Human microvascular endothelial cell activation by IL-1 and TNF-alpha stimulates the adhesion and transendothelial migration of circulating human CD14+ monocytes that develop with RANKL into functional osteoclasts.
Circulating pre-OCs may be recruited to locally inflamed sites through specific interactions with activated microvasculature. We found that HMVECs stimulated the adhesion and TEM of circulating pre-OCs, in an ICAM-1- and CD44-dependent manner, leading to greater RANKL-induced OC formation and bone pit resorption.


BACKGROUND
Inflammation is critical for healing processes but causes severe tissue destruction when chronic. Local osteoclast (OC) formation and bone resorption may increase at inflammatory sites through multiple mechanisms, including direct stimulation by inflamed microvasculature of circulating OC precursor (pre-OC) migration through a blood vessel barrier into bone or joint tissue. How this might occur is not yet well understood.


METHODS
Cytokine-activated human microvascular endothelial cell (HMVEC) monolayers, with or without IL-1 and TNF-alpha preactivation (24 h), were incubated in adhesion (1-3 h) or porous transwell transendothelial migration (TEM; 3 h) assays with human peripheral blood mononuclear cells (hPBMCs) or CD14+ monocyte or CD14- lymphocyte subsets. The number of cells that adhered or transmigrated, and their ability to thereafter develop with macrophage-colony stimulating factor (M-CSF) + RANKL into bone pit-resorbing OCs, were analyzed. Immunostaining and neutralizing antibodies to key cell adhesion molecules were used to determine their potential involvement in stimulated CD14+ monocyte TEM.


RESULTS
M-CSF + RANKL caused OC and bone pit formation only from hPBMCs and CD14+ cells but not CD14- cells. Adhesion of hPBMCs or CD14+ cells but not CD14- cells was stimulated by cytokine preactivation of HMVECs and led to the full capture of all circulating pre-OCs capable of developing into OCs. Cytokine-preactivated HMVECs also promoted the postadhesion TEM of hPBMCs and CD14+ populations, resulting in markedly greater OC formation and bone pit resorption by transmigrated cells. Immunodetectable vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and CD44 levels increased on cytokine-treated HMVEC surfaces, and neutralizing antibodies to ICAM-1 or CD44, but not VCAM-1 or platelet endothelial cell adhesion molecule (PECAM-1), inhibited stimulated CD14+ cell TEM through activated HMVECs.


CONCLUSIONS
This is the first demonstration that cytokine-activated HMVECs efficiently capture and promote the TEM of circulating pre-OCs capable of differentiating into bone-resorbing OCs. Thus, direct pre-OC recruitment by activated microvasculature at inflammatory sites may significantly contribute to normal OC bone remodeling during fracture healing or exacerbate pathological bone loss in various chronic inflammatory disorders.
Kindle
Libby
L
Department of Biology, Washington University, St Louis, Missouri, USA.
Rothe
Linda
L
Kriss
Michael
M
Osdoby
Philip
P
Collin-Osdoby
Patricia
P
D016723:Bone Remodeling; D001862:Bone Resorption; D002196:Capillaries / Q000166:cytology / Q000187:drug effects; D002352:Carrier Proteins / Q000494:pharmacology; D002448:Cell Adhesion; D002454:Cell Differentiation; D002465:Cell Movement; D004730:Endothelium, Vascular / Q000187:drug effects*; D005260:Female; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology*; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D010010:Osteoclasts / Q000166:cytology*; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D002352:Carrier Proteins; D018960:Hyaluronan Receptors; D007375:Interleukin-1; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D016173:Macrophage Colony-Stimulating Factor