• View in gallery
    Figure 1—

    Representative results of simultaneous flow cytometric analyses of phagocytic capacity and OBA in PMNs collected from blood samples obtained from healthy Beagles treated by IV injection with saline (0.9% NaCl) solution or MPSS administered at various points throughout a 26-hour period. Blood samples were collected from a jugular vein 2 hours after injections were completed. Isolated PMNs were cultured for 2 hours (1 × 106 cells/mL in each well). Cells were supplemented with fluorescent microspheres for the final 1 hour and with dihydrorhodamine 123 for the final 15 minutes of culture. During cytometric analysis, PMNs were gated to channel FL1 for evaluation of phagocytosis and channel FL3 for evaluation of ROS production. A—Dot plot of forward scatter (FSC), which is related to size of cells, and side scatter (SSC), which is related to granularity of cells. B—Logarithmic histogram of the proportion of red fluorescent microspheres phagocytosed by MPSS-naive PMNs. Proportion of phagocytosing cells (M1) was defined as the percentage of gated cells that contained microspheres. C—Logarithmic histogram of the proportion of red fluorescent microspheres phagocytosed by MPSS-exposed PMNs. D—Logarithmic histogram of amounts of fluorescent rhodamine 123 produced by ROS in MPSS-exposed PMNs (dotted line) or MPSS-naive PMNs (solid line).

  • View in gallery
    Figure 2—

    Mean ± SD phagocytic capacity (A) and OBA (B; arbitrary units) of peripheral blood PMNs collected from blood samples obtained from healthy Beagles treated by IV injection with saline (0.9% NaCl) solution (n = 6; white bars) or MPSS (n = 6; gray bars) administered at various time points throughout a 26-hour period. Blood samples were collected from a jugular vein before injections began (time 0) and 2, 12, and 24 hours after injections were completed. *Mean value for MPSS-treated group at time 0 is significantly (P < 0.05) different from the mean value at 2 hours after completion of injections (1-way ANOVA followed by a Dunnett test). †Mean value at 2 hours after completion of injections is significantly (P < 0.05) different between the treatment groups (2-sample t test). See Figure 1 for remainder of key.

  • View in gallery
    Figure 3—

    Mean ± SD phagocytic capacity (A and C; arbitrary units) and OBA (B and D; arbitrary units) of MPSS-naive peripheral blood PMNs isolated from blood samples collected from 6 Beagles and incubated with various compounds for 1, 2, 3, or 5 hours. In panels A and B, PMNs (1 × 106 cells/mL in each well) were incubated with 10μM t10c12-CLA (black bars) or a control vehicle (gray bars). Cells were supplemented with fluorescent microspheres for the final 1 hour and with dihydrorhodamine 123 for the final 15 minutes of culture. *Mean value for the cells incubated with t10c12-CLA is significantly (P < 0.05) different from the value for vehicle-incubated cells (2-sample t test). In panels C and D, PMNs were incubated with 10μM t10c12-CLA, linoleic acid (LA), NAC, NAC and t10c12-CLA, or a control vehicle for 2 hours. Cells were incubated with (gray bars) or without (white bars) fluorescent microspheres for the final 1 hour and with dihydrorhodamine 123 for the final 15 minutes of culture. *Mean value for the cells is significantly (P < 0.05) different. †Mean value for the cells incubated with microspheres differs significantly (P < 0.05) from the value for cells incubated with the same compound but without microspheres.

  • View in gallery
    Figure 4—

    Mean ± SD phagocytic capacity (A and C) and OBA (B and D; arbitrary units) of peripheral blood PMNs collected from blood samples obtained from 6 Beagles at 2 hours after completion of MPSS injections and incubated with various compounds for 1, 2, 3, or 5 hours. In panels A and B, PMNs (1 × 106 cells/mL in each well) were incubated with 10μM t10c12-CLA (black bars) or a control vehicle (gray bars). In panels C and D, PMNs were incubated for 2 hours with t10c12-CLA alone, NAC alone, t10c12-CLA in combination with NAC, or a control vehicle. Those cells were incubated with (gray bars) or without (white bars) fluorescent microspheres for the final 1 hour and with dihydrorhodamine 123 for the final 15 minutes of culture. See Figure 3 for remainder of key.

