Initiation of SIRS as a result of the transmural movement of LPS from the intestinal lumen into circulation is a fairly common occurrence in horses with severe forms of gastrointestinal disease (ie, those with the highest morbidity and mortality rates).1–3 Despite recent advances in our understanding of SIRS, effective treatments remain to be identified. Therefore, the response of the equine immune system to proinflammatory substances such as LPS deserves further investigation.
In other animal species, complications associated with SIRS develop when leukocytes are activated, leave the circulation, and enter the tissues, resulting in an increase in local expression of proinflammatory mediators.4,5 Limited evidence suggests that the same occurs in horses with gastrointestinal disease and in horses with experimentally induced endotoxemia and laminitis.6–11 A major limitation to studying SIRS in horses is the lack of reagents needed to measure plasma concentrations of cytokines and other inflammatory mediators. New molecular biology tools such as real-time RT-qPCR assays can be used to overcome this problem.12 When information about the equine genome is used to synthesize primers for RT-qPCR, it is possible to indirectly measure the expression of inflammatory mediators in circulating leukocytes and other cells on the basis of the concentrations of transcripts for the genes encoding those mediators.12–15 This approach has been extensively used in humans and laboratory animals to research SIRS, but only a few studies16,17 have been conducted in horses.
In humans and rodents, sepsis and experimental endotoxemia can lead to a state in which the inflammatory response to a subsequent exposure to LPS is blunted. Several terms have been used for this condition, including tolerance to LPS, endotoxin tolerance, immunosuppression, immunoparalysis, leukocyte de-activation, leukocyte adaptation, leukocyte desensitization, leukocyte anergy leukocyte refractoriness, and leukocyte reprogramming.18–20 This condition is characterized by reduced expression of proinflammatory cytokines such as IL-lβ, I-L6, and TNF-α in response to ex vivo exposure to LPS.21–24 In a few studies22,23 involving humans with severe sepsis, patients with a blunted production of proinflammatory cytokines by peripheral blood leukocytes after ex vivo exposure to LPS had higher morbidity (ie, longer duration of mechanical ventilation, prolonged stay in the intensive care unit, and a higher incidence of clinical infection) and mortality rates than did patients whose leukocytes responded as usual to LPS. To date, there are no equine studies in which the development of immunoparalysis has been investigated, either in experimental conditions or in horses with naturally occurring disease.
We have used RT-qPCR assays to monitor the expression of genes associated with inflammation in peripheral blood leukocytes collected from horses with gastrointestinal disease at admission to a referral hospital. In that study,25 different patterns of gene expression were detected in leukocytes frozen immediately after collection when horses were grouped by disease type and outcome. The purpose of the study reported here was to compare changes in gene expression in peripheral blood leukocytes from healthy horses and horses with gastrointestinal disease after ex vivo incubation with LPS to those measured in leukocytes incubated with the same culture conditions in the absence of LPS. We hypothesized that the expression of a set of 14 genes encoding cytokines, cell receptors, and enzymes associated with the inflammatory response in peripheral blood leukocytes after ex vivo exposure to LPS would reflect the disease type and outcome. We also hypothesized that 4 hours of ex vivo incubation of peripheral blood leukocytes in the absence of LPS would alter gene expression when compared with expression patterns measured in leukocytes frozen immediately after collection. Finally, we hypothesized that the changes in gene expression in peripheral blood leukocytes incubated with LPS ex vivo would be blunted in horses with severe gastrointestinal disease, compared with changes in leukocytes isolated from healthy horses.
Materials and Methods
Animals—The study included horses aged 1 year or older that were admitted to the University of Georgia's Large Animal Teaching Hospital for evaluation of acute gastrointestinal disease (ie, colic or diarrhea) from July 5 through September 8, 2007. During that same period, a blood sample was collected from each of 10 university-owned healthy adult horses (4 to 12 years old) that lacked clinical signs of any substantial inflammatory process, had no history of gastrointestinal disease for ≥ 6 months, and had not received any medication for at least 30 days. These 10 healthy horses were housed indoors during the day and on pasture during the night. Consent was obtained from all owners, and the study protocol was approved by the University of Georgia's Institutional Animal Care and Use Committee and the College of Veterinary Medicine's Clinical Research Committee.
Climatological conditions during the study—Daily weather data for the period of study were retrieved from official sources. Local daily minimal and maximal environmental temperature and humidity were obtained from the university's Climatology Research Laboratory. Daily air pollen counts from the closest counting station (Atlanta, Ga) were obtained from the daily Pollen and Mold Report of the National Allergy Bureau, American Academy of Allergy, Asthma & Immunology.
