DTIC ADA629617: Reduction of Burn Progression with Topical Delivery of (Antitumor Necrosis Factor-alpha )-Hyaluronic Acid Conjugates

2.3 MB·English

by  Defense Technical Information Center

Reduction of burn progression with topical delivery of
(antitumor necrosis factor-a)-hyaluronic acid conjugates
Liang Tso Sun, PhD1; Emily Friedrich, MS1; Joshua L. Heuslein, BS1; Rachel E. Pferdehirt, BS2;
Nicole M. Dangelo, BS2; Shanmugasundaram Natesan, PhD3; Robert J. Christy, PhD3;
Newell R. Washburn, PhD1,2,4
1.DepartmentofBiomedicalEngineering,CarnegieMellonUniversity,Pittsburgh,Pennsylvania,
2.DepartmentofChemistry,CarnegieMellonUniversity,Pittsburgh,Pennsylvania,
3.UnitedStatesArmyInstituteforSurgicalResearch,FortSamHouston,Texas,and
4.McGowanInstituteforRegenerativeMedicine,UniversityofPittsburgh,Pittsburgh,Pennsylvania
Reprintrequests: ABSTRACT
Prof.N.R.Washburn,CarnegieMellon
Inthisstudy,weexploredwhethertopicalapplicationofantibodiestargetingtumor
University—ChemistryandBiomedical
Engineering,4400FifthAvenue, necrosisfactor-a(TNF-a)orinterleukin-6(IL-6)conjugatedtohyaluronicacid(HA)
Pittsburgh,PA15213,USA. could reduce the extension of necrosis by modulating inflammation locally in a
Tel:[phone]; partial-thickness rat burn model. Partial-thickness to deep partial-thickness burn
Fax:[phone]; injuriespresentsignificantchallengesinhealing,astheseburnsoftenprogressfollow-
Email:[email protected] ingtheinitialthermalinsult,resultinginnecroticexpansionandincreasedlikelihood
ofsecondarycomplications.Necroticexpansionisdrivenbyamicroenvironmentwith
Manuscriptreceived:March25,2011 elevatedlevelsofpro-inflammatorymediators,andlocalneutralizationoftheseusing
Acceptedinfinalform:February29,2012 antibodyconjugatescouldreduceburnprogression.Trichrome-stainedtissuesections
indicatedtheleastnecrotictissuein(anti-TNF-a)-HA-treatedsites,while(anti-IL-6)-
DOI:10.1111/j.1524-475X.2012.00813.x HA-treatedsitesdisplayedsimilaroutcomestosalinecontrols.Thiswasconfirmedby
vimentinimmunostaining,whichdemonstratedthatHAtreatmentalonereducedburn
progressionbynearly30%,but(anti-TNF-a)-HAreduceditbyapproximately70%.
At all time points, (anti-TNF-a)-HA-treated sites showed reduced tissue levels of
IL-1b compared to controls, suggesting inhibition of a downstream mediator of
inflammation. Decreased macrophage infiltration in (anti-TNF-a)-HA-treated sites
comparedtocontrolswaselucidatedbyimmunohistochemicalstainingofmacroph-
ages,suggestingareductioninoverallinflammationinalltimepoints.Theseresults
suggest that local targeting of TNF-a may be an effective strategy for preventing
progressionofpartial-thicknessburns.
Burns are unique among acute injuries in the progressive have been reported both systemically and locally in burn
natureoftissuenecrosisfollowingtheinitialinsult.Theinitial patients,whichsuggestthatthesemediatorsmaybecentrally
injury induces a central zone of coagulation, a region of involved in establishing the pathophysiological environment
irreversible tissue loss due to protein denaturation and cell ofburns.
death.1Overthecourseofseveraldays,thesurroundingzone In this study, we investigated the effects of inhibiting
of stasis experiences continuing and potentially irreversible two of these pro-inflammatory mediators: TNF-a and IL-6.
necrotic conversion that expands wound size and depth, TNF-aisknowntosignalahostofdownstreaminflammatory
leading to delayed healing and increased likelihood of sec- mediators,includingIL-6,8andneutralizingthistargetcould
ondarycomplicationsandpatientmorbidity.2,3Earlyinterven- effectively modulate the resultant complex inflammatory
tionstomodifythepathophysiologicalenvironment,thereby cascade. TNF-a activates keratinocytes and macrophages to
abating tissue destruction in the zone of stasis, could poten- releasereactiveoxygenspecies(ROS),whichcaninducefree
tiallyreducetheextentofburninjury,andhenceofferclinical radical damages.9–11 Increases in inducible nitric oxide syn-
relevance.4 thase, IL-1b, and prostanoids, all downstream effects of
Thermaltraumainducessignificanttissuedamage,leading TNF-aup-regulation,candifferentiallypromotevasodilation
totissueischemia,edema,andthereleaseofoxidizingagents, orvasoconstrictions,12–14whileTNF-aitselfcaninduceendo-
whichfurtherextendthetissueinjury.5Burnedtissueisoften thelial permeability.15–17 TNF-a-induced prostaglandins can
described as being in a cytotoxic and degenerative state.6 also increase the risk of hypoperfusion in burn wounds.14
Several inflammation-mediated mechanisms for continued Ultimately,thesepathologicalmechanismsmayleadtonecro-
tissue destruction have been proposed, such as oxidative tictissueformationandincreasetheseverityofinjury.