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In vitro evaluation of the effect of trans-10, cis-12 conjugated linoleic acid on phagocytosis by canine peripheral blood polymorphonuclear neutrophilic leukocytes exposed to methylprednisolone sodium succinate

Ji-Houn KangLaboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea.

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Mhan-Pyo YangLaboratory of Veterinary Internal Medicine, Department of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea.

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Abstract

Objective—To examine whether in vitro treatment with trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) restores the phagocytic capacity and oxidative burst activity (OBA) of canine polymorphonuclear neutrophilic leukocytes (PMNs) exposed to methylprednisolone sodium succinate (MPSS).

Sample Population—Peripheral blood PMNs obtained from 12 healthy Beagles.

Procedures—The experimental design involved administration of a high dose of MPSS, which is the recommended protocol for dogs with acute spinal cord injury. To evaluate PMN function, blood samples were collected from dogs before IV injections of doses of MPSS or saline (0.9% NaCl) solution (time 0) and 2, 12, and 24 hours after injections ceased. Polymorphonuclear neutrophilic leukocytes were isolated from blood samples and incubated with t10c12-CLA alone or t10c12-CLA in combination with N-acetylcysteine (an antioxidant agent). Phagocytic capacity and OBA were measured simultaneously by use of flow cytometry.

Results—The phagocytic capacity and OBA of PMNs were suppressed by IV injection of MPSS and restored 12 hours after injection ceased. In vitro treatment with t10c12-CLA enhanced the phagocytic capacity and OBA of PMNs, regardless of whether dogs had been treated with MPSS. Effects of t10c12-CLA on OBA were detected only when phagocytosis was stimulated by microspheres. Use of N-acetylcysteine attenuated the stimulatory effects of t10c12-CLA.

Conclusions and Clinical Relevance—Exposure to t10c12-CLA enhanced the phagocytic capacity and OBA of canine PMNs, and this effect may have involved t10c12-CLA–induced generation of reactive oxygen species.

Abstract

Objective—To examine whether in vitro treatment with trans-10, cis-12 conjugated linoleic acid (t10c12-CLA) restores the phagocytic capacity and oxidative burst activity (OBA) of canine polymorphonuclear neutrophilic leukocytes (PMNs) exposed to methylprednisolone sodium succinate (MPSS).

Sample Population—Peripheral blood PMNs obtained from 12 healthy Beagles.

Procedures—The experimental design involved administration of a high dose of MPSS, which is the recommended protocol for dogs with acute spinal cord injury. To evaluate PMN function, blood samples were collected from dogs before IV injections of doses of MPSS or saline (0.9% NaCl) solution (time 0) and 2, 12, and 24 hours after injections ceased. Polymorphonuclear neutrophilic leukocytes were isolated from blood samples and incubated with t10c12-CLA alone or t10c12-CLA in combination with N-acetylcysteine (an antioxidant agent). Phagocytic capacity and OBA were measured simultaneously by use of flow cytometry.

Results—The phagocytic capacity and OBA of PMNs were suppressed by IV injection of MPSS and restored 12 hours after injection ceased. In vitro treatment with t10c12-CLA enhanced the phagocytic capacity and OBA of PMNs, regardless of whether dogs had been treated with MPSS. Effects of t10c12-CLA on OBA were detected only when phagocytosis was stimulated by microspheres. Use of N-acetylcysteine attenuated the stimulatory effects of t10c12-CLA.

Conclusions and Clinical Relevance—Exposure to t10c12-CLA enhanced the phagocytic capacity and OBA of canine PMNs, and this effect may have involved t10c12-CLA–induced generation of reactive oxygen species.

