Kathleen Wardwell

Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor CD163

CD163, a monocyte and macrophage‐specific surface glycoprotein, which is increased by interleukin‐10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia‐associated rise in plasma CD163 in vivo. Studies using the inhibitor TAPI‐0 indicate that a metalloproteinase is responsible for LPS‐mediated shedding of CD163. Finally, we demonstrate a marked increase in surface CD163 expression on circulating monocytes 24 h following experimental endotoxemia. These findings show that CD163 is rapidly mobilized in response to bacterial endotoxin. As hemoglobin can bind LPS and enhance its toxicity, it will be important to determine how cell surface and soluble CD163 influence inflammatory processes during sepsis.

Pivotal Advance: Activation of cell surface Toll‐like receptors causes shedding of the hemoglobin scavenger receptor CD163

The hemoglobin scavenger receptor (HbSR) CD163 is a monocyte/macrophage‐specific glycoprotein that binds and facilitates uptake of haptoglobin‐hemoglobin (Hp‐Hb) complexes, which are rapidly formed in the circulation upon hemolysis of red blood cells. Hemolysis can be caused by a diverse range of infectious agents and provides pathogens a source of iron to enhance their survival and replication. Previous work demonstrated that lipopolysaccharide (LPS) activates monocytes to cleave cell‐bound HbSR into a soluble mediator that retains the capacity to bind Hp‐Hb complexes. We report that blocking LPS activation of Toll‐like receptor 4 prevents LPS‐mediated shedding of CD163. Furthermore, activation of two other cell surface Toll‐like receptors (TLR), TLR2 and TLR5, induces shedding of the HbSR from human monocytes. In contrast, treatment of monocytes with intracellular TLR3, TLR7, and TLR9 agonists failed to cause HbSR shedding, suggesting that this shedding event is selective to cell surface TLR activation. These data demonstrate that the soluble HbSR is released from monocytic cells in response to TLR signaling as an acute innate immune response to extracellular pathogen infections.

Up-regulation of human monocyte CD163 upon activation of cell-surface Toll-like receptors

The hemoglobin (Hb) scavenger receptor, CD163, is a cell‐surface glycoprotein that is expressed exclusively on monocytes and macrophages. It binds and internalizes haptoglobin‐Hb complexes and has been implicated in the resolution of inflammation. Furthermore, the regulation of CD163 during an innate immune response implies an important role for this molecule in the host defense against infection. LPS, derived from the outer membrane of Gram‐negative bacteria, activates TLR4 to cause acute shedding of CD163 from human monocytes, followed by recovery and induction of surface CD163 to higher levels than observed on untreated monocytes. We now report that the TLR2 and TLR5 agonists—Pam3Cys and bacterial flagellin—have similar effects on CD163 surface expression. Up‐regulation of CD163 following treatment of human PBMC with TLR2, TLR4, and TLR5 agonists parallels increased production of IL‐6 and IL‐10, and neutralization of IL‐6 and/or IL‐10 blocks CD163 up‐regulation. Furthermore, simultaneous stimulation of TLR2 or TLR5 in combination with TLR4 activation results in enhanced up‐regulation of CD163. It is notable that exogenous recombinant IFN‐γ (rIFN‐γ) suppresses cell‐surface, TLR‐mediated IL‐10 production as well as CD163 up‐regulation. Sustained down‐regulation of CD163 mediated by rIFN‐γ can be partially rescued with exogenous rIL‐10 but not with exogenous rIL‐6. This divergent regulation of CD163 by cytokines demonstrates that human monocytes react differently to infectious signals depending on the cytokine milieu they encounter. Thus, surface CD163 expression on mononuclear phagocytes is a carefully regulated component of the innate immune response to infection.

Development of an ELISA to measure soluble CD163 in biological fluids

CD163 is a monocyte/macrophage restricted transmembrane glycoprotein and a member of the scavenger receptor cysteine-rich (SRCR) family of proteins. SRCR proteins are typically associated with the immune system. The regulation of CD163 by cytokines and glucocorticoids suggests that it plays a role in inflammatory processes. While CD163 is expressed as a membrane-bound protein, it has been shown to be actively shed from the surface of monocytes in a protease-dependent fashion when cells are stimulated with a phorbol ester. To better elucidate the function and biological importance of CD163, we have developed a solid-phase sandwich enzyme linked immunosorbant assay (ELISA) for the detection of soluble CD163 in biological fluids. This assay has good repeatability both within and between runs (coefficients of variation (CVs) of 3.2% and 7.1% or better, respectively). While detection of CD163 was inhibited by ethylenediamine tetraacetic acid (EDTA), CD163 immunoreactivity was not altered by the addition of heparin or hemoglobin. This report details the development of this novel assay for soluble CD163 and provides the first evidence of CD163 immunoreactivity in normal plasma and serum samples.