stress, cell death, hypoperfusion, and vasodilation, leading In contrast, IL-6 is a pleiotropic cytokine with a broad
to tissue ischemia and necrosis.7 Abnormal levels of pro- spectrumofpro-andanti-inflammatoryactivitiesintheburn
inflammatorymediators,suchastumornecrosisfactoralpha site,alsomakingitastrongcandidateforcytokineneutraliz-
(TNF-a), interleukin-1b (IL-1b), and interleukin-6 (IL-6), ing therapy. It is produced by T cells, B cells, monocytes,
WoundRepReg(2012)20563–572©2012bytheWoundHealingSociety 563
Report Documentation Page Form Approved
OMB No. 0704-0188
Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and
maintaining the data needed, and completing and reviewing the collection of information Send comments regarding this burden estimate or any other aspect of this collection of information,
including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington
VA 22202-4302 Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it

does not display a currently valid OMB control number
1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
01 JUL 2012 N/A -
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Reduction of burn progression with topical delivery of (antitumor
5b. GRANT NUMBER
necrosis factorâα)âhyaluronic acid conjugates
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Sun L. T., Friedrich E., Heuslein J. L., Pferdehirt R. E., Dangelo N. M.,
5e. TASK NUMBER
Natesan S., Christy R. J., Washburn N. R.,
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
United States Army Institute of Surgical Research, JBSA Fort Sam REPORT NUMBER
Houston, TX
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release, distribution unlimited
13. SUPPLEMENTARY NOTES
14. ABSTRACT
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON

a REPORT b ABSTRACT c THIS PAGE UU 10
unclassified unclassified unclassified
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
(anti-TNF-a)-hyaluronicacidconjugates Sunetal.
Figure1. Synthesis of (anti-TNF-a)-HA and (anti-IL-6)-HA conjugates. Carboxylic acid groups on HA were partially activated
followedbycouplingreactionwiththeaminegroupsoncytokine-neutralizingmonoclonalantibodies.
fibroblasts, keratinocytes, and endothelial cells.18,19 The atthesiteofinjury.30HAhasbeenshowntopromotewound
general physiological functions of IL-6 are complex; IL-6 healing in chronic wounds and other compromised tissues,31
induces T-cell proliferation and cytotoxic T-cell differentia- and synergistic effects with cytokine-neutralizing antibodies
tion, and terminal macrophage differentiation, making it maybeanticipated.Inthisstudy,wecomparedtheeffectsof
important in a broad range of inflammatory settings.20 The locally inhibiting TNF-a, a potent upstream promoter of
concentrationofcirculatingIL-6canbeelevatedinburnand inflammation, with inhibiting IL-6, a downstream mediator
septic patients 100-fold,21 and it is thought to be a central of inflammation that is known to be important in burn etio-
biochemical mediator in responses to trauma. It has gained logy but has a range of functions bridging pro- and anti-
attention as a therapeutic target to modulate post-trauma inflammatoryresponses.Thehypothesisofthisstudywasthat
symptoms and increase survival rates.22 Studies have found localized neutralization of TNF-a, an early mediator of
that excessive and prolonged circulating levels of IL-6 have inflammation,wouldbemoreeffectiveatinhibitingburnpro-
decreased survival rate,23,24 and progressive decline of IL-6 gressionthanlocalizedneutralizationofIL-6,alatemediator
levels after 3 days of initial burn increases the chance of ofinflammation.
recoveryinseverelyburnpatients.25Althoughitisstillunclear
wheretheexcessIL-6isproduced,Changandcoworkershave
suggested that the production of IL-6 is influenced by some MATERIALS AND METHODS
factorsreleasedinthelocalburntissue.26
Modifying the inflammatory microenvironment through
modulation of these cytokines, or the resultant activated Materials
immunecells,isthereforeanappealingtherapeuticstrategyto HA (M =1.6MDa) and 4-(dimethylamino)pyridine were
w
rescue viable tissue from burn pathogenesis. In a rat model, purchased from Sigma-Aldrich (St. Louis, MO) and used as
topicalapplicationofinhibitorofp38MAPK,apotentstimu- received. N-hydroxysulfosuccinimide sodium salt and N-(3-
lator of pro-inflammatory cytokine production and promoter dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
ofapoptosis,onburninjurieshasshownsignificantreduction (EDC) were purchased from Pierce (Rockford, IL). Anti-
of local TNF-a, IL-1b, and IL-6 expression, attenuation of rTNF-aandanti-rIL-6,bothfrompurifiedmousemonoclonal
dermal neutrophil sequestration, and a lower level of hair IgG,werepurchasedfromR&DSystems,Inc.(Minneapolis,
follicle cell apoptosis within 24 hours of the initial burn MN).Allreagentswerereconstitutedandstoredaccordingto
injury.27 Intravenous injection of semapimod, a selective themanufacturer’sinstructions.
