cell biochemistry and function

Cell Biochem Funct 2008; 26: 197–204.

Published online 4 October 2007 in Wiley InterScience

(www.interscience.wiley.com) DOI: 10.1002/cbf.1432

Effect of oleic and linoleic acids on the inflammatory phaseof wound healing in rats

Leonardo M. Pereira 1,4, Elaine Hatanaka 1*, Edgair F. Martins 1, Flavia Oliveira 1,Edson A. Liberti 2, Sandra H. Farsky 3, Rui Curi 1 and Tania C. Pithon-Curi 1,5

1Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Brazil2Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Brazil3Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Brazil4Faculty of Health Sciences, Methodist University of Piracicaba, SP, Brazil5Cruzeiro do Sul University, Sao Paulo, Brazil

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatoryresponse of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitrowas investigated. Awound in the dorsal surface of adult rats was performed and fatty acids were then topically administered.Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the woundswere increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascularpermeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of thenecrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growthfactor-alpha (VEGF-a) and interleukin-1beta (IL-1b) by neutrophils incubated in the presence of oleic and linoleic acid wasobserved. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant ininflammation 2 alpha/beta (CINC-2a/b). This pro-inflammatory effect of oleic and linoleic acids may speed up the woundhealing process. Copyright # 2007 John Wiley & Sons, Ltd.

key words—fatty acids; oleic acid; linoleic acid; wound healing; neutrophils; inflammation

INTRODUCTION

Cutaneous wound healing is a highly coordinatedprocess that restores skin integrity. This processinvolves production of soluble mediators and extra-cellular matrix components by resident cells (kerati-nocytes, fibroblasts, endothelial cells, nerve cells), andinfiltrating leukocytes. The healing process has fourphases: blood clotting, inflammation, new tissueformation and tissue remodelling.1–3

Local vasodilatation, fluid leakage into the extra-vascular space and blocking of lymphatic drainage

*Correspondence to: Dr E. Hatanaka, Departamento de Fisiologia eBiofısica, Instituto de Ciencias Biomedicas, Universidade de SaoPaulo, Avenida Prof. Lineu Prestes, 1524, 05508-900, Butanta, SaoPaulo, SP, Brasil. Tel: 55 11 3091 7245. Fax: 55 11 3091 7285.E-mail: ehata@usp.br

Copyright # 2007 John Wiley & Sons, Ltd.

produce three signs of inflammation that includeredness, swelling and heat. The fourth sign, pain, iscaused by distension of tissue spaces due to swellingand pressure, and also through nociceptor receptors.4

These events attract neutrophils and monocytes to theinflamed area. Neutrophils destroy bacteria andremove dead and dying cells from tissue spaces.Neutrophils and macrophages play a key role in theinflammation phase of wound repair by secretingcytokines and a great variety of growth factors.5–7

Fatty acids have important roles in immune andinflammatory responses.8–10 They are importantcomponents of the cell membrane. The nature ofthe fatty acids in membrane phospholipids contributesgreatly to the fluidity of the membrane and this isbelieved to play a role in regulating the activity ofmembrane proteins.11,12 Membrane phospholipids arealso a source of second messenger molecules, such as

Received 12 March 2007Revised 23 May 2007Accepted 1 June 2007

198 l. m. pereira ET AL.

diacylglycerol (DAG), phosphatic acid, inositol1,4,5-triphosphate, ceramide and arachidonic acid(AA) that are involved in activation of signallingpathways from the membrane. Some fatty acids aresubstrates for the synthesis of molecules such asprostaglandins (PG), thomboxanes (TX) and leuko-trienes (LT).13,14

Essential fatty acids (EFA), such as linoleic acid,have been employed for the prevention and treatment ofpressure ulcers.15 EFA are required to ensure epidermalintegrity and to maintain the water barrier in the skin.Linoleic acid is a precursor of AA and PG in theepidermis.16 The deficiency of EFA impairs cutaneouswound healing in mice, rats17–20 and infants.21

Research on EFA and wound healing is primarilydescriptive and restricted to case studies. There are onlyfew studies of single fatty acids to treat wounds.15–20