Neutrophils are the first line of defense against many infectious microorganisms. They are dynamic, motile cells that have the unique capacity to phagocytize and thereby eliminate pathogens and cell debris.1 Neutrophils generate ROS by producing the oxidase of the reduced form of nicotinamide adenine dinucleotide phosphate and other microbicidal substances that destroy microorganisms.2,3 The stimulation of ROS release by foreign bodies is called the oxidative burst.4 Reactive oxygen species function as potent antimicrobial agents and as signaling molecules that can regulate neutrophil function.5

Glucocorticoids have deleterious effects that limit their use in many clinical situations. Treatment with glucocorticoids can cause impaired neutrophil function in humans,6 but this effect in dogs has not been reported. Methylprednisolone sodium succinate is a glucocorticoid that has free radical–scavenging properties at high doses.7 High-dose treatment with MPSS is the only neuroprotective regimen supported by results of the National Acute Spinal Cord Injury Studies in human medicine.8 Experimental evidence in cats with spinal cord injury suggests that MPSS may be useful in minimizing the damaging sequelae.9 However, the use of MPSS in the treatment of animals with spinal cord injury remains controversial because a paucity of evidence exists to support the efficacy of MPSS in limiting the damaging effects and because of reports on immunosuppressive complications in both humans10 and dogs11 that receive MPSS. In humans with spinal cord injuries, treatment with MPSS is associated with an increased risk of infection12 and the incidence of pneumonia is higher in methylpredisolone-treated patients, compared with the incidence in those who did not receive corticosteroids.13

Conjugated linoleic acid is the collective term used for positional and geometric isomers of linoleic acid with conjugated double bonds.14 Numerous studies15–18 have focused on CLA because it can trigger modification of immune-cell function and reportedly has properties that may protect against adipogenesis, diabetes, carcinogenesis, and atherosclerosis. In particular, t10c12-CLA, a CLA isomer, alters immune function.19 Specifically, t10c12-CLA has an immunostimulatory effect on porcine peripheral blood PMNs20 and RAW 264.7 cells21 (mouse leukemic monocyte macrophage cell line) in vitro, and this effect is mediated by tumor necrosis factor-α.20,21 The purpose of the study reported here was to examine whether in vitro treatment with t10c12-CLA would restore the phagocytic capacity and OBA of canine PMNs exposed to MPSS. In addition, we sought to examine the effects of in vitro exposure of MPSS-exposed PMNs to t10c12-CLA.

Materials and Methods

Sample population—Blood samples were collected from twelve 3-year-old laboratory Beagles. Dogs weighed 9.42 ± 0.946 kg (mean ± SD). All dogs used were healthy, as judged by evaluation of the results of physical examination, indirect measurement of systolic blood pressure, parasitologic examination of fecal specimens via a flotation technique, heartworm antigen test, CBC, serum biochemical analysis, urinalysis, ACTH response test, and diagnostic imaging. All dogs were housed separately in cages with a 12-hour light:12-hour dark cycle. Dogs were fed a commercial dieta and provided tap water. All experimental procedures were approved by the ethics committee of the Chungbuk National University.

Experimental protocol—Dogs were randomly assigned to 1 of 2 groups. Six dogs received physiologic saline (0.9% NaCl) solution (control group), and the other 6 received MPSS (treatment group). A high dose of MPSS was used in accordance with the recommended protocol for canine patients with acute spinal cord injury.22 Dogs in the treatment group received an initial bolus dose of MPSSb (30 mg/kg, IV, for 5 minutes) through an over-the-needle catheter inserted into a cephalic vein, followed by a second bolus dose 2 hours later (15 mg/kg, IV). Additional doses of MPSS (10 mg/ kg, IV) were injected 8, 14, 20, and 26 hours after the first dose, for a total dose of 85 mg/kg during a 26-hour period. In the control group, an equivalent volume of saline solution was injected at the same time points. To evaluate PMN function, blood samples were collected by jugular venipuncture immediately before the initial doses were injected (time 0) and 2, 12, and 24 hours after the end of the injections (which corresponded to 28, 38, and 50 hours after the first dose was administered).

