Treatment with recombinant human tumor
necrosis factor-alpha protects rats against the
lethality, hypotension, and hypothermia of
gram-negative sepsis.
H R Alexander, … , M C Stovroff, J A Norton
J Clin Invest.
1991;
88(1)
:34-39.
https://doi.org/10.1172/JCI115298
.
Tumor necrosis factor (TNF) is a peptide secreted by macrophages in response to
endotoxin that can produce many of the changes seen in septic shock. After cecal ligation
and puncture (CLP) rats gradually develop tachycardia, hypotension, tachypnea, and
hypothermia. At 5 h post-CLP, rats have a peak in serum levels of endotoxin and 60% of
rats have blood cultures that grow Gram-negative rods (Escherichia coli and Klebsiella
pneumonia). At 20 h post-CLP all rats develop positive blood cultures. Serum levels of TNF
are not reproducibly measurable in rats following CLP. Rats undergoing CLP have a
50-80% mortality with deaths usually occurring 24-72 h postinjury. Repetitive (twice daily x 6 d)
i.p. injection of sublethal doses of recombinant human TNF-alpha (100 micrograms/kg) to
rats undergoing CLP 1 d after the treatment period resulted in a significant reduction in
mortality compared to control rats previously unexposed to rTNF (P less than 0.03). Animals
treated with rTNF had no hypotension or hypothermia after CLP and regained normal food
intake faster than control rats. 12 h after CLP the gene expression for manganous
superoxide dismutase (MnSOD), an inducible mitochondrial metalloenzyme responsible for
cellular resistance to injury from toxic reactive oxygen species, was higher in livers of rats
treated with rTNF suggesting that the TNF treatment augmented expression of this
protective enzyme. Unlike MnSOD, expression […]
Research Article
Treatment with Recombinant
Human Tumor Necrosis Factor-Alpha Protects Rats
Against the Lethality, Hypotension,
and
Hypothermia
of
Gram-Negative Sepsis
H.Richard Alexander,*Brett C.Sheppard,*J.Christian Jensen,* Howard N. Langstein,* Carolyn M. Buresh,*
David Venzon,*ElijahC.
Walker,$
Douglas L.Fraker,* Mark C.Stovroff,*andJeffrey A. Norton**Surgical Metabolism Section, Surgery Branch, and thetBiostatisticsandDataManagementBranchoftheClinicalOncology Program ofthe National CancerInstitute;andthe
OBiomedical
Engineeringand Instrumentation Branch ofthe Division ofResearch Resources, NationalInstitutesofHealth, Bethesda, Maryland20892
Abstract
Tumor necrosis factor (TNF) isapeptide secreted by
macro-phages inresponsetoendotoxin thatcanproducemanyofthe changesseeninsepticshock. After cecal ligation andpuncture
(CLP) rats gradually develop tachycardia, hypotension,
ta-chypnea, and hypothermia. At 5 h post-CLP,ratshaveapeak
inserumlevels of endotoxinand60%ofratshave blood cultures
that
growGram-negative
rods(Escherichia
coli
andKiebsiellapneumonia).
At20
h post-CLP allratsdeveloppositive blood cultures. Serum levelsof TNFarenotreproducibly measurable in rats following CLP. Rats undergoing CLP havea 50-80%mortality with deaths usually occurring 24-72 h postinjury. Repetitive (twice dailyX6 d) i.p. injection of sublethal doses of recombinant human TNF-alpha (100
jg/kg)
to ratsundergoing CLP 1 d after the treatment period resulted ina significant reduction in mortality comparedtocontrolratspreviouslyun-exposedtorTNF (P<0.03). Animals treated with rTNF had
nohypotensionorhypothermia after CLP andregainednormal food intake faster than controlrats. 12 hafterCLP thegene
expression formanganoussuperoxide dismutase (MnSOD),an
inducible mitochondrial metalloenzyme responsible for cellular resistance to injury from toxic reactive oxygen species, was
higherinlivers ofratstreated withrTNFsuggesting that the TNF treatment augmented expression of this protective
en-zyme.Unlike MnSOD, expression ofthegenefor copper-zinc SODwasnotaffectedbyCLPorrTNF treatment. The results suggest thatpriortreatmentwith recombinant TNFcan
amelio-ratethe lethality, hypotension, hypothermia, and anorexiaof Gram-negativesepsis in rats and that the mechanismmaybe related to enhanced hepatic expression of the gene for MnSOD. Repeated administration of recombinant TNFmay bea strategy to minimize mortality and morbidity of
Gram-negative sepsis. (J. Clin.
Invest. 1991.88:34-39.) Key
words: cachectin* cytokine *toleranceIntroduction
Arecentseries ofinvestigationshassuggestedthatcachectin/
tumor necrosis factor (TNF)' plays a prominentrole in the
AddresscorrespondencetoDr.JeffreyA.Norton, Head, Surgical Me-tabolismSection, Surgery Branch,National CancerInstitute/National
Institutes ofHealth, Building 10,Room2B07, Bethesda,MD20892. Receivedfor publication 15 March 1989 andinrevisedform 8 Jan-uary1991.