inhibitor of macrophage activation, 1 hour after burn in a
swinemodelreduceddepthofcellularnecrosisandthrombo-
sis,resultinginfasterreepithelialization.28Thesestudieshave
MonoclonalantibodyconjugationtoHA
successfully demonstrated that early intervention of inflam-
matorymodulationcanpotentiallyhaltburnwoundprogres- The conjugation chemistry followed a method previously
sion,butdevelopingatargetedmethodforaccomplishingthis developed in our laboratory. Briefly, HA(12mg) was modi-
without significant risks of systemic complications has not fiedwithanactiveestergroup,followedbyadditionofanti-
beenachieved. rTNF-aoranti-rIL6mAb(1mg),asshowninFigure1.The
In order to achieve targeted modulation of the inflamma- coupling reaction proceeded at 4°C overnight. The product
tory microenvironment, we have conjugated TNF-a or IL-6 was dialyzed (M cutoff 300kDa, Next Group, Southbor-
W
neutralizing antibodies with high-molecular-weight hyalu- ough, MA) against pure phosphate buffered saline solution
ronicacid(HA),andappliedthesematerialstopicallyinarat (PBS)for24hourswithfourchangesofPBSsolutionat4°C.
deeppartial-thicknessburnmodel.Ratswerechosenforthis Thefinalproductconsistedofroughly1%(w/v)HAsolution.
study because it was possible to obtain commercial rat anti- Inordertoachieveahigherviscosityofsolutionthatismore
mouseantibodiesthatarespecies-matched,avoidingpotential suitable for open wound applications, conjugate solution
complications associated with immunological responses was made to contain 5% HA by mixing three parts of the
to unmatched antibodies.29 These cytokines were targeted conjugateproductwithfourpartsof8%HAsolution,andthe
becauseoftheirestablishedimportanceinburnetiology,and final concentration of the cytokine-neutralizing antibody is
antibody conjugation to high-molecular-weight polysaccha- roughly 400mg/mL.Acontrol of 5% HAsolution was also
ridesprovidesameansforinhibitinginflammatoryresponses prepared.
564 WoundRepReg(2012)20563–572©2012bytheWoundHealingSociety
Sunetal. (anti-TNF-a)-hyaluronicacidconjugates
Figure2. Ratpartial-thicknesscontactburnmodel.(A)Theburnunitwasassembledwithamodifiedsolderingunitthatprovided
heatenergyforaØ17mmbrassdisk.Inordertoprovideconsistentpressureagainstskin,theburnunitwasweighted500g,and
thebrassdiskwasconnectedtoathermometertomonitortheactualtemperatureoftheunit.(B)Arepresentativeimageofa
shaved rat skin that just underwent 10 seconds of contact with an 85°C brass disk. (C) After 24 hours, the burn area has
developedintoaneschar.(D)Theescharwassurgicallyremovedandtheunderlyingskintissuewasexposed.(E)Treatmentswere
appliedandthewoundsweredressedwithTegaderm™(3M).
Ratdeeppartial-thicknessburnmodel debride the necrotic tissue and not cause further injury, and
was done blinded to the treatment specification until after
All animal experiments were performed following the poli-
the procedure was complete. Surgical debridement is a very
cies and procedures of the Institutional Animal Care and
common technique in burn wound care to prevent infection
Use Committee at Institute of Surgical Research, Fort Sam
andaidhealingbecauseitissimpleandselective.Burndepth
Houston, TX. Rats received a single partial-thickness burn,
wasvisuallyassessedafterescharremovaltoensurethecon-
treatedwithsalinesolution,HA,(anti-TNF-a)-HA,or(anti-
sistency of the injury. The viable tissue underneath was
IL-6)-HA (n=4 for all treatments). The rats were anesthe-
exposed and the treatments were applied immediately after.
tizedthroughouttheproceduresandallsubsequenttreatments
TheprocedureisshowninFigure2B–E.
and dressing changes. Buprenorphine was given as preemp-
Ratswererandomizedfordifferenttreatmentsanddifferent
tiveanalgesia(0.1–0.2mg/kgintraperitoneal[IP]orsubcuta-
timepoints.Escharremovaldatewasdefinedasday0,atwhich
neous[SQ])andisoflurane(2–5%inO viafacemask)given
2 pointthefirst200mLtreatmentwasapplied.Thewoundswere
to effect anesthesia. Buprenorphine (0.1 to 0.2mg/kg) was
dressedwithcommerciallyavailableTegaderm™andsealed
provided at 12 and 24 hours postsurgery and continued as
with Vetbond (3M, St. Paul, MN), and remained moist
necessary upon the observation of signs of continued pain
throughout the experiments with no further loss of tissue
based on clinical assessment, using the pain/distress assess-
observedindressingchanges.Treatmentswereappliedatday
mentscoringsheets.
0,2,and4,andratswereeuthanizedondays1,4,or7providing
A commercially available soldering unit was used as a
tissue samples from rats that received 1, 2, or 3 treatments,
controlled heat source. The soldering iron was modified by
respectively. The tissues were sectioned into three: one was
weldinga17-mmdiameter,2.5mm-thickbrassdiskonthetip
fixedwith10%formaldehydeforhistologyanalysis,another
and weighted with 500g to induce sufficient pressure for
wasflashfrozenintissueembeddingmediaforfurthersection-
efficientheattransfer.Thenoncontactingsideofthediscwas
ing and immunohistochemistry, and the other was stored in
connectedtoathermalcouplertomonitortheactualtempera-
RNAlater solution (Ambion,Austin, TX) at 4°C overnight,
ture of the disk, as shown in Figure2A. Burn injuries were
followedbylong-termstorageat-80°C.