Blood sample collection—One jugular groove of each horse was scrubbed with chlorhexidine and 70% isopropyl alcohol. Then, a 60-mL blood sample was obtained via jugular venipuncture by use of an 18-gauge needle and a syringe containing 1.5 mL of 100mM EDTA. Blood samples were collected over a period of 66 days. During this period, the environmental temperature ranged from 17.5° to 40.5°C (mean, 27.6°C), relative humidity ranged from 22% to 100% (mean, 68.3%), and pollen count ranged from 3 to 16 U/m3 (mean, 7.9 U/m3).
Leukocyte isolation and incubation—Within 30 minutes after blood sample collection, the syringe was positioned vertically on its plunger for 20 minutes to allow erythrocytes to sediment. Leukocyte-rich plasma was expressed into a sterile tube, diluted with an equal volume of Dulbecco PBS solution, and centrifuged (800 × g) at 20°C for 10 minutes. After discarding the supernatant, the erythrocytes were removed by use of hypotonie lysis and the leukocytes were washed with PBS solution and collected by use of centrifugation (800 × g) at 20°C for 5 minutes. The leukocytes were then suspended in 20 mL of PBS solution, and a 50-μL aliquot of this cell suspension was transferred to a vial containing 450 μL of 0.04% trypan bluea for viability assessment based on dye exclusion. The viable leukocytes (ie, nonstained leukocytes) were viewed with a light microscope and counted by use of a hemocytometer. With the cell count taken into account, leukocytes were suspended to a final concentration of 2 × 107 viable cells/mL in RPMI 1640 medium without phenol redb containing 1% commercial equine serum,c 2mM L-glutamine,d and 50 μg of gentamicin/mL.e One-milliliter portions of the final leukocyte suspension were transferred to sterile microcentrifuge tubes. To 1 tube, 1 ng of 055:B5 Escherichia coli LPSf was added. Cells in this tube and in 1 tube without LPS were incubated in a humidified atmosphere at 37°C and 5% C02 for 4 hours. The remaining vials with the leukocyte suspension were immediately centrifuged in a microcentrifuge at maximum speed (14,000 × g) for 1 minute. After the supernatant was discarded, the tubes with the cell pellets were stored at −80°C. These same protocols for cell harvesting and storage were used for the leukocyte samples incubated with or without LPS.
Real-time RT-qPCR—The RNA in cell samples was extracted by use of a commercial kit,g in accordance with the manufacturer's protocol, with inclusion of a deoxyribonuclease Ih digestion step to remove any contaminating genomic DNA. Assessment of RNA concentration and quality was performed with a spec-trophotometer.i Complementary DNA was synthesized by use of a thermal cyclerj and a commercial kit.k The RT-qPCR assay was performed with SYBR greenl in a sequence detection system,m with 18S rRNA used as an endogenous control sample.
Expression of 14 genes associated with systemic inflammatory responses was assessed: A2AR, COX-2, IL-lβ, IL-1RA, IL-6, IL-8, IL-10, L-selectin, MCP-3, MMP-9, P-selectin, Mn-SOD, TLR-4, and TNF-α. The RT-qPCR oligonucleotide primers were designed with commercial softwaren by use of sequences obtained from GenBank.o
Full validation of the SYBR green RT-qPCR assays for 12 of the 14 genes had previously been performed with RNA isolated from LPS-stimulated equine leukocytes. These validation assays included demonstration of the amplification of a single template, optimization of primer concentration, concordance of PCR amplification efficiency between the 12 genes and the 18S housekeeping gene, and linearity of amplification over at least 4 orders of template concentration.26 The housekeeping gene 18S was used as the internal control standard, and ΔCT was calculated as CTgene - CT18S.
To interpret the effect of incubation with and without LPS on gene expression, the ΔΔCT approach was used, with gene expression in the fresh samples serving as the calibrator. Therefore, ΔΔCT was calculated according to the following equations:
and
The mean values of ΔΔCT were used to calculate fold change in gene expression produced by incubation with and without LPS (fold change = 2-mean ΔΔCT).