1.Abbreviations usedinthispaper:CLP,cecalligationandpuncture;
CuZnSOD, copper-zinc superoxide; MnSOD,manganoussuperoxide dismutase; ROS, reactive oxygenspecies; rTNF, recombinantTNF; TNF,tumornecrosisfactor.
The Journal of Clinical
Investigation,
Inc. Volume88,July
1991,34-39toxic and lethal effects of septicemia and endotoxemia
(1-6).
Intravenous administration of recombinant human TNFre-sults in the
hemodynamic, hormonal,
and metabolicchanges
ofseptic
shock(1, 2).
Intravenous administration ofendotoxintohumans results in subsequent detection of circulating TNF levels
(5),
and antibodiestoTNFprotectmiceagainst
alethal dose of endotoxin(3)
andprotectbaboons againstalethalin-jection
of
Escherichia
coli (4).
Our laboratory (6, 7) and others (8-10) have noted toler-ancetothe anorectic effects of TNF with repetitive administra-tion.In
addition,
wehave noted thatprior
administration of recombinant (r) TNF-alpha alsoprotectsagainstalethal injec-tion of TNFor alethal injection of endotoxin (6). TNF treat-menthasbeen showntoinduce thegeneexpression forman-ganous superoxide dismutase (MnSOD) in vitro and in vivo (11-13). This enzyme is ubiquitously present in
eukaryotic
cells and is induced during conditions thatgeneratetoxicsu-peroxide radicals(O°) (14). MnSODisresponsibleforthe con-version of °2tothe less reactive H202, and 02 and is thoughtto beamajor protective mechanism against cellular injury from reactive oxygenspecies (ROS). This study demonstrates that
prior
treatmentwith recombinant human TNF-alphaprotects rodents against the lethal, hypotensive, hypothermic, andan-orectic effects ofaclinically relevant model of
Gram-negative
sepsis, cecal ligation andpuncture(CLP),andthatthis protec-tion is associated with the enhanced livergene expression of MnSOD.Methods
Cecal
ligation
and
punctureThisGram-negative septic challenge was conducted as previously de-scribed(15). A 4-cm abdominal midline incisionwas made in rats previously anesthetized (35 mg/kg pentobarbital i.p.), shaved, and prepped with Betadine. The cecum wasidentified and the avascular portionof the mesenterysharplyincised. Stoolwasthen milked from the proximal bowel into thececumanda3-0 silkligaturewasplaced just below the ileocecal valve. An 18-gauge angiocath (Abbott Laborato-ries, Inc., North Chicago, IL)wasthen usedtomakeasingle, antimes-enteric puncturemidwaybetween theligatureandproximalcecum.
The cecum was then returnedtothe abdominalcavityand the
abdo-men wasapproximated. Beforeclosingtheskin,normal saline(5 cm3/ kg)wasinjectedintraperitoneally.Thissepticmodelreliablyresulted in
a50-80%mortalitywith deathsoccurring24-72 hpostinjury.A few late deaths occurred - 8-9 d after CLP.Survivingratsslowlyreturned
tonormal appearance and food intake.
Model
characterization
Eight male F344rats wereanesthetized withintraperitoneal pentobar-bital (35 mg/kg) and underwent catheterization of therightcarotid artery.Polyethylene tubing (Clay Adams,Div. ofBecton,Dickinson & Co., Parsippany, NJ) waspreviously fashioned intoan arterial line catheterbywarmingandmeldingPE-100toPE-10tubing.The arterial catheterwasconnectedto aswivel apparatus that allowed unrestrained
movementandcontinuousmeasurementof blood pressure and heart
rate.Rats thenunderwent either cecalligationand puncture (n=5)or
sham laparotomy (n = 3) and wereplaced in individual metabolic cages.Continuous back pressurewasmaintainedonthearterial lineby theuse of a Medex pressure bag that flushed 2 ml of heparin saline solution (2
A/ml)
every hour. Vitalsignswererecorded every 8 h. Deter-minationsof blood pressure and heartrate weremadedirectlyfrom the physiograph arterial line tracing. Respiratory rate wasobtained by counting for 60 s, and rectal temperaturewasmeasuredbyadigital rectalthermometer(FisherScientific Co., Pittsburgh, PA).Ina secondexperiment, 160 rats underwent CLP andwerekilled eitherimmediatelyor at1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, 15, 20, 24, 25, 30, 36, 40, and 50 hafter CLPtoobtain blood for assay and culture. 20 rats were killed andexsanguinatedattheinitial time point(time0). Blood was obtained underlightpentobarbital anesthesia (15 mg/kg i.p.) by asepticinfrarenal aortic cannulation and cultured for bacteria. Serumwasseparated and assayed for TNFactivity bythe L929 assay and endotoxin level by the limulus assay. For purpose ofanalysisrats
weregrouped at nearesttime point and blood results were compared to
rats that werekilled immediately (time 0) after CLP as the control group.
L929TNFbioassay. The TNF-sensitive L929 cells were plated on 96-well plates (Costar Corp., Cambridge, MA) and cultured overnight
at37.5-C in 5%CO2toestablishamonolayer. On parallel plates, serial dilutions ofserum samples weremade in complete media with 1.5
Mg/ml
actinomycinD(Merck, SharpandDohme,WestPoint, PA)and 100Al
of this solution was transferred to plates with L929 cells giving a final concentration of 0.75 Mg/mlofactinomycin D. After 18 h of furtherincubation,surviving cellswerefixed,stained with 0.5% crystal violet (Sigma Chemical Co., St. Louis, MO), and the plates were counted inatitertek at 570nm.One unitofactivitywasdefinedasthe reciprocal of the dilution required to produce a 50% decrease in absor-bancerelativetocontrol cells exposedtoactinomycinDalone. The 50%decrease in absorbance wasassessedbyinterpolation of absor-bancemeasurements on atitertek. Each samplewasassayed intripli-cateand thethird sample in each serieswastreatedwithapolyclonal neutralizing antibodytohumanTNF(Endogen, Inc., Boston, MA) to
removeTNFactivity. Samplesweremixed ina1:1 vol of1:100 dilu-tion ofantibodyand allowedtoequilibrate for 90 minat4°C before L929 assay. The lowerlimit ofdetection ofTNFactivity inserum was 4
U/ml. Each U/ml ofTNFactivitywastheequivalentof 20 pg/ml of rhTNF (Cetus Corp., Emeryville, CA). Therefore, the detection limit of theL929 assay was 80 pg/ml.
Limulus endotoxinassay. Determination ofendotoxinlevels was performed usingastandard limulustest(SigmaChemical Co.).Briefly, endotoxin-free glassware was prepared by autoclaving material at
121°Cfor 1 h followed by heating for 3 h at 175°Cin an oven. Initial screening ofserum samples suggested the presence ofan inhibitor. Therefore all samples were heated at 75°C for 5 min and then diluted 1:10inendotoxin-free water(16).Then100Ml of previously obtained
serum wascoincubated with 100 Ml of limulus amebocyte lysateat
37.5°C for 1hinaclosedwaterbath. The presenceofahardgel, which didnotdisruptoninversion,wastakenas apositivetest.Samples that testedpositivewerethenserially diluted inendotoxin-freewaterand incubatedasabove. Theendotoxin level was determined by multiply-ingthe inverse of the highest dilution found positive by the lowest concentration of endotoxin standardfoundpositive. The lower limits of endotoxin detection in serum was 40-60 pg/ml.
Bacteriology. 1 ml of whole blood was inoculated into 8 ml enrichedthioglycollate broth and 8 ml ofenriched brain-heart broth (BBLMicrobiology Systems, Becton, Dickinson & Co., Cockeysville, MD) andincubated for 72 h at 37.5°C in 5%CO2. Allsamples were then Gram stained (FisherScientific). Gram-negative sampleswere
thenplatedontoMac-Conkey's agar
(Difco
Laboratories Inc.,Detroit, MI)and incubatedunderaerobic conditions, orplated ontospecial anaerobebloodplates (BBL) and incubated under anaerobic condi-tions(GaspakPlus; BBL) for 48 h.Singlecoloniesweretheninoculated intoaself-contained battery ofbiochemical tests (EnterotubeII;RocheDiagnostics,
Nutley, NJ).
TNF
treatmentand
its
effect
onCLP
20 male F344 ratsweighingbetween 200 and 280 gwereplaced in individually housed cages and allowed freeaccess to water.A
nonscat-terablecasein-based paste diet(C-21;ICN NutritionalBiochemicals, Cleveland,OH)wasusedtoenableaccuratemeasurementof food in-take (17). Before study, rats wereallowedto accommodateto their environment for 5 d. Body weight and food intake weremeasured daily. Whenfood intake andbody weight gainstabilized, ratswere
randomized in equal groupstoreceive either 100 Mg/kg ofrTNF(Cetus Corp.)i.p.twicedaily (BID)for 6 dorcontrolvehicle(PBSwith0.5% BSA). HumanrTNFhadaspactof betweenIand 2.5X I07 U/mgas
measuredby the L929bioassay andanendotoxin level of between 30 and50pg/6.5X106U asmeasuredbythe limulus assay(6,18). These
measurements wereprovided byCetus andwereconfirmed inour
as-says.
In two additional experiments, similarly treatedratsthen under-went CLP to determine theeffectsof TNF treatmentonthe lethality andnutritional morbidity of CLP.Inthese experiments, CLPwas per-formed 24hafter the last dose of TNF. After CLP, survival, food in-take, andbodyweightweremeasureddaily for 20 d. Whenan individ-ual ratregained normal food intake (definedasthe averageamount
consumed during the 3 d before the administration ofTNF)orsuffered death, it completed the observation period. This survival experiment was initially performed using 19 rats, 9 randomized to TNF and 10 control. It was thenrepeatedusing 25 rats, 10TNFand 15 control.
Otherexperimentswereperformedtodetermine whether measured physiological and blood parameters were different in control survivors, controlnonsurvivors, rTNF-treated survivors, and rTNF-treated
non-survivors after CLP. rTNF-treated rats (100 Mg/kg i.p. BIDX6 d, n=5) andsaline-treatedrats(control, n = 11) underwent CLP after place-mentof a carotid artery catheter 1 d after the 6-d treatment period. Each rat was housed in an individual metabolic cage aspreviously described.Vital signs were obtained every 6 h for between 24 and 36 h. 0.5-ml blood samples were collected through the arterial catheter at
time 0, 6, 12, and 18 h for culture, and for serum levels of LPS, TNF activity by the L929 assay, and TNF by acommercially available ELISA for the measurement of murine TNF (Genzyme Corp., Cam-bridge, MA). Saline was reinfused to correct for phlebotomy volumes. Rats were allowedfree access to food and water after the initial 24 h and survival was assessed daily for 1 wk. Two additional rats weregiven LPS(Sigma) 15 mg/kg i.p. andserum wascollectedbefore and 90min after LPS and assayed for level of LPS, TNF activity, andTNFlevel to serve as apositive control.Additional experiments wereperformedin similarly treated rats to again measure TNF levels by the two assays after CLP.
Northern blot hybridization
18 rats were treated with rTNF or saline and underwent CLP as de-scribed.Livers from rats were harvested under pentobarbital anesthesia attime 0 (no CLP), 12, and 24 h, and frozen in liquid nitrogen. Livers were harvested at each time point (n =3),crushed with a mortar and pestleprecooled in liquid nitrogen, homogenized at 4°C in guanidium isothiocyanate, and RNA separated over a cesium chloride gradient. Methods used for RNA extraction, preparation, electrophoresis, and hybridization have been previously described (19). RNA was hybrid-ized with human cDNA probes for MnSOD (a generous gift from G. H. Wong, Genentech, San Francisco, CA) (1 1), copper-zinc SOD (CuZn-SOD; a generous gift from A. Singh, Genentech, San Francisco, CA) (11), and beta-actin (19). Gel densitometry (Beckman Instruments, Carlsbad, CA) was performed to quantitate image density.
Statistics
Propor-tions were compared by the Fisher's exact test. In the final experiment data were analyzedusing the test for linear trend in a repeated measures analysis of variance.
Results
Characterization of cecal ligation and puncture. There
weresignificant increases
in heart rateof
ratsundergoing
CLPcom-pared
tosham-operated
control rats at 16 and 24 h postinjury(Fig.
1). Heart rateof CLP rats then returned to levels similar tosham-operated
controlanimals until
anothersignificant
latetachycardia
occurred at 56 h postinjury. Levels ofmean arterial pressure weresignificantly
decreased in rats undergoing CLPcompared
tosham-operated
controlsfrom
24 hpostinjury
un-til
death(Fig. 1). Respiratory
rateof
ratsundergoing
CLP wassignificantly increased
at24, 56, and
72 hafter injury
com-pared
tosham-operated
controls(Fig.
1). Ratsin
both groups wereinitially hypothermic
and becameeuthermic
by 8 hpost-operatively.
CLPanimals demonstrated
significant
hypother-mia
at 24 hpostinjury
compared toeuthermia
insham-oper-ated control
rats.Significant hypothermia persisted until
deathof
CLP rats(Fig. 1).
When20 rats werebled
immediately after
CLP(time
0), noanimal had detectable
serumlevels
of
TNFactivity, endotoxin,
orblood levels of
bacteria
(Fig. 2). These animals served
asthecontrol
groupfor
theanalysis of
serumlevels
of LPS
and TNFactivity and blood culture
resultsafter CLP. Serum
levelsof
endotoxin reached
anearly
significant
peak of
1.4±0.6ng/ml
at5 hpost-CLP compared
toundetectable levels
atthetime of
CLPand
returned to nearbaseline
levelsuntil
asecondsimilar
significant peak
at40
h.Serum levels
of
TNFactivity (L929
50
38
32
8 24 40 56 72
HOURS FOLLOWING CLP
Figure 1.Physiological measurementsfollowing cecalligationand
punc-ture orshamoperation. Five rats underwent CLP(closed circles)(see Methods) and threerats
(opencircles)underwent shamlaparotomy and hadcarotid artery
cath-etersplacedattime 0. Physiological
measure-ments wereobtained every 8 h. The combi-nation of CLP and
ca-rotid artery catheteriza-tion resulted in the deathofsomeof the CLPrats at - 72 h.
