), but lacked CD11c and BDCA-1. They expressed the macrophage scavenger receptor CD163 and stained weakly for HLA-DR and CD45. Isolated CD163+ dermal cells were inactive in stimulating T cell proliferation, but in biopsies of tattoos, these cells were selectively laden with granular pigments. Plasmacytoid DCs were also present in the dermis, marked by CD123 and BDCA-2. In summary, the normal dermis contains typical immunostimulatory myeloid DCs identified by CD11c and BDCA-1, as well as an additional population of poorly stimulatory macrophages marked by CD163 and FXIIIA.

Proteolytic shedding of the macrophage scavenger receptor CD163 in multiple sclerosis

The scavenger receptor CD163 is selectively expressed on tissue macrophages and human monocytes. CD163 has been implicated to play a role in the clearance of hemoglobin and in the regulation of cytokine production by macrophages. Membrane CD163 can be cleaved by matrix metalloproteinases (MMP) resulting in soluble CD163 (sCD163). In the present report the shedding of CD163 was investigated in multiple sclerosis (MS). An upregulation of plasma sCD163 and a down regulation of membrane CD163 in MS patients compared to healthy controls was observed. The levels of plasma sCD163 correlated with plasma MMP-9 levels in controls, but not in MS patients. Moreover, evidence was obtained for CD163-cleaving MMP activity in plasma of MS patients. Finally, the increased proteolytic shedding of CD163 correlated to reduced plasma levels of circulating inflammatory cytokines. Collectively, our results provide evidence for proteolytic shedding of CD163 in MS and suggest a possible link to cytokine production.

Conformation of an Endogenous Ligand in a Membrane Bilayer for the Macrophage Scavenger Receptor CD36

Phagocytic removal of aged or oxidatively damaged cells and macromolecules is an indispensable homeostatic function of the innate immune system. A structurally conserved family of oxidized phospholipids that serve as endogenous high-affinity ligands for the macrophage scavenger receptor CD36 (oxPC(CD36)) was recently identified. Enriched within atherosclerotic plaque and senescent cell membranes, oxPC(CD36) promote the uptake of oxidized lipoproteins and cell membranes by macrophages when present at only a few molecules per particle. How macrophages recognize oxPC(CD36) within cellular membranes and lipoprotein surfaces remains unknown. Herein, we deduce the conformation of oxPC(CD36) near the hydrophobic-hydrophilic interface within membrane bilayers by determining multiple critical internuclear distances using nuclear Overhauser enhancement spectroscopy. The molecular model reveals a unique conformation for oxPC(CD36) within bilayers whereby the distal end of the sn-2 acyl chain harboring the structurally conserved CD36 recognition motif protrudes into the aqueous phase. The remarkable conformation elucidated for oxPC(CD36) produces a surface accessible phagocytic "eat me signal" to facilitate senescent cell and oxidized lipoprotein recognition by the scavenger receptor CD36 as part of its immune surveillance function.

CD36 is a receptor for oxidized high density lipoprotein: Implications for the development of atherosclerosis

Atherosclerotic plaques result from the excessive deposition of cholesterol esters derived from lipoproteins and lipoprotein fragments. Tissue macrophage within the intimal space of major arterial vessels have been shown to play an important role in this process. We demonstrate in a transfection system using two human cell lines that the macrophage scavenger receptor CD36 selectively elicited lipid uptake from Cu 2+-oxidized high density lipoprotein (HDL) but not from native HDL or low density lipoprotein (LDL). The uptake of oxHDL displayed morphological and biochemical similarities with the CD36-dependent uptake of oxidized LDL. CD36-mediated uptake of oxidized HDL by macrophage may therefore contribute to atheroma formation.

A Unique Loop Extension in the Serine Protease Domain of Haptoglobin Is Essential for CD163 Recognition of the Haptoglobin-Hemoglobin Complex

Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (alpha-chain) and a serine protease domain (beta-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the beta-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin beta-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin.