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Cathelicidin Antimicrobial Peptides are Expressed in Salivary Glands and Saliva

Division of Dermatology, University of California San Diego; and VA San Diego Healthcare Center, San Diego, CA 92161, USA;

View larger version (70K): [in a new window]   Figure 1. CRAMP mRNA is detectable in salivary glands by RT-PCR. A 1-µg quantity of total RNA was used for each RT reaction. Bone marrow used as a positive control shows an intense band at the expected size. Salivary glands (submandibular) obtained from 3 separate dissections (#1, #2, #3) and tongue samples from 2 separate dissections, gingiva and palatal mucosa, show faint bands for CRAMP. CRAMP was not detected in buccal mucosa from 2 dissections or where water (DW) was used as a negative control. 18S RNA was amplified from duplicate samples. The ladder is 10 µL of the phi-X 174/HaeIII size marker.

View larger version (106K): [in a new window]   Figure 2. CRAMP mRNA is expressed in the submandibular (salivary) gland and oral mucosa. CRAMP mRNA is localized by in situ hybridization with specific anti-sense CRAMP cRNA probes. Tissues were obtained from normal adult mice (A,D), newborn Day-4 mice (B,E,G-L), and fetal mice at day 17 of gestation (E17) (C,F). All submandibular glands (adult, D-4, E-17) show CRAMP mRNA expression (A-C), as do the palatine minor salivary glands (G). In the salivary glands, CRAMP mRNA expression is seen in the acinar cells, but not in ductal cells. In gingiva and palatal mucosa, CRAMP mRNA is located in the basal cell layer and a few suprabasal cell layers (H, and upper layer in I), and the epithelium of the dorsum of the tongue shows mRNA in whole epidermis (I, lower layer). Sense cRNA probes show no signal in any tissue (D-F, J-L). Original magnification: x400.

View larger version (105K): [in a new window]   Figure 3. CRAMP protein is expressed in the submandibular (salivary) gland, and oral mucosa. CRAMP protein in submandibular glands of adult mouse (A), E-17 embryonic mouse (B), newborn Day-4 mouse (C), and palatine minor salivary gland of the newborn (G). Newborn gingiva (H) and tongue (I, lower layer) also show CRAMP expression. In newborn palatal mucosa, CRAMP is located in the basal cell layer and a few suprabasal cells (I, upper layer). For negative controls, pre-immune rabbit IgG (D-F, J-L) was used. Original magnifications: x400.

View larger version (63K): [in a new window]   Figure 4. Human saliva contains the cathelicidin LL-37. SDS-PAGE of human saliva extract and synthetic LL-37 peptide was evaluated by Western blot with rabbit antibody against full-length LL-37. Membrane was then stripped and re-probed with chicken antibody against the cathelin immature domain (CATH). Membrane was again stripped and re-probed with a unique chicken anti-LL-37 peptide antibody. Arrow at A corresponds to expected size of full-length human cathelicidin. Arrow at B (14 kDa) is intermediate size suggesting partial processing of cathelicidin. Arrow at C corresponds to 5-kDa mature LL-37 antimicrobial peptide.