Eachof the five CLP
ratssurvivedatleast 70 h. Noneof the sham-operatedratsdied. Dif-ferences between CLP
andsham-operatedrats
were assessedby the Wilcoxon rank sum test. Asterisks indicate that there isasignificant dif-ferenceattheP<0.05 level between thetwo
groups.
z
cX D
ES
z
W
-I-< cp c>cr U-oD o CLU ma
u
LUO
00.
1.25 0.75 0.25
100 (12/5) (10/10)
(12/20 /20)
(4/20) / (8/15)
20 (4/20)
I I I I
(0/20)8
24 40 56HOURSFOLLOWINGCLI
Figure 2. Blood param-etersfollowingcecal li-gation and puncture. Serumendotoxin and bloodculture resultsof rats (n = 160)killed im-mediately after (n = 20, time 0) and at various subsequenttimes after CLP. Data are mean±SEMfor serum levelsof endotoxin and 72 are
compared
tolevelsattime 0 by
indepen-P dent Student's t test. Asterisks indicate significant difference from time0 level (P <0.05). Percentagesfor rats with positive blood cultures are given. In paren-theses, numeratorindicates the numberof animals with positive bloodcultures and denominator indicates the total number of ani-malsstudied at each time point. Asterisks indicate that there is a sig-nificant difference in proportions compared to animals at time 0 by Fisher's exact test (P<0.01).
assay)
orimmunologic reactivity (ELISA)
werenotreproduci-bly measurable
in ratsfollowing
CLP.Blood cultures
did
not grow anyGram-positive bacteria orbacteroides
species.
Thepredominant bacteria isolated
wasE.coli (75% ofisolates) and
theremainder
was Klebsiellapneumo-nia. 20%(4/20) of
blood cultures grewbacteria
at 2and 10 h afterCLP which
was notdifferent from control. However,
at15 hpost-CLP 60% (12/20)of
bloodcultures becamepositive
and thisobservation
wasdifferent from the negative cultures
at time 0. It was at 24 h after CLP that the mean arterial pressure andthe
temperaturebegan its
progressive decline (Fig. 1)
and that asignificant proportion of blood cultures
becamepositive
(Fig.
2).
TNF
treatment
and its
effect
on CLP. Rats
given human
rTNFi.p. twice daily for 6 d decreased food intake and
atesignificantly less food than
control ratsgiven saline. However,
despite repetitive daily administration of rTNF,
ratsprogres-sively increased daily food intake
overlevels consumed onthe
previous day.
Onday 6 of
rTNF treatment, ratsconsumed the
same amountof
food
assaline-treated controls (data
notshown,
butsimilar
toreferences 6
and7).
Similarly,
ratsgiven
rTNFlost
body
weight only
onthe
initial day of
administra-tion, while saline control
ratsdid
not; butby
day 2,
ratsadmin-istered
rTNFbegan
togain body weight and
on treatmentdays
3-6
gained
similar
amountsof body
weight
ascontrols (data
notshownbut
similar
toreferences 6
and7).
In twoseparate
experiments,
ratstreatedwith
rTNFthatsubsequently underwent CLP had
significantly improved
sur-vival
compared to ratspreviously treated with saline
(Fig. 3).
Inaddition,
ratsfrom
the control group thatsurvived
CLP tooktwice
aslong
to return to normalfood intake
asratspreviously
treated
with
rTNF.Median days
tonormalfood
intake afterCLP
were8 d
for rTNF-treated
ratsand
15d for control
rats; thedifference in time
wassignificant
(P
<0.03)
andrepeatable
in the second
experiment (data
notshown).
Rats that weretreated
with
rTNFandsubsequently
under-wentcarotid
arterycatheter
placement
andCLP
hadsignifi-cantly
improved
survival
compared
tocontrols
(P
<0.01) (Fig.
4
a). Only
twoof eleven control
ratssurvived
while five of five TNF-treated ratssurvived.
Eight
of nine deaths in the control
36 Alexanderetal. 550
w
W .E
I
W E
cr
z2
U
n0
2
< C
°E E
w E
z 0
en
b
z
z
0 cr
0o
1.c0,
0.9 0.8
-0.7
-0.6
-0.5 - 0
0.4
-0.3
-0.2
-0.1 _
0.0
0 5 10 15 20
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
0 5 10 15 20
DAYSFOLLOWING CLP
Figure3. Mortality of cecalligation and puncture in control and rTNF-treated rats.(a) RatsweretreatedwithrTNF(100 ug/kgi.p. BID x 6 d) (n=9, closedcircles)orsaline vehicle (n= 10,open
cir-cles).24hlater CLPwasperformed (day0)and20-dsurvivalwas
as-sessed. Rats treatedwith rTNF hadsignificantly improvedsurvival(P
<0.03).(b)Repeatexperimentwith identical conditions except that there were 10 rats in therTNFgroup
(closed circles)
and 15ratsin the control group (opencircles).Ratstreated with rhTNFagainhad significantly improvedsurvival (P<0.02).group occurred by 48 h. There were no differences in pulse or
respirations
between thetwo groups.Immediately after CLP,
control
ratshad
significantly
higher
meanarterial
pressurethat
progressively decreased during the 30-h
measurementperiod
(P
<0.02,
Fig.