PMN isolation—The PMNs were isolated by density-gradient centrifugation immediately after collection of blood samples. Briefly, heparinized blood samples were diluted with an equal volume of PBS solution without calcium and magnesium and added (1:1 ratio) to polypropylene conical centrifuge tubes containing a polysaccharide solutionc that had been adjusted to a specific gravity of 1.077. Tubes were centrifuged at 400 × g for 45 minutes at 20°C. The PMNs were subsequently harvested from the upper layer of sedimented erythrocytes. To purify the PMNs, erythrocytes were allowed to sediment for 60 minutes in 1.5% dextrand (molecular weight, 200 kd) in PBS solution. Floating cells were gently collected and then pelleted by centrifugation at 400 × g for 5 minutes. Residual erythrocytes were lysed by treatment with 0.83% NH4Cl in a tri(hydroxymethyl)aminomethane buffer (pH, 7.2) for 5 minutes. Purity of PMNs in the final cell suspension was verified to be > 95%, as determined by analysis of a blood film obtained by use of cytocentrifugation and Wright-Giemsa staining analysis. Resulting PMNs were resuspended in RPMI 1640 mediume supplemented with 2mM L-glutamine, 0.02 mg of gentamicin/mL, and 5% heat-inactivated fetal bovine serum.f

Figure 1—
Figure 1—

Representative results of simultaneous flow cytometric analyses of phagocytic capacity and OBA in PMNs collected from blood samples obtained from healthy Beagles treated by IV injection with saline (0.9% NaCl) solution or MPSS administered at various points throughout a 26-hour period. Blood samples were collected from a jugular vein 2 hours after injections were completed. Isolated PMNs were cultured for 2 hours (1 × 106 cells/mL in each well). Cells were supplemented with fluorescent microspheres for the final 1 hour and with dihydrorhodamine 123 for the final 15 minutes of culture. During cytometric analysis, PMNs were gated to channel FL1 for evaluation of phagocytosis and channel FL3 for evaluation of ROS production. A—Dot plot of forward scatter (FSC), which is related to size of cells, and side scatter (SSC), which is related to granularity of cells. B—Logarithmic histogram of the proportion of red fluorescent microspheres phagocytosed by MPSS-naive PMNs. Proportion of phagocytosing cells (M1) was defined as the percentage of gated cells that contained microspheres. C—Logarithmic histogram of the proportion of red fluorescent microspheres phagocytosed by MPSS-exposed PMNs. D—Logarithmic histogram of amounts of fluorescent rhodamine 123 produced by ROS in MPSS-exposed PMNs (dotted line) or MPSS-naive PMNs (solid line).

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.494

PMN culture—Linoleic acid (> 99% purity)e and t10c12-CLA (> 98% purity)g were dissolved in ethanol to a final concentration of 50mM, and the stock solution was passed through a 0.45-μm membrane filter.h To examine the phagocytic capacity and OBA of PMNs in response to t10c12-CLA, PMNs isolated from the blood samples obtained from control dogs (MPSS-naive PMNs) and MPSS-treated dogs (MPSS-exposed PMNs) were placed in 24-well plates at a density of 1 × 106 cells/mL, then incubated with 10μM t10c12-CLA for 1, 2, 3, or 5 hours. To determine whether any effect of t10c12CLA was attributable to a general effect of n-6 polyunsaturated fatty acids, MPSS-naive cells were incubated with 10μM linoleic acid by use of the same protocol. To examine whether t10c12-CLA–induced phagocytic activity was directly associated with an oxidative burst, MPSS-naive and MPSS-exposed PMNs were incubated with 10μM t10c12-CLA and 10μM NAC,e which is an antioxidant agent. All cultures were performed at 37°C in a humidified atmosphere containing 5% carbon dioxide. Linoleic acid or t10c12-CLA was added to cells in a minimal volume (< 0.1%) of ethanol. An equivalent amount of ethanol was added to vehicle control cells, which were subsequently cultured by use of the aforementioned protocol. Viability of the PMNs was verified to be > 98% on the basis of evaluation of their ability to exclude trypan blue dye.

Simultaneous measurement of phagocytic capacity and OBA—Phagocytic capacity and OBA were evaluated simultaneously as described elsewhere.23,24 Briefly, 20 μL of a carboxylate-modified polystyrene fluorescent microspherei (size, 1.0 μm) suspension was adjusted to 1 × 109 beads/mL and added to wells containing PMNs for the final 1 hour of culture. To determine whether microspheres affected OBA, cells incubated with t10c12-CLA, linoleic acid, or t10c12-CLA and NAC were treated with and without the addition of microspheres. When 15 minutes of culture time remained, 1μM dihydrorhodamine 123e was added. Conversion of nonfluorescent dihydrorhodamine 123 into fluorescent rhodamine 123 by ROS was used to measure OBA,25 and phagocytic capacity was determined by estimating the numbers of PMN with phagocytosed fluorescent microspheres in the gated cell population per sample.