Fatty acids have been shown to control the functions ofneutrophils. As mentioned above, these cells play a keyrole in the healing process by releasing variouscytokines. Neutrophils typically infiltrate inflammatorylesions early and massively.22,23 Therefore, the effect offatty acids on neutrophils may play an important role inthe healing process, in particular, on the control ofangiogenesis and cell proliferation.The aim of this study was to investigate the effect of

linoleic and oleic acids on the inflammatory phase ofthe wound healing process in rats through in vivo andin vitro experiments. The effect of topically adminis-tered linoleic and oleic acids on the healing process,24 h after cutaneous wounding in rats, was investi-gated. The following parameters were measured in thewounded tissue: wet weights, vascular permeability,total protein and DNA contents, thickness of the outerlayer with necrosed cells and neutrophil infiltration towounded area. Production of vascular endothelialgrowth factor-alpha (VEGF-a), interleukin-1beta(IL-1b), rat cytokine-induced neutrophil chemoat-tractant in inflammation 2 alpha/beta (CINC-2a/b)and tumor necrosis factor-alpha (TNF-a) by ratneutrophils cultured in the presence of oleic andlinoleic acids was also evaluated.

MATERIALS AND METHODS

Materials

Bovine foetal serum, Hepes, penicillin, RPMI 1640medium supplemented with L-glutamine, sodiumbicarbonate, oleic and linoleic acids, oyster glycogenand streptomycin were supplied by Sigma ChemicalCo. (St. Louis, MO, USA). Stock solutions of fattyacids were prepared in ethanol. For use, the stock

Copyright # 2007 John Wiley & Sons, Ltd.

solution was dissolved in appropriate vehicles (PBS orRPMI medium), according to the experiments. Thefinal concentration of ethanol in the culture mediumdid not exceed 0.05%. This concentration of ethanolwas not toxic to the cells as also observed in ourprevious studies.24,25

Animals

Male Wistar rats weighing 180� 20 g were obtainedfrom the Department of Physiology and Biophysics,Institute of Biomedical Sciences, University of SaoPaulo, Brazil. The rats were maintained at 238C undera light: dark cycle of 12:12 h. Food and water weregiven ad libitum. The Animal Care Committee ofthe Institute of Biomedical Sciences approved theexperimental procedure of this study.

Wound healing preparation

The animals were anaesthetized with an intraperito-neal injection of 65mg/kg thiopental (Abbot, SaoPaulo, Brazil). A tricotomy was made in the dorsalsurface of the rats using a blade. The dorsal surfacewas rinsed with ethanol at 70%. The wound (one perrat) consisted of an excision of 1.5 cm� 2.5 cm fullthickness area of skin from the underlying fascia.Following surgery, the wound was immediatelytreated with topical application of 300ml of purefatty acids or the same volume of sterile PBS solution.The rats were kept in individual cages under awarming lamp and allowed to recover fully fromanaesthesia. The rats were divided into three groups:control (C) and linoleic acid (L) and oleic acid (O)treated.

Treatment

To investigate the in vivo effect of linoleic and oleicacids on the inflammatory phase of wound healingprocess, a topical and single dose of pure oil (98–99%purity) composed of linoleic or oleic acid (300ml)(LM Farma, Sao Jose dos Campos, Brazil) wereapplied immediately after the surgery. This volumefully covered the wound healing area. The controlgroup was treated with the same volume of sterilePBS.

Morphometric analysis

The wound healing area was measured immediatelyafter surgery and again 24 h later. The area wasphotographed (digital camera MVC-FD97 FD Mavica,

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DOI: 10.1002/cbf

effect of fatty acids on wound healing 199

SONY) concomitantly. The surgery areawas calculatedby using a paquimeter of 0.01mm precision (VernierCaliper 6, LEEtools1, USA). The height (H)(l) andwidth (L)($) were determined and the area (A)calculated by the formula: A¼H�L. The areasmeasured 24 h after the injury were compared withthose determined immediately after surgery in thecontrol (C), linoleic acid (L) and oleic acid (O) treatedgroups.

Determinations of the wound wet weight

Twenty-four hours after the injury, the animals wereanaesthetized as described above. The injured areawas surgically removed and the subcutaneous layerand the tissue surrounding were dissected. Thesamples were delimited by the edges of the woundand the layer most superficial of the skin inregeneration. The wet weight of the wounds was thendetermined.

Vascular permeability determination

Vascular permeability changes were evaluated by theEvans blue (EB) dye extravasation method. The EB(25mg per ml in 0.9% NaCl; 40mg/kg) was injectedinto the penian vein of the rats immediately after thesurgery, and 24 h later. Then the animals were killedby decapitation and exsanguinated during 15min. Thewounded area was removed and the concentration ofEB was determined after extraction using formamide(4ml per g) at 368C. The concentration of EB wasdetermined by spectrophotometry at 620 nm wave-length (SPECTRA MAX Plus, Sunnyvale, USA).

