Veterinary Immunology and Immunopathology 144 (2011) 120–128

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Veterinary Immunology and Immunopathology

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Research Paper

Lamellar leukocyte infiltration and involvement of IL-6 duringoligofructose-induced equine laminitis development

Michelle B. Visser ∗, Christopher C. PollittThe Australian Equine Laminitis Research Unit, School of Veterinary Science, The University of Queensland,Gatton 4343, Australia

a r t i c l e i n f o

Article history:Received 26 January 2011Received in revised form 20 June 2011Accepted 20 July 2011

Keywords:InflammationLaminitisLeukocyteIL-6CytokineProtease

a b s t r a c t

Laminitis is known to involve deregulation of proteases and destruction of the lamellarbasement membrane with the host inflammatory response also playing a role. Leukocyteinfiltration has been well characterized in the black walnut model of laminitis induction,but not in carbohydrate induced models. Increased gene expression of multiple cytokines,including IL-6, has also been implicated in laminitis development. Using real time PCR,immunohistochemistry and zymography methods, we characterize leukocyte infiltrationand IL-6 gene expression in oligofructose (OF) induced laminitis. As well, we use two in vitromodels to investigate a role for IL-6 in protease regulation. Laminitis was induced in normalstandardbred horses (n = 5) by alimentary OF dosing and lamellar biopsies were obtainedthroughout the 48 h experimental period. Lamellar explants and keratinocytes were alsoisolated from clinically normal horses for in vitro experiments. We found infiltration ofcalprotectin-positive leukocytes (monocytes and neutrophils) at 18–24 h post oligofructosedosing, while IL-6 gene expression was increased as early as 12 h post dosing. Additionally,while we found that IL-6 did not cause significant BM damage in vitro, it did result in

increased secreted proMMP-9 levels from lamellar explants. Thus, we find that leukocyteinfiltration does occur during oligofructose-induced laminitis development, however, IL-6gene expression in the lamellae may precede leukocyte infiltration. Additionally, we showIL-6 plays a role in increasing the level of proMMP-9 in vivo in a manner that does notinvolve keratinocytes.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Equine laminitis is characterized by failure of the lamel-lar connection to the distal phalanx of the hoof (Pollitt,

Abbreviations: OF, oligofructose; BWE, black walnut extract; ADAMTS,A Disintegrin And Metalloproteinase with Thrombospondin Motifs; APES,3-aminopropyltriethoxysilane; H&E, haematoxylin and eosin; PAS, peri-odic acid-Schiff; PDL, primary dermal lamellae; PEL, primary epidermallamellae.

∗ Corresponding author. Present address: Faculty of Dentistry, Univer-sity of Toronto, 124 Edward St. Toronto, Ontario, Canada M5G 1G6.Tel.: +1 416 979 4900x4452; fax: +1 416 979 4936.

E-mail address: michelle.visser@utoronto.ca (M.B. Visser).

0165-2427/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.vetimm.2011.07.016

1996). Current experimental models use alimentary over-load of carbohydrate (CHO) (starch or oligofructose (OF)),black walnut heartwood extract (BWE), or intravenousinfusion of insulin (Asplin et al., 2007; Galey et al., 1991;Garner et al., 1975; van Eps and Pollitt, 2006). These modelsclosely represent natural causes of laminitis such as over-consumption of grain or pasture grass (Garner et al., 1975;Geor, 2009; Goetz, 1989) as well as endocrine disorderscharacterized by insulin resistance and hyperinsulinaemia(Johnson et al., 2004). However, laminitis is also associated

with diseases such as colitis, metritis, enteritis, pleurop-neumonia and retained placenta (Heymering, 2010; Sloetvan Oldruitenborgh-Oosterbaan, 1999); all of which havean inflammatory component in common and can be likened

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o human sepsis, characterized by systemic inflammationnd multiple organ failure (Belknap et al., 2009).

