Recent investigations of the pathophysiologic events occurring in the digital laminae of horses during the early stages of laminitis have indicated the simultaneous occurrence of numerous pathologic events including vascular dysregulation,1-3 inflammation,4-9 and gene expression and activation of MMPs.10-13 Although these events have been studied by use of several methods, events have been most clearly defined for horses with black walnut–induced laminitis, in which 2 time points, the developmental and the acute onset of lameness, have been most frequently examined.2-6,9,14,15 The DTP, defined by a 30% decrease in WBC count,2 is the earliest time point examined in most studies on horses with black walnut–induced laminitis; this time point typically occurs 3 to 4 hours subsequent to BWE administration.16 The ALTP occurs between 9 to 12 hours after BWE administration in responsive individuals.16 Although the DTP of horses with black walnut–induced laminitis occurs in the prodromal phase many hours before clinical signs of digital pathologic events take place, several pathologic events are reported to be occurring, including vascular dysregulation,2,3 leukocyte emigration (extravasation of leukocytes) into laminae,6 inflammatory mediator signaling,4,5,8 and MMP accumulation.15 Therefore, because of the simultaneous occurrence of numerous events at the DTP, it is necessary to assess earlier time points to dissect the initiating events from downstream events. Thus, the purpose of the study reported here was to examine laminar inflammatory gene expression, leukocyte emigration, and MMP regulation occurring at 1.5 hours following BWE administration, a time point prior to onset of systemic or digital signs of pathologic events.
Materials and Methods
All protocols were approved by the Institutional Animal Care and Use Committee of The Ohio State University or Auburn University.
Black walnut–induced laminitis—Black walnut extract was made for administration to horses by soaking 2 g of black walnut heartwood shavings/kg in 6 L of deionized water, as previously described.2 Six liters of BWE (principal horses) or water (3-hour control horses) was administered via a nasogastric tube. Physical examination including assessment of rectal temperature, heart rate, respiratory rate, abdominal sounds, digital pulses, and evaluation of the gait was performed hourly. Blood was collected for CBC determination every 30 minutes starting immediately prior to BWE administration.
After BWE administration, horses were sedated with xylazine (1.1 mg/kg, IV) 5 minutes prior to anesthetic induction with a combination of diazepam (0.11 mg/kg, IV) and ketamine (2.2 mg/kg, IV). Horses were intubated, and isoflurane inhalation was used to maintain a surgical plane of anesthesia. Intermittent positive-pressure ventilation (15 mL/kg, 6 breaths/min) was used to ensure a steady delivery of isoflurane to facilitate the early harvesting of laminar specimens. Anesthesia was induced at 1.5 hours after BWE administration in the ETP group (n = 5) and between 3 and 4 hours (ie, onset of a 30% decrease in leukocyte count, as previously described4) after BWE administration in the DTP group (5). Anesthesia was induced at 3 hours after water administration in the 3-hour control group (n = 5).7
Forelimbs were rapidly removed by disarticulation of the metacarpophalangeal joint after placement of a tourniquet, and 1.5-cm-thick sagittal sections of the digit were cut with a band saw. Laminae were rapidly dissected from the hoof and third phalanx, and sections were immediately snap-frozen or placed in neutral-buffered 10% formalin for paraffin embedding. Each horse was euthanatized with pentobarbital sodium containing phenytoin sodiuma (20 mg/kg, IV) following laminar specimen collection.
Archived laminar specimens from Auburn University were used for laminae obtained at the onset of Obel grade 1 lameness (ALTP group, n = 5) after BWE administration, and specimens were also obtained from a control group (10-hour control group, 5) in which horses were anesthetized 10 hours after administration of 6 L of water; the protocol was identical for these horses as described in the present study for ETP and DTP horses, except that general anesthesia was induced with thiopental sodium and maintained with dilute pentobarbital with phenytoin sodium, as previously described.4
RNA isolation and cDNA synthesis—Total RNA was extracted from the forelimb laminae of each horse by use of a commercial kit.b Poly A mRNA was then isolated and used to make cDNA for each horse via RT. The cDNA was used to perform an RT-qPCR procedure to determine the mRNA expression for different cytokines.