Cultured cells were gently harvested, centrifuged at 400 × g for 3 minutes at 4°C, and washed 3 times with PBS solution containing 3mM EDTA. Cells were then resuspended in fixation buffer,j in accordance with the manufacturer's instructions. All steps after the beginning of cultivation were conducted in the dark. Cells were then analyzed by use of a multipurpose flow cytometerk and analysis software,l with an argon laser set at 488 nm. Samples of 10,000 cells each were assayed in triplicate. The FL1 channel was set to 505 to 545 nm to detect green fluorescent rhodamine 123, and the FL3 channel was set to 630 to 660 nm to detect red fluorescent microspheres. Cells were gated on the basis of forward and side light– scattering characteristics, and cell viability was confirmed by staining with propidium iodidee and cytometric evaluation of the ability of cells to exclude propidium iodide.26 Phagocytic capacity and OBA were expressed as percentages and mean fluorescence intensities (arbitrary units), respectively.

Statistical analysis—All analyses were performed with statistical software.m Differences between treatment groups were evaluated by means of a 1-way ANOVA, which was followed by a Dunnett test. Twogroup comparisons were performed by use of a 2-sample t test. A value of P < 0.05 was considered significant. Results are reported as mean ± SD.

Results

Effect of MPSS on the phagocytic capacity and OBA of canine PMNs—Phagocytic capacity and OBA of canine PMNs were determined simultaneously (Figure 1). Phagocytic capacity of PMNs from dogs that received MPSS was significantly lower 2 hours after MPSS injections concluded, compared with values obtained from the same group before injections (P < 0.001) and with values for the control group measured 2 hours after injections were completed (P = 0.019; Figure 2). Values for the OBA of PMNs from the MPSS-treated group obtained 2 hours after completion of injections were also significantly (P < 0.001) lower than OBA values for the same group at time 0 and significantly (P = 0.042) lower than those of the control group at 2 hours after completion of injections. Phagocytic capacity and OBAs of PMNs in both groups returned to preinjection values 12 hours after injections were completed.

Figure 2—
Figure 2—

Mean ± SD phagocytic capacity (A) and OBA (B; arbitrary units) of peripheral blood PMNs collected from blood samples obtained from healthy Beagles treated by IV injection with saline (0.9% NaCl) solution (n = 6; white bars) or MPSS (n = 6; gray bars) administered at various time points throughout a 26-hour period. Blood samples were collected from a jugular vein before injections began (time 0) and 2, 12, and 24 hours after injections were completed. *Mean value for MPSS-treated group at time 0 is significantly (P < 0.05) different from the mean value at 2 hours after completion of injections (1-way ANOVA followed by a Dunnett test). †Mean value at 2 hours after completion of injections is significantly (P < 0.05) different between the treatment groups (2-sample t test). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.494

Figure 3—
Figure 3—

Mean ± SD phagocytic capacity (A and C; arbitrary units) and OBA (B and D; arbitrary units) of MPSS-naive peripheral blood PMNs isolated from blood samples collected from 6 Beagles and incubated with various compounds for 1, 2, 3, or 5 hours. In panels A and B, PMNs (1 × 106 cells/mL in each well) were incubated with 10μM t10c12-CLA (black bars) or a control vehicle (gray bars). Cells were supplemented with fluorescent microspheres for the final 1 hour and with dihydrorhodamine 123 for the final 15 minutes of culture. *Mean value for the cells incubated with t10c12-CLA is significantly (P < 0.05) different from the value for vehicle-incubated cells (2-sample t test). In panels C and D, PMNs were incubated with 10μM t10c12-CLA, linoleic acid (LA), NAC, NAC and t10c12-CLA, or a control vehicle for 2 hours. Cells were incubated with (gray bars) or without (white bars) fluorescent microspheres for the final 1 hour and with dihydrorhodamine 123 for the final 15 minutes of culture. *Mean value for the cells is significantly (P < 0.05) different. †Mean value for the cells incubated with microspheres differs significantly (P < 0.05) from the value for cells incubated with the same compound but without microspheres.