Changes in circulating leukocytes have been observedn multiple models of laminitis induction. It has been

ell documented that leukopenia occurs 3–4 h follow-ng BWE administration (Eaton et al., 1995; Galey et al.,991). Conversely, increased levels of circulating leuko-ytes have been observed in CHO induction models prioro the onset of lameness (Garner et al., 1975; Moore et al.,981; van Eps and Pollitt, 2006). Immigration of neu-rophils and monocytes/macrophages into the lamellarissue has been documented using sensitive immunohisto-hemical methods in early BWE-induced laminitis (Blackt al., 2006; Faleiros et al., 2009b, 2011b; Loftus et al.,006, 2007). Although leukocytes have been observed inarbohydrate and insulin models of laminitis induction bylectron microscopy (Nourian et al., 2009; Pollitt, 1996),nly recently have leukocytes been detected immunohis-ochemically in starch-induced laminitis (Faleiros et al.,011a).

Involvement of a cytokine response in laminitis devel-pment has also been well documented (Belknap et al.,007; Fontaine et al., 2001), although responses dif-er between experimental models. In the BWE model,ncreases in proinflammatory cytokines and chemokinesccur as early as 1.5 h following administration (Faleirost al., 2009a; Loftus et al., 2007), afterwhich some medi-tors peak while others continue to increase into theevelopmental phase and onset of lameness (10–12 h)Belknap et al., 2007; Faleiros et al., 2009a; Fontainet al., 2001; Waguespack et al., 2004). Conversely inHO-induced models of laminitis, inflammatory mediatorsppear to increase later during induction, primarily at thenset of lameness (20–48 h) (Belknap et al., 2007; Leiset al., 2011).

Degradation of the lamellar basement membrane (BM)s recognized to occur during early CHO-induced lamini-is (Pollitt, 1996; Pollitt and Daradka, 1998; Visser andollitt, 2011), likely due to increased proteolytic activ-ty. Traditionally, the gelatinases MMP-2 and MMP-9 haveeen thought to be major players (Coyne et al., 2009; de

a Rebiere de Pouyade et al., 2009; Johnson et al., 1998;yaw-Tanner and Pollitt, 2004; Pollitt et al., 1998), thoughecent work by our group (Visser and Pollitt, 2011) alongith others (Coyne et al., 2009; de la Rebiere de Pouyade

t al., 2009) has highlighted a role for additional proteasesuch as neutrophil elastase and A Disintegrin And Metal-oproteinase with Thrombospondin Motifs-4 (ADAMTS-4)n laminitis. Cytokines are able to regulate the transcrip-ion and production of other inflammatory mediators asell as proteases, including MMPs (Mauviel, 1993; Overall

nd Lopez-Otin, 2002; Ries and Petrides, 1995). While someMPs present during laminitis development are likely pro-

uced directly from activated leukocytes, others may benduced from lamellar cells by the cytokine response thatccurs during laminitis induction.

In this study we analyze lamellar infiltration of leuko-

ytes during OF-induced laminitis using temporal lamellariopsies. As well, we clarify the timeline of IL-6 genexpression as well as examine a possible role for thisytokine during laminitis development.