Clinical data and horse groups—For the horses with gastrointestinal disease, information about clinical history, clinical findings at admission to the hospital, treatment, outcome, and, when applicable, necropsy findings was obtained from the medical records. Horses with gastrointestinal disease were grouped on the basis of disease type (CUO, gastrointestinal inflammation or strangulation, or nonstrangulated gastrointestinal obstruction) and outcome (discharge, death or euthanasia due to disease severity, or euthanasia due to financial constraints). The criteria for including a horse in the CUO group were absence of a clinical finding (eg, no profuse diarrhea, hard mass in the large colon detected by rectal palpation, or increased cell count in the peritoneal fluid) that would allow inclusion in another disease category and rapid recovery after treatment with fluids or drugs. The criteria for including a horse in the group in which euthanasia was performed because of financial constraints were absence of a clinical finding (eg, no peritoneal fluid containing plant particles and bacteria or large segment of nonviable intestine noticed at surgery or necropsy) suggestive of terminal disease or at least a very poor prognosis and evidence in the medical records that euthanasia was performed because of financial constraints.
Statistical analysis—Data from horses euthanized because the owner refused surgery were excluded from the comparison between the outcomes. Values of ΔΔCT for healthy and sick horses categorized by disease type and outcome were compared. After evaluating the normality of all data by creating histograms of the distributions, comparisons between horse groups (healthy and disease groups or healthy and outcome groups) were conducted by use of ANOVA and post hoc comparisons with the Bonferroni procedure. Comparisons of the effects of the 2 methods of leukocyte incubation were performed with a paired t test. Fatality proportions were compared between disease categories by use of the Fisher exact test. For all tests, a 2-sided alternative hypothesis was used and values of P < 0.05 were considered significant. Values of ΔΔCT considered clinically relevant were arbitrarily established as > 1 or < −1, which corresponded to a fold change in expression of > 2.0 or < 0.5, respectively.
Results
Animals—Forty-nine horses with gastrointestinal disease were included in the study. Eight horses had CUO, 18 had gastrointestinal inflammation or strangulation, and 23 had nonstrangulated gastrointestinal obstruction. Signs of gastrointestinal disease had been first detected 4 hours to 72 hours (median, 8 hours) before admission at the referral hospital, and all horses had been treated at least once with analgesics (eg, flunixin, xylazine, or butorphanol) or other medicines (eg, laxatives and fluids) before blood samples were collected. Of all 49 horses, 22 underwent celiotomy 22 only received medical treatment, and 5 were euthanized because surgery was indicated but declined by the owners. Of the 44 horses treated as recommended by the clinicians, 38 were discharged from the teaching hospital and 6 died or were euthanized. All horses included in the CUO group were discharged from the hospital.
Mortality rates did not differ between horses with gastrointestinal inflammation or strangulation and horses with nonstrangulated gastrointestinal obstruction (P = 0.68). Among the 38 horses discharged from the hospital, interval to discharge ranged from < 24 hours to 13 days after admission (median, 6 days). Complications detected while these horses were hospitalized included diarrhea (n = 6), transient ileus (4), and mild infection of the surgical wound (2). Signs of laminitis were only evident in 1 horse, which was euthanized because of disease severity
Gene expression after incubation without LPS—Relative to gene expression in leukocytes frozen immediately after isolation, relevant changes in gene expression (fold change > 2 or < 0.5) after ex vivo incubation without LPS that were significantly different between horse groups were detected for several genes (Tables 1 and 2). After incubation without LPS, expression of COX-2 in healthy horses was lower than that in horses with nonstrangulated obstruction and horses that survived to discharge from the hospital. After incubation without LPS, expression of IL-1 RA was increased in all groups, but a smaller increase was detected in horses with strangulated obstruction or gastrointestinal inflammation than in healthy horses or horses with nonstrangulated obstruction. Incubation without LPS also resulted in an increase in the expression of IL6 in healthy horses, and this increase differed significantly from the smaller increase in expression detected in horses with CUO and the lack of change in all other sick horses grouped by disease type or outcome. After incubation without LPS, expression of IL-8 was increased in healthy horses and those with CUO, and this increase was not evident in horses with nonstrangulated intestinal obstruction or nonsurvivors. Incubation without LPS increased the expression of IL-10 in healthy horses and all horses with gastrointestinal disease, except those with strangulated obstruction or inflammation. After incubation without LPS, expression of MMP-9 was decreased in healthy horses, horses with strangulated obstruction or inflammation, and nonsurvivors. Incubation without LPS increased the expression of Mn-SOD in horses with nonstrangulated obstruction. After incubation without LPS, expression of P-selectin was decreased in all groups of horses with gastrointestinal disease. Finally, incubation without LPS resulted in a modest increase in the expression of TNF-α in healthy horses, which contrasted with the marked increase in the sick horses.
