Background
Antimicrobial peptides can kill or inhibit pathogenic bacteria and thus play an important role in the innate immune defence. Cathelicidin antimicrobial peptide (CAMP, mouse homologue = CRAMP) is one of these peptides; it binds lipopolysaccharide (LPS) and thereby neutralises the pathogenic effects of the Toll-like receptor 4 (TLR) ligand LPS, e.g. in sepsis-induced myocardial dysfunction (cardiomyopathy). Here, we investigate LPS-dependent and -independent effects of CRAMP on NF-κB activation, cytokine expression, endothelial adhesion and transmigration of monocytes.
Methods and Results
As expected, CRAMP almost completely blocked NF-κB activation in THP-1 reporter cells by LPS (p<0.001), but not by the TLR2/6 agonist Pam2 or the TLR 2/1 agonist Pam3. In endothelial cells (MyEND) and monocytes (J774A.1), real-time PCR analysis showed that Cramp expression was strongly upregulated by LPS (>250-fold, p<0.001) and remained significantly upregulated by Pam2 and Pam3 (2-50-fold, p<0.01-p<0.001). The LPS-dependent upregulation of the proinflammatory cytokines Il-1β, Il-6, Tnf-α, Ccl-2 in both MyEND and J774A.1 cells was markedly attenuated by the addition of CRAMP (real-time PCR, p<0.001). In MyEND cells, the LPS-induced expression of the adhesion molecules Vcam-1, Icam-1, E-selectin, P-selectin was likewise inhibited by CRAMP (real-time PCR, p<0.001), and subsequently, the adhesion of J774A.1 cells to a MyEND cell monolayer (425 vs. 505 cells/hpf, p<0.01).
In addition, we also observed LPS-independent effects of CRAMP. Thus, CRAMP led to a moderate induction of inflammatory genes in MyEND and J774A.1 cells and E-selectin and P-selectin in MyEND cells (real-time PCR, p<0.01-p<0.001). Accordingly, we observed a slightly increased adherence of J774A.1 cells to a MyEND cell monolayer with CRAMP and in transmigration assays with J774A.1 cells through a MyEND cell monolayer, CRAMP was even more potent than LPS as a chemoattractant.
Conclusion
CRAMP is a potent inhibitor of LPS-dependent cytokine induction in monocytes and endothelial cells and of adhesion of monocytes to the endothelium, with potential benefits in curbing the cytokine storm and blood monocyte activity in sepsis, including sepsis-induced cardiomyopathy.
Material and methods (separate)
Myocardial endothelial cells (MyEND) and monocytic cells (J774A.1) were stimulated with LPS +/- CRAMP or CRAMP alone. NF-κB activation was determined using the reporter cell line THP-1 blue. The relative expression of proinflammatory cytokines and adhesion molecules was analysed by real-time PCR. Subsequently, the adhesion and transmigration of monocytes onto/through an endothelial cell layer were investigated in the adhesion assay and in transwell inserts, respectively.
