High doses of MPSS have been used to treat dogs and humans with acute spinal cord injuries caused by trauma or intervertebral disk protrusion.1–3 Spinal cord injuries, including intervertebral disk disease, are common clinical problems in dogs and are frequently encountered in small animal practice. High-dose treatments with MPSS can effectively induce rapid neurologic recovery in animals with experimentally induced and naturally occurring spinal cord traumas.2–5 Unfortunately, glucocorticoid treatments can also cause undesired adverse effects, such as delayed wound healing, immunosuppression, and gastric ulcers.2,4,6,7 Immunosuppression is considered one of the most serious problems with regard to clinical relevance because experiments in humans have revealed an increased risk of infections associated with MPSS treatment.8 Neutrophils (ie, PMNs) are the most abundant leukocytes in the blood, and their active participation in the host innate immune defense is clearly established. However, when glucocorticoids are used as part of a treatment regimen,3 their immunosuppressive effects on PMNs (determined in both in vitro9–11 and in vivo12 studies) are regarded as undesirable. High doses of MPSS have been used to treat dogs in clinical situations; however, to our knowledge, the effect of high-dose MPSS treatment on canine PMN functions has not been investigated. The study reported here was conducted to evaluate the effects of high-dose MPSS treatment on selected PMN functions in healthy dogs.
Materials and Methods
Animals—Seven healthy male Beagles were used in the study. Body weight ranged from 10.5 to 15 kg, and dogs ranged from 2 to 4 years of age. All dogs were adequately vaccinated, and all had negative results when tested for dirofilariasis. Results of physical and neurologic examinations, a CBC, and serum biochemical analysis were all within the anticipated limits or reference ranges. During the experiment, all dogs were housed individually in cages. The study followed the Guidelines for Institution Laboratory Animal Care and Use of The School of Veterinary Medicine at Kitasato University, Tokyo, Japan.
Experimental protocol—Blood samples for baseline values were collected 2 days prior to the start of the experiment. The experiment began when each dog was administered an initial dose of MPSSa (30 mg/kg, IV), which was followed by additional doses of MPSS (15 mg/kg, IV) given at 2 and 6 hours and at 6-hour intervals therafter until 48 hours after the initial dose. Blood samples were collected at 6 time points (1 hour and 1, 2, 4, 7, and 14 days after completion of the MPSS administration). Blood samples (10 mL) were collected from a jugular vein by use of a 22-gauge needle into heparin-coated 10-mL syringes.
Isolation of PMNs—The PMNs were isolated from the venous blood. Briefly, heparinized blood was mixed with PBS solutionb containing dextranc (ratio of 4 parts heparinized blood to 1 part PBS solution with dextran) and then incubated for 40 minutes at 28°C. The leukocyte-rich supernatant was mixed with the same volume of PBS solution and layered on top of 15 mL of a Ficoll-sodium metrizoate gradient of specific density.d After centrifugation at 362 × g for 30 minutes, the layer of peripheral blood mononuclear cells was removed. The remaining supernatant containing the PMNs was incubated with NH4Cl lysis buffer (pH, 7.4) for 10 minutes to lyse RBCs. After centrifugation at 266 × g for 5 minutes, purified PMNs were suspended in HBSSe containing 10% autologous plasma buffered with 10mM HEPES (pH, 7.4). The resulting PMN suspension was used to assess phagocytosis, adhesion, and chemotaxis. For assessing ROS production, the PMNs were resuspended in 1% fetal bovine serumf buffered with l0mM HEPES (pH, 7.4). Prior to use, cell viability was assessed by use of the trypan blue dye exclusion method.
Adhesion—Adhesion of PMNs was determined on the basis of the quantity of expressed adhesion molecules. The PMNs were incubated at 4°C for 40 minutes with primary monoclonal antibodies (mouse anti-dog CD11bg and mouse anti-dog CD18g were used for CD11b and CD18 detection, respectively). Cells were then incubated at 4°C for 40 minutes with secondary antibodies that included R-phycoery-thrin–labeled goat anti-mouse IgGlg for CD11b detection and fluorescein isothiocyanate-labeled goat anti-mouse IgGlg for CD18 detection. After incubation, cells were rinsed with PBS solution and then analyzed by use of flow cytometry.h The PMN population was gated from the residual peripheral blood mononuclear cells on the basis of the forward- and side-scatter characteristics of a dot plot. Geometric mean fluorescent intensity in the histogram analysis was calculated as an index of expression.
