Adenosine infusion attenuates soluble RAGE in endotoxin-induced inflammation in human volunteers.

AIM: To evaluate possible anti-inflammatory effects of pre-treatment with adenosine in a human experimental inflammatory model. METHODS: The study design was double-blind, crossover, placebo-controlled and randomized. In the Intensive Care Unit of a university hospital, 16 healthy male volunteers were treated for 5.5 h with infusions of adenosine 40 microg kg(-1) min(-1) or placebo. Thirty minutes after the start of adenosine or placebo, 2 ng kg(-1)E-Coli endotoxin was administered. Heart rate, body temperature, blood pressure, plasma cytokines (TNF-alpha, IL-6 and IL-10), soluble RAGE and resistin, exhaled nitric oxide and nitrite/nitrate in urine were determined. RESULTS: Endotoxin elicited the expected clinical signs of an inflammatory reaction (tachycardia, fever) and led to prominent release of the cytokines studied (P < 0.001). Resistin in plasma increased after endotoxin (P < 0.001). After placebo treatment, soluble RAGE (sRAGE) in plasma increased 5 h after the endotoxin challenge (P < 0.001) but not after adenosine. After placebo, orally exhaled NO increased with a peak at 4 h (P < 0.001), although there was no statistically significant difference between the two treatments. Nitrite/nitrate in urine (n = 11) did not differ between adenosine and placebo treatments. CONCLUSION: In conclusion, adenosine infusion starting before endotoxin challenge in humans attenuated sRAGE significantly but otherwise had no clear anti-inflammatory effect. Adenosine as a potential anti-inflammatory treatment in humans needs further study, including use of higher doses. The mechanism underlying the effect of adenosines on sRAGE remains unknown.

RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages.

Abstract High-mobility group box chromosomal protein 1 (HMGB1) is a protein with both intranuclear functions and extracellular cytokine-like effects. In this report, we study possible candidate receptors for HMGB1 on macrophages (Mphi) and define pathways activated by HMGB1 binding. Bone marrow Mphi were prepared from Dark Agouti (DA) rats and stimulated in vitro with HMGB1. The kinetics of tumour necrosis factor (TNF) production, NO production, activation of p38 mitogen-activated protein kinase (MAPK), p44/42 MAPK- and SAPK/JNK-signalling pathways, nuclear translocation of nuclear factor kappa B (NF-kappaB) and HMGB1-induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mphi from interleukin (IL)-1 receptor type I-/-, Toll-like receptor 2 (TLR2-/-) and RAGE-/- mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB1 induced TNF and NO production by Mphi, phosphorylation of all investigated MAP kinase pathways and NF-kappaB translocation, and expression of MHC class II was increased. Mphi from RAGE-/- mice produced significantly lower amounts of TNF, IL-1beta and IL-6, while IL-1RI-/- and TLR2-/- Mphi produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation. We conclude that HMGB1 has the potential to induce a proinflammatory phenotype in Mphi, with RAGE as the major activation-inducing receptor.

Activation of human umbilical vein endothelial cells leads to relocation and release of high-mobility group box chromosomal protein 1.

The nuclear protein high-mobility group box chromosomal protein 1 (HMGB1) was recently described to act as a pro-inflammatory cytokine and as a late mediator of severe sepsis and septic shock. The protein is released from monocytes in response to endotoxin and activates monocytes and endothelial cells through nuclear factor kappa B. We have previously demonstrated that the B-box of HMGB1 mediates a pro-inflammatory effect on endothelial cells including the upregulation of cell-adhesion molecules and release of interleukin (IL)-8 and granulocyte colony-stimulating factor. Here, we report that HMGB1 is released from human umbilical vein endothelial cells (HUVEC) in response to lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-alpha. A nuclear relocation of HMGB1 to the cytoplasm was seen at 4 h. Subsequently, high amounts of HMGB1 could be seen in the supernatants from stimulated cells after 16 h. It was also observed that the pro-inflammatory activity of HMGB1 is sensitive to dexamethasone. Interestingly, the HMGB1-induced TNF-alpha release from monocytes could be inhibited by either the A-box of the protein or the p38 inhibitor CNI-1493, but neither had any inhibitory effects on the HMGB1-dependent upregulation of cell-adhesion molecules on HUVEC. Altogether, these results suggest that HUVEC may be an important source of HMGB1 secretion in response to systemic infection and that endothelial cells and monocytes may use different signalling pathways.