d Immunopathology 144 (2011) 120–128 121

2. Methods

2.1. Experimental models and tissue collection

Archived lamellar tissue specimens were used in thisstudy. Laminitis was experimentally induced in 5 normalstandardbred horses by alimentary carbohydrate over-load using oligofructose (OF, 10 g/kg) (van Eps and Pollitt,2006). Lamellar biopsies (approximately 10 mm × 10 mm)were collected from the dorsal forelimb hoof wall priorto OF dosing and at 12, 18, 24, 30, 36 h post dosing, withthe horse standing, sedated with the appropriate palmarnerve blocked. Three biopsy sites were located on eachhoof (medial, lateral and central), alternating betweenforefeet with each successive timepoint (Croser and Pollitt,2006). At 48 h post dosing, the animals were euthanizedby overdose of barbiturate, forefeet disarticulated at thefetlock and mid-wall samples were collected from lamel-lar regions ventral to earlier biopsy sites. A control groupof horses (n = 3) were given a water bolus and biopsied inthe same manner and timepoints as the laminitis group(sham treated with biopsy). Locations of biopsy sites havebeen described previously (Visser and Pollitt, 2011). Exper-iments were conducted according to the animal ethicsguidelines set by the University of Queensland AnimalEthics Committee. All animals were continuously moni-tored and inspected by the Consultant Veterinary Officer tothe Animal Welfare Unit at the University of Queensland.If pain could not be alleviated promptly, early euthana-sia, even if this was prior to the planned conclusion ofthe experiment, was a proviso of the protocol. None ofthe horses in this study required any pain medication orearly euthanasia. For in vitro experiments, clinically normalhorses were euthanized by overdose of barbiturate and tis-sue obtained as previously described (Pollitt, 1996; Visserand Pollitt, 2010). For protein extraction and RNA isola-tion, tissue samples were flash frozen in liquid nitrogenand stored at −80 ◦C until use. For immunohistochemistry,tissue samples were fixed in 10% formalin for 24 h, followedby transfer to 70% ETOH until embedded in paraffin.

2.2. Real time PCR

Total RNA was isolated from frozen hoof tissue usingTrizol (Invitrogen). Tissue samples were homogenized inTrizol reagent (1 ml/100 mg tissue) using a power homog-enizer (Omni TH homogenizer, Omni International Inc,Marietta, GA, USA). RNA samples were treated with RNAsefree DNAse (Promega) to remove genomic DNA prior tocDNA synthesis. cDNA was synthesized from 1 �g of totalRNA using the reverse transcription system (Promega) withrandom hexamer primers.

Gene expression in tissue samples was measured usingreal-time PCR analysis. Primer sequences were designed toequine-specific sequences using Primer Express software(Applied Biosystems, Foster City, CA, USA) and are as fol-lows: �-2 Microglobulin For 5′-ACCCAGCAGAGAATGGA-

AAGC-3′, �-2 Microglobulin Rev 5′-CATCTTCTCTCCATTCT-TTAGCAAATC-3′; IL-6 For 5′-AAACCACCTCAAATGGACCA-CTA-3′, IL-6 Rev 5′-TTTTTCAGGGCAGAGATTTTGC-3′.Oligonucleotides were synthesized by Sigma Genosys

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(Castle Hill, NSW). PCR reactions were performed in 10 �lreaction volumes in 384 well plates prepared using anepMotion 5075 robot (Eppendorf Hamburg, Germany).PCR reactions consisted of 5 �l SYBR Green Master Mix(Applied Biosystems), 2 �l cDNA (1:5), 2 �l primer mix and1 �l H2O. Reactions were carried out in triplicate using anAB7900 thermocycler (Applied Biosystems). Reaction con-ditions consisted of a initial denaturation step of 95 ◦C for10 min, followed by 50 cycles of denaturation 95 ◦C for 15 sand elongation of 60 ◦C for 1 min. Following amplification,melting curve analysis was subsequently performed byone cycle of 95 ◦C, 2 min, 60 ◦C, 15 s and 95 ◦C 15 s. Relativegene expression levels, normalized to �-2 microglobu-lin, were determined using the comparative Ct method(2−��Ct) (Livak and Schmittgen, 2001). PCR productswere analyzed by standard agarose gel electrophoresis(Sambrook et al., 1989) to confirm the expected size of PCRproduct. Samples were electrophoresed on 2% agarose gelsembedded with 0.5 �g/ml ethidium bromide followed byanalysis by UV illumination.

2.3. In vitro experiments

Recombinant equine IL-6 was purchased from R&D Sys-tems Inc. Equine keratinocytes were prepared as described(Visser and Pollitt, 2010), grown to confluence in 60 mmtissue culture plastic dishes and washed with dPBS. Cellswere subsequently incubated for 48 h in serum free DMEMcontaining recombinant equine IL-6 (300 ng/ml, 150 ng/ml,15 ng/ml, 1.5 ng/ml) at 37 ◦C with 5% CO2. Conditionedmedium was collected and frozen until analysis.

