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    Mean and 95% confidence limits of the percentage of apoptosis of equine neutrophils cultured for 24 hours with various concentrations of LPS. Data are for 3 separate experiments on neutrophils isolated from blood samples obtained from the same 3 horses at 3 separate times. Apoptosis was assessed by use of annexin V and propidium iodide staining with flow cytometric quantification. *†Value differs significantly (*P = 0.004; †P < 0.001) from the value for 0.01 μg of LPS/mL. ‡§Value differs significantly (‡P = 0.014; §P < 0.001) from the value for 0.001 μg of LPS/mL.‖¶ Value differs significantly (‖P = 0.001; ¶P < 0.001) from the value for 0.1 μg of LPS/mL. #Value differs significantly (P = 0.024) from the value for 0.1 μg of LPS/mL.

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    Flow cytometry data for apoptosis of equine neutrophils cultured without (A and D) or with (B and E) LPS for 12 (A and B) or 24 (D and E) hours and box-and-whisker plots of the results of culture for 12 (C) and 24 (F) hours. Panels A, B, D, and E are representative results for flow cytometric quantification (fluorescence) of annexin V and propidium iodide staining of neutrophils. Cells in Q1 are artifact. Dead neutrophils are stained with both annexin V and propidium iodide (Q2), live neutrophils are not stained with annexin V or propidium iodide (Q3), and apoptotic neutrophils are stained with annexin V but not stained with propidium iodide (Q4). Panels C and F represent results for equine neutrophils isolated from blood samples obtained from 40 horses and cultured without (black boxes) and with (gray boxes) 1 μg of LPS/mL. Each box represents the 25th to 75th percentiles, the horizontal line in each box represents the median, and the whiskers represent the range. *†Within a cell category, value differs significantly (*P < 0.001; †P < 0.05) between treatments.

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    Photomicrographs of representative cytologic preparations of equine neutrophils isolated from blood samples obtained from 8 horses and cultured without (A and C) or with (B and D) 1 μg of LPS/mL for 12 (A and B) or 24 (C and D) hours and plots of the percentage of neutrophils with apoptosis (E). In panels A through D, notice the apoptotic neutrophils (arrows). Modified Giemsa stain; bar = 20 μm. In panel E, each square represents results for samples obtained from 1 horse and cultured without (black squares) or with (gray squares) LPS. The horizontal bar within in each group represents the median value. *†Value differs significantly (*P = 0.008; †P < 0.001) between treatments.

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    Activity of caspase-3 (A), -8 (B), and -9 (C) in neutrophil lysates after culture without (black squares) or with (gray squares) 1 μg of LPS/mL for 12 hours. Activity was indirectly determined as the free p-nitroanilide content corrected for protein concentration. Neutrophils were isolated from blood samples obtained from 16 horses; each square represents results for 1 horse, and the horizontal bar in each group represents the median value. *Value differs (P < 0.05) significantly from the value for the untreated neutrophils.

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    Apoptosis of equine neutrophils after culture for 24 hours without (black squares) or with (gray squares) 1 μg of LPS/mL, with a TLR4 inhibitor alone (black triangles), or with a TLR4 inhibitor and LPS (gray triangles). Apoptosis was assessed with flow cytometric quantification of annexin V and propidium iodide staining. Neutrophils were isolated from blood samples obtained from 7 horses. Each symbol represents results for 1 horse, and the horizontal bar in each group represents the median value. a,bValues with different letters differs (P < 0.05) significantly.

  • View in gallery

    Apoptosis of equine neutrophils cultured for 12 hours with inducers of apoptosis gambogic acid (GA; A) or staurosporine (Stauro; B). A—Neutrophils were cultured without (black squares) or with (gray squares) 1 μg of LPS/mL, gambogic acid (2 μg/mL; black circles), or gambogic acid and LPS (gray circles). B—Neutrophils were cultured without (black squares) or with (gray squares) 1 μg of LPS/mL, staurosporine (2μM; black hexagons), or staurosporine and LPS (gray hexagons). Apoptosis was assessed with flow cytometric quantification of annexin V and propidium iodide staining. Neutrophils were isolated from blood samples obtained from 6 horses. See Figure 5 for remainder of key.

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Role of toll-like receptor 4 and caspase-3, -8, and -9 in lipopolysaccharide-induced delay of apoptosis in equine neutrophils

Stacy L. Anderson DVM, MVSC, PhD1, Hugh G.G. Townsend DVM, MSC2, and Baljit Singh BVSc&AH, MVSc, PhD3
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  • 1 Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.
  • | 2 Department of Large Animal Clinical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.
  • | 3 Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada.

