Circulating TREM2 as a noninvasive diagnostic biomarker for NASH in patients with elevated liver stiffness.

BACKGROUND AND AIMS: Reliable noninvasive biomarkers are an unmet clinical need for the diagnosis of NASH. This study investigates the diagnostic accuracy of the circulating triggering receptor expressed on myeloid cells 2 (plasma TREM2) as a biomarker for NASH in patients with NAFLD and elevated liver stiffness. APPROACH AND RESULTS: We collected cross-sectional, clinical data including liver biopsies from a derivation ( n = 48) and a validation cohort ( n = 170) of patients with elevated liver stiffness measurement (LSM ≥ 8.0 kPa). Patients with NAFLD activity scores (NAS) ≥4 were defined as having NASH. Plasma TREM2 levels were significantly elevated in patients with NASH of the derivation cohort, with an area under the receiver operating characteristics curve (AUROC) of 0.92 (95% confidence interval [CI], 0.84-0.99). In the validation cohort, plasma TREM2 level increased approximately two-fold in patients with NASH, and a strong diagnostic accuracy was confirmed (AUROC, 0.83; 95% CI, 0.77-0.89; p < 0.0001). Plasma TREM2 levels were associated with the individual histologic features of NAS: steatosis, lobular inflammation, and ballooning ( p < 0.0001), but only weakly with fibrosis stages. Dual cutoffs for rule-in and rule-out were explored: a plasma TREM2 level of ≤38 ng/ml was found to be an optimal NASH rule-out cutoff (sensitivity 90%; specificity 52%), whereas a plasma TREM2 level of ≥65 ng/ml was an optimal NASH rule-in cutoff (specificity 89%; sensitivity 54%). CONCLUSIONS: Plasma TREM2 is a plausible individual biomarker that can rule-in or rule-out the presence of NASH with high accuracy and thus has the potential to reduce the need for liver biopsies and to identify patients who are eligible for clinical trials in NASH.

Haptoglobin-related protein in human plasma correlates to haptoglobin concentrations and phenotypes.

Haptoglobin-related protein (Hpr) is a plasma protein with high sequence similarity to haptoglobin (Hp). Like Hp, Hpr also binds hemoglobin (Hb) with high affinity, but it does not bind to the Hb-Hp receptor CD163 on macrophages. The Hpr concentration is markedly lower than Hp in plasma and its regulation is not understood. In the present study, we have developed non-crossreactive antibodies to Hpr to analyze the Hpr concentration in 112 plasma samples from anonymized individuals and compared it to Hp. The results show that plasma Hpr correlated with Hp concentrations (rho = 0.46, p = .0001). Hpr accounts for on average 0.35% of the Hp/Hpr pool but up to 29% at low Hp levels. Furthermore, the Hpr concentrations were significantly lower in individuals with the Hp2-2 phenotype compared to those with the Hp2-1 or Hp1-1 phenotypes. Experimental binding analysis did not provide evidence that Hpr associates with Hp and in this way is removed via CD163. In conclusion, the Hpr concentration correlates to Hp concentrations and Hp-phenotypes by yet unknown mechanisms independent of CD163-mediated removal of Hb-Hp complexes.

Metformin directly suppresses atherosclerosis in normoglycaemic mice via haematopoietic adenosine monophosphate-activated protein kinase.

AIMS: Atherosclerotic vascular disease has an inflammatory pathogenesis. Heme from intraplaque haemorrhage may drive a protective and pro-resolving macrophage M2-like phenotype, Mhem, via AMPK and activating transcription factor 1 (ATF1). The antidiabetic drug metformin may also activate AMPK-dependent signalling. Hypothesis: Metformin systematically induces atheroprotective genes in macrophages via AMPK and ATF1, thereby suppresses atherogenesis. METHODS AND RESULTS: Normoglycaemic Ldlr-/- hyperlipidaemic mice were treated with oral metformin, which profoundly suppressed atherosclerotic lesion development (P < 5 × 10-11). Bone marrow transplantation from AMPK-deficient mice demonstrated that metformin-related atheroprotection required haematopoietic AMPK [analysis of variance (ANOVA), P < 0.03]. Metformin at a clinically relevant concentration (10 µM) evoked AMPK-dependent and ATF1-dependent increases in Hmox1, Nr1h2 (Lxrb), Abca1, Apoe, Igf1, and Pdgf, increases in several M2-markers and decreases in Nos2, in murine bone marrow macrophages. Similar effects were seen in human blood-derived macrophages, in which metformin-induced protective genes and M2-like genes, suppressible by si-ATF1-mediated knockdown. Microarray analysis comparing metformin with heme in human macrophages indicated that the transcriptomic effects of metformin were related to those of heme, but not identical. Metformin-induced lesional macrophage expression of p-AMPK, p-ATF1, and downstream M2-like protective effects. CONCLUSION: Metformin activates a conserved AMPK-ATF1-M2-like pathway in mouse and human macrophages, and results in highly suppressed atherogenesis in hyperlipidaemic mice via haematopoietic AMPK.

