A low balance between microparticles expressing tissue factor pathway inhibitor and tissue factor is associated with thrombosis in Behçet's Syndrome.

Thrombosis is common in Behçet's Syndrome (BS), and there is a need for better biomarkers for risk assessment. As microparticles expressing Tissue Factor (TF) can contribute to thrombosis in preclinical models, we investigated whether plasma microparticles expressing Tissue Factor (TF) are increased in BS. We compared blood plasma from 72 healthy controls with that from 88 BS patients (21 with a history of thrombosis (Th+) and 67 without (Th-). Using flow cytometry, we found that the total plasma MP numbers were increased in BS compared to HC, as were MPs expressing TF and Tissue Factor Pathway Inhibitor (TFPI) (all p < 0.0001). Amongst BS patients, the Th+ group had increased total and TF positive MP numbers (both p ≤ 0.0002) compared to the Th- group, but had a lower proportion of TFPI positive MPs (p < 0.05). Consequently, the ratio of TFPI positive to TF positive MP counts (TFPI/TF) was significantly lower in Th+ versus Th- BS patients (p = 0.0002), and no patient with a TFPI/TF MP ratio >0.7 had a history of clinical thrombosis. We conclude that TF-expressing MP are increased in BS and that an imbalance between microparticulate TF and TFPI may predispose to thrombosis.

Methotrexate-mediated activation of an AMPK-CREB-dependent pathway: a novel mechanism for vascular protection in chronic systemic inflammation.

AIMS: Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used disease-modifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4-carboxamide ribonucleotide which activates AMP-activated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPK-regulated protective genes. METHODS/RESULTS: In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1 mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKα(Thr172), and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivation-induced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)(Ser133) phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKα(Thr172) phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression. CONCLUSIONS: These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against cardiovascular events seen in patients with RA treated with MTX.

The exaggerated inflammatory response in Behçet's syndrome: identification of dysfunctional post-transcriptional regulation of the IFN-γ/CXCL10 IP-10 pathway.

The mechanisms underlying the exaggerated inflammatory response in Behçet's syndrome (BS) remain poorly understood. We investigated the response of CD14(+) blood monocytes to interferon (IFN)-γ, focusing on the chemokine CXCL10. Chemokine synthesis and release were analysed at a protein and mRNA level following stimulation with IFN-γ. Findings in BS patients were compared with 25 healthy controls (HC), 15 rheumatoid arthritis (RA) and 15 systemic lupus erythematosus (SLE) disease control patients. BS monocytes produced significantly more CXCL10 protein than HC monocytes from 2 h following IFN-γ stimulation, despite equivalent quantities of mRNA, suggesting more efficient translation. This was significantly more pronounced in BS with high disease activity and in those with ocular and neurological clinical manifestations. The imbalance between CXCL10 protein and mRNA expression was not observed in either RA or SLE patients, and was not seen with other chemokines studied (CXCL9, CXCL11 and CCL2). Furthermore, BS monocytes treated with an alternative stimulant (LPS) did not show abnormal tumour necrosis factor (TNF)-α release. Sucrose density gradients to segregate monocyte CXCL10 mRNA into free RNA or polysome-associated RNA showed equal proportions in BS and HC samples, suggesting that the difference between BS and HC may be due to reduced negative control of CXCL10 translation in BS at a post-initiation level. We conclude that BS monocytes have dysfunctional post-transcriptional regulation of CXCL10 mRNA, resulting in over-expression of CXCL10 protein upon IFN-γ stimulation. As CXCL10 is a chemokine that recruits mononuclear cells, this abnormality may contribute to the exaggerated inflammatory responses that characterizes BS.

Pegylated interferon-α-2b reduces corticosteroid requirement in patients with Behçet's disease with upregulation of circulating regulatory T cells and reduction of Th17.