Lamellar explants were prepared as described previ-ously (Pollitt, 1996). Explants were incubated in DMEMsupplemented with equine recombinant IL-6 (100 ng/ml,500 ng/ml, 1000 ng/ml) for 48 h at 37 ◦C with 5% CO2. Con-ditioned medium was collected as well as tissue explantsfixed in 10% formalin or snap frozen in liquid nitro-gen. Explant integrity was examined using a calibratedstrain gauge force transducer similar to previously reported(Mungall and Pollitt, 2002). The amount of force requiredto separate the tissue explant was measured using Chart 5software (ADInstruments).

2.4. Immunohistochemistry

Formalin fixed specimens were embedded in paraffin,sectioned at 5 �m and mounted on Superfrost Plus slides(Menzel) or APES treated Superfrost slides for immunohis-tochemical and general histology staining (haematoxylinand eosin (H&E) or periodic acid-Schiff (PAS) stains). Sec-tions were heated at 37 ◦C for 30 min to soften the waxfollowed by deparrafinization by incubation in xylene (3×3 min) followed by rehydration using a series of gradedETOH steps (100%, 2× 5 min; 90%, 1× 5 min; 80%, 1×5 min; 75% 1× 5 min) then rinsed in tap water. Antigenretrieval was performed using 0.05% Protease XXIV (Sigma)for 15 min at 37 ◦C followed by washing with 50 mM

Tris–HCl pH 7.6. Tissue sections were then blocked with 2%normal goat serum (Vector Laboratories, Burlingame, CA,USA) for 30 min at room temperature followed by incu-bation in primary antibody (mouse monoclonal antibody

d Immunopathology 144 (2011) 120–128

MAC387 recognizing human calprotectin, Abcam) dilutedin antibody dilution solution with background reducingcomponents (DAKO) 18 h at 4 ◦C. The antibody used hasbeen validated to recognize equine calprotectin in leuko-cytes (Grosche et al., 2008; Matyjaszek et al., 2009). As anegative control mouse IgG was used in place of primaryantibody. Following washing, sections were incubated ina biotin-conjugated secondary antibody prepared in 2%normal goat serum for 30 min at room temperature thenincubated in 3% H2O2 for 20 min to block endogenousperoxidase activity, followed by washing. Streptavidin-HRP was applied for 1 h at room temperature followedby washing. Development was performed using AEC+high sensitivity substrate (2–30 min) (DAKO) followed bynuclear staining with haematoxylin. Slides were rinsed inrunning tap water and mounted with aqueous medium(Faramount, DAKO). Stained tissue sections were viewedwith an Olympus BX-50 microscope and images capturedusing a CoolSNAP-Pro CF monochrome digital camera fit-ted with a CRI MicroColor RGB filter (Media Cybernetics)using Image-Pro Plus software.

2.5. Statistics

Relative gene expression levels of each biopsy timepoint compared to the time 0 h control sample werereported as median fold changes (n = 5) analyzed usingthe non-parametric Mann–Whitney test with significanceset at P < 0.05. In vitro IL-6 experiments were analyzedusing an unpaired t test (each IL-6 condition individu-ally compared to control) with significance set at 0.05.Correlation between the number of horses demonstratingcalprotectin expression (leukocyte infiltration) and time ofbiopsy sample (h) was analyzed using the Spearman coef-ficient. Analysis was performed using GraphPad Software(La Jolla, CA, USA)

3. Results

3.1. Leukocyte infiltration during laminitis development

Leukocytes, measured by calprotectin expression, wereinitially detected at 18 h in two horses, while by 30 hpost dosing all horses demonstrated positive staining(Spearman coefficient = 0.9728, P = 0.0028). Few calpro-tectin positive cells were present in lamellar samples priorto OF dosing (Fig. 1A). Reactivity was first observed inleukocytes around primary dermal lamellae (PDL) ves-sels (Fig. 1B, black arrows), followed by reactivity in thesub-lamellar dermal tissue (Fig. 1B and D, blue arrows)and larger dermal vascular beds (Fig. 1C, orange arrows).At later stages of dosing (24 h or later) strong cytoplas-mic reactivity was also associated with primary epidermallamellae (PEL) basal cells, rather than specific leukocytes(Fig. 1D). Histological staining analysis of serial tissue sec-tions indicates that calprotectin positive cells are of boththe monocyte and the neutrophil morphology (Fig. 1E

and F). Lamellar biopsy samples from the sham treatedwith biopsy group revealed minimal leukocyte infiltra-tion, detected by calprotectin positive cells primarily after36 h of laminitis induction in dermal tissue surrounding