Total protein determination

The total protein content of the tissue was deter-mined after surgical removal of the wound healingtissue.26 The samples were homogenized in salineusing an Ultra-Turrax T8 S8N-5G homogenizer(IKA1, Staufen, Germany). The samples were thencentrifuged at 211g for 30min at 48C. The proteinconcentration was determined by spectrophotometryusing bovine serum albumin as standard (BSA) at awavelength of 595 nm (SPECTRA MAX Plus,Sunnyvale, USA).26

Determination of DNA content usingdiphenylamine

The colour reaction of deoxyribose with diphenyla-mine was used for determination of DNA content. The

Copyright # 2007 John Wiley & Sons, Ltd.

tissue samples were homogenized in HCl 2N in a1:5 ratio (g/ml) using Ultra-Turrax T8 S8N-5Ghomogenizer (IKA1, Staufen, Germany). The hom-ogenate was warmed in a water-bath at 758C for30min to cause hydrolysis of the DNA and thencentrifuged at 845g for 20min at 48C. The supernatant(0.5ml) was added to 1ml of diphenylamine reagent(prepared as follows: 1.5 g diphenylamine; 100mlacetic acid and 1.5ml sulfuric acid) and boiled in awater-bath for 10min. The DNA concentration wasdetermined by spectrophotometry at 595 nm (SpectraMax Plus, Sunnyvale, USA). For the standardcurve, DNA from calf thymus (Sigma, St. Louis,USA) was dissolved in sodium hydroxide solution at0.05–0.4mg/ml concentrations.

Microscopic analysis

After 24 h of the treatment of the wounds, tissuesamples were collected and kept in Bouin reagent(75% picric acid, 25% formol and 5% acetic acid) for96 h. The samples were then washed in water for 12 hand then submitted to dehydration (alcohol at 70%,80%, 90%, 95% and absolute) being diaphanized byimmersion in xylol and enclosed in paraffin. Twelveserial transverse slices with 6mm thickness weretreated with haematoxylin eosin (HE) solution to stainthe nuclei of the cells. The thickness of the mostsuperficial edge containing necrotic cells was deter-mined in micrometers, using the KS-300 ImagingSystem program for histological analysis (Zeiss,Hallbergmoos, Germany). The values are presentedas mean and standard error means of five measure-ments.

Air pouch assay and exudate collection andprocessing (neutrophil migration analysis)

Rat skin air pouches were produced according toFarsky et al.27 A volume of 20ml sterile air (usingfluoropore filters, 0.22 pm) was injected into thesubcutaneous tissue of the back trunk of rats underanaesthesia. Seven days later an additional volume of10ml sterile air was then injected, and on the 8th day,1ml of a solution of oleic acid or linoleic acid(100mM) in sterile PBS was injected into the pouchunder anaesthesia and aseptic conditions. Stocksolutions of fatty acids in ethanol were dissolved inPBS. Negative control animals received 1ml of sterilePBS, and positive controls received 1ml of sterile PBSplus chemotactic factor N-formyl-Met-Leu-Phe(fMLP (10 nM)) through the same route.

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200 l. m. pereira ET AL.

Four hours after the injection of the fatty acids, theanimals were killed by decapitation and the inflam-matory exudate was collected after washing theintraperitoneal cavity with 20ml sterile PBS. Thesuspension was centrifuged at 500g for 10min at�48C. After that, the cells were suspended in 1mlPBS and the number of cells was determined using aNeubauer chamber.

Rat leukocyte preparation

Adult male rats were killed by decapitation withoutanaesthesia. Neutrophils were obtained by intraper-itoneal (i.p.) lavage using 40ml sterile PBS, 4 h afterthe i.p. injection of 20ml sterile oyster glycogensolution (Sigma, Type II) at 1% in PBS. Theprocedures were performed under endotoxin-freeconditions. The number of viable cells (>98%) wascounted in a Neubauer chamber using a lightmicroscope (Nikkon, Japan) and Trypan blue solutionat 1%.