RT-qPCR procedure—A commercial real-time thermocyclerc was used in the study, and quantification with external standards was performed with the fluorescent format for SYBR Green I dye, as previously described.4,5 Primers for IL-1β, TNF-α, IL-6, IL-8, IL-10, MMP-2, MMP-9, ICAM-1, E-selectin, and 4 housekeeping genes (ie, β-actin, β2-microglobulin, glyceraldehyde-3 phosphate dehydrogenase, and TATA-box binding protein) were designed from equine-specific sequences by use of computer programs, as previously described.5,17 Amplified cDNA fragments were ligated into a linearized vector for use as standard templates for production of a standard curve for the RT-qPCR reactions, as previously described.5
All PCR reactions were performed in glass capillaries in 20-μL volumes (5-μL sample DNA and 15-μL of 1.33× PCR master mixture). The master mix included Taq polymerase (1 unit), uracil N-glycosylase (0.2 units), SYBR Green stock solution (1:10,000 dilution), and PCR buffer. The PCR buffer (20mM Tris-HCL; pH, 8.4) contained 0.05% each of Tween 20 and a nonionic detergent. All primers were used at a 1μM concentration. The RT-qPCR amplification reactions were performed in a commercial real-time thermocycler as previously described,5 with amplification reactions of cDNA from individual control, ETP, or DTP horses and gene-specific templates for the standard curve being performed in the same run. Amplification reactions were performed in duplicate from the individual laminar cDNA samples from each horse (ie, laminar cDNA samples from the individual horses were not pooled). For the standard curves, 10-fold serial dilutions of template (linearized plasmids containing the different gene-specific cDNA inserts) were used at concentrations of 104 to 100 template molecules/reaction to create a standard curve. These curves were used for relative quantification of the target amplicon and the housekeeping genes in each cDNA sample for the normalization process. A normalization factor was determined for the laminar tissue as previously described.5 Briefly, RT-qPCR assay was performed for 4 housekeeping genes (β-actin, β2microglobulin, glyceraldehyde-3-phosphate dehydrogenase, and TATA-box binding protein). The resulting quantitative PCR data for the 4 genes were assessed by use of a computer program.d The 2 housekeeping genes that received the best score (and were reported as acceptable) by use of the computer program (β-actin and β2-microglobulin) were used to make a normalization factor.18 Amplification data obtained by RT-qPCR assay for the different genes were divided by the normalization factor of the housekeeping genes in the same sample.
CD13 immunohistochemistry—Laminar sections were placed in neutral-buffered 10% formalin for 24 hours followed by 70% ethanol until paraffin embedding. Routine immunohistochemical methods were performed on frontal laminar sections from each horse in control and ETP groups by use of citrate buffer for antigen retrieval and an anti-equine CD13 antibodye followed by use of a commercial immunoperoxidase kitf and 3′3-diaminobenzidine hydrochloride for localization of antigen, as previously described.6 Laminar leukocyte counts were performed by averaging the number of CD13-positive leukocytes per field under 40× magnification (10 counts/section, 4 to 6 sections from different paraffin blocks were counted/horse) in laminae with light microscopy.6