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Fig. 1. Leukocyte infiltration during laminitis development. No calprotectin expressing cells are observed in control biopsies prior to OF dosing (A).Calprotectin positive leukocytes are observed during early laminitis induction within (B, black arrows) and around vessels (B, blue arrows) as well as insub lamellar vascular beds (C, orange arrows). Calprotectin expression also observed in epidermal lamellae (D). (E) H&E histological stain of lamellar tissueof serial section shown in panel B. Neutrophils (white arrows) and monocytes (yellow arrows) are both observed. Inset square in panel (E) is shown in (F).Scale bar equals 30 �m. SEL: secondary epidermal lamellae, PDL: primary dermal lamellae, Vl: vessel, V: sub-lamellar vascular bed. (For interpretation ofthe references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1Timeline of initial lamellar IL-6 gene expression and leukocyte infiltrationduring oligofructose induction.

Horse IL-6 gene expression Calprotectin expression

1 12 h 18 h2 30 h 30 h

3 18 h 24 h4 12 h 24 h5 24 h 18 h

biopsy sites (data not shown). Minimal changes to lamellarlaminin and collagen type IV were also observed in shamtreated biopsy samples (Visser and Pollitt, 2011).

3.2. IL-6 expression during laminitis induction andin vitro activity

Increased levels of IL-6 gene expression were observedin lamellar biopsy tissue by RT-PCR as early as 12 h postinduction (2 of 5 horses, Table 1) with significant increasesup to 48 h (Table 2, *P < 0.05, **P < 0.01). The time of first IL-6 gene expression and leukocyte appearance, as measuredby calprotectin positive cells, is compared in Table 1. In 4 of5 horses, IL-6 gene expression either occurs simultaneouslywith or precedes the appearance of leukocytes by 6–12 h.

Further, in vitro experiments were performed on lamel-lar tissue explants from normal horses treated with varyingconcentrations of equine recombinant IL-6. No differencein the amount of force required to separate the tissuecompared to non-treated samples (Fig. 2A) was observed.Histological evaluation of PAS stained lamellar explant sec-tions also revealed no loss of BM staining (Fig. 2C, blackarrows) or significant tissue separation compared to tissuesincubated in DMEM (Fig. 2B) alone. Occasional focal areasof tissue separation (Fig. 2, white arrows) were observedin IL-6 treated samples but these effects are likely minimalas no change in the amount of force to separate the tissuewas observed. As a positive control, lamellar explants wereincubated in the presence of APMA, a MMP activator, result-ing in both decreased force required to separate explants(Fig. 2A) and clear tissue separation seen by microscopy(Fig. 2D, white arrows).

Gelatin zymography of lamellar explant medium incu-bated with increasing concentrations of IL-6 revealedincreased expression of pro-MMP-9 (P < 0.05); however, noconversion to the active form was seen. No change in theamount or activation of pro-MMP-2 was observed (Fig. 3A).A representative zymogram is shown in Fig. 3B.

As lamellar explants are full thickness tissue samplesconsisting of both epithelial and mesenchymal compo-nents, the effect of IL-6 on individual cell types cannot beestablished. The effect of IL-6 on gelatinases secreted by

Table 2IL-6 gene expression during laminitis induction.