Gene expression (mean ± SEM ΔΔCT) as measured via RT-qPCR assays in peripheral blood leukocytes isolated from blood samples of horses without (n = 10) and with (49) gastrointestinal disease (grouped by disease category) and incubated in medium with and without LPS.
| Gene | Incubation medium | Healthy horses | Horses with gastrointestinal disease | ||
|---|---|---|---|---|---|
| CUO(n = 8) | Strangulated obstruction or inflammation (n = 18) | Nonstrangulated obstruction (n = 23) | |||
| A2AR | Medium alone | -2.86 ± 0.28* (7.27) | -3.58 ± 0.30* (11.93) | -3.11 ± 0.25*(8.61) | -3.91 ± 0.23* (15.07) |
| A2AR | Medium + LPS | −1.04 ± 0.25* (2.06) | −1.04 ± 0.20* (2.05) | −0.92 ± 0.16* (1.90) | −0.54 ± 0.18* (1.46) |
| COX-2 | Medium alone | 1.41 ± 0.46*a (0.38) | 0.01 ± 0.56a,b (1.00) | 0.28 ± 0.37*a,b (0.83) | −0.18 ± 0.27b (1.13) |
| COX-2 | Medium + LPS | −0.54 ± 0.19* (1.45) | −0.77 ± 0.13 (1.70) | −0.97 ± 0.17* (1.96) | −0.54 ± 0.18 (1.46) |
| IL-1β | Medium alone | −2.03 ± 0.43 (4.08) | −2.84 ± 0.65 (7.18) | −1.40 ± 0.40* (2.64) | −2.34 ± 0.31 (5.06) |
| IL-1β | Medium + LPS | −2.43 ± 0.28 (5.39) | −2.21 ± 0.18 (4.63) | −2.55 ± 0.19* (5.85) | −1.98 ± 0.23 (3.94) |
| IL-1RA | Medium alone | −4.71 ± 0.22*a (26.19) | −4.24 ±0.36*ab (18.95) | −3.22 ± 0.37*b (9.29) | −4.32 ± 0.23*a (20.03) |
| IL-1RA | Medium + LPS | −1.92 ± 0.26* (3.78) | −1.85 ± 0.16* (3.60) | −2.03 ± 0.19* (4.10) | −1.59 ± 0.18* (3.02) |
| IL-6 | Medium alone | −3.61 ± 0.37*a (12.19) | −1.18 ± 0.48b (2.26) | 0.29 ± 0.57*b (0.82) | −0.39 ± 0.29*b (1.31) |
| IL-6 | Medium + LPS | −1.20 ± 0.25* (2.29) | −2.35 ± 0.39 (5.11) | −2.25 ± 0.38* (4.77) | −2.02 ± 0.28* (4.05) |
| IL-8 | Medium alone | −1.78 ± 0.65a (3.44) | −1.66 ± 1.00a,b (3.15) | −0.26 ± 0.39a,b (1.19) | 0.20 ± 0.29b (0.87) |
| IL-8 | Medium + LPS | −0.83 ± 0.17 (1.77) | −0.76 ± 0.23 (1.69) | −0.46 ± 0.12 (1.38) | −0.42 ± 0.13 (1.34) |
| IL-10 | Medium alone | −1.87 ± 0.37a,b (3.66) | −2.23 ± 0.17a,b (4.68) | −0.94 ± 0.33*a (1.92) | −2.10 ± 0.23*b (4.30) |
| IL-10 | Medium + LPS | −2.35 ± 0.36a (5.10) | −1.73 ± 0.20a,b (3.31) | −1.84 ± 0.20*a,b (3.58) | −1.36 ± 0.11*b (2.57) |
| L-selectin | Medium alone | 2.10 ± 0.22* (0.23) | 2.10 ± 0.15* (0.23) | 2.35 ± 0.16* (0.20) | 2.25 ± 0.12*(0.21) |
