Review for 10.1186/1471-230X-2-23 (2002)

Reviewer's□report 
□Expression□of□cytokine□mRNA□and□secretion□of□tumor□necrosis□factor-a□by□gallbladder□epithelial□ Title: 
cells:□Response□to□bacterial□lipopolysaccharides 
Authors: 
Christopher□E□Savard□( cesavard@u.washington.edu )
Thane□A□Blinman□( blinman@email.chop.edu )
Ho-Soon□Choi□( choihs@hanyang.ac.kr )
Sung-Koo□Lee□( sklee@amc.seoul.kr )
Stephen□J□Pandol□( Stephen.Pandol@med.va.gov )
Sum□P□Lee□( splee@u.washington.edu )
□1□ □9□Sep□2002 Version: Date: 
□Prof□Robert□V□Rege Reviewer: 
□A□paper□of□considerable□general□medical□or□scientific□interest Level□of□interest: 
□Accept□after□discretionary□revisions Advice□on□publication: 
Savard□et□al.□present□studies□examining□expression□of□cytokine□mRNA□and□secretion□of□TNFa□by□ 
gallbladder□epithelial□cells□with□and□without□exposure□to□bacterial□lipopolysaccharides□(LPS).□Mouse□ 
gallbladder□epithelial□cells□were□cultured□suspended□above□human□myo-fibroblast□feeder□cells,□but□ 
experiments□were□done□in□the□absence□of□feeder□cells□by□moving□the□gallbladder□cells□to□new□plates.□ 
This□removed□the□confounding□effects□of□non-epithelial□cells.□Quantitative□and□semi-quantitative□ 
RT-PCR□was□used□to□detect□expression□of□mRNA□on□cell□lysates□and□TNFa□protein□was□measured□in□ 
apical□media,□basolateral□media,□and□cell□lysates□using□an□immunoassay.□ 
Untreated□cells□expressed□mRNA□for□TNFa,□RANTES,□and□MIP-2,□but□did□not□secrete□TNFa□protein.□ LPS□(100□mg)□increased□expression□of□TNFa□and□MIP-2□mRNA,□secreted□TNFa□protein,□and□induced□ 
expression□of□IL-1b□and□MCP-1□mRNA.□LPS□from□E.□coli,□P.□aeruginosa,□or□Klebsiella□and□a□lower□dose□ 
(2□mg)□of□LPS□produced□similar□effects.□Lack□of□release□of□lactate□dehydrogenase□into□the□media□and□of□ 
increased□permeability□of□the□confluent□layers□to□mannitol□eliminated□non-specific□damage□to□the□cells□by 
LPS.□The□authors□conclude□that□gallbladder□epithelial□cells□are□capable□of□producing□cytokines□and□ 
responding□to□LPS.□They□speculate□that□gallbladder□epithelial□cells□gallbladder□play□an□active□role□in□ 
mediating□detection□of□and□defense□against□bacteria□in□bile.□They□allude□to□possible□roles□of□cytokines□in□ 
control□of□epithelial□cell□functions.□ 
COMMENTS□ 
The□strength□of□the□study□is□the□use□of□normal□gallbladder□epithelial□cells□grown□in□culture□to□eliminate□ 
stromal□and□immune□cells□in□the□gallbladder□wall.□This□eliminates□the□problem□of□identifying□the□source□ of□the□cytokine□mRNA□or□protein□in□experiments□performed□using□in□tact□gallbladder□wall.□The□results□ therefore□clearly□show□that□cultured□gallbladder□cells□express□mRNA□for□some□cytokines,□express□other□ 
cytokines□after□stimulation□with□LPS,□and□secrete□TNFa.□Although□the□authors□speculate□about□several□ 
functions□of□these□cytokines□based□on□findings□in□the□literature,□the□physiological□significance□of□these□ findings□was□not□addressed□in□these□studies.□ 
Although□the□results□strongly□suggest□that□gallbladder□epithelial□cells□have□the□same□capabilities□in□vivo,□ 
it□is□possible□that□cultured□cells□undergo□phenotypic□changes,□with□respect□to□their□ability□to□secrete□ 
cytokines,□when□passed□in□culture.□ 
The□authors□should□indicate□that□the□role□of□gallbladder□cells□in□inflammatory□and□infectious□diseases□is□ 
yet□to□be□determined.□Are□the□amounts□of□expression□and□secretion□of□cytokine□high□enough□to□produce□ the□paracrine□and□autocrine□effects□the□authors□allude□to?□Is□it□enough□to□communicate□with□immune□ 
cells□in□the□gallbladder□wall?□ 
Although□secretion□of□TNFa□was□verified□by□measuring□protein□secretion□using□immune□assay,□secretion 
of□other□cytokines□was□not□verified.□The□ability□to□synthesize□secrete□each□pertinent□cytokine□will□ 
eventually□have□to□be□demonstrated.□ 
The□methods□state□that□RT-PCR□was□performed□using□gene-specific□primers□for□"10□cytokines".□This□is□ 
not□correct.□Table□1□lists□primers□for□8□cytokines□and□for□two□internal□controls□-□GAPDH□and□ARP.□ 
TNFa□protein□is□reported□in□pg□per□106□cells.□Secretion□into□the□media□is□graphed□as□pg.□Shouldn't□the□ 
units□be□concentration□-□pg/volume□of□media?□Or□was□this□the□total□pg□secreted□into□the□apical□or□ basolateral□media?□ 
Competing□interests: 
N/A