Tissue-resident macrophages in omentum promote metastatic spread of ovarian cancer.

Experimental and clinical evidence suggests that tumor-associated macrophages (TAMs) play important roles in cancer progression. Here, we have characterized the ontogeny and function of TAM subsets in a mouse model of metastatic ovarian cancer that is representative for visceral peritoneal metastasis. We show that the omentum is a critical premetastatic niche for development of invasive disease in this model and define a unique subset of CD163+ Tim4+ resident omental macrophages responsible for metastatic spread of ovarian cancer cells. Transcriptomic analysis showed that resident CD163+ Tim4+ omental macrophages were phenotypically distinct and maintained their resident identity during tumor growth. Selective depletion of CD163+ Tim4+ macrophages in omentum using genetic and pharmacological tools prevented tumor progression and metastatic spread of disease. These studies describe a specific role for tissue-resident macrophages in the invasive progression of metastatic ovarian cancer. The molecular pathways of cross-talk between tissue-resident macrophages and disseminated cancer cells may represent new targets to prevent metastasis and disease recurrence.

Specific targeting of CD163+ TAMs mobilizes inflammatory monocytes and promotes T cell-mediated tumor regression.

Tumor-associated macrophages (TAMs) play critical roles in tumor progression but are also capable of contributing to antitumor immunity. Recent studies have revealed an unprecedented heterogeneity among TAMs in both human cancer and experimental models. Nevertheless, we still understand little about the contribution of different TAM subsets to tumor progression. Here, we demonstrate that CD163-expressing TAMs specifically maintain immune suppression in an experimental model of melanoma that is resistant to anti-PD-1 checkpoint therapy. Specific depletion of the CD163+ macrophages results in a massive infiltration of activated T cells and tumor regression. Importantly, the infiltration of cytotoxic T cells was accompanied by the mobilization of inflammatory monocytes that significantly contributed to tumor regression. Thus, the specific targeting of CD163+ TAMs reeducates the tumor immune microenvironment and promotes both myeloid and T cell-mediated antitumor immunity, illustrating the importance of selective targeting of tumor-associated myeloid cells in a therapeutic context.

The macrophage-related biomarkers sCD163 and sCD206 are released by different shedding mechanisms.

The hemoglobin receptor CD163 and the mannose receptor CD206 are both expressed on the surface of human macrophages. Upon inflammatory activation, the receptors are shed from the macrophage surface generating soluble products. The plasma concentration of both soluble CD163 (sCD163) and soluble CD206 (sCD206) are increased in several diseases, including inflammatory conditions and cancer. Here, we show that in contrast to CD163, LPS-mediated shedding of CD206 in humans is slow and a result of indirect signaling. Although both sCD163 and sCD206 were increased in response to LPS stimulation in vivo, only CD163 was shed from LPS-stimulated macrophages in vitro. Although both sCD163 and sCD206 were released from cultured macrophages stimulated with zymosan and PMA, shedding of CD206 was generally slower and less efficient and not reduced by inhibitors against the major protease classes. These data indicate that CD163 and CD206 are shed from the macrophages by very different mechanisms potentially involving distinctive inflammatory processes.

The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen.