Phagocytosis—Phagocytotic activity of PMNs was evaluated by use of the microassay method, with slight modifications.13 Briefly, samples were suspended in PBS solution and then incubated with fluorescence particlesi at 37°C for 30 minutes. Immediately after incubation, samples were cooled on ice for 10 minutes. Samples were then centrifuged at 415 × g for 5 minutes in 3mM EDTA-2Na solution. Precipitated PMNs were used for flow cytometric analysis.j Phagocytotic activity was expressed as the percentage of fluorescent (ie, positive) cells per 10,000 counts.
Migration—Chemotactic capability of the PMNs was determined by assessing the amount of polymerized actin filaments. The PMNs were suspended in PBS solution containing 90% methanol (vol/vol) and incubated for 15 minutes. Cells were rinsed with PBS solution and then incubated with fluorescein isothiocyanate-labeled phalloidink for 15 minutes. Samples were analyzed via the same flow cytometry method used in the adhesion experiment.
Preparation of serum-opsonized zymosan—Zymosan (zymosan A from Saccharomyces cerevisiae)l was boiled in ultrapure water for 60 minutes and then washed with sterilized ultrapure water. After centrifugation, pelleted zymosan was resuspended in fresh autologous serum at a concentration of 10 mg/mL and incubated for 60 minutes at 37°C. The suspension was washed twice in HBSS and then resuspended in HBSS at a concentration of 10 mg/mL.
ROS production—The PMNs (5 × 106 cells) in HBSS containing 0.5mM CaCl2, 1mM MgCl2, 10μM chemiluminescent solution,m and 50 μg of horseradish peroxidasen/mL were placed in each well of a 96-well microplate. The suspension (volume, 315 μL) was incubated for 5 minutes at 37°C. After incubation, PMNs were activated by adding 35 μL of serum-opzonized zymosan (10 mg/mL). Chemiluminescent measurements were performed every 0.2 seconds by use of a luminometero at 37°C; luminescence was recorded for 30 minutes. The response was expressed as the peak amount of luminescence.
Statistical analysis—All data were expressed as mean ± SEM. A paired t test was used to compare results at various time points with the baseline value. Values of P < 0.05 were considered significant.
Results
Changes were detected in PMN adhesion after high-dose MPSS treatment. There was a significant decrease in the mean ± SEM expression of CD11b on the PMNs on days 1 (MFI; 9.8 ± 2.6), 2 (MFI; 14.1 ± 4.0), and 4 (MFI; 14.4 ± 2.7) after MPSS treatment, compared with the baseline value (MFI; 16.7 ± 2.6). By day 7 after MPSS treatment, all of the expressions were similar to the baseline value. Although a significant decrease was also detected in the mean expression of CD18 on the PMNs on days 1 (MFI; 9.2 ± 2.3) and 2 (MFI; 12.2 ± 1.3) after MPSS treatment, compared with the mean baseline value (MFI; 14.4 ± 2.0), expression was similar to the baseline value by day 4 after MPSS treatment (Figure 1).
Mean ± SEM expression of CD11b (A) and CD18 (B) measured by use of flow cytometry in isolated PMNs of 7 dogs after a high-dose MPSS treatment. Blood samples were collected 2 days before the start of (baseline) and 1, 2, 4, 7, and 14 days after completion of MPSS treatment. *Value differs significantly (P < 0.05) from the baseline value.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Mean ± SEM expression of CD11b (A) and CD18 (B) measured by use of flow cytometry in isolated PMNs of 7 dogs after a high-dose MPSS treatment. Blood samples were collected 2 days before the start of (baseline) and 1, 2, 4, 7, and 14 days after completion of MPSS treatment. *Value differs significantly (P < 0.05) from the baseline value.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Mean ± SEM expression of CD11b (A) and CD18 (B) measured by use of flow cytometry in isolated PMNs of 7 dogs after a high-dose MPSS treatment. Blood samples were collected 2 days before the start of (baseline) and 1, 2, 4, 7, and 14 days after completion of MPSS treatment. *Value differs significantly (P < 0.05) from the baseline value.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Changes in PMN migration after high-dose MPSS treatment were evaluated by examining the expression of F-actin (Figure 2). There were no significant differences detected for F-actin expression.