Abstract

OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro.

SAMPLE Venous blood samples from 40 adult horses.

PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities.

RESULTS Treatment with LPS resulted in a significant delay in apoptosis after incubation for 12 and 24 hours (neutrophils from blood samples of 40 horses). There was a significant correlation between increases in LPS dose and decreases in apoptosis after incubation for 24 hours (3 experiments, each of which involved neutrophils obtained from the same 3 horses at 3 separate times). Caspase-9 activity, but not caspase-3 or -8 activity, was significantly reduced in LPS-treated neutrophils after incubation for 12 hours (neutrophils from blood samples of 17 horses). Treatment with a TLR4 inhibitor or intrinsic and extrinsic inducers of apoptosis prevented LPS-delayed apoptosis.

CONCLUSIONS AND CLINICAL RELEVANCE LPS treatment delayed apoptosis of equine neutrophils in vitro for up to 24 hours in a dose-dependent manner by alteration of the intrinsic pathway of apoptosis and was dependent on TLR4 signaling. Increased neutrophil life span may contribute to the development of a systemic inflammatory response syndrome in endotoxemic horses.

Abstract

OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro.

SAMPLE Venous blood samples from 40 adult horses.

PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities.

RESULTS Treatment with LPS resulted in a significant delay in apoptosis after incubation for 12 and 24 hours (neutrophils from blood samples of 40 horses). There was a significant correlation between increases in LPS dose and decreases in apoptosis after incubation for 24 hours (3 experiments, each of which involved neutrophils obtained from the same 3 horses at 3 separate times). Caspase-9 activity, but not caspase-3 or -8 activity, was significantly reduced in LPS-treated neutrophils after incubation for 12 hours (neutrophils from blood samples of 17 horses). Treatment with a TLR4 inhibitor or intrinsic and extrinsic inducers of apoptosis prevented LPS-delayed apoptosis.

CONCLUSIONS AND CLINICAL RELEVANCE LPS treatment delayed apoptosis of equine neutrophils in vitro for up to 24 hours in a dose-dependent manner by alteration of the intrinsic pathway of apoptosis and was dependent on TLR4 signaling. Increased neutrophil life span may contribute to the development of a systemic inflammatory response syndrome in endotoxemic horses.

Horses are extremely sensitive to endotoxin in the blood, and they generate a robust systemic inflammatory response after systemic administration of as little as 20 ng of LPS endotoxin/kg.1,2 Horses may become endotoxemic from various disease processes, with the most common being a result of conditions affecting the gastrointestinal tract, including colitis and strangulating intestinal lesions.1,3 Endotoxemia results from compromise of the intestinal epithelial barrier and subsequent bacterial translocation, which ultimately leads to a systemic inflammatory response.1,3,4 Neutrophils are important for the propagation of an inappropriate systemic inflammatory response to endotoxin because of their nonspecific methods of antigen neutralization, which often leads to collateral damage to adjacent cells and tissues.4

Cells may undergo apoptosis through 2 pathways. The extrinsic pathway involves activation of cell membrane receptors by specific ligands (eg, tumor necrosis factor-α), which is followed by activation of a cascade of intracellular proteases (referred to as caspases) that initiate apoptosis.5 Alternatively, the intrinsic pathway begins with an intracellular signal, such as irreparable damage to DNA or severe cell stress, which changes the balance of the Bcl-2 family of proteins to increase mitochondrial permeabilization and allows release of cytochrome c into the cytosol and activation of the apoptosome and initiator caspase-9. The extrinsic and intrinsic pathways are linked through activation of the proapoptotic Bcl-2 homology-3-interacting domain death agonist protein by caspase-8. As a final step in both pathways, the effector caspases, caspase-3 and -7, enter the nucleus and cause DNA fragmentation that leads to characteristic changes in nuclear morphology, including karyorrhexis and pyknosis.6,7 The effector caspases also activate proteins that dissemble components of the cytoskeleton (eg, actin), which results in cell shrinkage and blebbing.8,9