High mobility group 1 B-box mediates activation of human endothelium.

OBJECTIVES: Severe sepsis and septic shock is a consequence of a generalized inflammatory systemic response because of an invasive infection that may result in acute organ dysfunction. Mortality is high despite access to modern intensive care units. The nuclear DNA binding protein high mobility group 1 (HMGB1) protein has recently been suggested to act as a late mediator of septic shock via its function as a macrophage-derived pro-inflammatory cytokine (J Exp Med 2000; 192: 565, Science1999; 285: 248). We investigated the pro-inflammatory activities of the A-box and the B-box of HMGB1 on human umbilical venular endothelial cells (HUVEC). DESIGN: The HUVEC obtained from healthy donors were used for experiments. Recombinant human full-length HMGB1, A-box and B-box were cloned by polymerase chain reaction (PCR) amplification from a human brain quick-clone cDNA. The activation of HUVEC was studied regarding (i) upregulation of adhesion molecules, (ii) the release of cytokines and chemokines, (iii) the adhesion of neutrophils to HUVEC, (iv) the activation of signalling transduction pathways and (v) the involvement of the receptor for advanced glycation end-products (RAGE). RESULTS: The full-length protein and the B-box of HMGB1 dose-dependently activate HUVEC to upregulate adhesion molecules such as ICAM-1, VCAM-1 and E-selectin and to release IL-8 and G-CSF. The activation of HUVEC could be inhibited to 50% by antibodies directed towards the RAGE. HMGB1-mediated HUVEC stimulation resulted in phosphorylation of the ELK-1 signal transduction protein and a nuclear translocation of p65 plus c-Rel, suggesting that HMGB1 signalling is regulated in endothelial cells through NF-kappaB. CONCLUSIONS: The HMGB1 acts as a potent pro-inflammatory cytokine on HUVEC and the activity is mainly mediated through the B-box of the protein. HMGB1 may be a key factor mediating part of the pro-inflammatory response occurring in septic shock and severe inflammation.

Rouleaux-forming serum proteins are involved in the rosetting of Plasmodium falciparum-infected erythrocytes.

Excessive sequestration of Plasmodium falciparum-infected (pRBC) and uninfected erythrocytes (RBC) in the microvasculature, cytoadherence, and rosetting, have been suggested to be correlated with the development of cerebral malaria. P. falciparum erythrocyte membrane protein-1 (PfEMP1) is the parasite-derived adhesin which mediates rosetting. Herein we show that serum proteins are crucial for the rosette formation of four strains of parasites (FCR3S1, TM284, TM180, and R29), whereas the rosettes of a fifth strain (DD2) are serum independent. Some parasites, e.g., FCR3S1, can be depleted of all rosettes by washes in heparin and Na citrate and none of the rosettes remain when the parasite is grown in foetal calf serum or ALBUMAX. Rosettes of other parasites are less sensitive; e.g., 20% of TM180 and R29 and 70% of TM284 rosettes still prevail after cultivation. A serum fraction generated by ion-exchange chromatography and poly-ethylene-glycol precipitation restored 50% of FCR3S1 and approx 40 to 100% of TM180 rosettes. In FCR3S1, antibodies to fibrinogen reverted the effect of the serum fraction and stained fibrinogen bound to the pRBC surface in transmission electron microscopy. Normal, nonimmune IgM and/or IgG was also found attached to the pRBC of the four serum-dependent strains as seen by surface immunofluorescens. Our results suggest that serum proteins, known to participate in rouleaux formation of normal erythrocytes, produce stable rosettes in conjunction with the recently identified parasite-derived rosetting ligand PfEMP1.

Enrichment of immunoglobulin binding Plasmodium falciparum-infected erythrocytes using anti-immunoglobulin-coated magnetic beads.

