Human endogenous peptide p33 inhibits detrimental effects of LL-37 on osteoblast viability.
OBJECTIVE
High levels of the antimicrobial peptide, LL-37, are detected in gingival crevicular fluid from patients with chronic periodontitis. LL-37 not only shows antimicrobial activity but also affects host-cell viability. The objective of the present study was to identify endogenous mechanisms that antagonize the detrimental effects of LL-37 on osteoblast viability, focusing on the human peptide p33 expressed on the surface of various cell types.


METHODS
Human osteoblast-like MG63 cells and human hFOB1.19 osteoblasts were treated with or without LL-37 in the presence or absence of p33. Recombinant human p33 was expressed in an Escherichia coli expression system. Lactate dehydrogenase (LDH) was assessed using an enzymatic spectrophotometric assay. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation. Cell number was assessed by counting cells in a Bürker chamber. Intracellular Ca(2+) was monitored by recording Fluo 4-AM fluorescence using a laser scanning confocal microscope. Cellular expression of p33 was determined by western blotting.


RESULTS
LL-37 caused a concentration-dependent release of LDH from human osteoblasts, showing a half-maximal response value (EC50 ) of 4 μm and a rapid and sustained rise in the intracellular Ca(2+) concentration of osteoblasts, suggesting that LL-37 forms pores in the cell membrane. p33 (10 μm) inhibited the LL-37-induced LDH release and LL-37-evoked rise in intracellular Ca(2+) concentration, suggesting that p33 prevents LL-37-induced permeabilization of the cell membrane. Moreover, p33 blocked LL-37-induced attenuation of osteoblast numbers. Also, mucin antagonized, at concentrations representative for nonstimulated whole saliva, LL-37-evoked LDH release, whilst cationic endogenous polyamines had no impact on LL-37-induced LDH release from osteoblasts.


CONCLUSIONS
The endogenous peptide p33 prevents LL-37-induced reduction of human osteoblast viability. Importantly, this mechanism may protect the osteoblasts from LL-37-induced cell damage in patients suffering from chronic periodontitis associated with high levels of LL-37 locally.
Department of Experimental Medical Science, Lund University, Lund, Sweden.
D
D
Svensson
J
J
Westman
C
C
Wickström
D
D
Jönsson
H
H
Herwald
B-O
BO
Nilsson
D023181:Antimicrobial Cationic Peptides / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D002118:Calcium / Q000032:analysis; D002352:Carrier Proteins / Q000494:pharmacology; D002452:Cell Count; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015922:Complement C1q / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000037:antagonists & inhibitors; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D024101:Mitochondrial Proteins / Q000494:pharmacology; D009077:Mucins / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects*; D011951:Receptors, Complement; D054804:Cathelicidins
D023181:Antimicrobial Cationic Peptides; C087299:C1QBP protein, human; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D009077:Mucins; D011951:Receptors, Complement; C032402:complement 1q receptor; D015922:Complement C1q; D004247:DNA; D007770:L-Lactate Dehydrogenase; D002118:Calcium; D054804:Cathelicidins
antimicrobial peptide; calcium; cell number; lactate dehydrogenase release