Although it has not been specifically evaluated in horses, neutrophils in other species have a relatively short circulatory life span (half-life in circulation, 6 to 8 hours), which ends with the induction of apoptosis via the intrinsic pathway of apoptosis.10–12 The abbreviated life span of neutrophils may be a mechanism by which the body removes a population of potentially activated cells.13 Phagocytosis of apoptotic neutrophils by macrophages results in release of anti-inflammatory cytokines (eg, transforming growth factor-β) and the inhibition of proinflammatory cytokines (eg, interleukin-1β, interleukin-8, and tumor necrosis factor-α).14–16 In addition, neutrophil apoptosis is the primary signal for their removal from the blood by phagocytic cells, especially macrophages, located in the spleen, liver, and bone marrow.12,17

Delayed neutrophil apoptosis has been implicated in the development of an inappropriate systemic inflammatory response to endotoxin and prevention of the resolution of inflammation.17–20 Bacterial LPS or sepsis delays neutrophil apoptosis in laboratory mammals21 and humans11,18,22 in vitro and in vivo. It has been proposed that delayed apoptosis of neutrophils in response to LPS is influenced by activation of the nuclear factor-κB and phosphoinositide 3-kinase pathways via TLR4 signaling, which leads to an increase in antiapoptotic proteins (eg, Bcl-2, myeloid leukemia cell-1, and A1) that downregulates the intrinsic pathway of apoptosis.10,23

To the authors’ knowledge, only 1 study24 has been conducted to evaluate the effect of LPS on the life span of equine neutrophils in vitro. In that study,24 there was an increase in neutrophil apoptosis after LPS treatment, which is in direct contrast to results for all other species. Because horses are highly susceptible to the effects of endotoxin, further evaluation of the effects of LPS on neutrophil life span is needed. Therefore, the objective of the study reported here was to investigate the effect of LPS on apoptosis of equine neutrophils in vitro. We hypothesized that LPS would delay apoptosis of equine neutrophils in a dose-dependent manner via downregulation of the intrinsic pathway of apoptosis.

Materials and Methods

Sample

Neutrophils were isolated from blood samples obtained from 40 healthy horses. Blood samples (60 mL/sample) were collected aseptically from a jugular vein into evacuated tubes containing EDTA.a All procedures and experimental protocols that involved the use of animals were approved by the University of Saskatchewan Committee on Animal Care and Supply and the University of Saskatchewan Animal Research Ethics Board.

Neutrophil isolation and culture conditions

Neutrophils were isolated by use of a previously reported protocol,25 with minor modifications. Isolation was performed at room temperature (20° to 22°C). Immediately after blood samples were collected, the blood was allowed to separate (room temperature for 30 to 45 minutes) into plasma and red cell fractions. The plasma fraction was layered onto 10 mL of density gradient mediumb and centrifuged (400 × g for 30 minutes). Supernatant was aspirated, and the pellet (which contained erythrocytes and granulocytes) was washed with HBSS without phenol, magnesium, or calcium by use of centrifugation at 200 × g for 10 minutes. Erythrocytes were lysed by incubation with 2 mL of sterile distilled water (pH, 7.4) for 25 seconds and restored to normotonicity with an equal volume of hypertonic (2×) HBSS. Cells were washed 3 times with HBSS by use of centrifugation at 200 × g for 10 minutes, suspended in culture medium (RPMI 1640 medium containing 10% fetal bovine serum, 2mM l-glutamine, 100 U of penicillin/mL, 50 μg of streptomycin/mL, and 25mM HEPES), assessed for viability by use of trypan blue staining, and counted with a hemacytometer. Cell purity was determined on cytologic specimens prepared with cell concentrations diluted to 1 million cells/mL by use of a cytocentrifugec (1,000 × g for 4 minutes with medium acceleration) and stained with a modified Giemsa stain.d Experiments proceeded if cell viability was > 98% and cell purity was > 90%.

For all experiments, neutrophils were suspended in culture medium (as described previously) in plastic 24-well cell culture plates at a concentration of 2 × 106 cells/mL and incubated at 37°C in 5% CO2. Prior to performing apoptosis assays, neutrophils were removed from cultures, pelleted, and washed twice with HBSS by use of centrifugation at 400 × g for 8 minutes.