12 h 18 h 24

Mediana 1 297.7 57Minimum to maximum values 1/68.57 1/1712 1/P valueb NS <0.05 <0

a Relative normalized fold change in IL-6 gene expression compared to time 0 hb P value determined by the Mann–Whitney test, NS = not significant.

d Immunopathology 144 (2011) 120–128

lamellar keratinocytes was also determined. Addition ofvarying doses of IL-6 to keratinocyte cultures did not resultin any change in the amount of MMP-9 or MMP-2 secretednor MMP activation (Fig. 3C and D).

4. Discussion

It is well established that lamellar leukocyte infiltrationoccurs during BWE induced laminitis (Black et al., 2006;Faleiros et al., 2009b, 2011b; Loftus et al., 2007), althoughleukocyte infiltration in CHO-induced laminitis models hasbeen in question. Neutrophils have been observed cross-ing the lamellar BM in early electron microscopy studies ofCHO-induced acute tissue samples (Pollitt, 1996) and herewe report leukocyte infiltration as early as 18 h post dosingin OF-induced laminitis. A recent report has also docu-mented the presence of leukocytes at the onset of fever,10–20 h following starch administration (Faleiros et al.,2011a). The results of our study, together with the observedBM changes 12 h post OF dosing in the same archived tissuesamples (Visser and Pollitt, 2011), suggests that BM degra-dation precedes leukocyte infiltration during OF laminitisinduction.

The various experimental models used in these studiesmust be considered as they likely differ mechanistically.Along with differences in systemic leukocyte dynamics(Moore et al., 1981; van Eps and Pollitt, 2006), pathogenicevents occur earlier in the BWE model, as lameness isobserved as early as 12 h (Galey et al., 1991). Additionally, ithas been reported that lamellar BM failure rarely occurs inBWE laminitis (Black et al., 2006) compared to well-definedlamellar BM pathology of carbohydrate models (French andPollitt, 2004; Pollitt, 1996; Pollitt and Daradka, 1998; Visserand Pollitt, 2011).

Together with calprotectin localization associated withleukocytes, calprotectin is also located throughout thelamellar epidermal cell cytoplasm in the later stages ofOF-induced laminitis in our study, similar to other exper-imental laminitis models (Faleiros et al., 2011a, 2009b).While calprotectin localization in leukocytes is primar-ily associated with inflammatory conditions (Striz andTrebichavsky, 2004), localization in epithelial tissue is alsoassociated with tissue injury such as the hyperprolifera-tive keratinocytes of skin wounds (Thorey et al., 2001).Thus, the observed calprotection localization in the lamel-lar tissue may represent a stress and/or injury responseoccurring secondary to leukocyte infiltration during OFlaminitis induction.

Our data presented here, along with additional workfrom our group demonstrating BM damage as early as 12 hpost OF dosing in the same archived tissue samples (Visserand Pollitt, 2011), supports the notion that BM damage pre-

h 30 h 36 h 48 h

.39 89.84 213.2 387.91640 44.68/3642 90.28/5306 31.22/2803.05 <0.01 <0.01 <0.01

biopsy.

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Fig. 2. Effect of recombinant equine IL-6 on lamellar explants. (A) Graph represents the median (2 explants per condition, n = 2) force required to separate lamellar explants incubated in increasing concentrations ofrecombinant IL-6. Incubation of explants in APMA serves as a positive control for MMP activation and tissue separation. *P < 0.05 compared to no IL-6 by unpaired t test. PAS stained explant sections demonstratingBM localization in tissue incubated in (B) DMEM, (C) 500 ng/ml IL-6 or (D) APMA. Scale bar = 30 �m. SEL: secondary epidermal lamellae, PEL: primary epidermal lamellae. Black arrow denotes the intact BM,white arrow indicates separated BM.