TNF-a, IL-1b, CINC 2a/b and VEGF-a assay

Neutrophils (2.5� 106 cells/ml) were suspended inRPMI1640 medium and supplemented with 0.3 g/Lglutamine, 2.32 g/L Hepes, 2 g/L sodium bicarbonate,100mg/ml streptomycin, 100UI/ml penicillin and10% foetal serum and cultured at 378C and 5%CO2, inthe absence/presence of oleic acid or linoleic acid (5,25, 50, 100 and 200mM). Stock solutions of fatty acidsin ethanol were dissolved in RPMI 1640 medium. Thefinal concentration of ethanol in the culture mediumdid not exceed 0.05%. This concentration of ethanolwas not toxic to the cells. Control neutrophils werecultured in RPMI 1640 medium in the presence ofethanol (0.05%). After 4 or 18 h of cell culture, thesupernatants were collected and frozen at�808C untilcytokine determination by ELISA (Quantikine, R&DSystem, Minneapolis, MN, USA). IL-1b, CINC 2a/band VEGF-a were determined in the supernatant ofneutrophils (2.5� 106cells per ml) cultured for 18 h,

Table 1. Total area of the wound, wet weight of the wound tissue, neut

Control

Total area (cm2) 4.5� 0.Wet weight (g) 0.62� 0.Total protein (mg/g fresh weight tissue) 39.7� 1.DNA content (mg/g fresh weight tissue) 13.1� 0.Neutrophils count (number) 10.80� 0.

The values are presented as mean�SEM of eight rats.�p< 0.05 for comparison with the control group.

Copyright # 2007 John Wiley & Sons, Ltd.

whereas TNF-awas assayed in cells cultured for 4 h inabsence or presence of the fatty acids.

Statistical analysis

Statistical analysis was carried out by comparing thecontrol group (C) with the linoleic acid (L) and oleicacid (O) groups. Comparison between the linoleic acidand oleic acid groups was carried out by ANOVAusing the post-hoc Student-Newman-Keuls MultipleComparisons test and there was no difference betweenthe two groups. In addition, Dunnett’s test (INStat;Graph Pad Software, San Diego, CA, USA) was usedfor comparison between the treated and the controlgroups. The significance level was set at p< 0.05.

RESULTS

The inflammatory phase involves vascular and cellularevents and is best characterized by oedema, erythema,marked increase of blood supply and influx ofneutrophils. This phase occurs in the first hours(24–72 h) after the injury. To investigate in vivo theeffect of linoleic and oleic acids on the inflammatoryphase of wound healing process we analyzed the (i)total area and mass of the wound tissue treated withlinoleic or oleic acids, (ii) total protein and DNAcontents of the tissue samples after treatment with thefatty acids, (iii) vascular permeability and neutrophilmigration to the injured area. The topical treatment ofthewounds with oleic and linoleic acids was comparedwith the control group that received PBS by the sameroute.

There was no difference between the treatmentswith oleic and linoleic acids and the control groupconcerning the total area of thewounds (Table 1). Bothfatty acids however led to an increase of the woundhealing tissue mass as compared to the control group.The wounds topically treated with linoleic and oleicacids showed an increase of approximately 32% in wetweight (Table 1).

rophils counting, total protein and DNA content of the wound tissues

Oleic acid Linoleic acid

28 4.3� 0.42 5.1� 0.3404 0.82� 0.05� 0.83� 0.05�

00 41.2� 2.31 48.1� 2.41�

71 13.6� 0.82 15.3� 0.81�

65 24.6� 1.80� 19.33� 2.16�

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effect of fatty acids on wound healing 201

The treatment with linoleic acid caused a significantincrease of the protein (by 21%) and DNA (by 17%)contents compared with the control group (Table 1).The treatment with oleic acid had no significant effect.

The treatments with linoleic or oleic acids had noeffect on vascular permeability of the wounded area ascalculated on the wet weight basis. However, thetreatment with both fatty acids led to an increase in thenumber of neutrophils that migrated to the injuredarea. Linoleic and oleic acids caused an increase of79% and 126%, respectively, in the proportion ofneutrophils in relation to total cell number of thewounded tissue compared with the control group(Table 1).

To validate the hypothesis that fatty acids caninduce neutrophil migration to awound site we studiedthe neutrophil influx into air pounches in rats treated ornot with oleic and linoleic acid. The chemotacticfactor fMLP was used as a positive migration controland PBS solution was used as a negative control. Thenumber of neutrophils that migrated to air poucheswas determined 4 h after the injection of PBS, PBSplus fMLP and PBS plus oleic or linoleic acids(Figure 1). A significant neutrophil influx into thepouches was induced by both fatty acids.