4b).
Ratspreviously treated with
rTNFhad
nosignificant change
in meanarterial
pressureduring
the 30-hperiod.
Temperature
wassignificantly
decreased in control
ratsthat did
notsurvive CLP compared
to ratspreviously
treatedwith
rTNFandcontrol
rats thatdid survive CLP. This
differ-ence wasfirst
significant
at18
hafter CLP (P
<0.001)
(Fig.
4c).
Ratsfrom control and
rTNF treatment groups each hadimmediate positive
bloodcultures drawn through the carotid arteryline (- 45
minafter CLP) and cultures remained
posi-tive
ateachtime point.
Serumendotoxin
levels peakedat
12 hafter CLP
inthese
rats(675±125
pg/ml)
and
levels at 24 hremained
similar
butslightly reduced.
There were no signifi-cantdifferences in
serumendotoxin levels in
control orrTNF-treated
rats.Levels
of
serum TNFactivity
werebelow
thelimit
of detection of the
assayin all
rats.Control
ratstreated with
intraperitoneal
endotoxin
had serum endotoxin levels equal to 27±14ng/ml
andserum TNFactivity
levels equal to 640±0U/ml
at90
minafter endotoxin. Levels
of
each wereundetect-able
before endotoxin.
Before
CLP (t = 0) thegenefor
MnSOD was onlymini-mally detectable in
theliver
of
controlanimals
but wasslightlyamplified
in the liver of rTNF-treated
rats(Fig.
5).Expression
of
the genefor CuZnSOD
wasequal in the liver of control andrTNF-treated
rats anddid
not changeafter
CLP.However, in both rTNF- andsaline-treated
animals,
the genefor
MnSOD was augmented at 12 hafter CLP
compared to t=0 levels. Ina 1.0
0.9
0.8- Figure 4.
Mortality
andz 0.7-
physiological
parame-F 0.6- tersof control and
0.5
rTNF-treated ratsafterr 0.4 carotid arterial catheter
0.2
placementand CLP.(a)
0.1 Ratsweretreatedwith
0. 44 192 rTNF(100
gg/kg
i.p.b b120-120 BIDX6 d) (n = 5,
closed
circles)
orsaline110 vehicle(n=
11,
open100
circles)
and 1 d laterWE
-so9 underwentcarotid
ar-z s
90
terycatheterplacement
and CLP.
Physiological
X_ parametersandblood0 6
12
18 24 30 i wereobtainedevery6 hc 40 for 30 h and survival
wasassesseddaily for7
d. Ratstreatedwith
W35
D Y/ ~ * rTNFhadsignificantly
!i \improvedsurvival (P
L 30
\N<
0.01). (b)
Meanarte-rial pressure
measure-25 mentsofratsdescribed 0 6 12 18 24 30 36 42 in (a). Mean arterial
HOURSFOLLOWINGCLP pressure was
signifi-cantly increased in control rats (opencircles) comparedto rTNF-treated rats(closedcircles) immediatelyafter CLP
(*P
<0.02). Anal-ysisofvariancedemonstrated thatmeanarterial pressure didnotchange in TNF-treated rats, butsignificantlyprogressivelydecreased in controlrats over30 h(P=0.02). (c) Temperature ofratsdescribed in (a). Temperaturewassignificantlydecreased in controlratsthat did not survive(opencircles) comparedto TNF-treatedrats
(closed
circles)and control rats that survived (opentriangles). Analysis
of variancedemonstrated that thesefindingsweresignificantat 18 h post-CLP and remainedsignificant throughout(*P<0.01).addition,
12 h after CLPhepatic
geneexpression
of MnSOD wasincreased
much moremarkedly
in thelivers of rats treatedwith
rTNF. 1 d after CLP liver MnSOD geneexpression
re-turned to levelssimilar
tobaseline levels
except that theexpres-sion in control animals
appeared
slightly
greaterthan
rTNF-treated rats(Fig.
5).
Pretreatment Figure 5. Geneexpression of MnSOD
Saline rhTNFo A549 after CLP in theliver of rTNF- or sa-MSOOD line-treated rats.Animals were treated
^Kb hi * * withrTNF orsaline controlas
de-Kb fit * scribed and underwent CLP 24 h later. CuZnSOD Ad m RNA was extracted from liver at each
06 Kb 'i time point. At t=0, before CLP, there
2.1Kb
is evidenceof slightly increasedex-pression of the gene for MnSOD in
Hours after CLP the liver of rats treated with rTNF comparedtocontrols. 12hafterCLP thereis clearaugmentationof the gene for MnSOD observed in both thecontrol and rTNF-treatedratscomparedtolevelsat t=0.In
addition,at 12 h MnSOD geneexpression is further augmented in theliver of rTNF treated comparedtocontrolrats.After24 h
MnSOD geneexpressionreturns tobasal levels in both groups. The hepaticgeneexpression for CuZnSOD and beta-actin remains the
samebefore andateachtimepointafter CLP in eitherrTNF orsaline controlanimals.RNAextractedfrom A549 cells treated with 0.1 g LPS(Sigma) servedas apositivecontrol.