| L-selectin | Medium + LPS | 0.28 ± 0.19* (0.82) | 0.80 ± 0.18* (0.58) | 0.70 ± 0.09* (0.61) | 0.67 ± 0.05* (0.63) |
| MCP-3 | Medium alone | −6.28 ± 0.55* (77.86) | −6.91 ± 0.91* (120.48) | −5.58 ± 0.70* (47.94) | −6.32 ± 0.38* (79.69) |
| MCP-3 | Medium + LPS | −1.05 ± 0.17*a (2.07) | −2.17 ± 0.37*a,b (4.50) | −1.85 ± 0.31*a,b (3.61) | −2.38 ± 0.25*b (5.22) |
| MMP-9 | Medium alone | 1.15 ± 0.38*ab (0.45) | 0.43 ± 0.16a,b (0.74) | 1.17 ± 0.27a (0.45) | 0.18 ± 0.20b (0.88) |
| MMP-9 | Medium + LPS | −0.89 ± 0.72a* (1.85) | 0.16 ± 0.30a,b (0.90) | 0.55 ± 0.21b (0.69) | 0.55 ± 0.14b (0.68) |
| Mn-SOD | Medium alone | −0.15 ± 0.17a (1.11) | −0.68 ± 0.12a,b (1.60) | −0.51 ± 0.22a,b (1.42) | −1.16 ± 0.17b (2.24) |
| Mn-SOD | Medium + LPS | −0.76 ± 0.63 (1.69) | −1.08 ± 0.15 (2.11) | −1.13 ± 0.14 (2.19) | −0.91 ± 0.10(1.87) |
| P-selectin | Medium alone | 0.19 ± 0.24a (0.87) | 1.11 ± 0.18a,b (0.46) | 1.49 ± 0.24*b (0.36) | 1.53 ±0.19*b (0.35) |
| P-selectin | Medium + LPS | −0.59 ± 0.48a (1.50) | 0.87 ± 0.23b (0.55) | 0.60 ± 0.18*b (0.66) | 0.46 ± 0.17*b (0.73) |
| TLR-4 | Medium alone | 0.10 ± 0.39 (0.93) | 0.43 ± 0.51 (0.74) | 0.90 ± 0.16* (0.54) | 0.55 ± 0.15* (0.68) |
| TLR-4 | Medium + LPS | −0.61 ± 0.14 (1.53) | −0.59 ± 0.40 (1.50) | −0.25 ± 0.14* (1.19) | −0.15 ± 0.08* (1.11) |
| TNF-α | Medium alone | −1.42 ± 0.25a (2.67) | −4.00 ± 0.60b (15.99) | −4.67 ± 0.39*b (25.48) | −4.77 ± 0.35*b (27.30) |
| TNF-α | Medium + LPS | −1.89 ± 0.40 (3.72) | −2.08 ± 0.46 (4.21) | −1.63 ± 0.22* (3.10) | −1.41 ± 0.24* (2.66) |
Values in parentheses represent fold change in gene expression. Gray highlighting of a row indicates the simultaneous significant (P < 0.05) difference between groups and, for at least 1 group, a fold change in gene expression of > 2 or < 0.5, indicating a biologically important change.
For each gene, expression differed significantly (P < 0.05) between the 2 incubation conditions within a group.
Within each row, different superscript letters indicate a significant (P < 0.05) difference between values of horse groups.
Fold change in gene expression was calculated as 2−mean ΔΔCT. The ΔΔCT was calculated as follows: ΔΔCTincubation without LPS = ΔCTsample incubated without LPS − ΔCTnonincubated sample; ΔΔCTincubation with LPS = ΔCTsample incubated with LPS − ΔCTsample incubated without LPS.