induced on the back of shaved, anesthetized adult male
Sprague-Dawleyratswithmassesrangingfrom230to250g
(n=4). The brass disc was heated to 85°C, stabilized for 2
Extractionoftissuecytokines
minutes,andtheburninjuryinflictedbyplacingthebrassdisc
ontheskinfor10seconds. TheRNAlater-preservedtissueswerecutintoapproximately
Consistencyofwounddepthwasassessedinseparatepre- 0.1gpiecesthatwerethenhomogenizedin600mLofT-PER
liminary experiments using collagen IV immunostaining to TissueProteinExtractionsolution(ThermoScientific,Rock-
verify the reproducibility of deep partial-thickness burns at ford,IL),amilddetergent,fortotalproteinextraction.Homo-
thesites(datanotshown).1,32Escharwassurgicallyremoved genates were centrifuged at 10,000rcf for 5 minutes to
following the circumference of the nonviable tissue the next remove large tissue particles. The resulting supernatant was
day using surgical scissors taking extreme care to only removedandstoredat-80°C.
WoundRepReg(2012)20563–572©2012bytheWoundHealingSociety 565
(anti-TNF-a)-hyaluronicacidconjugates Sunetal.
Totalproteinandenzyme-linkedimmunosorbent were then dehydrated using the reverse of the deparaffiniza-
assaysfromburntissue tion treatment described above prior to cover slipping. Each
PBSrinseintheprotocolwasfor3minutesatroomtempera-
Thesupernatantswerethawedandquantitativelyassayedfor
ture,withoccasionalagitation.
total protein concentrations using the Pierce BCA Protein
Assay kit (Thermo Scientific). For this assay, samples were
diluted1:20inPBS.Theassaywascarriedoutaccordingto Immunohistochemicalanalysis
thekit’sprocedurewitha1:8ratioofdilutedsampletoBCA
Theimmunostainedslideswereexaminedandimagedusinga
workingreagentandanalyzedbySAFIREmicroplatereader
LeicaDMILLEDmicroscope(Germany).TheCD68-stained
(SAFIRE, San Jose, CA) with absorbance set at 562nm.
imageswerethenevaluatedquantitativelyinablindedfashion
Enzyme-linked immunosorbent assays (ELISA) for IL-1b
by two independent investigators (MR and JP). Quantitative
concentrations from tissue extracts were done using the
analysis was performed by counting the number of immu-
respectiveQuantikineRatELISAkits(R&DSystems)usinga
nopositive cells in three matched microscope fields at 20¥
96-well plate reader. Assay samples for IL-1b were diluted
magnification.Thenumberofpositivestainedcellswasthen
accordingtoprocedureatdilutionsof1:10.Allassayswere
averaged to obtain the final results. The vimentin-stained
doneaccordingtothekitproceduresandanalyzedat562nm
images were quantified in two separate rounds of blinded
with a 549nm correction. The resulting total protein and
analysis,andtheaveragethicknessofthenecrotictissuewas
cytokine concentrations were verified by using internal con-
measured at three different regions of the burn wounds: the
trols with a known concentration of the target protein, sup-
twoedgesandthecenter.Themeasurementswerethenaver-
pliedwitheachkit.Curvefittingtothestandardswasusedto
agedforthefinalresults.
expresstheconcentrationofbothtotalproteinandcytokines
inthetissuesamplesinpg/mgprotein.
Statisticalanalysis
Histologicalandimmunohistochemicalstaining One-way analysis of variance was performed for ELISA,
vimentin measurements, and macrophage counts using
The explanted burn site specimens were mounted on glass
Minitab statistical software. Data were log transformed in
slides and embedded in paraffin, then subsequently deparaf-
order to pass a normality test for ELISAand vimentin mea-
finized with xylene followed by a graded series of etha-
surements.Dunnett’sandTukey’smethodswereusedasthe
nol solutions (100-70%). Sections were then stained with
post-tests to determine which groups were significantly dif-
Masson’s trichrome using a microwave staining protocol
ferentfromsalinecontrolandfromeachother,respectively.
(Richard-AllenScientific,Kalamazoo,MI)formorphological
assessment. The slides were then cleared and dehydrated
usingthereverseofthedeparaffinizationtreatmentdescribed
RESULTS
above prior to coverslip mounting. Following deparaffiniza-
tion, the slides were immersed in citrate antigen retrieval
buffer (10mM citric acid monohydrate, pH 6.0, Spectrum, Evaluationoftrichromestaining:grossappearanceof
Gardena,CA)thatwasthenbroughttoaboil(95–100°C)for theburnsite
20min. The buffer was allowed to cool and the slides were
Trichromeimagesoftheburntissuetreatedwithsaline,HA,
thenwashedtwiceinTRIS-bufferedsaline-Tween20(Trizma
(anti-IL-6)-HA, and (anti-TNF-a)-HA were examined to
Base,Tween20,Sigma)solution(pH7.4)andtwiceinPBS.
evaluate gross appearance of the wound and the effect of
Thetissuesectionswereencircledwithahydrophobicbarrier
treatmentsonthehealingprogression,asshowninFigure3.