Determination of apoptosis by use of flow cytometry

In an initial experiment, neutrophils were isolated from samples collected from 3 horses 3 separate times. Cells were left untreated or were treated with serial 10-fold concentrations (0.001 to 10 μg/mL) of LPS from Escherichia coli O55:B5.e The experiment was repeated with neutrophils isolated from samples obtained from 40 horses and cultured for 12 or 24 hours with or without 1 μg of LPS/mL. Washing was performed as described previously, and neutrophils then were stained with fluorescent dye-conjugated annexin V and propidium iodide by use of a commercially available assay kitf used in accordance with the manufacturer's instructions. A flow cytometerg was used to quantify unstained cells (live cells), cells stained with annexin V (apoptotic cells), or cells stained with annexin V and propidium iodide (dead cells). Flow cytometry data were analyzed with commercial software.h The gated population was determined with unstained cells on the basis of size (forward scatter) and granularity (side scatter). Quadrants to set the threshold of fluorescence intensity for both annexin V and propidium iodide were determined by use of untreated cells stained with only annexin V or propidium iodide. Data were acquired on at least 10,000 gated events. All experiments were performed in duplicate, and mean values were calculated and recorded.

Determination of apoptosis by use of cytologic examination

Neutrophils isolated from samples obtained from 8 horses that were cultured for 12 or 24 hours with or without 1 μg of LPS/mL and evaluated by use of flow cytometry were concurrently evaluated by use of cytologic preparations, as described previously. Nuclear morphology of neutrophils was evaluated by use of light microscopy at 100× magnification by one of the investigators (SLA), who was unaware of the treatment applied to each of the cultures. Neutrophils were classified as apoptotic if they were karyorrhectic or pyknotic.5 A total of 500 neutrophils were counted in multiple arbitrarily selected fields to provide a percentage of apoptotic neutrophils.

Measurement of caspase activities

Caspase-3, -8, and -9 activities were indirectly measured in neutrophils isolated from blood samples obtained from 16 horses and cultured for 12 hours with or without 1 μg of LPS/mL; caspase activities were measured with a commercially available kiti used in accordance with the manufacturer's instructions. Caspase-3 activity was measured to determine overall apoptotic activity, caspase-8 activity was measured to assess the contribution of the extrinsic pathway on apoptosis of neutrophils, and caspase-9 activity was measured to assess the contribution of the intrinsic pathway on apoptosis of neutrophils. Neutrophils were cultured and washed as described previously and then lysed with the manufacturer's lysis buffer. Cell lysates were stored at −80°C until processing. Cell lysates were thawed, and protein concentration in cell lysates was quantified in duplicate by use of a commercial kit.j Cell lysates from each treatment were incubated in duplicate with a reaction buffer provided by the manufacturer that contained dithiothreitol and the substrate of each of the 3 caspases; samples were incubated in 96-well plates for 2 hours at 37°C. Immediately after incubation was completed, free p-nitroanilide content was quantified by measuring absorbance at 405 nm with a microtiter plate reader. Amounts of free p-nitroanilide were corrected for protein concentration in cell lysates, and mean values for free p-nitroanilide were calculated and recorded.

Determination of pathways involved in neutrophil apoptosis

To further determine the mechanisms involved in apoptosis, neutrophils were isolated from blood samples obtained from 6 horses and cultured for 12 hours with or without 1 μg of LPS/mL and with or without the apoptosis inducers gambogic acidk (2 μg/mL; apoptosis inducer via inhibition of antiapoptotic Bcl-2 proteins) or staurosporinel (2μM; apoptosis inducer via pan-caspase activation). To determine the involvement of TLR4 activation by LPS and the effects on apoptosis, neutrophils were isolated from blood samples obtained from 7 horses and cultured for 24 hours with or without 1 μg of LPS/mL and with or without a TLR4 inhibitorm (1 μg/mL) or the dissolving agent dimethyl sulfoxidee (1 μL/mL [control treatment]). Apoptosis was determined by use of labeling with annexin V and propidium iodide quantified with flow cytometry, as described previously.

Statistical analysis

Data were analyzed and graphed by use of 2 commercial software packages.n,o The Shapiro-Wilk test of normality was performed on all dependent variables. Distributions of the experimental data generally were non-Gaussian; therefore, nonparametric tests were used throughout the analysis. A mixed-effects maximum likelihood regression model controlling for the random effects of horse and experiment, followed by pairwise comparisons among the predicted outcomes, was used to assess the impact of LPS on the percentage of apoptosis. Spearman rank correlation was used to assess correlation of flow cytometry data with light microscopy data. Comparisons between paired outcomes (ie, effect of LPS-treated vs untreated neutrophils on apoptosis that was analyzed with flow cytometry or cytologic examination) were analyzed by use of the Wilcoxon rank sum test. Comparisons among > 2 outcomes were assessed by use of the Kruskal-Wallis test followed by the Dunn multiple comparisons test. Data were reported as the median and interquartile range. For all comparisons, values of P < 0.05 were considered significant.