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.494

Effect of incubation with other compounds on the phagocytic capacity and OBA of MPSS-naive canine PMNs in vitro—Phagocytic capacity and OBA of MPSSnaive PMNs were significantly (P = 0.003 and P = 0.018, respectively) higher 2 hours after PMNs were treated with t10c12-CLA, compared with results for vehicleincubated PMNs measured at the same point (Figure 3). In contrast, incubation of cells with 10μM linoleic acid had no significant effect on phagocytic capacity (P = 0.256) or OBA (P = 0.099), compared with results for vehicle-incubated cells. An increase in OBA in cells incubated with t10c12-CLA became evident only after cells were treated with microspheres (P < 0.001). When 10μM NAC was administered, phagocytic capacity and OBA were significantly (P < 0.001 and P = 0.009, respectively) reduced, compared with results for cells incubated with t10c12-CLA alone, which indicated that NAC diminished the stimulatory effect of t10c12-CLA.

Effect of t10c12-CLA on the phagocytic capacity and OBA of MPSS-exposed PMNs—Phagocytic capacity of MPSS-exposed PMNs was significantly higher 2 (P < 0.001), 3 (P < 0.001), and 5 (P = 0.001) hours after the onset of incubation with t10c12-CLA, compared with results for vehicle-incubated PMNs assayed at the same points (Figure 4). The effect of t10c12-CLA typically decreased as the duration of incubation increased. Similarly, the OBA of MPSS-exposed PMNs was significantly higher 2 (P < 0.001) and 3 (P = 0.002) hours after the onset of incubation with t10c12-CLA, compared with results for vehicle-incubated PMNs assayed at the same points. A significant (P < 0.001) increase in the OBA of MPSS-exposed PMNs became evident only after microspheres were added to cells incubated with t10c12-CLA. Phagocytic capacity and OBA of PMNs incubated with t10c12-CLA and NAC were significantly (P = 0.010) lower than those of cells incubated with t10c12-CLA alone.

Figure 4—
Figure 4—

Mean ± SD phagocytic capacity (A and C) and OBA (B and D; arbitrary units) of peripheral blood PMNs collected from blood samples obtained from 6 Beagles at 2 hours after completion of MPSS injections and incubated with various compounds for 1, 2, 3, or 5 hours. In panels A and B, PMNs (1 × 106 cells/mL in each well) were incubated with 10μM t10c12-CLA (black bars) or a control vehicle (gray bars). In panels C and D, PMNs were incubated for 2 hours with t10c12-CLA alone, NAC alone, t10c12-CLA in combination with NAC, or a control vehicle. Those cells were incubated with (gray bars) or without (white bars) fluorescent microspheres for the final 1 hour and with dihydrorhodamine 123 for the final 15 minutes of culture. See Figure 3 for remainder of key.

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.494

Discussion

The effect of glucocorticoids on the innate immunity associated with phagocytes has been extensively studied, and contradictory results have been reported. Glucocorticoids reduce the phagocytic capacity of sheep alveolar macrophages,27 and in vitro studies have revealed the inhibitory effect of glucocorticoids on the production of ROS by human monocytes28 and rat peritoneal phagocytes.29 In contrast, dexamethasone treatment has no effect on the release of reactive oxygen intermediates in cultures of macrophages derived from human blood.30 Stimulatory effects of glucocorticoids on phagocytic capacity in human monocytes have been reported.28 In our study, a high-dose course of treatment with MPSS reduced the phagocytic capacity and OBA of canine PMNs 2 hours after treatment concluded. We also found a suppressive effect of MPSS on the same variables after a single bolus injection of 30 mg of MPSS/kg (data not shown). These suppressive effects of MPSS were detected within a short period after the end of injection but were not detectable 24 hours later.

Traditionally, the effects of glucocorticoids on the immune system have been attributed to genomic events regulated through the modulation of nuclear transcription.31 However, it was reported32,33 that glucocorticoids can rapidly inhibit phagocytosis and superoxide anion production in mouse peritoneal macrophages in vitro through a nongenomic mechanism. The suppressive effects of MPSS on the phagocytic capacity and OBA of canine PMNs detected in the study reported here are most likely related to this rapid, nongenomic mechanism of action of glucocorticoids.

