Anders Etzerodt

CD163 and Inflammation: Biological, Diagnostic, and Therapeutic Aspects

SIGNIFICANCE The hemoglobin (Hb) scavenger receptor, CD163, is a macrophage-specific protein and the upregulated expression of this receptor is one of the major changes in the macrophage switch to alternative activated phenotypes in inflammation. Accordingly, a high CD163 expression in macrophages is a characteristic of tissues responding to inflammation. The scavenging of the oxidative and proinflammatory Hb leading to stimulation of the heme-oxygenase-1 and production of anti-inflammatory heme metabolites indicates that CD163 thereby indirectly contributes to the anti-inflammatory response. RECENT ADVANCES In addition to this biological role in inflammation, CD163 is a potential inflammation biomarker and a therapeutic target. The biomarker form of CD163 is the soluble plasma CD163 that arises from the increased shedding of CD163 mediated by the tumor necrosis factor-α (TNF-α) cleaving enzyme. This explains that a steadily increasing literature documents that the plasma level of soluble CD163 is increased in a large spectrum of acute and chronic inflammatory disorders. The nonshed membrane form of CD163 in macrophages constitutes a target for drugs to be directed to macrophages in inflammation. This approach has been used in an animal inflammation model to highly increase the apparent therapeutic index of anti-inflammatory glucocorticoid drug that was coupled to an anti-CD163 antibody. Furthermore, other recent animal data, which indirectly involve CD163 in macrophages, demonstrate that injections of haptoglobin attenuate Hb-induced damages after blood transfusion. CRITICAL ISSUES AND FUTURE DIRECTIONS The diagnostic and therapeutic properties of CD163 await further clinical studies and regulatory approval before implementation in the clinic.

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.

The Haptoglobin-CD163-Heme Oxygenase-1 Pathway for Hemoglobin Scavenging

The haptoglobin- (Hp-) CD163-heme oxygenase-1 (HO-1) pathway is an efficient captor-receptor-enzyme system to circumvent the hemoglobin (Hb)/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp leading to CD163-mediated uptake of the complex in macrophages followed by lysosomal Hp-Hb breakdown and HO-1-catalyzed conversion of heme into the metabolites carbon monoxide (CO), biliverdin, and iron. The plasma concentration of Hp is a limiting factor as evident during accelerated hemolysis, where the Hp depletion may cause serious Hb-induced toxicity and put pressure on backup protecting systems such as the hemopexin-CD91-HO pathway. The Hp-CD163-HO-1 pathway proteins are regulated by the acute phase mediator interleukin-6 (IL-6), but other regulatory factors indicate that this upregulation is a counteracting anti-inflammatory response during inflammation. The heme metabolites including bilirubin converted from biliverdin have overall an anti-inflammatory effect and thus reinforce the anti-inflammatory efficacy of the Hp-CD163-HO-1 pathway. Future studies of animal models of inflammation should further define the importance of the pathway in the anti-inflammatory response.

Targeting the Hemoglobin Scavenger receptor CD163 in Macrophages Highly Increases the Anti-inflammatory Potency of Dexamethasone

Synthetic glucocorticoids are potent anti-inflammatory drugs but serious side effects such as bone mobilization, muscle mass loss, immunosuppression, and metabolic alterations make glucocorticoid therapy a difficult balance. The therapeutic anti-inflammatory effect of glucocorticoids relies largely on the suppressed release of tumor-necrosis factor-α and other cytokines by macrophages at the sites of inflammation. We have now developed a new biodegradable anti-CD163 antibody-drug conjugate that specifically targets the glucocorticoid, dexamethasone to the hemoglobin scavenger receptor CD163 in macrophages. The conjugate, that in average contains four dexamethasone molecules per antibody, exhibits retained high functional affinity for CD163. In vitro studies in rat macrophages and in vivo studies of Lewis rats showed a strong anti-inflammatory effect of the conjugate measured as reduced lipopolysaccharide-induced secretion of tumor-necrosis factor-α. The in vivo potency of conjugated dexamethasone was about 50-fold that of nonconjugated dexamethasone. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent dose of the conjugate had no such effect, measured as thymus lymphocytes apoptosis, body weight loss, and suppression of endogenous cortisol levels. In conclusion, the study shows antibody-drug conjugates as a future approach in anti-inflammatory macrophage-directed therapy. Furthermore, the data demonstrate CD163 as an excellent macrophage target for anti-inflammatory drug delivery.