It has been shown that nonimmune, human immunoglobulins are bound to the surface of certain strains of Plasmodium falciparum-infected erythrocytes. We describe a novel way of enriching parasitized red blood cells (pRBC) for immunoglobulin binding/rosette formation using Dynabeads coated with antibodies raised against human immunoglobulins. Whole P. falciparum cultures were mixed with the precoated beads for approximately 120 min at room temperature, and the bound pRBC were isolated by magnetic force. The nonbound cell fraction contained ring-infected pRBC, immunoglobulin-negative, trophozoite-infected pRBC, and uninfected erythrocytes. A consistent elevation in the immunofluorescence and rosette formation rates of 100% and 86% respectively, was detected after the first enrichment and subcultivation. Protein A or G were also found to support binding of pRBC through surface-expressed immunoglobulin. The Dynabead technique is a novel way of enriching pRBC based on the immunoglobulin-binding capacity of the infected erythrocyte.

The time course of cytoadhesion, immunoglobulin binding, rosette formation, and serum-induced agglutination of Plasmodium falciparum-infected erythrocytes.

We describe morphologic characteristics of acridine orange-stained Plasmodium falciparum-infected erythrocytes and the sequential expression of several adhesion phenomena. In particular, we have studied when the adhesive and antigenic modifications appear on the infected erythrocyte surface that mediate binding to C32 melanoma cells (cytoadherence) or to erythrocytes (rosette formation) during a complete 48-hr life cycle of the parasite. The C32 melanoma cell binding started at about 12 hr and was seen during the whole life cycle with a peak around 28 hr (650 infected erythrocytes/100 C32 melanoma cells). Rosettes started to appear and immunoglobulin was found bound to the parasitized red blood cell (PRBC) somewhat later (16-20 hr). These adhesive events culminated at the mid-trophozoite/schizont stage (24-36 hr) with rosette formation and an immunoglobulin binding rate of about 50%, which decreased to about half of the peak values at the end of the life cycle. Serum-induced agglutination of the infected erythrocytes was also most extensive at 24-36 hr, but agglutination was seen with all late stage parasites, i.e., both trophozoites and schizonts at 24-48 hr of age. Taken together, adhesion to C32 melanoma cells starts prior to that of rosette formation, immunoglobulin binding, or serum-induced agglutination.

Multiple adhesive phenotypes linked to rosetting binding of erythrocytes in Plasmodium falciparum malaria.

The cerebral form of severe malaria is associated with excessive intravascular sequestration of Plasmodium falciparum-infected erythrocytes (PRBC). Retention and accumulation of PRBC may lead to occlusion of brain microvessels and direct the triggering of acute pathologic changes. Here we report that by selection, cloning, and subcloning, we have identified rare P. falciparum parasites expressing a pan-adhesive phenotype linked to erythrocyte rosetting, a previously identified correlate of cerebral malaria. Rosetting PRBC not only bound uninfected erythrocytes but also formed autoagglutinates, adhered to endothelial cells, and bound to CD36, immunoglobulins, and the blood group A antigen. The linkage of rosetting, autoagglutination, and cytoadherence involved the coexpression on a single PRBC of ligands with multiple specificities and the binding to two or more receptors on erythrocytes and to at least two other cell adhesion molecules, including a new endothelial cell receptor for P. falciparum-infected erythrocytes. Limited proteolysis that differentially cleaved the rosetting ligand PfEMP1 from the PRBC surface abrogated all the binding phenotypes of these parasites, implicating the variant antigen PfEMP1 as a carrier of multiple ligand specificities. The results encourage the further study of pan-adhesion as a potentially important parasite phenotype in the pathogenesis of severe P. falciparum malaria.

PECAM-1/CD31, an endothelial receptor for binding Plasmodium falciparum-infected erythrocytes.