Gene expression (mean ± SEM ΔΔCT) as measured via RT-qPCR assays in peripheral blood leukocytes isolated from blood samples of horses without (n = 10) and with (44) gastrointestinal disease with (grouped by disease category) and incubated in medium with and without LPS
| Gene | Incubation medium | Healthy horses | Horses with gastrointestinal disease | |
|---|---|---|---|---|
| Discharged (n = 38) | Euthanized or died (n = 6) | |||
| A2AR | Medium alone | −2.86 ± 0.28* (7.27) | −3.64 ± 0.16* (12.44) | −2.78 ± 0.64 (6.87) |
| A2AR | Medium + LPS | −1.04 ± 0.25* (2.06) | −0.80 ± 0.10* (1.75) | −0.91 ± 0.23 (1.87) |
| COX-2 | Medium alone | 1.41 ± 0.46*a (0.38) | −0.02 ± 0.24*b (1.01) | 0.33 ± 0.60a,b (0.80) |
| COX-2 | Medium + LPS | −0.54 ±0.19* (1.45) | −0.62 ±0.12* (1.54) | −1.27 ± 0.34 (2.41) |
| IL-1β | Medium alone | −2.03 ± 0.43 (4.08) | −2.23 ± 0.25 (4.69) | −1.70 ± 1.14 (3.24) |
| IL-1β | Medium + LPS | −2.43 ± 0.28 (5.39) | −2.15 ± 0.16 (4.42) | −2.30 ± 0.20 (4.93) |
| IL-1RA | Medium alone | −4.71 ± 0.22* (26.19) | −4.09 ± 0.21* (17.02) | −3.30 ± 0.82 (9.87) |
| IL-1RA | Medium + LPS | −1.92 ± 0.26* (3.78) | −1.71 ± 0.14*(3.27) | −1.97 ± 0.32 (3.92) |
| IL-6 | Medium alone | −3.61 ± 0.37*a (12.19) | −0.45 ± 0.29*b (1.37) | 0.66 ± 1.08b (0.63) |
| IL-6 | Medium + LPS | −1.20 ± 0.25* (2.29) | −2.11 ± 0.22* (4.32) | −1.84 ± 0.79 (3.58) |
| IL-8 | Medium alone | −1.78 ± 0.65a (3.44) | −0.50 ± 0.31a,b (1.41) | 0.73 ± 0.80b (0.60) |
| IL-8 | Medium + LPS | −0.83 ± 0.17 (1.77) | −0.45 ± 0.10 (1.37) | −0.64 ± 0.21 (1.56) |
| IL-10 | Medium alone | −1.87 ± 0.37 (3.66) | −1.89 ± 0.19 (3.72) | −0.62 ± 0.60 (1.53) |
| IL-10 | Medium + LPS | −2.35 ± 0.36a (5.10) | −1.62 ± 0.12b (3.07) | −1.52 ± 0.29a,b (2.86) |
| L-selectin | Medium alone | 2.10 ± 0.22* (0.23) | 2.27 ± 0.10* (0.21) | 2.34 ± 0.21* (0.20) |
| L-selectin | Medium + LPS | 0.28 ± 0.19*a (0.82) | 0.70 ± 0.06*b (0.62) | 0.64 ± 0.15*a,b (0.64) |
| MCP-3 | Medium alone | −6.28 ± 0.55* (77.86) | −6.19 ± 0.37* (72.94) | −6.21 ± 1.28*(73.95) |
| MCP-3 | Medium + LPS | −1.05 ± 0.17*a (2.07) | −2.23 ± 0.20*b (4.68) | −1.11 ± 0.35*a,b (2.16) |
| MMP-9 | Medium alone | 1.15 ± 0.38* (0.45) | 0.54 ± 0.17 (0.69) | 1.05 ± 0.47 (0.48) |
| MMP-9 | Medium + LPS | −0.89 ± 0.72*a (1.85) | 0.40 ± 0.14b (0.76) | 0.64 ± 0.26ab (0.64) |
| Mn-SOD | Medium alone | −0.15 ± 0.17a (1.11) | −0.91 ± 0.14b (1.88) | −0.29 ± 0.25*a,b (1.23) |
| Mn-SOD | Medium + LPS | −0.76 ± 0.63 (1.69) | −0.96 ± 0.09 (1.94) | −1.28 ± 0.14* (2.42) |
| P-selectin | Medium alone | 0.19 ±0.24a (0.87) | 1.48 ±0.14*b (0.36) | 1.02 ± 0.46a,b (0.49) |
| P-selectin | Medium + LPS | −0.59 ± 0.48a (1.50) | 0.56 ± 0.14*b (0.68) | 0.49 ± 0.23a,b (0.71) |
| TLR-4 | Medium alone | 0.10 ± 0.39 (0.93) | 0.60 ± 0.15* (0.66) | 1.13 ± 0.26* (0.46) |
| TLR-4 | Medium + LPS | −0.61 ± 0.14 (1.53) | −0.29 ± 0.11* (1.23) | −0.39 ± 0.08* (1.31) |
| TNF-α | Medium alone | −1.42 ± 0.25a (2.67) | −4.48 ± 0.24*b (22.34) | −4.15 ± 0.93*b (17.79) |
| TNF-α | Medium + LPS | −1.89 ± 0.40 (3.72) | −1.57 ± 0.20* (2.97) | −1.69 ± 0.29* (3.24) |
See Table 1 for key.
Gene expression with versus without LPS incubation—Relative to gene expression in leukocytes after ex vivo incubation without LPS, relevant changes in gene expression (fold change > 2 or < 0.5) after ex vivo incubation with LPS that were also significantly different between horse groups were evident for only 2 genes. An increase in LPS-induced expression of IL-10 was detected in healthy and sick horses, but this change was significantly less pronounced in horses with nonstrangulated obstruction and horses that survived relative to the change in healthy horses. An increase in LPS-induced expression of MCP-3 was detected in healthy and all groups of sick horses, but this change was significantly more pronounced in horses with nonstrangulated obstruction and horses that survived, relative to that in healthy horses.