OBJECTIVE: To determine whether the addition of 26 weeks of subcutaneous peginterferon-α-2b could reduce the requirement for systemic corticosteroids and conventional immunosuppressive medication in patients with Behçet's disease (BD). METHODS: We conducted a multicentre randomised trial in patients with BD requiring systemic therapy. Patients were randomised to 26 weeks of peginterferon-α-2b in addition to their standard care or to standard care only and followed 6-monthly for 3 years with BD activity scores and quality of life questionnaires. Patients at one centre had blood taken to measure regulatory T cells (Tregs) and Th17 cells. RESULTS: 72 patients were included. At months 10-12, while among the entire patient population there was no difference in the corticosteroid dose or immunosuppression use between the treatment groups (adjusted OR 1.04, 95% CI 0.34 to 3.19), post hoc analysis revealed that in patients who were on corticosteroids at baseline the corticosteroid requirement was significantly lower in the peginterferon-α-2b (6.5 (5-15) mg/day) compared with the non-interferon group (10 (8.25-16.5) mg/day, p=0.039). Furthermore, there was a trend towards an improved quality of life that became significant by 36 months (p=0.008). This was associated with a significant rise in Tregs and a decrease in Th17 cells which was still present at 1 year and 6 months after the interferon was stopped. The safety profile was similar with adverse events in 10% in both groups. CONCLUSIONS: The addition of peginterferon-α-2b to the drug regime of BD patients did not significantly reduce their corticosteroid dose required at 1 year. However, in those on corticosteroids at baseline post hoc analysis demonstrated that the addition of peginterferon-α-2b did result in a significant reduction in corticosteroid dose with a significantly improved quality of life and trend to reduce other required immunosuppressive agents. This effect was seen at 1 year and associated with a rise in Tregs suggesting a possible mode for interferon action. TRIAL REGISTRATION NUMBER: ISRCTN 36354474; EudraCT 2004-004301-18.

Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma.

Regulation of cell survival is a key part of the pathogenesis of multiple myeloma (MM). Jun N-terminal kinase (JNK) signaling has been implicated in MM pathogenesis, but its function is unclear. To elucidate the role of JNK in MM, we evaluated the specific functions of the two major JNK proteins, JNK1 and JNK2. We show here that JNK2 is constitutively activated in a panel of MM cell lines and primary tumors. Using loss-of-function studies, we demonstrate that JNK2 is required for the survival of myeloma cells and constitutively suppresses JNK1-mediated apoptosis by affecting expression of poly(ADP-ribose) polymerase (PARP)14, a key regulator of B-cell survival. Strikingly, we found that PARP14 is highly expressed in myeloma plasma cells and associated with disease progression and poor survival. Overexpression of PARP14 completely rescued myeloma cells from apoptosis induced by JNK2 knockdown, indicating that PARP14 is critically involved in JNK2-dependent survival. Mechanistically, PARP14 was found to promote the survival of myeloma cells by binding and inhibiting JNK1. Moreover, inhibition of PARP14 enhances the sensitization of MM cells to anti-myeloma agents. Our findings reveal a novel regulatory pathway in myeloma cells through which JNK2 signals cell survival via PARP14, and identify PARP14 as a potential therapeutic target in myeloma.

Wireless capsule endoscopy in the investigation of intestinal Behçet's syndrome.

OBJECTIVE: Intestinal Behçet's Syndrome (BS) is a difficult diagnosis to establish. We describe the use of wireless capsule endoscopy (WCE) in the investigation of 11 patients with suspected intestinal BS. METHODS: Out of 11 patients, 10 with suspected intestinal BS were found to have small intestinal ulcers on capsule endoscopy. Each case was retrospectively assessed for symptoms, signs, anaemia, other investigations, treatment and complications. RESULTS: All 11 patients had established diagnoses of BS as defined by the International Study Group criteria. Central abdominal pain and change in bowel habit were the predominant symptoms, both occurring in seven patients. Upper gastrointestinal (GI) endoscopy and colonoscopy identified duodenitis, ileitis and colitis in three patients. Barium studies and CT were normal in all cases. WCE revealed small intestinal ulcers throughout the ileum in five patients and ulcers located either in the proximal and/or distal ileum in five other patients. One patient had significant symptoms, signs and ulcers leading to a change in treatment to infliximab, and this resulted in resolution of symptoms and ulcers. Ten age- and sex-matched controls investigated for unexplained GI symptoms had no intestinal lesions on capsule endoscopy. CONCLUSION: WCE is useful in the investigation of GI symptoms in BS. It is particularly helpful in those patients in whom conventional investigations have been normal or fail to account for symptoms and signs. This technique may guide treatment and provide a better understanding of intestinal pathology in BS.