Excessive binding of Plasmodium falciparum-infected red blood cells (pRBCs) to the vascular endothelium (cytoadherence) and to uninfected erythrocytes (rosetting) may lead to occlusion of the microvasculature and thereby contribute directly to the acute pathology of severe human malaria. A number of endothelial receptors have been identified as targets for the pRBCs, including CD36, intercellular adhesion molecule-1 (ICAM-1) and chondroitin-4-sulfate (CSA). In vitro, CD36 is the most frequent target of strains from patients with mild as well as severe P. falciparum malaria, but is expressed at low levels on the cerebral microvasculature and therefore seems unlikely to be involved in the evolution of cerebral disease. Strains of P. falciparum that form rosettes are associated both with the occurrence of cerebral malaria and severe anemia. Here we report that malaria-infected RBCs adhere to platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) on the vascular endothelium. pRBCs bind to endothelial cells, to PECAM-1/CD31 transfected cells, and directly to recombinant PECAM-1/CD31 absorbed onto plastic. Soluble PECAM-1/CD31 and monoclonal antibodies specific for the amino-terminal segment of PECAM-1/CD31 (domains 1-4) blocked the binding. Interferon-gamma (IFN-gamma)-essential for the development of cerebral malaria in the mouse-was found to augment adhesion of human pRBCs to PECAM-1/CD31 on endothelial cell monolayers. Our results suggest that PECAM-1/CD31 is a virulence-associated endothelial receptor of P. falciparum-infected RBCs.

Novel fibrillar structure confers adhesive property to malaria-infected erythrocytes.

Infections with the malaria parasite Plasmodium falciparum are characterized by sequestration of erythrocytes infected by mature forms of the parasite. Sequestration seems critical for the survival of the parasite, but may lead to excessive binding in the microvasculature and death of the human host. We report here that a novel electrondense fibrillar structure, containing immunoglobulins M or M and G, is found at the surface of infected erythrocytes that adhere to host cells. In cases of cerebral malaria, fibrillar strands are also seen in the microvasculature at autopsy. Our findings may explain the adhesive mechanism by which malaria-infected erythrocytes cause the vascular obstruction seen in complicated malaria infections.

Rosette formation in Plasmodium falciparum isolates and anti-rosette activity of sera from Gambians with cerebral or uncomplicated malaria.

The ability of Plasmodium falciparum-infected red blood cells (RBC) to form spontaneous erythrocyte rosettes was studied in 130 fresh isolates from Gambian children with cerebral or uncomplicated malaria from August to November 1990. All isolates (24 of 24) from patients with cerebral malaria formed rosettes, but only 61 of 106 isolates from children with uncomplicated malaria formed rosettes. The mean rate of rosette formation in isolates from children with cerebral malaria (28.3%) was significantly greater than that in isolates from children with uncomplicated malaria (8.5%). Giant rosettes were more frequently formed in isolates from patients with cerebral malaria than in those from patients with uncomplicated malaria. Sera of children with cerebral disease generally lacked anti-rosette activity, while many sera from children with uncomplicated malaria showed strong anti-rosette activity when tested against the patients' ow parasites. Some sera that were devoid of autologous rosette-disrupting activity were able to disrupt rosettes formed in other isolates, indicating the presence of different rosette formation mechanisms. Forty percent (6 of 15) of the sera from patients with cerebral malaria caused microagglutination of the patients' own uninfected and infected RBC, while only 10% (3 of 31) of sera from children with uncomplicated disease caused microagglutination. The ability of infected RBC to bind to melanoma cells grown in vitro did not differ between patients with cerebral or uncomplicated malaria. The results of this study, taken in conjunction with our previous findings, establish a strong association between rosette formation in P. falciparum-infected RBC and cerebral malaria.

Molecular mechanisms and biological importance of Plasmodium falciparum erythrocyte rosetting.

Rosetting, i.e. the spontaneous binding of uninfected to malaria infected erythrocytes and endothelial cytoadherence may hinder the blood flow and lead to severe Plasmodium falciparum malaria. Falciparum isolates obtained from unconscious patients all form rosettes and/or express a significantly higher mean rosetting rate than isolates from patients with uncomplicated malaria. Furthermore, sera of patients with cerebral malaria are devoid of anti-rosetting activity while sera from patients with mild disease carry high levels of anti-rosetting antibodies. The presence of anti-rosetting antibodies also seems important for the efficient interaction of rosetting infected rbc and leukocytes. Two parasite derived rosetting ligands of Mr 22K and Mr 28K named "rosettins", have been found on the surface of rosetting infected erythrocytes. CD36 has in at least some strains of parasites been found to function as a rosetting receptor on the uninfected erythrocyte. Heparin disrupts rosettes of P. falciparum in vitro and inhibits the sequestration of rosetting cells ex vivo. In conclusion, rosetting seems a crucial factor in the development of cerebral malaria and treatment of patients with anti-rosetting substances might become an effective adjunct in the treatment of severe malaria.