Discussion
Because of the known effects of LPS on leukocytes,7,27,28 the changes in gene expression resulting from LPS incubation of peripheral blood leukocytes from diseased and healthy horses in the present study were expected. However, as was evident in this study, leukocyte isolation and incubation without LPS can also induce significant changes in leukocyte gene expression. In the horses with gastrointestinal disease, leukocytes may have been already activated in vivo, and the 4-hour incubation without additional LPS may have simply allowed time for the changes in gene expression to be manifested. Furthermore, the possibility that physical or chemical stimulation of the leukocytes during isolation and culture resulted in activation cannot be excluded and may help explain the changes in gene expression in leukocytes from healthy horses after ex vivo incubation without LPS. The magnitude of the changes in gene expression during incubation without LPS highlighted the importance of distinguishing the effects of ex vivo stimulation by LPS from the effects of ex vivo manipulation and incubation. Our findings also highlighted the limitations of comparing results obtained in other studies conducted with different methodologies. These observations and conclusions are in agreement with those of recent studies28,29 in which gene expression in mononuclear cells was affected by cell isolation and ex vivo incubation.
A blunted leukocyte response to LPS in the study horses with gastrointestinal disease was not apparent in the present study. The magnitude of the change in expression of 8 of the 10 proinflammatory genes in response to LPS relative to incubation without LPS was not different or was greater than the change resulting from incubation without LPS relative to the expression in fresh leukocytes. Furthermore, in all groups of horses with gastrointestinal disease, incubation of leukocytes with LPS resulted in > 2-fold increases in the expression of 4 of the 10 proinflammatory genes (for IL-1β, IL-6, MCP-3, and TNF-α).
In this study, different patterns of expression were identified for each horse group and gene. The changes in expression of IL-10 and MCP-3 were likely the most relevant because marked changes in expression resulted from incubation with and without LPS and significant differences between horse groups were evident after leukocyte incubation with LPS. The other interesting aspect of the behavior of these 2 genes was the fact that their responses to LPS were inversely related to one another.
An anti-inflammatory cytokine, IL-10, is produced primarily by monocytes and, to a lesser extent, by lymphocytes.20,30 This cytokine suppresses the synthesis of proinflammatory cytokines30–32 and is one of the principal modulators of the inflammatory response.33,34 In peripherally obtained blood from septic humans, an increase in plasma concentration and leukocyte expression of IL-10 is associated with immunosuppression and a poor prognosis.35–37 Similarly, an increase in IL-10 expression by leukocytes has been associated with a poor outcome in foals with septic and nonseptic diseases.17 In contrast, IL-10 expression by nonincubated peripheral blood leukocytes from the horses with gastrointestinal disease in the present study did not differ from expression in healthy horses and was not associated with outcome.25 There is evidence that IL-10 is neither the main factor nor an absolute requirement for the development of immunosuppression associated with severe inflammation and sepsis.19,34,38,39 In the present study, the increased expression of IL-10 in leukocytes from both healthy and sick horses after 4-hour incubation without LPS may have resulted from ex vivo physical or chemical cell stimulation, as has been reported.29
The LPS-induced increase in IL-10 expression evident in all horse groups in the present study contrasts with a previous report27 that IL-10 expression by peripheral blood monocytes obtained from healthy horses did not increase in response to LPS. Differences in study design (eg, total leukocytes were used in the present study rather than monocytes used in the other study27) may explain these differences. Conversely, the findings of the present study are in agreement with reports of studies35,40–42 in other animal species in which serum concentration and gene expression of IL-10 increased after in vivo and ex vivo exposure of peripheral blood leukocytes to LPS. The other interesting finding in the present study regarding IL-10 was the less pronounced increase in expression in leukocytes from horses with nonstrangulated obstruction and horses that were discharged from the hospital. This finding suggested that the leukocytes from horses that survived had a relatively more proinflammatory phenotype when compared with the phenotype in healthy horses, which is in agreement with findings in septic humans and foals.17,37