The inflammatory effect of cardiopulmonary bypass on leukocyte extravasation in vivo.

OBJECTIVE: Extravascular trafficking of leukocytes into organs is thought to play a major role in the pathophysiologic mechanisms of the inflammatory response to cardiopulmonary bypass, yet leukocyte extravasation is difficult to study clinically. Here we have tested the hypothesis that leukocyte emigration into skin blisters can provide a way to monitor the inflammatory effect of cardiopulmonary bypass that allows testing of anti-inflammatory interventions (exemplified by aprotinin). METHODS: Patients undergoing primary elective coronary artery bypass grafting (n = 14) were randomized into 2 equal groups to receive saline infusion during cardiopulmonary bypass (control group) or high-dose aprotinin. Experimental skin blisters (in duplicate) were induced on the forearm by means of topical application of the vesicant cantharidin, and blister fluid was sampled at 5 hours postoperatively. Inflammatory leukocyte subsets in blister fluid were analyzed by means of flow cytometry by using expression of CD11b and CD62L as a phenotypic marker of activation. RESULTS: In the control group of patients, cardiopulmonary bypass surgery triggered a 381% increase in leukocyte extravasation into the skin compared with reference blisters carried out before surgical intervention, with neutrophil (P = .014), monocyte (P = .014), and eosinophil (P = .009) levels all statistically significantly increased. In the aprotinin group there was no statistically significant increase during cardiopulmonary bypass surgery in any inflammatory leukocyte subset. The activation phenotype of extravascular leukocytes was not significantly altered between surgical groups. CONCLUSIONS: This study introduces the cantharidin blister technique as a powerful new research tool for analyzing the inflammatory effect of cardiopulmonary bypass in vivo. It has provided detailed molecular insight into the extravascular leukocyte population during cardiopulmonary bypass. Although aprotinin blocked cardiopulmonary bypass-dependent extravasation of leukocytes, there was no change in their CD11b/CD62L activation status. The cantharidin skin test thus represents a novel research tool for evaluating future anti-inflammatory interventions in cardiothoracic surgery.

Statin-mediated cytoprotection of human vascular endothelial cells: a role for Kruppel-like factor 2-dependent induction of heme oxygenase-1.

BACKGROUND: Heme oxygenase-1 (HO-1), by exerting anti-inflammatory, antiproliferative, antiapoptotic and antioxidant effects in the vasculature, protects against atherosclerosis and post-transplant vasculopathy. We noted the overlap between the effects of HO-1 and those attributed to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). This led to an investigation of the role of HO-1 in statin-mediated cytoprotection in primary human endothelial cells (ECs), and the ability of Kruppel-like factor 2 (KLF2) to regulate HO-1 function. METHODS/RESULTS: Treatment of human umbilical vein and aortic ECs with atorvastatin significantly upregulated HO-1 promoter activity, mRNA expression and protein expression, increasing HO-1 enzymatic activity as shown by raised intracellular bilirubin IXalpha. This effect was indirect, dependent upon inhibition of HMG-CoA reductase and geranylgeranylation, and independent of nitric oxide or changes in mRNA stability. Atorvastatin protected ECs against the generation of reactive oxygen species and H(2)O(2)-induced injury. HO-1 inhibition, with small interfering RNA (siRNA) or zinc protoporphyrin IX, abrogated atorvastatin-mediated cytoprotection. Atorvastatin upregulated KLF2 expression, whereas KLF2 siRNA attenuated statin-induced HO-1 and its associated antioxidant cytoprotective effects. Iron chelation, adenoviral-mediated overexpression of ferritin or supplementation of culture media with biliverdin reversed the inhibitory effects of HO-1 and KLF2 siRNA, suggesting that bile pigments and ferritin mediate the antioxidant actions of statin-induced HO-1. CONCLUSIONS: We have identified a novel link between KLF2 and HO-1 in human vascular ECs, demonstrating that atorvastatin-mediated HO-1 upregulation, and its associated antioxidant effect, is KLF2-dependent. The relationship between KLF2 and HO-1 is likely to represent an important component of the vasculoprotective profile of statins.

Both Fcgamma and complement receptors mediate transfer of immune complexes from erythrocytes to human macrophages under physiological flow conditions in vitro.

