Induction of cytokine messenger RNA transcripts in mouse macrophages by Listeria monocytogenes isolated from channel catfish

Abstract

Objective: To determine whether differences exist in induction and quantity of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)1 beta, and IL-10 mRNA transcripts when mouse J774A.1 macrophages are infected with Listeria monocytogenes, including 2 isolates originating from channel catfish, the wild-type virulent (EGD) strain, and a nonhemolytic strain (ATCC 15313).

Samples: Listeria monocytogenes isolates from kidneys or fillets of channel catfish were used to stimulate cytokine production from mouse macrophages. The RNA from the infected macrophages was collected.

Procedure: Four hours after infection with L monocytogenes, total cellular RNA was extracted from the J774A.1 cells and reversed transcribed to cDNA, which was amplified, using specific primers for TNF-alpha, IL-1 beta, or IL-10. The specific amplified DNA fragments were detected on polyacrylamide gels and quantified, using a reverse transcription polymerase chain reaction (PCR)-mediated ELISA.

Results: The wild-type hemolytic EGD strain and the 2 hemolytic catfish isolates of L monocytogenes induced higher amounts of TNF-alpha-, IL-1 beta-, and IL-10-specific mRNA in J774A.1 cells than did the nonhemolytic strain.

Conclusions: Hemolysin-associated induction of TNF-alpha, IL-1 beta, and IL-10 cytokines may be related to survival and replication of L monocytogenes in macrophages. It also suggests that the PCR-mediated ELISA procedure is a sensitive test to quantify cytokines from cell cultures.