penandwereincubatedinT20blockingbuffer(Pierce)for1
The apical surface of the wound bed was created by eschar
houratroomtemperatureinahumidifiedchambertoinhibit
removal1dayfollowingprimaryinjury.Newlyformedgranu-
nonspecificbindingoftheprimaryantibody.Followingincu-
lation tissue started to appear underneath the intact dermis,
bation in blocking serum, the sections were incubated in
and significant amounts of granulation tissue were observed
primary antibody, mouse anti-rat CD68 (Serotec, Raleigh,
in(anti-TNF-a)-HA-treatedwoundsonday7.Auniquelayer
NC), diluted to 1:100 in PBS, in a humidified chamber at
of tissue, stained in red, can be identified above the wound
4°Covernight.Followingtheovernightincubation,theslides
edges at later time points. This intensely stained layer of
were washed three times in PBS. Sections were then incu-
tissue,whichwaspostulatedtobethedermaltissuethathad
batedinasolutionof3%HO inmethanolfor20minutesat
2 2 undergone secondary necrosis after burn, grew significantly
roomtemperaturetoquenchendogenousperoxidaseactivity.
under most of the treatments, shown on the day 7 images,
TheslideswerewashedthreetimesinPBSbeforeincubation
except (anti-TNF-a)-HA treatment group. In spite of the
in secondary antibody, biotinylated horse anti-mouse IgG
growing necrosis, granulation tissues occupied most of the
(Vector,Burlingame,CA),diluted1:150inPBSfor1hourin
defect zone in HA-treated burn wounds, while saline- and
a humidified chamber at room temperature, and then sub-
(anti-IL-6)-HA-treated wounds have shown less granulation
jected to three more washes in PBS.The sections were then
tissueformation.
incubatedinVectastainABC(EliteABCkit,Vector)reagent
for 30 minutes in a humidified 37°C chamber, again rinsed
three times in PBS, and incubated in 4% diaminobenzadine
Vimentinimmunohistochemistryandnecrotic
substrate solution (DAB substrate peroxide kit, Vector) at
tissuequantification
roomtemperature.Theslideswererinsedinwatertostopthe
developmentofthediaminobenzadinesubstrateandcounter- Inordertoinvestigatefurthertheextentofnecrosis,vimentin
stained using Harris hematoxylin stain (Vector). The slides immunostaining was utilized to visualize viable tissues, and
566 WoundRepReg(2012)20563–572©2012bytheWoundHealingSociety
Sunetal. (anti-TNF-a)-hyaluronicacidconjugates
Figure3. Masson’strichromeimagesoftheedgesoftheburnsitearrangedaccordingtotreatmentsandtimepoints.Allimages
areat4¥magnification.Clearexcisionlinesarepresentintheday1images,andsomeevidenceofmicrothrombosiscausedby
theinitialburnisvisible,especiallyinimagesofsalinecontrols.Granulationtissuesstarttoappear4daysafterescharremoval
in the deep dermal region. By day 7, granulation tissue has grown to fill most of the defect space in HA- and (anti-TNF-a)-HA-
treatedsites,whilesalineand(anti-IL-6)-HA-treatedwoundsitesseemtodisplayslowergranulationtissueformation.Onday4,
alayerofnecrotictissueonthesurfaceofthewoundsisobservedinmostofthewoundsites,andonday7,necrosishasgrown
significantlyparticularlyinsaline,HA,and(anti-IL-6)-HA-treatedburnsites.Inseveralinstances,thislayerofnecrosisisseparated
fromtheunderlyinghealthytissueduringhistologyprocess.(*necrosis;#granulationtissue).
WoundRepReg(2012)20563–572©2012bytheWoundHealingSociety 567
(anti-TNF-a)-hyaluronicacidconjugates Sunetal.
Figure4. VimentinIHCstained(pink)burnwoundimagesat4¥magnificationarrangedaccordingtotreatmentsandtimepoints.
Dashedlinesrepresenttheseparationofnecrosisandviabletissue.Onday1,thereisnovisibleevidenceofnecrosis,duetothe
excisionofescharthedaybefore.Onday4,alayerofnon-stainedtissueisobserved,suggestingthatwoundprogressionhas
penetratedintothedeeperlayerofskin.Onday7,thesizeofthenecrosisissignificantlysmallerin(anti-TNF-a)-HA-treatedwound
sitesthanthatunderothertreatments.
nonstainedregionswereidentifiedasnecrotic.Vimentinisa approximate separation of healthy dermis and nonviable
cytoskeletalproteinthatNanneyandcoworkersestablishedas tissue.Therewasnovisualevidenceofnecrosisintheimages
amarkerforviabletissueatburnsites.33InFigure4,adashed of day 1 samples, due to the eschar removal the day before.
line was carefully drawn in each image to indicate the However, the growing depth of nonviable tissues is clearly
568 WoundRepReg(2012)20563–572©2012bytheWoundHealingSociety
Sunetal. (anti-TNF-a)-hyaluronicacidconjugates
Figure5. Thicknessofnecrotictissueonday7.(Anti-TNF-a)-
HA-treatedburnsiteshavesignificantlythinnernecroticzones Figure6. IL-1b concentration in the local burn tissue. IL-1b
thantheothercompositionstreatedsites.HAtreatmentalso concentrationpeaksatday1,andattenuateonday4insaline-
appears to reduce necrotic conversion comparing to saline treated controls. (Anti-TNF-a)-HA-treated wound sites show
treatedcontrols.(§p<0.001). similar trend of IL-1b response, while the IL-1b levels are
significantly lower than those in saline-treated sites through-
out the experimental period. HA initially inhibits the IL-1b
observableintheimagesofday4andday7inalltreatment expression on day 1, and the level of IL-1bis kept about the
groups,except(anti-TNF-a)-HA. sameleveluntiltheendoftheexperimentalperiod,suggest-
Thethicknessofnecrotictissuewasmeasuredmanuallyby ing that HA has limited effect in suppressing inflammation.
