Morphological and immunological evidence of a unique selective production and endoplasmic reticular accumulation of interleukin-1a in rat peritoneal macrophages induced by Pseudomonas aeruginosa exotoxin A Yen-Te Huang, a,b Chian-Ren Jeng, a Chiung-Hsiang Cheng, a Ling-Ling Chueh, a Jiuan Judy Liu, a,c and Victor Fei Pang a, * a Graduate Institute of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan, ROC b National Laboratory Animal Breeding and Research Center, National Science Council, Taipei 115-29, Taiwan, ROC c Developmental Center for Biotechnology, Taipei 106, Taiwan, ROC Received 12 July 2002; accepted 18 March 2003 Abstract The immunotoxicity of Pseudomonas aeruginosa exotoxin A (ETA) on macrophages was evaluated by incubating rat peritoneal macrophages(RPM)with1–100ng/mlETAfor3–60h.AlthoughtheoverallchangesincellviabilityandDNA,RNA,andprotein synthesisoftheETA-treatedRPM(E-RPM)werereducedinadose-andtime-dependentmanner,therewasatransientbutevident rebound in RNA and/or protein synthesis at 24–36h post-incubation (HPI) at 1–50ng/ml ETA. However, a more apparent en- hancementappearedinRNAandproteinsynthesisat36–48HPIin10and50ng/mlE-RPMafternormalizedonthebasisofviable cell.Most50–100ng/mlE-RPMunderwentnecrosis/apoptosisbefore24HPI.By36HPI,41%of10ng/mlE-RPMremainedviable but were full of cytoplasmic granules due to the accumulation of glycoprotein in segmentally dilated endoplasmic reticulum. Im- munological staining of the granules revealed strong IL-1a but weak or no signals for IL-1b, IL-1 receptor antagonist, IL-6, and TNF-a.Atime-dependentincreaseinIL-1a butnoIL-1b wasdetectedincelllysateof10ng/mlE-RPM;however,neitherIL-1a nor IL-1b was detected in culture supernatant. Thus, besides cytopathic and functional effects, ETA could induce a unique selective production and endoplasmic reticular accumulation of IL-1a in RPM. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: IL-1a; Accumulation; Endoplasmic reticulum; Exotoxin A; Pseudomonas aeruginosa; Peritoneal macrophage; Rat 1. Introduction Pseudomonas aeruginosa produces several substances that may influence its infectivity [1,2]. Among them, exotoxin A (ETA) is considered the major and the most lethalvirulentfactorproducedbythemajorityofclinical isolates [3,4]. There is a significant decrease in the viru- lenceofeitherETA-deficientmutant[5,6]orETAexcre- tion-deficientmutant[7].Inaddition,antibodiestoETA provide protection in both experimental and clinical Pseudomonas infections [6,8]. It is well known that ETA inhibits protein synthesis in mammalian cells by trans- ferring the ADP–ribose moiety of NAD þ to the elonga- tionfactor2leadingtocelldeath[9,10].However,amore recent study has shown that point mutations in the gene encoding ETA resulted in mutant ETAs with markedly reducedADP–ribosyltransferaseactivitybutnoeffectson cytotoxicity[11]. ExotoxinAcanexertamodulatoryeffectonthehost immune system by altering the functions of B and T lymphocytes, macrophages, and/or granulocytes [12– 16]. Earlier studies have shown that ETA could induce cell death, reduce ½ 3 Hthymidine uptake and phagocy- tosis in human macrophages [13,15], and inhibit protein Cellular Immunology 221 (2003) 143–156 www.elsevier.com/locate/ycimm * Corresponding author. Present address: Department of Veteri- nary Medicine, College of Bio-resources and Agriculture, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan. Fax: +886-2-23661475. E-mail address: pang@ccms.ntu.edu.tw (V.F. Pang). 0008-8749/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0008-8749(03)00076-5