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


METHODS
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.
Treutiger
C J
CJ
Department of Medicine, Center for Infectious Medicine, Karolinska Institute at Huddinge University Hospital, Stockholm, Sweden. carl.johan.treutiger@medhs.ki.se
Mullins
G E
GE
Johansson
A-S M
AS
Rouhiainen
A
A
Rauvala
H M E
HM
Erlandsson-Harris
H
H
Andersson
U
U
Yang
H
H
Tracey
K J
KJ
Andersson
J
J
Palmblad
J E W
JE
D015153:Blotting, Western / Q000379:methods; D015815:Cell Adhesion Molecules / Q000032:analysis; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D019040:E-Selectin / Q000235:genetics; D004730:Endothelium, Vascular / Q000187:drug effects*; D024243:HMGB1 Protein / Q000494:pharmacology*; D006801:Humans; D007249:Inflammation / Q000503:physiopathology; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis; D016328:NF-kappa B / Q000235:genetics; D009504:Neutrophils / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000494:pharmacology*; D018805:Sepsis / Q000503:physiopathology; D015398:Signal Transduction / Q000502:physiology; D014178:Translocation, Genetic / Q000235:genetics; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis
D015815:Cell Adhesion Molecules; D016207:Cytokines; D019040:E-Selectin; D024243:HMGB1 Protein; D016328:NF-kappa B; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1