A chemokine produced by monocytes, MCP-3, has potent chemotactic activity for most leukocytes including monocytes, lymphocytes, and neutrophils,43–46 although little is known regarding the role of MCP-3 in sepsis or endotoxemia. Gene expression of MCP-3 in human monocytes stimulated ex vivo by LPS has been reported,46 but this response was inconsistent in other in vivo and ex vivo studies44,47 with LPS. For example, MCP-3 gene expression increased in laminar tissue during the first 24 hours of experimentally induced laminitis in horses48 but not in peripheral blood leukocytes isolated at admission from horses with gastrointestinal disease.25 In the present study, the marked increase in MCP-3 expression by leukocytes after incubation in the absence of LPS contrasts with the relatively modest additional increase in MCP-3 gene expression in leukocytes incubated with LPS. These findings suggest that LPS is not a strong stimulus for MCP-3 gene expression, as has been reported for human monocytes,44,47 or that the response initiated by the techniques used to isolate and incubate leukocytes may have overshadowed the response to LPS. In any case, gene expression of MCP-3 induced by LPS was more pronounced in leukocytes from horses with nonstrangulated obstruction and from horses that survived, relative to the observations regarding leukocytes from healthy horses. These findings are the inverse of what was found for IL-10, which is a cytokine that has a direct inhibitory effect on the expression of the MCP-3 gene by human monocytes.45,46
The various disease types, stages, and severity as well as treatments (before and after sample collection) were likely major sources of variability in the present study. These factors, combined with the relatively small number of horses, likely limited the power of this study to detect significant differences if they truly existed. To interpret the results, it is also important to consider that most horses with gastrointestinal disease included in the study were in relatively good condition as indicated by the clinical findings at admission to the hospital, the low complication and fatality rates, and the short duration of hospitalization. Conversely, in studies22,23 involving humans in which patterns of cytokine expression in leukocytes after ex vivo exposure to LPS correspond to disease type or outcome, there were large numbers of patients in critical condition. Similarly, endotoxin tolerance in laboratory animals is typically investigated through the use of experimental models of severe sepsis.49 On the basis of these findings, it is possible that immunoparalysis would only occur in horses with extremely severe conditions, which, at this time, are typically euthanized because of the high expense of providing intensive care for such a large patient and the poor prognosis. Regardless, the present study of equine leukocytes incubated ex vivo with LPS revealed other patterns of gene expression that appear to be related to pathogenesis and prognosis of equine gastrointestinal disease. Leukocyte response to ex vivo incubation with LPS consequently deserves additional investigation as a potential tool for early decision making in the treatment of horses with gastrointestinal disease.
Abbreviations
| A2AR | Adenosine 2A receptor |
| COX-2 | Cyclooxygenase-2 |
| CT | Number of cycles to threshold |
| CUO | Colic of unknown origin |
| IL | Interleukin |
| IL-1RA | Interleukin-1 receptor antagonist |
| LPS | Lipopolysaccharide |
| MCP-3 | Monocyte chemotactic protein-3 |
| MMP-9 | Matrix metalloproteinase-9 |
| Mn-SOD | Mitochondrial Superoxide dismutase |
| RT-qPCR | Reverse transcription quantitative PCR |
| SIRS | Systemic inflammatory response syndrome |
| TLR-4 | Toll-like receptor-4 |
| TNF | Tumor necrosis factor |
Trypan Blue, Sigma-Aldrich, St Louis, Mo.
RPMI 1640 (1×), Mediatech Ine, Herndon, Va.
Donor Equine Serum, US Origin, HyClone Laboratories Inc, Logan, Utah.
L-glutamine, Mediatech Inc, Herndon, Va.
Gentamicin sulfate, Mediatech Inc, Herndon, Va.
LPS from Escherichica coli 055:b5, List Biological Laboratories Inc, Campbell, Calif.
RNeasy, QIAGEN Inc, Germantown, Md.
TURBO DNase, Applied Biosystems Inc, Foster City Calif
ND-1000 UV-Vis Spectrophotometer, NanoDrop Technologies Inc, Wilmington, Del.
Mastercycler Gradient, Eppendorf Inc, Westbury NY.
High Capacity cDNA Reverse Transcription Kit, Applied Biosystems Inc, Foster City Calif.
SYBR Green PCR Master Mix, Applied Biosystems Inc, Foster City, Calif.
7900HT Fast Real-Time PCR System, Applied Biosystems Inc, Foster City Calif.
Primer Express, Applied Biosystems Inc, Foster City Calif.
Genbank [database online]. Bethesda, Md: National Center for Biotechnology Information, 2010. Available at: www.ncbi.nlm.nih.gov/Genbank/. Accessed May 2007.
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