Gelatin zymography—Ten percent SDS-polyacrylamide gels impregnated with 0.1% gelatin were prepared as described,13,15 with 30 μg of protein content/track of the gel. For qualitative comparison of gelatinase content in digital laminae of control, ETP, and DTP horses, laminar specimens (5 horses/group) were pooled. Individual laminar specimens from ETP horses (5 horses) were also assessed to determine variability among horses. Laminar specimens were boiled in SDS– laminar specimen buffer (350mM Tris-Base, 10% SDS, 38% glycerol, and 12% bromphenol blue) for 5 minutes, loaded into gels, and electrophoresed at 150 V for 1 hour. Gels were transferred to 100 mL of 2.5% Triton X-100 and washed for 1 hour at room temperature (approx 20° to 23°C) on a rotary shaker, after which time the Triton X-100 solution was replaced with 100 mL of enzyme buffer (50mM Tris [pH, 7.5], 200mM NaCl, and 5mM CaCl2) and the gels were incubated at 37°C for 28 hours. Denaturation with SDS followed by renaturation with Triton X-100 confers gelatinolytic activity to zymogens, thus allowing for detection of both pro and active forms of the enzyme. Following incubation, gels were stained with 100 mL of 0.5% Coomassie blue staing in 30% methanol and 10% acetic acid for 3 hours and then destained with 25% methanol and 10% acetic acid. Gels were examined by obtaining digital photographs. Intensities of MMP bands were quantified by use of a software program.h
Statistical analysis—To determine the relationships between selected variables, pairwise comparisons (with values of 5 horses/group) were performed between RT-qPCR results, CD13-positive leukocyte counts, and band intensity for MMPs-2 and -9. Linear regression analysis was performed by use of a software program.i Fold changes (increase or decrease) in cytokine gene expression were determined by comparing nontransformed-normalized values between control and principal horses. Once cDNA data were normalized, values were log transformed prior to analysis. A 2-tailed t test was performed on the transformed data of each normalized cDNA value for individual cytokines from each horse, and values of P < 0.05 were considered significant.
Results
ETP horses—Real-time quantitative PCR values (mean ± SE, cDNA copies per normalization factor) of control horses versus principal horses were determined for cytokines, COX isoforms, enzymes, and adhesion molecules (Tables 1 and 2). Laminar mRNA concentrations for cytokines IL-1β, IL-6, and IL-8 were significantly increased in ETP horses, compared with control horses, whereas a moderate increase in COX-2 mRNA was observed for ETP horses. Adhesion molecules E-selectin and ICAM-1 also underwent increases in mRNA concentrations in ETP horses; the expression of these adhesion molecule genes appeared to peak at this early time point, compared with expression in DTP and ALTP horses. In contrast, no significant change was present in mRNA concentrations of TNF-α, IL-10, COX-1, or MMP-9 in ETP horses. Concentrations of MMP-2 mRNA underwent a significant decrease in ETP horses, compared with control horses.
Changes in laminar mRNA concentrations* of cytokines and enzymes at 1.5 hours after BWE administration in horses.
Changes in laminar adhesion molecule mRNA concentrations* after BWE administration in horses.
DTP and ALTP horses—E-selectin and ICAM-1 mRNA concentrations were significantly increased in DTP horses, compared with control horses (Table 2). Expression of the gene encoding E-selectin was significantly increased in ALTP horses, compared with control horses, while gene expression for ICAM-1 in ALTP horses returned to baseline at the onset of Obel grade 1 lameness.
WBC count and CD13 immunohistochemistry—No significant changes in WBC count were present at 1.5 hours following BWE administration in the ETP horses (8,516 ± 754 cells/mL), compared with WBC count at the 0 time point in ETP horses (ie, prior to BWE administration; 8,746 ± 807 cells/mL) or at the 1.5-hour time point in the control horses (8,440 ± 1,308 cells/mL).
Laminar CD13-positive leukocyte counts were significantly (P = 0.020) increased in ETP horses (6.98 ± 4.82 cells/field; magnification of 40×), compared with control horses (0.056 ± 0.034 cells/field; 40×), which had a consistent lack of CD13-positive leukocytes in contrast to ETP horses. Most of the CD13-positive leukocytes were identified as neutrophils on the basis of nuclear morphology.