Hemolysis is a complication in septic infections with Staphylococcus aureus, which utilizes the released Hb as an iron source. S. aureus can acquire heme in vitro from hemoglobin (Hb) by a heme-sequestering mechanism that involves proteins from the S. aureus iron-regulated surface determinant (Isd) system. However, the host has its own mechanism to recapture the free Hb via haptoglobin (Hp) binding and uptake of Hb-Hp by the CD163 receptor in macrophages. It has so far remained unclear how the Isd system competes with this host iron recycling system in situ to obtain the important nutrient. By binding and uptake studies, we now show that the IsdH protein, which serves as an Hb receptor in the Isd system, directly interferes with the CD163-mediated clearance by binding the Hb-Hp complex and inhibiting CD163 recognition. Analysis of truncated IsdH variants including one or more of three near iron transporter domains, IsdHN1, IsdHN2, and IsdHN3, revealed that Hb binding of IsdHN1 and IsdHN2 accounted for the high affinity for Hb-Hp complexes. The third near iron transporter domain, IsdHN3, exhibited redox-dependent heme extraction, when Hb in the Hb-Hp complex was in the oxidized met form but not in the reduced oxy form. IsdB, the other S. aureus Hb receptor, failed to extract heme from Hb-Hp, and it was a poor competitor for Hb-Hp binding to CD163. This indicates that Hb recognition by IsdH, but not by IsdB, sterically inhibits the receptor recognition of Hb-Hp. This function of IsdH may have an overall stimulatory effect on S. aureus heme acquisition and growth.

Anti-CD163-dexamethasone protects against apoptosis after ischemia/reperfusion injuries in the rat liver.

AIM: The Pringle maneuver is a way to reduce blood loss during liver surgery. However, this may result in ischemia/reperfusion injury in the development of which Kupffer cells play a central role. Corticosteroids are known to have anti-inflammatory effects. Our aim was to investigate whether a conjugate of dexamethasone and antibody against the CD163 macrophage cell surface receptor could reduce ischemia/reperfusion injury in the rat liver. METHODS: Thirty-six male Wistar rats were used for the experiments. Animals were randomly divided into four groups of eight receiving anti-CD163-dexamethasone, high dose dexamethasone, low dose dexamethasone or placebo intravenously 18 h before laparotomy with subsequent 60 min of liver ischemia. After reperfusion for 24 h the animals had their liver removed. Bloods were drawn 30 min and 24 h post ischemia induction. Liver cell apoptosis and necrosis were analyzed by stereological quantification. RESULTS: After 24 h' reperfusion, the fraction of cell in non-necrotic tissues exhibiting apoptotic profiles was significantly lower in the high dose dexamethasone (p = 0.03) and anti-CD163-dex (p = 0.03) groups compared with the low dose dexamethasone and placebo groups. There was no difference in necrotic cell volume between groups. After 30 min of reperfusion, levels of haptoglobin were significantly higher in the anti-CD163-dex and high dose dexamethasone groups. Alanine aminotransferase and alkaline phosphatase were significantly higher in the high dose dexamethasone group compared to controls after 24 h' reperfusion. CONCLUSIONS: We show that pharmacological preconditioning with anti-CD163-dex and high dose dexamethasone reduces the number of apoptotic cells following ischemia/reperfusion injury.

Drug Trafficking into Macrophages via the Endocytotic Receptor CD163.

In inflammatory diseases, macrophages are a main producer of a range of cytokines regulating the inflammatory state. This also includes inflammation induced by tumor growth, which recruits so-called tumor-associated macrophages supporting tumor growth. Macrophages are therefore relevant targets for cytotoxic or phenotype-modulating drugs in the treatment of inflammatory and cancerous diseases. Such targeting of macrophages has been tried using the natural propensity of macrophages to non-specifically phagocytose circulating foreign particulate material. In addition, the specific targeting of macrophage-expressed receptors has been used in order to obtain a selective uptake in macrophages and reduce adverse effects of off-target delivery of drugs. CD163 is a highly expressed macrophage-specific endocytic receptor that has been studied for intracellular delivery of small molecule drugs to macrophages using targeted liposomes or antibody drug conjugates. This review will focus on the biology of CD163 and its potential role as a target for selective macrophage targeting compared with other macrophage targeting approaches.

Anti-inflammatory liposomes have no impact on liver regeneration in rats.