Abnormal clearance by the mononuclear phagocytic system of immune complexes (IC) is important in the pathogenesis of systemic lupus erythematosus (SLE). We have developed an in vitro model to investigate the cellular mechanisms involved in the transfer of soluble IC from erythrocytes to human macrophages under physiological flow conditions. In this assay, erythrocytes bearing fluorescently labelled IC are perfused over monolayers of human monocytes or monocyte-derived macrophages in a parallel-plate flow chamber, and transfer quantified using confocal microscopy and flow cytometry. Using aggregated human IgG as a model IC, we have been able to demonstrate transfer of IC from erythrocytes to macrophages. Blocking studies with specific neutralizing antibodies have shown that both complement and Fcgamma receptors are required for IC transfer. Blockade of CR4 (alpha(x)beta(2) integrin), FcgammaRIIa or FcgammaRIII reduced transfer, while anti-CR3 (alpha(m)beta(2) integrin) had no effect. Blockade of CR3, FcgammaRIIa or FcgammaRIII also reduced the number of adhesive interactions between fluorescently labelled IC-bearing erythrocytes and macrophage monolayers. Taken together with the transfer data, this suggests differing roles for these receptors in the human IC transfer reaction that includes an adhesive function which facilitates IC processing by mononuclear phagocytes. Finally, a functional effect of the FcgammaRIIa R131/H131 polymorphism, important in susceptibility to SLE, has also been demonstrated using this model. Uptake of IgG(2) but not IgG(1)-containing soluble IC was reduced by macrophages from individuals homozygous for the R131 allelic variant of the receptor.

A rapid method for labelling CD4+ T cells with ultrasmall paramagnetic iron oxide nanoparticles for magnetic resonance imaging that preserves proliferative, regulatory and migratory behaviour in vitro.

A number of techniques have been developed to track the migration of T cells in vivo, but they all suffer significant shortcomings, including the examination of selected organs rather than the organism as a whole--thus precluding longitudinal studies--or limitations imposed by poor spatial resolution and the application of ionizing radiation. By conjugating the HIV tat peptide to ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles in a reaction yielding a mean valence of 45, a magnetic resonance (MR) contrast agent was synthesised that allowed T cells to be efficiently labelled within just 5 min. The USPIO nanoparticles were incorporated into both the cytoplasm and nucleus of labelled cells, which retained normal in vitro proliferative responses to a polyclonal stimulus; suppressive responses mediated by labelled CD4(+) CD25(+) regulatory T cells; chemotactic responses to the chemokine CXCL-12; and transmigration of an activated endothelial monolayer. We believe that this rapid, efficient and essentially non-toxic approach to labelling both murine and human T cells for MRI holds considerable promise, paving the way for the wider immunological application of this exciting technology.

Atorvastatin affects leukocyte gene expression in dyslipidemia patients: in vivo regulation of hemostasis, inflammation and apoptosis.

BACKGROUND: The beneficial effect of HMG-CoA reductase inhibitors (statins) on coronary artery disease has been linked to mechanisms beyond their lipid-lowering effect. However the existence of direct, lipid-independent effects of statin in humans is still controversial. OBJECTIVE: To investigate the early effect of atorvastatin on peripheral blood mononuclear cells (PBMC) in dyslipidemic patients using gene arrays. PATIENTS AND METHODS: Eleven male patients with primary hyperlipidemia received 20 mg atorvastatin daily for 4 weeks. Blood was collected at baseline, 12 h, 36 h, 1 and 4 weeks after the start of treatment. RESULTS: Human microarrays containing 12 650 genes were used to study the effect of atorvastatin on PBMC gene expression at all time-points. Two hundred and forty genes were significantly regulated by atorvastatin treatment, several of which are involved in hemostasis, inflammation and other processes critical to atherosclerosis. Different patterns of response over time suggested both lipid-dependent and independent effects of atorvastatin on gene expression. CONCLUSIONS: This study demonstrates for the first time that atorvastatin regulates gene expression in PBMC in man before changes in the lipid profile are detectable in serum. Using blood leukocytes as a pharmacogenomic surrogate, we have identified new in vivo targets of atorvastatin treatment.

Accelerated atherosclerosis in inflammatory rheumatic diseases.

