Antimicrobial peptide LL‐37 and its pro‐form, hCAP18, in desquamated epithelial cells of human whole saliva

Abstract

The antimicrobial peptide LL‐37 is active against oral bacteria and has been demonstrated to be present in human saliva, but its distribution in different fractions of saliva is not known. LL‐37 is formed from its intracellular pro‐form, hCAP18, in an extracellular enzymatic reaction catalyzed by proteinase 3 and kallikrein 5. Here, we prepared cell‐containing and cell‐free fractions of unstimulated human whole saliva by centrifugation after depolymerization of mucins with dithiothreitol, and measured the levels of hCAP18/LL‐37 in these fractions using ELISA. Cellular expression of hCAP18/LL‐37 was determined by western blotting and immunocytochemistry. The ELISA analyses demonstrated that both cells and cell‐free saliva contained hCAP18/LL‐37. Western blot analysis of cell‐pellet homogenates showed a strong band corresponding to hCAP18 at the correct molecular weight and a weak band corresponding to LL‐37. Phase‐contrast and light microscopy revealed that the cells consisted of desquamated epithelial cells. These cells expressed cytoplasmic immunoreactivity for hCAP18/LL‐37. The peripheral part of the cytoplasm, corresponding to the plasma membrane, was particularly rich in hCAP18/LL‐37 immunoreactivity. No immunoreactivity was observed after omission of the primary antibody. We conclude that desquamated epithelial cells of human whole saliva contain antimicrobial hCAP18/LL‐37, suggesting that these cells may take part in the innate immune system by harboring and releasing these peptides.

Desquamated Epithelial Cells of Unstimulated Human Whole Saliva Express Both EGF Transcript and Protein

“…Te same cells were double stained for nuclei using the nuclear marker DAPI and for EGF immunoreactivity. As seen in Figure 2, DOECs are big cells with a small nucleus and extensive cytoplasm as described before [6]. Strong immunoreactivity for EGF was Interestingly, EGF contents were several folds higher in DOECs compared to HaCaT cells, both expressed per cell (Figure 4(a)) and per mg total protein (Figure 4(b)).…”

“…The same cells were double stained for nuclei using the nuclear marker DAPI and for EGF immunoreactivity. As seen in Figure 2 , DOECs are big cells with a small nucleus and extensive cytoplasm as described before [ 6 ]. Strong immunoreactivity for EGF was detected in the cytoplasm of many cells, whereas some cells showed a weak cytoplasmic signal (Figures 2(a) – 2(c) ).…”

“…HaCaT Cells. Unstimulated whole saliva was collected from four systemically healthy, nonsmoking volunteers (27-60 years of age), and DOECs were isolated as described previously [6]. Neither the periodontal status of the volunteers nor their use of drugs was determined.…”

“…Notably, the number of leukocytes is very low in saliva from individuals with healthy gingiva, and hence, desquamated epithelial cells represent the principal cell type of whole saliva collected from healthy individuals [ 2 ]. The desquamated oral epithelial cells (DOECs) are large cells, and they show morphological signs typical for aged epithelial cells such as small nuclei and large cytoplasm [ 5 , 6 ]. We have previously demonstrated that DOECs harbor the human antimicrobial peptide LL-37, suggesting that these cells also may synthesize and/or act as containers for other biologically important salivary peptides and proteins [ 6 , 7 ].…”

Antimicrobial peptides: mechanism of action, activity and clinical potential

“…Most cathelicidins are amphiphilic α-helical AMPs [ 6 ], in which cecropins, magainins and LL-37 have been well studied. LL-37 is the only human cathelicidin of an active fragment released from hCAP18 by serine protease 3 in neutrophils with a net charge of + 6 at a neutral pH [ 94 , 95 ]. The circular dichroism of LL-37 shows a disordered structure in water and is transformed into an α-helical structure in the presence of HCO 3 − , SO 4 2− , or CF 3 CO 2 − at the concentration of 15 mmol/L [ 96 ].…”