two personnel on blinded samples, and average thickness of (Anti-IL-6)-HA-treated wounds sites demonstrate no specific
three different locations, two on each of the edges and one trendstoIL-1bexpression.(#p<0.05).
onthecenteroftheburn,oneachtissueslidewasreportedin
Figure 5. On day 7, the difference in thickness of necrotic
zonesfrom(anti-TNF-a)-HA-treatedgroupwassignificantly
the numbers of positively stained cells and nonstained cells
lowerthaninanyothergroup(p=0.001).
were counted in a blinded manner by three independent
personnel. The results from quantification are shown in
Figure7E. (Anti-TNF-a)-HA-treated wound sites have
Tissuecytokineconcentration
clearly demonstrated a reduction of macrophage numbers
Proteins in the local burn tissue were extracted from throughout the experimental duration (day 1: p<0.001, day
RNAlater-preservedtissue.ELISAassayswereperformedto 4:p<0.05,day7:p=0.005).Interestingly,HA-treatedsites
measure the tissue concentration of IL-1b in pg/mL, which did not have measurably different numbers of CD68+ cells
was then converted to pg IL-1b/mg total protein. IL-1b is comparedtosalinetreatment.Thiscontrastswiththeresults
centrally involved in inflammatory responses, making it a observedinthetreatmentofincisionalwoundswithHA,for
good marker for the overall state of inflammation following which we observed a significant increase in numbers of
burn injury.34 Measured IL-1b concentrations are shown in recruited macrophages.30 In addition, (anti-IL-6)-HA-treated
Figure6. IL-1b expression was significantly suppressed by injurysitesdidnotdemonstrateanyobservableeffectonthe
(anti-TNF-a)-HA treatments compared to saline on day 4 numberofCD68+cellsattheburnsite,suggestingthatneu-
(p<0.05) and day 7 (p<0.05). IL-1b concentrations trend tralization of this cytokine did little to reduce the state of
lower in (anti-TNF-a)-HA-treated burn wounds compared inflammationatburnsites.
toHAtreatmentonday4and7aswell,withHAtreatments
alsoshowingatrendtowarddecreasedIL-1blevelscompared
tosalineonlyonday1.However,(anti-IL-6)-HAtreatments DISCUSSION
didnothavesignificanteffectonIL-1bexpression,beyonda
Inthisresearch,wecomparedtheeffectsoflocallyinhibiting
lessrobustandnonsignificantdecreasecomparedtosalineon
TNF-aandIL-6inaratpartial-thicknessburnmodel,andthe
days1and4,suggestingthatneutralizationofIL-6hadlittle
resultsindicatethatmodulatingtheactivitiesofacytokineat
effectontheoverallinflammatorymicroenvironment.
theapexofthesignalingcascade(TNF-a)wasmoreeffective
in reducing necrosis than targeting a downstream mediator
(IL-6)inthecontextofburnwoundhealing.
Macrophageinfiltration
There are conflicting reports on the levels of TNF-a fol-
Inordertoinvestigatefurthertheanti-inflammatoryeffectsof lowingburns,primarilybecausethebaselineserumlevelsin
the cytokine-neutralizing HAconjugates, paraffin-embedded healthy patients are low (< 1pg/mL) and only increase by a
tissue sections were stained for a pan-macrophage marker, factorof5inthefirstweekfollowingburns.21Nevertheless,it
CD68.30 High-power images (20¥) were taken at the subcu- is known thatTNF-a is a potent stimulator of inflammation
taneous regions at the wound edges, shown in Figure7, and andoccupiesacentralpositionintheinflammatorycascade,
WoundRepReg(2012)20563–572©2012bytheWoundHealingSociety 569
(anti-TNF-a)-hyaluronicacidconjugates Sunetal.
Figure7. CD68IHCstainedburnwoundimagesofday4treatedwith(A)saline,(B)HA,(C)(anti-TNF-a)-HA,and(D)(anti-IL-6)-HA
at 20¥ magnification and the result of CD68+ cell counts. The arrows indicate the positively stained cells and (anti-TNF-a)-HA-
treatedwoundsexhibitgrossreductionofCD68staining,andthequantificationresultverifiesthesignificantreductionofCD68+
cellnumbersin(anti-TNF-a)-HAtreatmentcomparingtoalltheothertreatmentsatallthreetimepoints.HA-treatedwoundsshow
monotonicdecreaseofCD68+cellsfromday1to7withoutstatisticalsignificance.Theothertwotreatmentsshowhighlevelof
CD68+cellsthroughouttheexperiments.(#p<0.05;*p<0.005;§p<0.001).