Results

Effect of LPS treatment on apoptosis of equine neutrophils

The dose-dependent effects of LPS on apoptosis of neutrophils were determined. Neutrophils were isolated for 3 separate experiments with samples from each of the same 3 horses for each experiment. Neutrophils were untreated or treated with 0.001, 0.01, 0.1, 1, or 10 μg of LPS/mL; neutrophils were cultured for 24 hours. After adjustments were made for the random effects of horse and experiment, there was a significant (P < 0.001) decrease in the percentage of apoptosis associated with increasing concentrations of LPS (Figure 1).

Figure 1—
Figure 1—

Mean and 95% confidence limits of the percentage of apoptosis of equine neutrophils cultured for 24 hours with various concentrations of LPS. Data are for 3 separate experiments on neutrophils isolated from blood samples obtained from the same 3 horses at 3 separate times. Apoptosis was assessed by use of annexin V and propidium iodide staining with flow cytometric quantification. *†Value differs significantly (*P = 0.004; †P < 0.001) from the value for 0.01 μg of LPS/mL. ‡§Value differs significantly (‡P = 0.014; §P < 0.001) from the value for 0.001 μg of LPS/mL.‖¶ Value differs significantly (‖P = 0.001; ¶P < 0.001) from the value for 0.1 μg of LPS/mL. #Value differs significantly (P = 0.024) from the value for 0.1 μg of LPS/mL.

Citation: American Journal of Veterinary Research 79, 4; 10.2460/ajvr.79.4.424

Effects of LPS on apoptosis were evaluated further with neutrophils isolated from blood samples obtained from 40 horses. Isolation procedures yielded neutrophils with viability > 99% and mean ± SD purity of 93.5 ± 2.3%; eosinophils were the primary contaminating cell type. Neutrophils were cultured with or without 1 μg of LPS/mL for 12 or 24 hours. There was a significant (P < 0.001) reduction in the percentage of apoptosis, as indicated by staining with annexin V and no staining with propidium iodide, at both time points for cells cultured with LPS, compared with the results for cells cultured without LPS (Figure 2). After neutrophils were cultured for 12 and 24 hours, there were significantly (P < 0.001) more live cells for the LPS treatment at both time points. After neutrophils were cultured for 12 hours, there were significantly (P = 0.02) more dead cells for the LPS treatment, but there was no significant difference in the percentage of dead cells between treatments after culture for 24 hours.

Figure 2—
Figure 2—

Flow cytometry data for apoptosis of equine neutrophils cultured without (A and D) or with (B and E) LPS for 12 (A and B) or 24 (D and E) hours and box-and-whisker plots of the results of culture for 12 (C) and 24 (F) hours. Panels A, B, D, and E are representative results for flow cytometric quantification (fluorescence) of annexin V and propidium iodide staining of neutrophils. Cells in Q1 are artifact. Dead neutrophils are stained with both annexin V and propidium iodide (Q2), live neutrophils are not stained with annexin V or propidium iodide (Q3), and apoptotic neutrophils are stained with annexin V but not stained with propidium iodide (Q4). Panels C and F represent results for equine neutrophils isolated from blood samples obtained from 40 horses and cultured without (black boxes) and with (gray boxes) 1 μg of LPS/mL. Each box represents the 25th to 75th percentiles, the horizontal line in each box represents the median, and the whiskers represent the range. *†Within a cell category, value differs significantly (*P < 0.001; †P < 0.05) between treatments.

Citation: American Journal of Veterinary Research 79, 4; 10.2460/ajvr.79.4.424

Nuclear morphology was concurrently assessed in neutrophils isolated from blood samples obtained from 8 horses that had been cultured with or without 1 μg of LPS/mL for 12 or 24 hours. There was a significant reduction in the percentage of apoptotic neutrophils, as measured by altered neutrophil nuclear morphology, at 12 (P = 0.008) and 24 (P < 0.001) hours (Figure 3). Light microscopy results were highly correlated with flow cytometry results at 12 (r = 0.66; P = 0.006) and 24 (r = 0.90; P < 0.001) hours.

Figure 3—
Figure 3—

Photomicrographs of representative cytologic preparations of equine neutrophils isolated from blood samples obtained from 8 horses and cultured without (A and C) or with (B and D) 1 μg of LPS/mL for 12 (A and B) or 24 (C and D) hours and plots of the percentage of neutrophils with apoptosis (E). In panels A through D, notice the apoptotic neutrophils (arrows). Modified Giemsa stain; bar = 20 μm. In panel E, each square represents results for samples obtained from 1 horse and cultured without (black squares) or with (gray squares) LPS. The horizontal bar within in each group represents the median value. *†Value differs significantly (*P = 0.008; †P < 0.001) between treatments.