Ideally, a healthy, well-perfused wound willdemonstrate a red and granular bed. A wound withabundant fibrotic or necrotic tissue or a wound with adry desiccated appearance may indicate a poorprognostic. Here we studied the thickness of thenecrotic cell layer after treatment of the wound witholeic and linoleic acids. For this purpose, fiveperpendicular random measures of the superficialedge of the wound containing necrotic cells weremade. The animals treated with linoleic or oleic acids

LOfMLPcontrol

20

40

60

*

*

***

mig

ratio

n (x

104 c

ells

)

Figure 1. Cellular influx into air pouches after injection of oleic orlinoleic acids (100mM). fMLP (10 nM) was used as a positivecontrol. The values are presented as mean�SEM of five animalsper group. �p< 0.05, ���p< 0.001 for comparison between thetreatments with fatty acids and control as indicated by ANOVAand Dunnett’s test

Copyright # 2007 John Wiley & Sons, Ltd.

showed a 60% decrease in the thickness of the necroticcell layer compared with the control group (Figure 2).Neutrophils regulate the initial phase of the wound

healing process and orchestrate the subsequent phasesthrough: (i) migration to the inflammatory focus; (ii)phagocytosis of cellular debris and microorganisms;(iii) release of pro-inflammatory cytokines, chemo-kines and angiogenic/growth factors, (iv) productionof reactive oxygen species. These events are closelycorrelated and depend in some instance of signallinginitiated by cytokines and growth factors. Herein westudied in vitro the release of TNF-a, IL-1b, CINC 2a/b and VEGF-a by neutrophils treated in vitro witholeic and linoleic acids. A dose-dependent increase ofVEGF-a and IL-1b production by neutrophils treatedwith oleic and linoleic acids was observed (Figure 3).Oleic acid was able to stimulate also the production ofCINC-2a/b by incubated neutrophils. These fattyacids did not modify TNF-a production by neutrophilsin the conditions used.

Figure 2. Thickness of the necrotic cell edge of the wound.histological analysis of tissue treated with haematoxylin esolution (left column). Necrotic cells are shown in the right coluthe image was obtained using the Imaging System program.values represent the mean�SEM of eight animals per gr���p< 0.001 for comparison between control and treatmentthe linoleic and oleic acids, using Dunnett’s test. Groups: con(C), linoleic acid (L) and oleic acid (O)

Cell Biochem Funct 2008; 26: 197–

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Theosinmn;Theoup.withtrol

204.

/cbf

dicA cieloniL dicA cielOR= 0.91; p=0.01

200150100500

0,6

0,8

1,0

VE

GF

(ng

/mL)

oleic acid (µM)

R=0.84; p= 0.03

200150100500

0,4

0,5

0,6

VE

GF

(ng

/mL)

linoleic acid (µM)

R= 0.93; p= 0.005

200150100500

0,2

0,4

0,6

0,8

CIN

C 2

α/β

(ng

/mL)

oleic acid (µM)

R = -0.61; p = 0.19

200150100500

0,2

0,3

0,4

0,5

CIN

C 2

α/β

(ng

/mL)

linoleic acid (µM)

R=0.99; p= 0.0001

200150100500

0,20

0,25

0,30

IL-1

β (n

g/m

L)

oleic acid (µM)

R= 0.96; p= 0.0006

200150100500

0,1

0,2

0,3

IL-1

β (n

g/m

L)

linoleic acid (µM)

R = -0.53; p = 0.27

200150100500

0,21

0,22

0,23

0,24

0,25

TN

F- α

(ng/

mL)

oleic acid (µM)

R = -0.22; p = 0.66

200150100500

0,20

0,22

0,24

TN

F-α

(ng

/mL)

linoleic acid (µM)

Figure 3. Levels of VEGF-a, CINC-2a/b, IL-1b and TNF-a in the supernatant of neutrophils cultured in the absence and in the presence ofdifferent concentrations of oleic and linoleic acids (5, 25, 50, 100 and 200mM). The measurements of TNF-a were carried out after 4 hculture whereas determination of the remaining cytokines was performed after 18 h. The data represent the average�SEM of at least fourexperiments. p represents the values of Pearson correlation

202 l. m. pereira ET AL.