beingamongthefirstinflammatoryresponsemoleculestobe Pathogenic inflammation leads to increased blood vessel
up-regulatedfollowinginjuryandstimulatingtheproduction permeability and increased levels of blood vessel damaging
ofnumerousdownstreammediatorsthataremeasuredinburn ROS.22,41Degenerationofbloodvesselsandcapillariesoccurs
sites.36Someofthemostsuccessfuldrugscurrentlymarketed through several mechanisms in burn wound etiology related
arebasedonTNF-ainhibition35andareusedtotreatabroad to pro-inflammatory mediators such as TNF-a and IL-6.4,42
rangeofinflammatoryconditions,suchaspsoriasis,rheuma- TNF-a is considered to be a more potent pro-inflammatory
toid arthritis, and inflammatory bowel disease. That each of cytokinethanIL-6,though,andiscapableofinducingawide
these widely varying disease states can be treated through varietyoffactorsthatleadstosubsequentpathology.Thiswas
inhibitionofthesamesignalingproteinspeakstothefunda- observed histologically, where blood vessels had a dramati-
mental importance of TNF-a in inflammatory processes. callydifferentvisualappearanceafterinjury,suggestingthat
Effects of mediating inflammation in burn wounds, specifi- these are signs of vasodilation or blood clots. Furthermore,
cally through local TNF-a inhibition, have yet to be thor- damage to blood vessels appeared to be minimized by the
oughly explored. Our data support a first insight into yet treatmentwith(anti-TNF-a)-HAbutnotby(anti-IL-6)-HA.
another disease state that can potentially be treated through Cellular contributions to wound progression include
inhibitionofTNF-a. neutrophil and macrophage dysfunctions. Neutrophils are
In contrast to TNF-a, IL-6 increases measurably in burn attractedtotheinjurysitewithincreasingexpressionofadhe-
patients.34 Signaling by IL-6 can support inflammatory sionmoleculesstimulatedbypro-inflammatorycytokinesand
responses,suchasrecruitmentofmononuclearcells,andinhi- abundant chemotactic signals in the wound.43 Macrophages
bition of T-cell apoptosis and T differentiation.37 It is also are hyperactivated post-burn, and release a host of pro-
reg
known to play a central role in anti-inflammatory signaling, inflammatory mediators, which also have been suggested
in some settings even inhibiting TNF-a19,38 and stimulating to lead to the down-stream immune-suppression state.44,45
interleukin-1 receptor antagonist and interleukin-10 produc- Several studies have differentially inhibited the interaction
tion.39,40Itisfurtherdownstreaminthesignalingcascadeand between cell adhesion molecules and integrins and demon-
thoughttobeabridgebetweeninflammationandhealing.The strated that minimizing neutrophil and macrophage infil-
pleiotropicnatureofIL-6mayprovideonlyasmalltherapeu- tration reduces wound progression.46,47 TNF-a is a potent
ticwindowtowhichinhibitionofthecytokinewouldproduce activator of endothelial cells to induce expression of cell
an effective anti-inflammatory effect. In light of this, it may adhesionmolecules,andmodulatingtheactivitiesofTNF-a
not be surprising that we saw no measurable effect in IL-1b has shown to decrease macrophage infiltration,30 which sup-
levels and amounts of necrotic tissue in (anti-IL-6)-HA- ports our data indicating lower CD68 macrophage counts in
treatedwounds. (anti-TNF-a)-HA-treatedrats.
570 WoundRepReg(2012)20563–572©2012bytheWoundHealingSociety
Sunetal. (anti-TNF-a)-hyaluronicacidconjugates
As previously mentioned, modulating acute inflammation deathindeeppartialthicknessburnsbycoexpressionanalysisof
usingp38MAPKinhibitorwasshowntobeeffectiveinreduc- TUNELandFas.Surgery2006;139:854–5.
ingcellularapoptosis,27butthelong-termeffectofsuchtreat- 4. Singh V, Devgan L, Bhat S, Milner SM. The pathogenesis of
ment was unclear. Singer and coworkers have also burnwoundconversion.AnnPlastSurg2007;59:109–15.
demonstrated30%reductionindepthofhairfolliclenecrosis 5. Kao CC, Garner WL. Acute burns. Plast Reconstr Surg 2000;
aftersystemicdeliveryofsemapimodinapigmodel,28while 101:2482–93.
systemicdeliveryofcurcuminhasshownapositiveeffectin 6. ArtursonG.Pathophysiologyoftheburnwoundandpharmaco-
reducingburnwoundprogressionby50%inaratcombburn logical treatment. The Rudi Hermans Lecture, 1995. Burns
model7daysafterinitialburn.48However,systemicdelivery 1996;22:255–74.
of anti-inflammatory agents in a burn patient increases the 7. ShuppJW,NasabzadehTJ,RosenthalDS,JordanMH,FidlerP,
riskofsecondarycomplications,andlocaldeliveryofTNF-a JengJC.Areviewofthelocalpathophysiologicbasesofburn
neutralizing agent from a HA delivery vehicle has reduced woundprogression.JBurnCareRes2010;31:849–73.
necrosisbyupto70%post-burninaratmodel.Inadditionto 8. Piguet PF, Grau GE, Vassalli P. Tumor necrosis factor and
providingameansoflocaldelivery,webelievethattheremay immunopathology.ImmunolRes1991;10:122–40.