INTRODUCTION: Surgical resection is the gold standard in treatment of hepatic malignancies, giving the patient the best chance to be cured. The liver has a unique capacity to regenerate. However, an inflammatory response occurs during resection, in part mediated by Kupffer cells, that influences the speed of regeneration. The aim of this study was to investigate the effect of a Kupffer cell targeted anti-inflammatory treatment on liver regeneration in rats. METHODS: Two sets of animals, each including four groups of eight rats, were included. Paired groups from each set received treatment with placebo, low dose dexamethasone, high dose dexamethasone or low dose anti-CD163 dexamethasone. Subsequently, the rats underwent 70% partial hepatectomy. The two sets were evaluated on postoperative day 2 or 5, respectively. Blood was drawn for circulating markers of inflammation and liver cell damage; liver tissue was sampled for analysis of regeneration rate and proliferation index. RESULTS: The high dose dexamethasone group had significantly lower body and liver weight than the placebo and anti-CD163-dex groups. There were no differences in liver regeneration rates between groups. Hepatocyte proliferation was completed faster in the placebo group, although this was not significant. The anti-CD163-dex group showed increased blood levels of albumin and alanine aminotransferase and a diminished inflammatory response in terms of significantly reduced haptoglobin, α2-macroglobulin and Interleukine-6. CONCLUSION: Low dose dexamethasone targeted to Kupffer cells does not affect histological liver cell regeneration after 70% hepatectomy in rats, but reduces the inflammatory response judged by circulating markers of inflammation.

Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion.

Cultures of human CD34(pos) cells stimulated with erythroid growth factors plus dexamethasone, a model for stress erythropoiesis, generate numerous erythroid cells plus a few macrophages (approx. 3%; 3:1 positive and negative for CD169). Interactions occurring between erythroblasts and macrophages in these cultures and the biological effects associated with these interactions were documented by live phase-contrast videomicroscopy. Macrophages expressed high motility interacting with hundreds/thousands of erythroblasts per hour. CD169(pos) macrophages established multiple rapid 'loose' interactions with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169(neg) macrophages established 'tight' interactions with mature erythroblasts and phagocytosed these cells. 'Loose' interactions of CD169(pos) macrophages were associated with proerythroblast cytokinesis (the M phase of the cell cycle) suggesting that these interactions may promote proerythroblast duplication. This hypothesis was tested by experiments that showed that as few as 103 macrophages significantly increased levels of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide incorporation frequency in S/G2/M and cytokinesis expressed by proerythroblasts over 24 h of culture. These effects were observed also when macrophages were co-cultured with dexamethasone directly conjugated to a macrophage-specific CD163 antibody. In conclusion, in addition to promoting proerythroblast proliferation directly, dexamethasone stimulates expansion of these cells indirectly by stimulating maturation and cytokinesis supporting activity of macrophages.

Structural basis for inflammation-driven shedding of CD163 ectodomain and tumor necrosis factor-α in macrophages.

The haptoglobin-hemoglobin receptor CD163 and proTNF-α are transmembrane macrophage proteins subjected to cleavage by the inflammation-responsive protease ADAM17. This leads to release of soluble CD163 (sCD163) and bioactive TNF-α. Sequence comparison of the juxtamembrane region identified similar palindromic sequences in human CD163 ((1044)Arg-Ser-Ser-Arg) and proTNF-α ((78)Arg-Ser-Ser-Ser-Arg). In proTNF-α the Arg-Ser-Ser-Ser-Arg sequence is situated next to the previously established ADAM17 cleavage site. Site-directed mutagenesis revealed that the sequences harbor essential information for efficient cleavage of the two proteins upon ADAM17 stimulation. This was further evidenced by analysis of mouse CD163 that, like CD163 in other non-primates, does not contain the palindromic CD163 sequence in the juxtamembrane region. Mouse CD163 resisted endotoxin- and phorbol ester-induced shedding, and ex vivo analysis of knock-in of the Arg-Ser-Ser-Arg sequence in mouse CD163 revealed a receptor shedding comparable with that of human CD163. In conclusion, we have identified an essential substrate motif for ADAM17-mediated CD163 and proTNF-α cleavage in macrophages. In addition, the present data indicate that CD163, by incorporation of this motif in late evolution, underwent a modification that allows for an instant down-regulation of surface CD163 expression and inhibition of hemoglobin uptake. This regulatory modality seems to have coincided with the evolution of an enhanced hemoglobin-protecting role of the haptoglobin-CD163 system in primates.