Laminar gelatinase content—No detectable 92-kd gelatinase, corresponding to pro–MMP-9, was found in the pooled control laminar specimen (0.089 arbitrary units). An intermediate amount was detected in the pooled laminar specimen of ETP horses (0.804 arbitrary units) in comparison to the pooled laminar specimen of DTP horses (0.966 arbitrary units; Figure 1). The pro–MMP-9 content was variable between individual laminae extracts from each horse in the ETP group, with 2 of 5 laminar specimens having positive results for the protein. In contrast, zymogen and the active form of MMP-2 remained consistent among all laminar specimens from control and ETP horses.
Gelatin zymography of laminar tissue extracts. Equivalent concentrations of protein were subjected to SDS–renaturing zymography. A—Laminar specimens from control horses (n = 5), ETP horses (5), and DTP horses (5) were pooled. B— Laminar specimens from the ETP group were analyzed individually. STD = Human MMP-9/MMP-2 positive control sample.
Citation: American Journal of Veterinary Research 68, 11; 10.2460/ajvr.68.11.1205
Linear regression analysis—At 1.5 hours after BWE administration, positive correlations existed between IL-8 and IL-1β gene expression (P < 0.001; Figure 2) as well as between CD13-positive leukocytes and IL-8 mRNA (P = 0.009), which was consistent with the role of IL-8 as the predominant neutrophil chemokine. Based on linear regression analysis, MMP-9 protein concentrations did not correlate with MMP-9 gene expression at 1.5 hours (P = 0.065).
Linear regression analysis of selected variables. Correlations were performed between (A) IL-8 versus IL-1β gene expression (P < 0.001), (B) MMP-9 versus CD13-positive leukocytes (P = 0.009), and (C) MMP-9 protein versus MMP-9 gene expression (P = 0.064). NF = Normalization factor.
Citation: American Journal of Veterinary Research 68, 11; 10.2460/ajvr.68.11.1205
Discussion
The 2 most popular hypothetic causes of laminar failure in the past decade have been ischemic damage to laminae resulting in epidermal necrosis and separation of the epidermal laminae19,20 and the more recent metabolic hypothesis in which laminar breakdown is proposed to occur as a result of stimulation of MMP activity by circulating toxins,11 suggested to be worsened by a possible increase in laminar blood flow in the prodromal stages of the disease.11,21 Another recent theory centers around inflammatory damage to the laminar tissue occurring as a result of local digital cytokine gene expression and infiltration of the laminar tissue with leukocytes.5,6,8,22 As all events encompassed by these 3 theories (ie, digital hemodynamic changes, inflammation, and increased MMP concentrations) are reported at the time points previously examined for horses with black walnut–induced laminitis,2,3,15 investigation of inflammatory events at an earlier time point was crucial to further delineate the initiating pathologic events in the affected digit. In the present study, several distinct differences were observed between the ETP and the better described DTP occurring 3 to 4 hours after administration of BWE.
In regard to proinflammatory cytokine gene expression, the same general immune response was detected at the ETP as was recently reported for the DTP, including increased gene expression for cytokines characteristic of the innate immune response (IL-1β, IL-6, and IL-8) and the inflammatory enzyme COX-2.4,7 Compared with the other inflammatory molecules assessed, IL-6 underwent much greater variability in gene expression among individual horses (up to a 200-fold difference between horses for IL-6 vs 10-fold differences among horses for other molecules), making it difficult to compare means at different time points when only 5 horses are assessed in each group. Interleukin-1β and IL-8 appeared to consistently peak at the ETP, compared with previously reported results for the DTP and ALTP. The peak in IL-1β concentration at the ETP is not surprising as IL-1β and TNF-α is known to peak early in inflammatory processes including sepsis.23,24
The lack of increased TNF-α mRNA concentrations at the ETP may be the result of several factors. Although TNF-α may peak earlier than at 1.5 hours, this is not likely to be the case because expression of the cytokine gene typically remains increased at 1 to 2 hours after the onset of systemic inflammation in other disease processes.23 The lack of TNF-α induction may also be caused by the previously reported effect of the IκB protein, MAIL/Iκβζ, on increasing IL-6 gene expression while decreasing TNF-α gene expression.7,25 However, it is also possible that the low TNF-α mRNA concentrations are attributable to the fact that no resident population of leukocytes of the monocyte lineage (the main producers of TNF-α)24 exists in digital laminae of horses, and the primary emigrating cell type is the neutrophil, a cell that has been previously described to produce high amounts of IL-1β without increased TNF-α gene expression.26,27