Citation: American Journal of Veterinary Research 79, 4; 10.2460/ajvr.79.4.424

Effects of LPS treatment on the intrinsic pathway of equine neutrophil apoptosis and TLR4 signaling

Activity of caspases was used to identify involvement of the intrinsic and extrinsic pathways. Activities of caspase-8 and -9, both of which are initiator caspases, were chosen to identify activation of the extrinsic and intrinsic pathways, respectively. Caspase-3 activity was used to identify overall apoptotic activity because caspase-3 is an effector caspase for both the extrinsic and intrinsic pathways. Caspase activity was compared between untreated and LPS-treated cells isolated from blood samples obtained from 17 horses. After neutrophils were cultured for 12 hours, there was no difference in caspase-3 or caspase-8 activity between untreated and LPS-treated cells, but there was a significant (P = 0.012) reduction in caspase-9 activity in LPS-treated cells, compared with untreated cells (Figure 4). Coculture of neutrophils with LPS and a TLR4 inhibitor (a chemical that blocked the intracellular domain of TLR4 and prevented TLR4 signaling) for 24 hours significantly increased neutrophil apoptosis, compared with results for treatment with LPS alone (1 experiment with blood samples obtained from 7 horses; Figure 5). Coculture of neutrophils with staurosporine, an inducer of apoptosis through pan-caspase activation, with or without LPS for 12 hours caused a significant increase in apoptosis, compared with results for cells treated with LPS alone (1 experiment with blood samples obtained from 6 horses; Figure 6). Similarly, coculture of gambogic acid, an inducer of apoptosis through regulation of the proapoptotic Bcl-2 protein Bax and the antiapoptotic protein Bcl-2, with or without LPS for 12 hours caused a significant increase in apoptosis, compared with results for cells treated with LPS alone (1 experiment with blood samples obtained from 6 horses).

Figure 4—
Figure 4—

Activity of caspase-3 (A), -8 (B), and -9 (C) in neutrophil lysates after culture without (black squares) or with (gray squares) 1 μg of LPS/mL for 12 hours. Activity was indirectly determined as the free p-nitroanilide content corrected for protein concentration. Neutrophils were isolated from blood samples obtained from 16 horses; each square represents results for 1 horse, and the horizontal bar in each group represents the median value. *Value differs (P < 0.05) significantly from the value for the untreated neutrophils.

Citation: American Journal of Veterinary Research 79, 4; 10.2460/ajvr.79.4.424

Figure 5—
Figure 5—

Apoptosis of equine neutrophils after culture for 24 hours without (black squares) or with (gray squares) 1 μg of LPS/mL, with a TLR4 inhibitor alone (black triangles), or with a TLR4 inhibitor and LPS (gray triangles). Apoptosis was assessed with flow cytometric quantification of annexin V and propidium iodide staining. Neutrophils were isolated from blood samples obtained from 7 horses. Each symbol represents results for 1 horse, and the horizontal bar in each group represents the median value. a,bValues with different letters differs (P < 0.05) significantly.

Citation: American Journal of Veterinary Research 79, 4; 10.2460/ajvr.79.4.424

Figure 6—
Figure 6—

Apoptosis of equine neutrophils cultured for 12 hours with inducers of apoptosis gambogic acid (GA; A) or staurosporine (Stauro; B). A—Neutrophils were cultured without (black squares) or with (gray squares) 1 μg of LPS/mL, gambogic acid (2 μg/mL; black circles), or gambogic acid and LPS (gray circles). B—Neutrophils were cultured without (black squares) or with (gray squares) 1 μg of LPS/mL, staurosporine (2μM; black hexagons), or staurosporine and LPS (gray hexagons). Apoptosis was assessed with flow cytometric quantification of annexin V and propidium iodide staining. Neutrophils were isolated from blood samples obtained from 6 horses. See Figure 5 for remainder of key.

Citation: American Journal of Veterinary Research 79, 4; 10.2460/ajvr.79.4.424

Discussion

Results of the study reported here supported the hypothesis that LPS would delay apoptosis of equine neutrophils in a dose-dependent manner in vitro. Although the mechanism by which LPS delayed apoptosis of equine neutrophils was not fully elucidated, results suggested that LPS treatment interfered with the intrinsic pathway of apoptosis through reduced caspase-9 activity and was dependent on TLR4 signaling.