Cross‐linking of FcγR triggers shedding of the hemoglobin‐haptoglobin scavenger receptor CD163

CD163, the hemoglobin (Hb)‐haptoglobin scavenger receptor, is a monocyte/macrophage‐restricted member of the scavenger receptor, cysteine‐rich family of proteins. In addition to being expressed on the cell surface, a soluble form of CD163 has also been reported. Like tumor necrosis factor α (TNF‐α), surface CD163 is proteolytically cleaved from the plasma membrane in response to lipopolysaccharide (LPS) stimulation. As cross‐linking of the Fcγ receptor (FcγR) is similarly known to induce TNF‐α shedding, the effect of FcγR stimulation on CD163 shedding was investigated. We found that FcγR stimulation resulted in a rapid release of surface CD163 into the supernatant that was blocked by inhibitors of protein kinase C and tyrosine kinases. Although LPS and FcγR stimulation in short‐term cultures suppressed CD163 mRNA expression, long‐term cultures of monocytes treated with LPS—but not with a FcγR cross‐linking reagent—resulted in an interleukin‐10‐dependent recovery of surface CD163 expression. These studies suggest that the presence of immune complexes in infection or autoimmunity may radically alter the nature of CD163‐dependent monocyte/macrophage processes. This may be particularly important in disease states in which immune complexes and high levels of free Hb are present, such as in autoimmune hemolytic anemia, transfusion reactions, or infections by hemolytic bacteria.

TGF-β regulation of human macrophage scavenger receptor CD163 is Smad3-dependent

Tight regulation of the inflammatory response is essential for the maintenance of physiologic homeostasis. A potentially important mediator of this process is CD163, a macrophage‐specific member of the scavenger receptor cysteine‐rich family. CD163 surface expression is up‐regulated by glucocorticoids and the anti‐inflammatory cytokine interleukin‐10, and CD163 is shed acutely from the cell surface in response to lipopolysaccharide. We now demonstrate that transforming growth factor‐β (TGF‐β) markedly reduces expression of CD163. Treatment of primary human monocytes with TGF‐β inhibited basal as well as dexamethasone‐induced CD163 mRNA and protein expression. De novo protein synthesis was not required for this inhibition, suggesting that TGF‐β regulates CD163 expression transcriptionally. To delineate this transcriptional regulation, a 2.5‐kb fragment of the CD163 promoter was isolated. This promoter was inhibited by TGF‐β, and suppression was dependent on Smad3 expression. These results define a novel function for TGF‐β and implicate an important role for CD163 in the host response to inflammation.

Increase in plasma and surface CD163 levels in patients undergoing coronary artery bypass graft surgery

Although haptoglobin polymorphism has been shown to be a genetic risk factor in coronary artery disease, its mechanisms of action are incompletely defined. Recently, a macrophage scavenger receptor for the uptake of haptoglobin-hemoglobin (Hp-Hb) complexes was cloned and designated CD163. Macrophage expression of CD163 is increased by glucocorticoids, IL-10 and IL-6. To better understand the in vivo response of CD163 to an inflammatory stimulus and glucocorticoid treatment, we studied 18 patients who underwent elective coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass (CPB). We report a rapid increase in plasma levels of soluble CD163 by 1 h post-declamping the aorta during CABG surgery with CPB. Furthermore, we demonstrate significant increases in monocyte CD163 on post-operative day 1; 14-fold for patients pre-treated with methylprednisolone and 3-fold for those who did not receive exogenous glucocorticoids. These findings show CD163 to be rapidly mobilized in response to systemic inflammatory stimuli and to be affected significantly by glucocorticoids in vivo. The proposed role of CD163 as a Hp-Hb scavenger and anti-inflammatory molecule, in conjunction with the results of this study, make CD163 an intriguing target for potential manipulation of the acute response to inflammation.

In Vivo Exposure to High or Low Cortisol Has Biphasic Effects on Inflammatory Response Pathways of Human Monocytes

BACKGROUND: Recent studies demonstrate that glucocorticoids (GCs) have both supportive (stimulatory) and suppressive effects on immune responses, depending upon the GC concentration. Since some GC effects on inflammation are stimulatory, we hypothesized that acute in vivo GC depletion would decrease inflammatory responses of human monocytes. METHODS: Monocytes were isolated from healthy volunteer participants before and after in vivo treatment with; 1) IV saline, 2) IV high dose hydrocortisone (8 &mgr;g·kg−1·min−1) followed by oral hydrocortisone overnight, and 3) oral RU486 (200 mg at 0400 and 1600 h) to block the intracellular GC receptor and IV etomidate (1.5 mg·kg−1·h−1) for 12 h to prevent compensatory adrenal cortisol synthesis. Plasma adrenocorticotropic hormone, plasma, and salivary cortisol were measured serially. Monocytes were tested for; 1) cytokine responses, 2) expression of CD163, CD119, and CD54, and 3) mRNA levels of GC-responsive inflammatory mediators. All measurements were made with and without in vitro stimulation of monocytes by lipopolysaccharide. RESULTS: Cortisol and adrenocorticotropic hormone measurements demonstrated effective manipulation of in vivo cortisol. In vivo hypercortisolemia and in vivo GC depletion had reciprocal effects on monocyte mRNA levels of 4 important GC-responsive molecules: 1) GC receptor, CD163, interleukin-10, and suppressor of the cytokine synthesis-3. Monocyte cytokine responses and protein expression were not affected by GC depletion. CD163 expression was increased by hypercortisolemia. CONCLUSIONS: Short-term GC depletion affects mRNA levels of GC-responsive molecules but does not affect monocyte protein expression or cytokine responses.