Although TNF-α concentrations are commonly reported to increase in sepsis, most of the studies assess concentrations in the blood, lungs, or liver (all with high amounts of monocyte-lineage cells [ie, Kupffer cells and pulmonary macrophages]).24,28 When TNF-α gene expression in tissues without a resident macrophage population is assessed in sepsis, mRNA concentrations commonly do not change, whereas other cytokines including IL-1 isoforms and IL-6 are increased.29,30 In a previous study9 on horses with black walnut–induced laminitis, we found that laminar cells undergoing marked IL-1β gene expression at the DTP did not appear to be normal constituents of the vessel wall, but were cells commonly found in the perivascular interstitium that are likely to be emigrating neutrophils because of their similar spatial distribution as that found for neutrophils present in affected laminae at the DTP. Thus, the main reason for increased IL-1β mRNA concentrations in the face of normal TNF-α concentrations in affected laminae is likely to be the unique lack of cells of monocyte lineage in the laminar tissue.
The peak in mRNA concentrations of the central neutrophil chemokine IL-8 at the ETP (142-fold increase), compared with later time points (20- and 10-fold increase at the DTP and ALTP, respectively), most likely results in a significant increase in IL-8 localization on the luminal surface of the endothelium.31 Contact of leukocytes rolling on the endothelial surface with IL-8 results in instantaneous activation of integrins on leukocytes, resulting in binding with their cognate ligands on the endothelium, an essential step in leukocyte emigration into tissues.23 The positive correlation between IL-8 gene expression and CD13-positive leukocyte counts corroborates the central role of this chemokine in neutrophil extravasation. Interleukin-8 gene expression may be induced by IL-1β, as IL-8 is known to be induced by central proinflammatory cytokines.32 However, it is also possible that IL-1β and IL-8 are induced by the same signaling event, which is most likely the activation of endothelial pattern recognition receptors (ie, toll-like receptors) by circulating toxins. The strong correlation between IL-1β and IL-8 supports either possibility.
Because of the important role that adhesion molecules play in IL-8–mediated neutrophil adhesion and extravasation, we investigated the expression of 2 central endothelial adhesion molecule genes, E-selectin and ICAM-1.33 As their gene expression pattern had not previously been reported at the DTP or ALTP, we determined mRNA concentrations of the 2 adhesion molecules at all 3 time points. Both E-selectin and ICAM-1 mRNA concentrations had a distinct peak at the ETP, with ICAM-1 actually decreasing from a 56-fold increase at the ETP to no significant increase at the ALTP. Thus, the simultaneous peak of IL-8, ICAM-1, and E-selectin at the ETP suggests that endothelial activation is peaking early in the disease process, likely contributing to leukocyte emigration into the laminar tissues as is observed in organ damage in human sepsis.33,34
Even with minimal change in WBC count, an increase in incidence of laminar leukocyte emigration occurred at the ETP as was previously reported at the DTP (a time point characterized by decreased WBC counts in BWE-treated horses).2 These results suggest that laminar endothelial activation occurs prior to systemic leukocyte activation and is responsible for initial leukocyte emigration; activated endothelium is capable of inducing normal (inactivated) leukocytes to adhere and extravasate. However, some indices of systemic leukocyte activation have recently been reported to increase by 1 hour after BWE administration, indicating that early emigration may be a combination of early leukocyte activation and endothelial activation.14
Laminar MMP-9 concentrations were detectable in only 2 of 5 horses at the ETP, whereas we previously reported MMP-9 concentrations to be increased in all laminae at the DTP of BWE-treated horses versus control horses.15 In that study,15 MMP-9 concentrations appeared to continue to increase in digital laminae of horses until the onset of clinical signs of acute lameness. The presence of laminar MMP-9 in only 2 of 5 horses at 1.5 hours, compared with its presence in all horses at the DTP, suggests that MMP accumulation often occurs between these 2 time points. It is unlikely that the lack of MMP-9 in the other 3 horses is attributable to an aberrant response to BWE administration. On the basis of development of leukopenia as a positive response, a high percentage (approx 80%) of BWE-treated horses responded to the extract with our current protocol. Interestingly, a decrease in MMP-2 mRNA concentration was found at the ETP. Although IL-10 can inhibit transcription of MMP-2,35,36 IL-10 mRNA expression was not inversely correlated with MMP-2 mRNA expression (data not shown). We must concede, however, that IL-10 gene expression may indeed be responsible for the reduction of MMP-2 gene expression detected.