In our study, in vitro application of t10c12-CLA enhanced the phagocytic capacity and OBA of MPSS-naive and MPSS-exposed PMNs. Effects of t10c12-CLA on MPSS-exposed PMNs were sustained for longer periods, compared with effects on MPSS-naive PMNs. Of interest, the effect of t10c12-CLA on the OBA of PMNs was detected only when phagocytosis was stimulated by microspheres. Thus, the stimulatory effect of t10c12-CLA on the OBA of MPSS-naive and MPSS-suppressed PMNs may be dependent on the formation of the phagosome and activation of receptors associated with internalization of particles. The antioxidant agent NAC can modulate neutrophil activity when used at high concentrations (ie, 10mM).34 We found that treatment with NAC attenuated the effects of t10c12-CLA on the OBA of MPSS-exposed and MPSS-naive PMNs. Treatment with NAC also diminished the ability of t10c12-CLA to enhance the phagocytic capacity of PMNs. Analysis of these results indicated that t10c12-CLA may mediate effects on phagocytosis by directly inducing the production of ROS.

In clinical medicine, nutritional therapy may be important for immunoregulation, inflammation, neoplasia, and vascular diseases.35 Mechanisms for modulation of the immune system by fatty acids include the regulation of arachidonate metabolism and eicosanoid production, modification of membrane fluidity, and transcriptional regulation of gene expression.36–39 Fatty acid composition of phagocytes may influence phagocytosis and the ability to generate an oxidative burst.40,41 Conjugated linoleic acid can modulate the accumulation of arachidonate in cells.42 Thus, the increased phagocytic capacity and OBA of canine PMNs incubated with t10c12-CLA may be related to the modulation of fatty acid composition by t10c12-CLA. However, additional experiments are necessary to determine whether t10c12-CLA directly modulates nongenomic or genomic mechanisms of MPSS-mediated downregulation of immune function. To expand on the potential therapeutic properties of t10c12-CLA identified in our study, additional research by use of oxidative burst–deficient neutrophils from animals or humans with an immunodeficiency disorder such as chronic granulomatous disease43 will also be necessary.

In the study reported here, t10c12-CLA directly stimulated the phagocytic capacity and OBA of canine PMNs, regardless of whether dogs were treated with MPSS beforehand. Furthermore, enhancement of PMN phagocytic capacity by t10c12-CLA may have involved augmentation of ROS.

ABBREVIATIONS

ROS

Reactive oxygen species

MPSS

Methylprednisolone sodium succinate

CLA

Conjugated linoleic acid

t10c12-CLA

Trans-10, cis-12 conjugated linoleic acid

PMN

Polymorphonuclear neutrophilic leukocyte

OBA

Oxidative burst activity

NAC

N-acetylcysteine

a.

ProPlan, Nestlé Purina PetCare Korea Ltd, Seoul, Republic of Korea.

b.

SOLU-MEDROL INJ, Pfizer Pharmaceuticals Korea, Seoul, Republic of Korea.

c.

Histopaque-1077, Sigma Chemical Co, St Louis, Mo.

d.

Wako Pure Chemical Industries Ltd, Osaka, Japan.

e.

Sigma Chemical Co, St Louis, Mo.

f.

Invitrogen Co, Grand Island, NY.

g.

10(E),12(Z)-Octadecadienoic acid, Matreya LLC, Pleasant Gap, Pa.

h.

Minisart, Sartorius, Hannover, Germany.

i.

TransFluoSpheres, Molecular Probes Inc, Eugene, Ore.

j.

BD Cytofix, BD Biosciences, San Jose, Calif.

k.

FACSCalibur system, Becton Dickinson Immunocytometry Systems, San Jose, Calif.

l.

CELLQuest, version 3.336, Becton Dickinson Immunocytometry Systems, San Jose, Calif.

m.

SigmaStat, version 2.0, SPSS Inc, Chicago, Ill.

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Contributor Notes

Supported by a 2007 research grant of the Chungbuk National University.

Address correspondence to Dr. Yang.