Over recent years it has become clear that patients with inflammatory rheumatic diseases are at increased risk of developing atherosclerosis. The exact causes for this are probably related in part to a general adverse effect of inflammation on atherogenesis, and in part to immune mechanisms specific to individual rheumatic diseases. This review discusses proposed mechanisms of accelerated atherosclerosis, including abnormal lipid and lipoprotein profiles, oxidative stress, enhanced apoptosis, thrombophilia, immune complexes, and increased mononuclear cell infiltration of atherosclerotic lesions, and local generation of cytokines.

Clinical inhibition of the seven-transmembrane thrombin receptor (PAR1) by intravenous aprotinin during cardiothoracic surgery.

BACKGROUND: Protease-activated receptor-1 (PAR1) is the principal thrombin receptor in the vasculature, and antagonists against this receptor are in preclinical trials. Aprotinin, already approved for clinical use to reduce transfusion requirements in cardiopulmonary bypass (CPB) surgery, has been shown to inhibit PAR1 activation in vitro. Here, we exploit CPB as a model for thrombin generation in humans to examine whether aprotinin can inhibit platelet PAR1 activation clinically. METHODS AND RESULTS: PAR1 expression and function on platelets was examined in coronary artery bypass grafting (CABG) patients randomized into 2 groups: (1) those receiving saline infusion during CPB (n=17) and (2) those receiving aprotinin (2x10(6) kallikrein inhibitor units [KIU] in pump prime, 2x10(6) KIU loading dose, followed by 0.5x10(6) KIU/h [n=13]). Platelets in the saline group showed loss of PAR1-specific function at 2 hours after CPB, but this was preserved in the aprotinin group (P<0.001). These effects were most likely targeted at PAR1 receptor cleavage, because (1) the level of thrombin generated during CPB did not vary significantly between groups, (2) expression of SPAN12, which detects only uncleaved PAR1 receptors, was preserved in the aprotinin but not the placebo group (P<0.05), and (3) supporting evidence in vitro showed reduced thrombin-induced PAR1 cleavage (P<0.001) and platelet aggregation (P<0.001) in the presence of aprotinin. CONCLUSIONS: This study demonstrates that platelet PAR1 activation by thrombin can be inhibited by aprotinin. Our results extend the clinical mechanism of action of aprotinin and provide the first proof of principle that PAR1 can be inhibited clinically. This has implications beyond cardiac surgery for the development of therapeutic PAR1 blockade.

Non-invasive imaging in the diagnosis and management of Takayasu's arteritis.

OBJECTIVES: Takayasu's arteritis (TA) is a rare disease, in which early diagnosis and assessment of treatment efficacy remain a problem. Signs and symptoms may be non-specific and conventional blood tests unreliable, with vascular inflammation often persisting in the face of a normal acute phase response. The current "gold standard" investigation, x ray angiography, is invasive and only identifies late, structural changes in vessels. Recently, non-invasive imaging methods have shown promise in the assessment of patients with TA. METHODS: The invasive and non-invasive imaging performed on all patients in the rheumatology department at the Hammersmith Hospital between May 1996 and May 2002 who fulfilled the ACR criteria for TA were reviewed. All patients were clinically active at diagnosis and were treated with high dose oral prednisolone and additional oral or intravenous immunosuppression. RESULTS: Non-invasive imaging methods ([(18)F]fluorodeoxyglucose positron emission tomography ([(18)F]FDG-PET) and magnetic resonance imaging (MRI)) provided important additional information about disease activity ([(18)F]FDG-PET) and progression of vessel wall thickening (MRI) when compared with x ray angiography. CONCLUSIONS: Non-invasive imaging methods provide useful additional information towards the diagnosis and management of TA. Such techniques may allow earlier diagnosis and more accurate assessment of response to treatment than conventional clinical assessment and/or angiography. Non-invasive imaging is likely to be useful in the management of other large vessel vasculitides.

Distinct yet complementary mechanisms of heparin and glycoprotein IIb/IIIa inhibitors on platelet activation and aggregation: implications for restenosis during percutaneous coronary intervention.