Targeting dexamethasone to macrophages in a porcine endotoxemic model.

OBJECTIVES: Macrophages are important cells in immunity and the main producers of pro-inflammatory cytokines. The main objective was to evaluate if specific delivery of glucocorticoid to the macrophage receptor CD163 is superior to systemic glucocorticoid therapy in dampening the cytokine response to lipopolysaccharide infusion in pigs. DESIGN: Two randomized, placebo-controlled trials. SETTING: University hospital laboratory. SUBJECTS: Female farm-bred pigs (26-31 kg). DESIGN: A humanized antibody that binds to pig and human CD163 was produced, characterized, and conjugated with dexamethasone. In the first study (total n = 12), pigs were randomly assigned to four groups: 1) saline; 2) dexamethasone (1.0 mg/kg); 3) dexamethasone (0.02 mg/kg); and 4) anti-CD163-conjugated dexamethasone (0.02 mg/kg). In the second study (total n = 36), two additional groups were included in addition to the four original groups: 5) anti-CD163-conjugated dexamethasone (0.005 mg/kg); 6) unconjugated anti-CD163. Treatments were given 20 hours prior to infusion of lipopolysaccharide (1 µg × kg × h) for 5 hours. Blood samples were analyzed for cytokines, cortisol, and adrenocorticotropic hormone. RESULTS: In the saline group, lipopolysaccharide increased cytokine and plasma cortisol levels. In both studies, dexamethasone (1 mg/kg) and anti-CD163 dexamethasone (0.02 mg/kg) uniformly attenuated tumor necrosis factor-α peak levels (both p < 0.05) compared with low-dose dexamethasone (0.02 mg/kg). However, dexamethasone 1 mg/kg significantly suppressed plasma cortisol and adrenocorticotropic hormone levels compared with anti-CD163 dexamethasone (0.02 mg/kg; p < 0.05). No significant hemodynamic difference existed between groups. The anti-CD163 dexamethasone drug conjugate exhibited a fast plasma clearance, with a half-life of approximately 5-8 minutes. CONCLUSION: Targeted delivery of dexamethasone to macrophages using a humanized CD163 antibody as carrier exhibits anti-inflammatory effects comparable with 50 times higher concentrations of free dexamethasone and does not inhibit endogenous cortisol production. This antibody-drug complex showing similar affinity and specificity for human CD163 is, therefore, a promising drug candidate in this novel type of anti-inflammatory therapy.

CD163 binding to haptoglobin-hemoglobin complexes involves a dual-point electrostatic receptor-ligand pairing.

Formation of the haptoglobin (Hp)-hemoglobin (Hb) complex in human plasma leads to a high affinity recognition by the endocytic macrophage receptor CD163. A fast segregation of Hp-Hb from CD163 occurs at endosomal conditions (pH <6.5). The ligand binding site of CD163 has previously been shown to involve the scavenger receptor cysteine-rich (SRCR) domain 3. This domain and the adjacent SRCR domain 2 of CD163 contain a consensus motif for a calcium-coordinated acidic amino acid triad cluster as originally identified in the SRCR domain of the scavenger receptor MARCO. Here we show that site-directed mutagenesis in each of these acidic triads of SRCR domains 2 and 3 abrogates the high affinity binding of recombinant CD163 to Hp-Hb. In the ligand, Hp Arg-252 and Lys-262, both present in a previously identified CD163 binding loop of Hp, were revealed as essential residues for the high affinity receptor binding. These findings are in accordance with pairing of the calcium-coordinated acidic clusters in SRCR domains 2 and 3 with the two basic Arg/Lys residues in the Hp loop. Such a two-point electrostatic pairing is mechanistically similar to the pH-sensitive pairings disclosed in crystal structures of ligands in complex with tandem LDL receptor repeats or tandem CUB domains in other endocytic receptors.

Is classical pseudoxanthoma elasticum a consequence of hepatic 'intoxication' due to ABCC6 substrate accumulation in the liver?