I
Discussion
Previous
studies in our laboratory havedemonstrated
that resis-tance tothe
anorectic
andweight
losseffects
of recombinant human TNF/cachectin develops during a repeated twice dailydosage schedule. Additionally, similar
rTNFtreatment
regi-mensconfer
protection when rats or mice were subsequentlychallenged with
alethal dose of endotoxin
orTNF (6, 21). Invitro
and in vivo TNF has been shown to induce MnSOD, a metalloenzyme that reduces ROS to less toxic H202 and02
(1
1). This enzymeis induced
in a variety of tissues, including ratliver, during conditions
thatgenerate a free radical stressand
is thought
tobe
responsible for cellular resistance
to injuryfrom
ROS(14,
22).In
this work
we haveexamined
thepossibility
that prior treatmentwith recombinant
human TNF mayoffer protection
from
deathand minimize
the toxicityof
a septic challenge. While thisGram-negative
sepsis model is not as well quantified asabacterial innoculum, cecal ligation and puncture is
advan-tageous asit develops slowly and simulates
apolymicrobial
enteric insult
thatis
similar
to that seenin
patients with colonic
perforations.
When compared tosham-operated
controls, ratsundergoing CLP develop tachycardia
at16
hpostinjury,
fol-lowed
byhypotension, hypothermia,
and tachypnea at 24 hpostinjury (Fig. 1).
Inratsbled and killed after CLP,
peaklevels
of endotoxin in
serumoccurred
5 hafter
injury and then levels
rapidly decreased.
Serum
levels
of
TNFactivity
were notreproducibly
measur-able in
animals
despite
the
endotoxin peak.
At20
hafter CLP
there
wasasignificant
rise in the number of
ratswith
positive
blood cultures
(60%) and the
proportion
laterfurther increased
to100%
at50
hpostinjury (Fig.
2).
Circulating
levels of
TNFhave been detected by others in
alimited number of
heteroge-neousstudies
of infection. Serum levels of
TNFhave been
ele-vatedin
patients with fatal infectious diseases, including AIDS
(23),
meningococcemia
(24, 25), and fulminant
hepatic
failure
secondary
tosepsis (26). Similarly,
serumlevels of
TNFhave
beendetectable in
experimental sepsis
with
Listeria(27) and
E.coli
(28). However, in other
experimental
models of
infection,
such
asinfected burns
(29),
Listeriameningitis (30),
orSal-monellosis (31),
serumlevels of
TNFhave beenslightly
ele-vated in occasional animals (28)
ornotdetectable
(29, 30).
Thefact that
TNF was notreproducibly
measurable
mayimply
that
thereis
anepisodic
and
notcontinuous secretion of
TNFafter
CLP.
Because oneis limited
by the
amountand number of
blood
samples that
can bedrawn in
asmall animal like
the rat,it
is possible
tomiss
anepisodic
peak in
serumTNFactivity.
Another
recentstudy in mice
alsodid
not detectcirculating
levels
of
TNFafter CLP
(32).
Asothers have described in
aninfected
burn
model
(29),
inratsthat haveundergone CLP
we haverecently observed
aninduction of
TNF geneexpression
ina
variety
of
tissues,
including
lung, liver,
and
spleen,
suggesting
that
production
of
this
cytokine
isaugmented (Alexander,
H.
R., unpublished observations).
The recent
observation that tolerance
orresistance
tothe
toxic and lethal effects of recombinant
tumornecrosis
factor/
cachectin
canoccurhas been made
concomitantly by
several
groupsworking with
recombinant
TNF(6-9, 16, 33).
Theob-servation that
treatmentwith
rTNFcan protectagainst
the
morbidity
and
mortality of
Gram-negative sepsis
described
here mayhave clinical relevance. Previous
treatmentwith
TNF notonly reduces
thelethality
(Figs.
3
and 4a) of
theseinfec-tions,
butit
significantly improves
themarked
hypothermia
and
hypotension
of
thesepsis (Fig. 4,
b
andc).
Temperature
changes
(fever
in
higher mammals)
is
oneof
themostprevalent
clinical
features of infection. Prior
treatmentwith
TNF appears toabrogate
theequivalent
tofever and shock in
septic
rats.Blood pressure and temperature is maintained in the normal
range,while control
rats becomehypotensive
andhypother-mic.