OBJECTIVE: To study the effect of unfractionated heparin (UFH) versus low molecular weight heparin (LMWH) in combination with glycoprotein (Gp) IIb/IIIa blockers on platelet activation and aggregation. METHODS: Washed platelets were stimulated with thrombin in the presence or absence of UFH (monoparin), LMWH (enoxaparin), and a Gp IIb/IIIa blocker (abciximab, eptifibatide, or tirofiban). RESULTS: Although Gp IIb/IIIa antagonists blocked the final common pathway of thrombin induced platelet aggregation, UFH and LMWH were better at blocking upstream platelet activation. UFH was significantly more effective than LMWH at inhibiting P selectin expression (p = 0.001) and platelet derived growth factor release from thrombin activated platelets (p = 0.012). CONCLUSIONS: UFH and LMWH exert complementary effects to Gp IIb/IIIa blockers by inhibiting afferent pathways of platelet activation. Coadministration of heparin with Gp IIb/IIIa blockers provides improved protection against persistent platelet activation, thereby improving outcome after percutaneous coronary intervention. Judging from these data, UFH may be more effective in this regard than LMWH, at least in vitro. The use of LMWH in preference to UFH during percutaneous coronary intervention, although initially attractive, may inadequately protect against platelet activation despite the presence of Gp IIb/IIIa blockers.

Phenotypic and functional differences between human saphenous vein (HSVEC) and umbilical vein (HUVEC) endothelial cells.

The vascular endothelial cell (EC) plays an essential role in the pathogenesis of inflammation, transplant rejection and tumour metastasis. Most research on vascular ECs uses human umbilical vein endothelial cells (HUVECs). However, HUVECs are derived from immune-naive foetal tissue, and show significant functional differences from adult vascular endothelium. In this paper, we characterise an alternative model based on human saphenous vein ECs (HSVECs), describe their culture conditions and provide a detailed functional comparison with HUVECs. Compared with HUVECs, HSVECs show an increased sensitivity to ox-LDL and a reduced response to cytokines, as indicated by adhesion molecule expression as well as leukocyte adhesion and transmigration. With respect to their ability to present antigen, HSVECs have a higher level of HLA-DR, CD40 and ICOS-L following cytokine stimulation. In addition, HSVECs upregulate the costimulatory ligand CD80 (B7.1) following CD40 ligation, and support allogeneic T cell proliferation, while HUVECs fail to express CD80. Due to differential expression of adhesion molecules, poorly differentiated tumour cell lines also showed more adhesion to HSVECs than to HUVECs. These results indicate that HSVECs have advantages over HUVECs for studying adult vascular endothelial pathology in vitro.

Hemoglobin scavenger receptor CD163 mediates interleukin-10 release and heme oxygenase-1 synthesis: antiinflammatory monocyte-macrophage responses in vitro, in resolving skin blisters in vivo, and after cardiopulmonary bypass surgery.

The recently described hemoglobin scavenger receptor CD163 mediates the endocytosis of hemoglobin:haptoglobin (Hb:Hp) complexes and thereby counters Hb-induced oxidative tissue damage after hemolysis. Although CD163 has been indirectly associated with antiinflammatory and atheroprotective activity, no ligand-receptor-effector pathway has yet been described for this receptor. To understand the significance of CD163 and more clearly define downstream pathways linked to inflammatory resolution, we studied the expression and function of CD163 in human monocytes/macrophages using both in vitro and in vivo models. Differentiation of human blood monocytes into macrophages either by in vitro culture or in resolving cantharidin-induced skin blisters led to an equivalent increase (>15x) in CD163 expression. Elevated CD163 levels were also noted on circulating monocytes in cardiac surgical patients during the resolution phase of the systemic inflammatory response to cardiopulmonary bypass surgery. In each case, binding of Hb:Hp to CD163-bearing cells elicited potent interleukin-10 secretion, and this was inhibited by the anti-CD163 antibody RM3/1. Release of interleukin-10, in turn, induced heme oxygenase-1 stress protein synthesis via an autocrine mechanism. Such induction of heme oxygenase-1 was observed in vivo 24 to 48 hours after the onset of cardiopulmonary bypass surgery. These results identify novel antiinflammatory and cytoprotective effector pathways in human monocytes/macrophages related to Hb scavenging and metabolism, which may have relevance in atheroprotection, wound healing, and patient recovery postoperatively.