Pseudoxanthoma elasticum (PXE) is a serious genetic disorder with ectopic mineralization affecting the skin, the eye and the cardiovascular system. The disease is predominantly caused by mutations in the transmembrane ABC protein ABCC6, a putative small substrate transporter. Interestingly, ABCC6 seems virtually absent in the affected organs, whereas a high expression is seen in hepatocytes. This and further published experimental evidence indicate that PXE is a systemic, metabolic liver disease where circulatory changes affect the peripheral mineralization process. Owing to the well-characterized transport of organic substrates by related ABC proteins, it has been proposed that PXE is caused by impaired export of an antimineralization compound to the blood. The authors here present an alternative hypothesis that explains ectopic mineralization in PXE as a consequence of hepatic accumulation of ABCC6 substrate(s) that via gene-regulating effects leads to altered hepatic secretion and activation of antimineralization/anticalcification proteins such as fetuin-A and Gla proteins.

Tumor-promoting macrophages induce the expression of the macrophage-specific receptor CD163 in malignant cells.

Tumor-associated macrophages (TAMs) represent a distinct malignancy-promoting phenotype suggested to play a key role in tumor formation and metastasis. We aimed to investigate the expression of the monocyte/macrophage-restricted receptor CD163 in bladder tumor biopsies and assess the potential mechanism inducing the CD163 expression in tumor cells. A high CD163 mRNA expression (n = 87) was significantly associated with a poor 13-year overall survival (log-rank test, χ(2) = 8.931; p = 0.0028). Moreover, CD163 mRNA expression was significantly increased in muscle 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 <0.0001) and IL-10 (r = 0.75; p <0.0001), mediators known to induce CD163 expression in vitro. CD163 immunostaining (n = 46) confirmed the association between dense TAM infiltration and histologically advanced disease. In 39% of the biopsies, CD163 immunoreactivity was also observed in tumor cells, and CD163-expressing metastatic cells were identified in lymph node biopsies (n = 8). Bladder cancer cell lines did not express CD163; however, when cocultured with macrophages the bladder cancer cell expression of CD163 was significantly induced in an IL-6/IL-10 independent manner. In conclusion, we show a strong association between CD163 mRNA expression in bladder cancer biopsies and poor patient outcome. CD163 expression was not confined to the infiltrating TAMs, but was also expressed by a significant portion of the malignant cells in both tumors and lymph nodes. CD163 expressing tumor cells may constitute a subpopulation of tumor cells with a phenotypic shift associated with epithelial-to-mesenchymal transition (EMT) and increased metastatic activity induced by TAMs.

Efficient intracellular drug-targeting of macrophages using stealth liposomes directed to the hemoglobin scavenger receptor CD163.

The hemoglobin scavenger receptor CD163 is exclusively expressed in the monocytic lineage and preferentially in tissue resident macrophages of the M2 phenotype and in macrophages in sites of inflammation and tumor growth. In the present study we have designed liposomes specifically targeting CD163 by hydrophobic linkage of CD163-binding monoclonal antibodies to polyethylene glycol-coated liposomes ('stealth liposomes'). Targeting to the endocytic CD163 protein greatly increased the uptake of liposomes in CD163 transfected cells and macrophages as visualized by confocal microscopy and flow cytometry of cells exposed to CD163 targeting liposomes loaded with calcein. Strong cytotoxic effects were seen in CD163-expressing human monocytes by using the chemotherapeutic agent doxorubicin as cargo of the liposomes. In conclusion, the use of stealth liposomes modified to recognize CD163 is a potential way to target drugs to macrophages that support inflammatory and malignant processes.

The conserved scavenger receptor cysteine-rich superfamily in therapy and diagnosis.

The scavenger receptor cysteine-rich (SRCR) superfamily of soluble or membrane-bound protein receptors is characterized by the presence of one or several repeats of an ancient and highly conserved protein module, the SRCR domain. This superfamily (SRCR-SF) has been in constant and progressive expansion, now up to more than 30 members. The study of these members is attracting growing interest, which parallels that in innate immunity. No unifying function has been described to date for the SRCR domains, this being the result of the limited knowledge still available on the physiology of most members of the SRCR-SF, but also of the sequence versatility of the SRCR domains. Indeed, involvement of SRCR-SF members in quite different functions, such as pathogen recognition, modulation of the immune response, epithelial homeostasis, stem cell biology, and tumor development, have all been described. This has brought to us new information, unveiling the possibility that targeting or supplementing SRCR-SF proteins could result in diagnostic and/or therapeutic benefit for a number of physiologic and pathologic states. Recent research has provided structural and functional insight into these proteins, facilitating the development of means to modulate the activity of SRCR-SF members. Indeed, some of these approaches are already in use, paving the way for a more comprehensive use of SRCR-SF members in the clinic. The present review will illustrate some available evidence on the potential of well known and new members of the SRCR-SF in this regard.