Inaddition,
the time
torecoveryof normal
food intake is
significantly
shorter
inTNF-treated
ratsthan control
survi-vors.Previous work in
ourlaboratory demonstrated
that simi-lar TNFtreatmentalsominimized the marked cellular
damage
associated with
high
doses of
endotoxin
and that this
protec-tion lasted between
4and
14 dafter the
discontinuation
of
treatment(6).
Inaddition,
theprotection
doesnotappeartobe
induced
by
endotoxin
contamination
of
therecombinant
hTNFpreparations
becausethe
rTNFis
verylow in
endotoxin,
and
macrophages
from TNF-treated animals
respond
differ-ently
than
macrophages
from
LPS-treated
animals, implying
that the mechanisms
aredifferent
(16). Finally,
recent work suggeststhat
similar
protection
tothe lethal effects of
TNF,
endotoxin,
and CLP
canalso
beinduced
by
asingle
intrave-nousinjection
of rTNF, further
supporting possible
clinical
applications (34).
In
vitro,
TNFinduction
of MnSOD
has beenimplicated
asthe
protective
mechanism
against
cellular
injury
from free
radi-calstress(
13).
Inthe liver of rTNF-treated
rats(compared
tocontrol
animals)
there is
aslight
enhancement of the
lowlevel
of
expression
of the gene for MnSOD
24 hafter the
treatmentperiod just
before CLP
(t
=0)
(Fig. 5).
This indicates
thatin
themultiple
doseexperiments
described
here,
rTNFtreatment,
unlike the
single
intravenous dose model
previously
reported
(11),
did
notmarkedly
induce
MnSOD,
but there
wasstill
someevidence of
TNFinduction of the
genefor
hepatic
MnSOD
suggesting
thatit
maybe part ofthe
protective
mecha-nism.
12 hafter CLP
thereis
aclear increase in
hepatic
MnSOD
geneexpression
incontrol animals
implying
asignifi-cant
free radical
stress asaresult of CLP.
Inthe
liver ofanimals
treated with
rTNF,
there is
amarked additional
augmentation
of
the genefor this
protective
enzyme.Our
experiments
do
notattemptto
dissect
theregulation
of
hepatic
MnSOD
geneex-pression
butit
appearsclear
that
at12 hafter CLP
transcription
ofthis
geneis increased
by
prior
treatmentwith
rTNF.Interest-ingly,
this is
just
before the time
(18
hpost-CLP)
when
physio-logical
differences between the
TNFand
control
grouparefirst
observed
(Fig. 4).
Incontrast,
aspreviously
described
CuZn-SOD
is
anoninducible
cytosolic
enzymethat is
constitutively
expressed
in
tissue
(14),
and theresults demonstrated
herealso
clearly
indicate
thatCuZnSOD is
notresponsible
for
anyTNF-induced differences.
Inthese studies neither
TNFtreatmentnor the
subsequent
CLP
changed
the
geneexpression
of
CuZnSOD.
The
fact
thatMnSOD
expression
is
very lowat24
hafter
CLP
(in fact,
TNF-treatedarelessthancontrol)
suggests
thatthe
geneexpression
seen at12 h hasgenerated enough
enzymeactivity
towithstand
theoxidant
stressof CLP.
Experiments
in
a
murine model of endotoxin shock
arecurrently being
per-formed in
ourlaboratory
toevaluate this
possibility.
The
marked
improvement
in
survival
seenwith
rTNFtreatmentand its dramatic
improvement
in
hypotension
and
hypother-mia
after
CLP maysuggest
thatinduction of the
genefor
MnSOD
in theliver is
notthe
only
mechanism of
TNF-in-duced
protection.
Furthermore,
TNFincreases
neutrophil
tivity (35, 36)
which may also be a protective action of thecytokine. Finally,
recent work suggests that there exists asolu-ble
fragment of the
TNFreceptor molecule that may serve as a TNFinhibitor
(37). Partof
the explanation of the mechanismof
thedramatic protective
effectsof TNF treatment described here may be theinduction of
asoluble TNF receptor fragment that reducescirculating
levels of TNF activity. Our inability to measurecirculating
levelsof TNFactivity
may beconsistent
with this hypothesis. However, there did not appear to be adifference in ability
to measurelevels of
serum TNFactivity
after CLP in control
orrTNF-treated
rats.Insummary, rats treated with
recombinant
humanTNF-alpha develop resistance
tothe
anorectic
andweight
losseffects
of
TNFand
protection against
thelethal, hypotensive,
hypo-thermic,
andanorectic effects of
anexperimental model of
sep-sis,
cecalligation
and puncture. Theprotective mechanism
may bemediated
by aTNF-induced
augmentation of
expres-sion
of
the genefor MnSOD,
an enzyme thatis
maximally
expressed
at 12 hafter CLP.
This work documents that treat-ment with rTNF may have usein minimizing
themorbidity
and
mortality of Gram-negative sepsis.
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