Obesity Alters Adipose Tissue Macrophage Iron Content and Tissue Iron Distribution

Adipose tissue (AT) expansion is accompanied by the infiltration and accumulation of AT macrophages (ATMs), as well as a shift in ATM polarization. Several studies have implicated recruited M1 ATMs in the metabolic consequences of obesity; however, little is known regarding the role of alternatively activated resident M2 ATMs in AT homeostasis or how their function is altered in obesity. Herein, we report the discovery of a population of alternatively activated ATMs with elevated cellular iron content and an iron-recycling gene expression profile. These iron-rich ATMs are referred to as MFehi, and the remaining ATMs are referred to as MFelo. In lean mice, ~25% of the ATMs are MFehi; this percentage decreases in obesity owing to the recruitment of MFelo macrophages. Similar to MFelo cells, MFehi ATMs undergo an inflammatory shift in obesity. In vivo, obesity reduces the iron content of MFehi ATMs and the gene expression of iron importers as well as the iron exporter, ferroportin, suggesting an impaired ability to handle iron. In vitro, exposure of primary peritoneal macrophages to saturated fatty acids also alters iron metabolism gene expression. Finally, the impaired MFehi iron handling coincides with adipocyte iron overload in obese mice. In conclusion, in obesity, iron distribution is altered both at the cellular and tissue levels, with AT playing a predominant role in this change. An increased availability of fatty acids during obesity may contribute to the observed changes in MFehi ATM phenotype and their reduced capacity to handle iron.

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, χ2 = 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.

Plasma Clearance of Hemoglobin and Haptoglobin in Mice and Effect of CD163 Gene Targeting Disruption

AIM In humans, plasma haptoglobin (Hp) and the macrophage receptor CD163 promote a fast scavenging of hemoglobin (Hb). In the present study, we have compared the mouse and human CD163-mediated binding and uptake of Hb and HpHb complex in vitro and characterized the CD163-mediated plasma clearance of Hb in CD163 gene knockout mice and controls. RESULTS Contrary to human Hp, mouse Hp did not promote high-affinity binding to CD163. This difference between mouse and man was evident both by analysis of the binding of purified proteins and by ligand uptake studies in CD163-transfected cells. Plasma clearance studies in mice showed a fast clearance (half-life few minutes) of fluorescently labeled mouse Hb with the highest uptake in the kidney and liver. HPLC analysis of serum showed that the clearance curve exhibited a two-phase decay with a faster clearance of Hb than plasma-formed HpHb. In CD163-deficient mice, the overall clearance of Hb was slightly slower and followed a one-phase decay. INNOVATION AND CONCLUSION In conclusion, mouse Hp does not promote high-affinity binding of mouse Hb to CD163, and noncomplexed mouse Hb has a higher CD163 affinity than human Hb has. Moreover, CD163-mediated uptake in mice seems to only account for a part of the Hb clearance. The new data further underscore the fact that the Hp system in man seems to have a broader and more sophisticated role. This has major implications in the translation of data on Hb metabolism from mouse to man.

Structural Basis for Inflammation-driven Shedding of CD163 Ectodomain and Tumor Necrosis Factor-α in Macrophages

Background: ADAM17 mediates shedding of CD163 and tumor necrosis factor-α (TNF-α) during inflammation. Results: Similar substrate sequence motifs in proTNF-α and CD163 are essential for ADAM17-mediated cleavage. Conclusion: The structural basis for shedding of CD163 and TNF-α is disclosed. Significance: The data provide new molecular information on the inflammatory response and explain evolution of a regulatory mechanism for CD163 expression. 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 (1044Arg-Ser-Ser-Arg) and proTNF-α (78Arg-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.

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.

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−1 × h−1) 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-&agr; 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.