DISCUSSION

Evidence is presented here that oleic and linoleic acidscause marked changes in the wound during theinflammation period of the healing process. These

Copyright # 2007 John Wiley & Sons, Ltd.

fatty acids showed a pro-inflammatory effect oncutaneous wound healing. This conclusion is based onthe increase in the wound mass, protein and DNAcontents (cellularity), neutrophil infiltration, neutro-

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effect of fatty acids on wound healing 203

phil influx into the air pouches and cytokineproduction by neutrophils. The increased migrationratio of neutrophils into air pounches and the increasedcytokine release by neutrophils treated with fatty acidsare not due to cell death, since cell membrane integrityloss (necrosis) and DNA fragmentation (apoptosis)were not modified in basal and treated neutrophils asshown in our previous studies.24

Our results add important mechanistic informationto the observations of Cardoso et al. These authorsshowed that linoleic and oleic acids can modulate theclosure of surgically induced skin wounds. A mildimprovement in wound closure was observed in thelinoleic acid-treated animals concurrent with a peak ofnitric oxide production at 48 h post-surgery. However,oleic acid strongly inhibited the production of nitricoxide at the wound site. The authors suggested arelevant role and potential therapeutic use of fattyacids in skin wound healing.18

One of the techniques used for the treatment ofchronic wounds is the topical application of exogen-ous growth factors and cytokines. For example, atreatment of wounds with colony-stimulating factor(GM-CSF), a growth protein for haematopoietic cells,stimulates proliferation, maturation and recruitment ofneutrophils/monocytes and endothelial cells andinduces keratinocyte proliferation.28

Neutrophils play a central role in tissue damage.29,30

The invading neutrophils are capable of synthesizingand releasing a variety of pro-inflammatory mediatorsincluding cytokines, chemokines, growth factors,angiogenic factors and AA. PG can be synthesizedfromAAby the skin of humans and rats in the first stageof the inflammation phase in epidermal healing. PG areinvolved in tissue repair, cell spreading and migrationand epidermal cell proliferation.31 AA and itsmetabolites are mediators of several events duringwound healing, such as cell growth, angiogenesis andsynthesis of extracellular matrix. Linoleic acid can beused for the biosynthesis of AA and PG in theepidermis.

In the present study, oleic and linoleic acidsprovoked a marked increase in the number ofneutrophils that migrated to the wound healing area.The increase in the skin wound mass observed may bea consequence of the effect of the fatty acids on cellmigration. The capacity of oleic and linoleic acids toinduce neutrophil migration was confirmed byneutrophil influx into air pouches.

The three cytokines determined in this study,TNF-a, IL-1b and CINC-2a/b, exert profound effectson immune and inflammatory responses. TNF-a andIL-1b are important in the activation of endothelial

Copyright # 2007 John Wiley & Sons, Ltd.

cells and in the expression of adhesion molecules,leukocyte integrins and endothelial selectins contri-buting to accumulation of phagocytes in the inflamedsite.32 CINC-2a/b (cytokine-induced neutrophil che-moattractants in inflammation) also contributes to therecruitment and accumulation of phagocytes in theinflamed tissue.33 VEGF is a prime regulator ofangiogenesis and vasculogenesis.34,35

Conceivably, these findings place neutrophils at apivotal position where they regulate initial phase ofwound healing process and orchestrate not onlythe acute inflammatory phase but also subsequentphases.32,34–37 These effects of oleic and linoleic acidseffects can be potentially important in situations thatexhibit neutrophil dysfunction and a non-healingwound, as occurs in glycogen storage disease-1b(GSD-1b),38 chronic granulomatous disease(CGD),28,39 leukocyte adhesion deficiency type 1(LAD-1)38,40 and diabetes.41

The nutritional deficiency of EFA has also beenassociated with deficient cutaneous wound healing asreported in mice, rats17–20 and infants.21 In Brazil,fatty acid mixtures containing oleic and linoleic acidshave been used for prevention and treatment ofpressure ulcers.14–16 The topical application of fattyacids also improves hydration, elasticity and preventsskin breakdown in patients under poor nutritionalconditions.15,16,18

In conclusion, evidence is presented herein thatoleic and linoleic acids present a pro-inflammatoryeffect as indicated by the increase in wound mass,protein and DNA contents and neutrophil migration towound tissue and air pouches. These fatty acids canalso stimulate neutrophils to release VEGF-a, IL-1band CINC-2a/b. This may be an important mechanismfor the effect of linoleic and oleic acids in speeding upthe wound healing process.

ACKNOWLEDGEMENTS

This research is supported by the Fundacao deAmparo a Pesquisa do Estado de Sao Paulo (FAPESP),Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior (CAPES) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq).The authors are indebted to the constant technicalassistance of G. de Souza, J.R. Mendonca and E.P.Portioli.

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DOI: 10.1002/cbf