A multitude of assays are available for assessing apoptosis in cells. We chose to use annexin V and propidium iodide staining in live cells because it was possible to identify cells undergoing early apoptotic changes attributable to phosphatidylserine exposure on the outer cell membrane. This alteration in cell membrane composition occurs before alteration in nuclear morphology. Therefore, although the light microscopy results correlated well with results for annexin V and propidium iodide staining, these methods identified cells at different points in the process of apoptosis.

It was recently reported16 that treatment of equine neutrophils with multiple types of LPS in vitro promoted apoptosis of neutrophils, which is in direct contrast to results of the study reported here and findings for other species.11,19,21–23,26,27 It must be stated that there was substantial interhorse and, to a lesser extent, interexperiment variability with regard to the effect of LPS treatment on apoptosis of equine neutrophils in the present study. It is known that there is significant interindividual variation in the response to LPS exposure, primarily because of polymorphism in cell surface receptors (namely TLR4 and CD14) that recognize LPS.28 Polymorphism of TLR4 or CD14 on the surface of equine neutrophils could have been a potential cause of the variation in results of the experiments among horses of the present study. Therefore, it is possible that a lower number of experimental units (especially biological units represented by the horses) could have led to the opposite result, such as that reported for triplicate experiments with 10 horses in the aforementioned study.24

Regardless of the experimental design that was used, the discrepancy in results for the present study and that other study24 was most likely attributable to the challenges encountered when working with neutrophils. Neutrophils are highly reactive cells, so small differences in health status of a horse, neutrophil isolation procedures,29–32 contamination during isolation,33 type of LPS, and culture conditions34,35 could have a profound effect on their short life span in culture. Activation (or lack thereof) may have an effect on neutrophil responsiveness to LPS, which can lead to differential apoptosis responses in vitro. For example, in another study36 conducted by our research group, we found that neutrophils isolated from blood samples of horses with systemic inflammatory response syndrome are refractory to the LPS-induced delay in apoptosis in vitro. Thus, we propose that activation of neutrophils prior to exposure to LPS causes tolerance that results in reduced LPS stimulation in vitro. To the authors’ knowledge, no studies have been conducted that specifically addressed apoptosis of neutrophils in vitro after prestimulation with an activating substance (eg, LPS).

In the study reported here, neutrophil apoptosis was delayed in a dose-dependent manner, which has also been reported for human neutrophils.27 However, the concentration of LPS (1 μg/mL) to which the neutrophils were exposed in prolonged culture conditions to reliably delay apoptosis in the present study would be difficult to achieve systemically in a horse. First, LPS is rapidly removed from the equine blood compartment after IV infusion.37 Second, if such a concentration was achieved in the blood, it would be approaching the lethal dose of LPS for a horse (150 to 175 μg/kg [approx 2 μg/mL of blood volume]).38 However, it is possible that activation of a small population of neutrophils by a relatively high concentration of LPS could lead to a global effect on neutrophil life span in vivo through intercellular signaling via cytokine production and mononuclear cell activation. Further investigation is required to evaluate ex vivo neutrophil life span following in vivo challenge exposure with LPS.

Mechanisms of LPS-delayed apoptosis of neutrophils were evaluated in the present study. As expected on the basis of the known pathways of LPS activation, delayed apoptosis of equine neutrophils was dependent on TLR4 signaling. These results support the mechanism for LPS-delayed apoptosis of neutrophils currently proposed for other species.10,23 In the present study, we used a TLR4 inhibitor that is a cyclohexene derivative. This substance was first identified for its ability to modulate sepsis, and it subsequently was found to suppress TLR4 signaling by blocking TLR4 interaction with Toll-interleukin-1R.39 To our knowledge, this inhibitor has not been used to evaluate neutrophil apoptosis, but it has been used to evaluate other neutrophil signaling pathways involving TLR4.40

