Human beta-defensins in dental pulp

Objectives: Human beta-defensins (hBD-1, -2, -3) are recently discovered members of a family of small antimicrobial peptides that have been identified in epithelia. The objective of this study was to investigate the expression of human beta-defensins in the dental pulp. Methods: A total of 25 dental pulps were obtained from patients having teeth extracted during routine surgical procedures. The samples were immediately frozen in liquid nitrogen and stored at –70°C. RNA was extracted from homogenized biopsies according to standard protocols using TRIzol reagent (WAK-Chemie, Germany). Complementary DNA (cDNA) was synthesized from 1 µg of RNA using the AMV RT-Kit (Promega, Germany). Expression of transcripts for antimicrobial peptides was determined by means of PCR using 4 µl of a 1:4 dilution of cDNA-solution as template; 35 cycles of PCR amplification were performed using GoldStat Taq DNA polymerase (Eurogenetec, Belgium). All PCR reactions were done in duplicate. Immunohistochemistry on 5 formalin fixed and paraffin embedded pulps was performed with rabbit-antisera to hBD-1 and hBD-2 as well as preimmune serum as control. For immunostaining of parallel sections the streptavidin-biotin-complex technique was employed. Antibodies were used at a final concentration of 10 µg/ml in 4% normal human serum. The primary antibody was detected with rabbit anti-mouse peroxidase using DAB as chromogene. Results: An expression of transcripts for the antimicrobial peptides hBD-1, -2 and -3 was found in the majority of the investigated samples (hBD-1: 18/25, hBD-2: 13/25, hBD-3: 11/25). Immunostaining revealed a strong nuclear reaction for the hBD-1 and -2 peptides in odontoblasts. Conclusions: The present expression profiling of newly discovered human beta-defensins indicates that these peptides are produced by odontoblasts and suggests a role for hBDs in the innate host defense of human pulps.