Thus, the peak in indicators of endothelial activation (ie, E-selectin and ICAM-1), proinflammatory cytokines such as IL-1β and IL-8, and neutrophil influx prior to consistent increases in MMP-9 concentrations suggests that MMP synthesis and accumulation are likely downstream events induced by the proinflammatory cytokine milieu occurring early in the disease process. At 1.5 hours, it is likely that the primary source of MMP-9 zymogen present in digital laminae of horses is the emigrating neutrophil, a cell well-characterized to carry high concentrations of MMP-9 in cytoplasmic granules. Whereas no correlation was found between CD13-positive leukocyte counts and MMP-9 zymogen at the ETP, this assertion is supported by a significant correlation between laminar leukocyte counts and MMP-9 zymogen at later time points for horses with black walnut-induced laminitis. Also, no increase in laminar MMP-9 gene expression was found at the ETP. Increased MMP-9 concentrations in digital laminae of horses at later time points are likely caused by the presence of neutrophils and increased MMP-9 gene expression in local laminar cells at the ALTP.
Together, these results suggest a similar early induction of the innate branch of the immune system in response to BWE administration, as has been previously characterized in early human sepsis–systemic inflammatory response syndrome. The dramatic peak in mRNA concentrations for IL-8, ICAM-1, and E-selectin at the ETP with subsequent peaks in MMP concentrations at later time points indicate that endothelial inflammation and activation may be the initiating event leading to laminar pathologic events. Together with the results of other studies,4-10,12-15 these data indicate that proinflammatory cytokine induction, endothelial activation leading to neutrophil extravasation, COX-2 activity, and MMP accumulation all represent potential targets for therapeutic intervention for horses during the early stages of laminitis.
ABBREVIATIONS
| MMP | Matrix metalloproteinase |
| DTP | Developmental time point |
| BWE | Black walnut extract |
| ALTP | Acute onset of lameness time point |
| ETP | Early time point |
| RT-qPCR | Reverse transcription–quantitative PCR |
| IL | Interleukin |
| TNF | Tumor necrosis factor |
| ICAM | Intercellular adhesion molecule |
| COX | Cyclooxygenase |
Beuthanasia-D, Schering-Plough, Union, NJ.
Absolutely RNA miniprep kit, Stratagene, La Jolla, Calif.
mRNA isolation kit, Roche Molecular Biochemical, Indianapolis, Ind.
geNorm computer program, Ghent University, Ghent, Belgium.
Courtesy of Dr. D. Paul Lunn, Department of Clinical Sciences, College of Veterinary menicine and Biomedical Sciences, Colorado State University, Fort Collins, Colo.
Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, Calif.
Brilliant Blue R, Sigma Chemical Co, St Louis, Mo.
ImageJ software, Rasband WS, ImageJ, US National Institutes of Health, Bethesda, Md. Available at: rsb.info.nih.gov/ij/. Accessed Jun 26, 2006.
GraphPad Prism, GraphPad Software Inc, San Diego, Calif.
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