126 M.B. Visser, C.C. Pollitt / Veterinary Immunology and Immunopathology 144 (2011) 120–128

e densitmellartive gel

Conflict of interest statement

Fig. 3. Effect of recombinant equine IL-6 on gelatinase expression. Averagn = 2)) of secreted gelatinase protein levels following incubation of (A) launpaired t test (each IL-6 concentration compared to no IL-6). Representa

cedes leukocyte infiltration. MMPs including gelatinases aswell as fragments derived from ECM components, are allchemotatic for leukocytes (Adair-Kirk and Senior, 2008;Mydel et al., 2008; Senior et al., 1989; Steadman et al.,1993) and some of these molecules are degraded duringlaminitis development (Johnson et al., 2000; Pollitt, 1998;Visser and Pollitt, 2011). MMP-9 has been found to increaseneutrophil recruitment and migration in post-ischemicliver (Khandoga et al., 2006), while chemical inhibition ofgelatinases prevented leukocyte adhesion and migration(Reichel et al., 2008).

In both BWE and CHO-overload laminitis induction,changes in cytokine profiles of lamellar tissue have beenanalyzed (Belknap et al., 2007; Faleiros et al., 2009a; Leiseet al., 2011; Loftus et al., 2007; Waguespack et al., 2004).IL-6 is increased in both models and at multiple time-points, suggesting a prominent role for this cytokine. Ourresults presented here add to the evidence of IL-6 involve-ment in laminitis, as we found IL-6 gene transcription tobe increased as early as our first sampling point at 12 hpost dosing in some horses, yet before neutrophil infil-tration. While the function of IL-6 during laminitis is notknown, IL-6 has been suggested be involved in leuko-cyte recruitment in some tissues. For example, instillationof IL-6 has been demonstrated to induce recruitment ofneutrophils in rat lungs (Hierholzer et al., 1998), whilerecruitment of neutrophils, monocytes and lymphocytesinto air pouches was decreased in IL-6 knockout mice(Romano et al., 1997). Although IL-6 is not traditionallythought of as a chemokine, it may be involved albeit indi-rectly in recruitment of leukocytes during laminitis; as 4 of5 horses demonstrated IL-6 expression prior to or simul-

taneous with the presence of leukocytes, although furtherexperimentation is required to confirm this. It has also beenreported that cytokine gene expression including IL-6 isincreased as early as 1.5 h following BWE administration

ometry units (median with interquatrile range (2 explants per condition,explants or (C) keratinocytes in varying IL-6 concentrations. *P < 0.05 byatin zymograms are shown for explants (B) and keratinocytes (D).

in both the lung and the liver, while leukocyte numbersdid not increase until a later developmental point (Stewartet al., 2009).

Cytokines are known to regulate protease productionand activation (Ries and Petrides, 1995). Using an in vitrolamellar explant model, no major change in the lamellarBM structure or amount of force to separate the tissuewas observed following IL-6 incubation, suggesting thatthe lamellar BM structure was relatively intact. However,increased levels of secreted pro MMP-9 was observed inIL-6 treated samples indicating that IL-6 increases produc-tion of this MMP, but not its activation. Additional studiesusing cultured normal equine keratinocytes did not repro-duce the IL-6 induced MMP-9 increase, suggesting thatthis is not the primary cell type involved. However, onemust consider that in vitro cell line experimentation maynot completely recapitulate the complex environment ofthe lamellae. Recently, it has been reported that MMP-9protein localizes to leukocytes in laminitis tissue samples(Loftus et al., 2008), however, additional studies are neededto confirm the connective role between IL-6 and MMP-9.

5. Conclusion

In summary we confirm mixed leukocyte immigrationto the lamellae during OF-induced laminitis. Meanwhile,lamellar IL-6 gene expression is increased as early as 12 hpost OF dosing, and in vitro studies suggest that IL-6 playsa role in increasing MMP-9 production.

None of the authors has a financial or personal rela-tionship with other people or organizations that couldinappropriately influence or bias this work.

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cknowledgements

Funding was provided by the Rural Industries Researchnd Development Corporation of Australia and the Ani-al Health Foundation of St. Louis, Missouri. M.B.V. was

he recipient of an Endeavour International Postgraduateesearch Scholarship from the University of Queensland.e thank Emma Croser, Christopher Owens, Katie Asplin,

li Nourian and Marianne Keller for assistance with tis-ue collection and animal experiments. The study sponsorsad no role in study design, data collection, analysis or

nterpretation or the decision to submit this manuscriptor publication.

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