Comparative assessment of the recognition of domain-specific CD163 monoclonal antibodies in human monocytes explains wide discrepancy in reported levels of cellular surface CD163 expression.

BACKGROUND: CD163 is expressed exclusively on cells of the monocyte/macrophage lineage and is widely used as a marker of human macrophages. Further, it has been suggested as a diagnostic marker of monocyte/macrophage activity in inflammatory conditions and as a therapeutic target. However, studies continue to exhibit great discrepancy in the measured percentage of CD163-expressing blood monocytes in healthy individuals. In this study we sought to clarify this inconsistency in reported levels of CD163 surface expression by a detailed analysis of a panel of CD163 antibodies used in previous studies. MATERIALS AND METHODS: The cellular distribution of CD163 on human peripheral blood monocytes in freshly drawn blood and peripheral blood mononuclear cells isolated from buffy-coats was investigated by flow cytometry using CD163 monoclonal antibodies recognizing scavenger receptor cysteine-rich (SRCR) domain 1 (MAC2-158), domain 4 (R-20), domain 7 (GHI/61), and domain 9 (RM3/1). The CD163 monoclonal antibodies were characterized in binding and endocytosis experiments in human macrophages and CD163-transfected Flp-In CHO cells. Calcium-dependent ligand binding was assessed using surface plasmon resonance, and the specificity of the CD163 monoclonal antibodies was analyzed by western blotting. RESULTS AND DISCUSSION: Flow cytometric analysis revealed that the estimated proportion of CD163-expressing human peripheral blood monocytes increased when using CD163 monoclonal antibodies recognizing epitopes in the N-terminal part of CD163, remote from the membrane surface. Moreover, the proportion of CD163 positive monocytes observed was highly dependent on free calcium. GHI/61 did not exhibit CD163 binding in the presence of calcium as measured by surface plasmon resonance, which was in agreement with the concordant loss of binding in heparin-stabilized whole blood observed by flow cytometry. In contrast, RM3/1 exhibited weak binding to CD163 in the absence of calcium but high affinity binding to CD163 in the presence of calcium. R-20 and MAC2-158 were unaffected by extracellular calcium levels. The latter SRCR domain 1mAb consistently recognized more than 80% CD163-positive monocytes in human peripheral blood. CONCLUSION: Epitope accessibility and extracellular calcium dependence elucidate discrepancies in reported levels of monocytic CD163 expression. Utilizing monoclonal antibodies to the N-terminal part of CD163 more than 80% monocytes in human peripheral blood could be identified as CD163 positive, indicating that most, and conceivably all, human peripheral blood monocytes do express CD163.

Tumor necrosis factor α-converting enzyme (TACE/ADAM17) mediates ectodomain shedding of the scavenger receptor CD163.

CD163 is expressed specifically in the monocyte/macrophage lineage, where it mediates uptake of haptoglobin-hemoglobin complexes, leading to metabolism of the oxidative heme molecule. Shedding of the CD163 ectodomain from the cell surface produces a sCD163 plasma protein, and a positive correlation is seen between the sCD163 plasma level and the severity of various infectious and inflammatory diseases. In the present analysis of the phorbol ester-induced shedding of sCD163 in CD163 cDNA-transfected HEK293 cells, we used metalloproteinase inhibitors and siRNA-mediated inhibition of metalloproteinases to identify TACE/ADAM17 as an enzyme responsible for PMA-induced cleavage of the membrane-proximal region of CD163. As TACE/ADAM17-mediated shedding of TNF-α is up-regulated in macrophages subjected to inflammatory stimuli, the present results now provide a likely explanation for the strong empirical relationship between the sCD163 plasma level and infectious/inflammatory diseases relating to macrophage activity.