For the study reported here, we chose to evaluate the activity of the caspases, which are the primary proteins involved in apoptotic pathways. Measuring the relative activity of caspases that are involved in the various apoptotic pathways allows for the identification of the primary pathway that a cell undergoes during apoptosis. The reduction in caspase-9 activity and increase in apoptosis after concurrent treatment with LPS and gambogic acid suggested that the intrinsic pathway was affected by LPS treatment, as has been reported for other species.10,23 Caspase-9 is the main initiator caspase for the intrinsic pathway of apoptosis. It has been reported41 that caspase-9 activity is reduced in neutrophils isolated from blood samples obtained from septic human patients, which supports the importance of caspase-9 in LPS-delayed apoptosis of neutrophils. However, to the authors’ knowledge, the study reported here was the first in which reduced caspase-9 activity was detected after treatment of neutrophils with LPS in vitro. Gambogic acid induces apoptosis in a human melanoma cell line through regulation of the proapoptotic Bcl-2 protein Bax and the antiapoptotic protein Bcl-2.42 However, to the authors’ knowledge, there have been no studies conducted to evaluate the effect of gambogic acid treatment on apoptosis of neutrophils in vitro. Results for use of gambogic acid to induce apoptosis in LPS-treated equine neutrophils in the present study potentially supported the findings in other studies10,23 that LPS-delayed apoptosis of neutrophils involves a change in the balance of the Bcl-2 family of proteins. Further investigation to identify the effect of gambogic acid on expression of individual members of the Bcl-2 family of proteins in equine neutrophils is warranted. Nevertheless, the data reported here revealed a role for caspase-9 and the intrinsic pathway of apoptosis in LPS-induced delay of apoptosis in equine neutrophils.

It is apparent that apoptosis or delayed apoptosis of neutrophils occurs via alternative pathways in the face of various substances. For example, granulocyte colony-stimulating factor delays neutrophil apoptosis independent of the Bcl-2 family of proteins by inhibiting calcium-dependent cysteine proteases (ie, calpains) while also stabilizing an X-linked inhibitor of apoptosis (an enzyme that inhibits caspase-3 and -9 in human neutrophils).43 It is possible that there is an alternative method of apoptosis inhibition of equine neutrophils attributable to LPS treatment. Further experiments are needed to investigate apoptosis of equine neutrophils and should include the evaluation of specific proteins that play an important role in cellular apoptosis.

For the study reported here, apoptosis of equine neutrophils was delayed in vitro after treatment with LPS. This delay was as a result of reduced capase-9 activity and dependent on TLR4 signaling. Further studies are necessary to fully elucidate the mechanisms involved in LPS-delayed apoptosis of equine neutrophils. On the basis of the results reported here, it can be considered that alteration of the classical intrinsic apoptotic pathway is not the only cause of LPS-delayed apoptosis of neutrophils.

Acknowledgments

This manuscript represents a portion of a thesis submitted by Dr. Anderson to the University of Saskatchewan College of Graduate Studies and Research as partial fulfillment of the requirements for a Doctor of Philosophy degree.

Supported by the Western College of Veterinary Medicine Equine Health Research Fund.

Presented as a poster at the American College of Veterinary Surgeons Surgical Summit, Nashville, Tenn, October 2015, and the International Equine Colic Symposium, Lexington, Ky, July 2017.

The authors thank Dr. Cheryl Waldner for assistance with the statistical analysis.

ABBREVIATIONS

Bcl

B-cell lymphoma

HBSS

Hank buffered salt solution

LPS

Lipopolysaccharide

TLR

Toll-like receptor

Footnotes

a.

Vacutainer, Becton Dickinson, Mississauga, ON, Canada.

b.

Ficoll-Paque Plus, GE Healthcare, Mississauga, ON, Canada.

c.

Shandon cytospin 4, Thermo Scientific, Waltham, Mass.

d.

Hemacolor, EMD Chemical, Gibbstown, NJ.

e.

Sigma-Aldrich Canada, Oakville, ON, Canada.

f.

CF 488A-annexin V and propidium iodide apoptosis assay kit, Biotium Inc, Hayward, Calif.

g.

CyFlow, Partec, Swedesboro, NJ.

h.

FlowMax software, version 2.6, Quantum Analysis GmbH, Münster, Germany.

i.

Caspase-3, -8, and -9 colorimetric assay kit, Biovision, Milpitas, Calif.

j.

DC protein assay, Bio-Rad Laboratories, Mississauga, ON, Canada.

k.

Enzo, Farmingdale, NY

l.

Biovision, Milpitas, Calif.

m.

CLI-095, Invivogen, San Diego, Calif.

n.

Stata, version 14, StataCorp LP, College Station, Tex.

o.

Graphpad Prism, version 7.02, Graphpad Software Inc, La Jolla, Calif.

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Contributor Notes

Dr. Anderson's present address is College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN 37752.

Dr. Singh's present address is Office of the Dean, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4Z6, Canada.

Address correspondence to Dr. Singh (baljit.singhl@ucalgary.ca).