besynergisticeffectsonhealingbetweenHA,thedegradation 9. Diegelmann RF, Evans MC. Wound healing: an overview of
products of which are known to function as damage- acute,fibroticanddelayedhealing.FrontBiosci2004;9:283–9.
associated molecular patterns and theTNF-a antibody.This 10. HortonJW.Freeradicalsandlipidperoxidationmediatedinjury
willbeexploredfurtherinsubsequentstudies. inburntrauma:theroleofantioxidanttherapy.Toxicology2003;
Localized neutralization of TNF-a with antibody- 189:75–88.
polysaccharideconjugatesisanappealingstrategyforlocally 11. YoungCN,KoepkeJI,TerleckyLJ,BorkinMS,BoydSavoyL,
controlling the inflammatory processes associated with burn TerleckySR.Reactiveoxygenspeciesintumornecrosisfactor-
progression. Our results suggest that inhibition of a central, alpha-activated primary human keratinocytes: implications for
upstream mediator of inflammation is more effective than psoriasis and inflammatory skin disease. J Invest Dermatol
downstreamcytokines,suchasIL-6.Thedosesusedinthese 2008;128:2606–14.
studies followed the antibody doses used in our previous 12. CamachoM,Lopez-BelmonteJ,VilaL.Rateofvasoconstrictor
studyonreducinginflammationinincisionalwounds.30While prostanoids released by endothelial cells depends on
many of the systemic side effects associated with TNF-a cyclooxygenase-2expressionandprostaglandinIsynthaseactiv-
inhibitors can be avoided with this approach, there is still ity.CircRes1998;83:353–65.
clearlyanincreasedriskforinfectionatinjurysites,although 13. Koide M, Kawahara Y, Tsuda T, Yokoyama M. Cytokine-
none have been observed in either incisional or burn studies inducedexpressionofaninducibletypeofnitricoxidesynthase
thusfar.Understandingthedose–responsebehavioratafun- geneinculturedvascularsmoothmusclecells.FEBSLett1993;
damental level will be critical for evaluating the potential 318:213–7.
clinicalefficacyofthesematerials. 14. O’NeillLA,LewisGP.Interleukin-1potentiatesbradykinin-and
TNFalpha-inducedPGE2release.EurJPharmacol1989;166:
131–7.
ACKNOWLEDGMENTS 15. Gao X, Xu X, Belmadani S, Park Y, Tang Z, Feldman AM,
Chilian WM, Zhang C. TNF-alpha contributes to endothelial
The authors acknowledge support from the Armed Forces
dysfunction by upregulating arginase in ischemia/reperfusion
Institute of Regenerative Medicine (W81XWH-08-2-0032)
injury.ArteriosclerThrombVascBiol2007;27:1269–75.
andtheNationalInstitutesofHealth(R43GM085897).NRW
16. Johns DG, Webb RC. TNF-alpha-induced endothelium-
gratefully acknowledges support from a 3M Non-Tenured
independent vasodilation: a role for phospholipase
Faculty Grant, the Wallace H. Coulter Foundation Transla-
A2-dependent ceramide signaling. Am J Physiol 1998; 275:
tional ResearchAward program, and the Heinz Endowment
H1592–8.
(C1747). REPand NMD wish to thank the Howard Hughes
17. ZhangC,XuX,PotterBJ,WangW,KuoL,MichaelL,Bagby
Medical Institute for support through the Undergraduate
GJ,ChilianWM.TNF-alphacontributestoendothelialdysfunc-
Science Education Program at CMU.52005865 The authors
tion in ischemia/reperfusion injury. Arterioscler Thromb Vasc
also wish to thank Dr. Yoram Vodovotz and Dr. Lillian
Biol2006;26:475–80.
Nanney for helpful discussions, and John Walters for help
18. Kishimoto T. The biology of interleukin-6. Blood 1989; 74:
withstatisticalanalysis.
1–10.
Conflict of Interest: NRW has started a company to com-
19. Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC,
mercialize aspects of this research and wishes to disclose a
DinarelloCA.Correlationsandinteractionsintheproductionof
potentialconflictofinterest.
interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in
humanbloodmononuclearcells:IL-6suppressesIL-1andTNF.
Blood1990;75:40–7.
REFERENCES
20. NishimotoN,KishimotoT.Interleukin6:frombenchtobedside.
1. JacksonDM.Thediagnosisofthedepthofburning.BrJSurg NatClinPractRheumatol2006;2:619–26.
1953;40:588–96. 21. FinnertyCC,HerndonDN,PrzkoraR,PereiraCT,OliveiraHM,
2. GravanteG,PalmieriMB,EspositoG,DeloguD,Santeusanio Queiroz DM, Rocha AM, Jeschke MG. Cytokine expression
G, Filingeri V, Montone A. Apoptotic death in deep partial profile over time in severely burned pediatric patients. Shock
thicknessburnsvs.normalskinofburnedpatients.JSurgRes 2006;26:13–9.
2007;141:141–5. 22. BifflWL,MooreEE,MooreFA,PetersonVM.Interleukin-6in
3. Gravante G, Filingeri V, Delogu D, Santeusanio G, Palmieri theinjuredpatient.Markerofinjuryormediatorofinflamma-
MB, Esposito G, Montone A, Sconocchia G. Apoptotic cell tion?AnnSurg1996;224:647–64.
WoundRepReg(2012)20563–572©2012bytheWoundHealingSociety 571