Mean ± SEM expression of F-actin in isolated PMNs of 7 dogs after a high-dose MPSS treatment. The F-actin expression was determined via flow cytometric measurement of phalloidine content represented by fluorescent intensity. See Figure 1 for key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Mean ± SEM expression of F-actin in isolated PMNs of 7 dogs after a high-dose MPSS treatment. The F-actin expression was determined via flow cytometric measurement of phalloidine content represented by fluorescent intensity. See Figure 1 for key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Mean ± SEM expression of F-actin in isolated PMNs of 7 dogs after a high-dose MPSS treatment. The F-actin expression was determined via flow cytometric measurement of phalloidine content represented by fluorescent intensity. See Figure 1 for key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Changes were detected in phagocytotic activity of the PMNs after high-dose MPSS treatment (Figure 3). Mean ± SEM percentages of the microbead-engulfing PMNs were significantly decreased on days 1 (31.6 ± 9.5%), 2 (36.8 ± 8.9%), and 7 (45.9 ± 6.4%) after MPSS treatment, compared with the mean baseline value (50.1 ± 7.2%). Phagocytotic activity was similar to baseline values by day 14 after MPSS treatment.
Mean ± SEM phagocytotic capacity measured by use of flow cytometry in isolated PMNs of 7 dogs after a high-dose MPSS treatment. See Figure 1 for key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Mean ± SEM phagocytotic capacity measured by use of flow cytometry in isolated PMNs of 7 dogs after a high-dose MPSS treatment. See Figure 1 for key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Mean ± SEM phagocytotic capacity measured by use of flow cytometry in isolated PMNs of 7 dogs after a high-dose MPSS treatment. See Figure 1 for key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Changes were detected in the PMN oxidative burst after high-dose MPSS treatment (Figure 4). Oxidative burst was evaluated by use of the chemiluminescent method during stimulation with serum-opzonized zymosan. A significant decrease in the mean ± SEM chemiluminescent response was detected on day 7 after MPSS treatment (0.2 ± 0.1 reactive light units), compared with the baseline value (1.0 ± 0.5 reactive light units).
Mean ± SEM chemiluminescence as an index of oxidative burst measured by use of a luminometer in isolated PMNs of 7 dogs after a high-dose MPSS treatment. RLUs = Reactive light units. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Mean ± SEM chemiluminescence as an index of oxidative burst measured by use of a luminometer in isolated PMNs of 7 dogs after a high-dose MPSS treatment. RLUs = Reactive light units. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Mean ± SEM chemiluminescence as an index of oxidative burst measured by use of a luminometer in isolated PMNs of 7 dogs after a high-dose MPSS treatment. RLUs = Reactive light units. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.541
Discussion
The primary objective of the study reported here was to investigate canine PMN response after high-dose MPSS treatment. Therapeutic regimens that include the use of MPSS are routinely recommended for the treatment of neurologic disease and acute spinal cord injury. In fact, an initial administration of a high dose of MPSS within 8 hours after spinal cord injury is critical to secure an optimal outcome.3 Prevention of paralysis as a result of a spinal cord injury is considered to be fairly important because this condition could lead to deterioration in the quality of life, which would create a substantial burden for both patients and clients. Merits of MPSS administration have been clearly identified; unfortunately, glucocorticoids (including MPSS) are immune suppressors. Therefore, a better understanding of how PMN functions are altered by MPSS treatment would be beneficial in helping to achieve more effective clinical management of affected patients.
Glucocorticoids inhibit production of proinflammatory factors (prostaglandins, thromboxanes, and leukotrienes).13,14 Because the effects of glucocorticoids result in cascading responses, they have the potential to influence a wide range of anti-inflammatory processes that are independent of the original cause. However, inhibition of these inflammatory processes can also lead to the suppression of immune function. This is a major concern because infectious diseases develop more frequently during glucocorticoid treatments as a result of immune suppression.8
Phagocytosis by PMNs is the direct defense approach that protects hosts from infectious microorganisms. Activation of the oxidative burst produces ROS, such as superoxide anion, that aid in the elimination of these microorganisms.15 To achieve the desired quality of host defense, it is essential that the serial actions of adhesion, migration, phagocytosis, and oxidative burst are appropriately applied.
Adhesion of PMNs is partly attributable to mechanisms in which cytokines and chemotactic factors from cells of inflammatory sites enhance Mac-1 (CD11b-CD18) expression on PMNs16–18; Mac-1 is a strong adhesive molecule that is a counterpart of intercellular adhesion molecule on the vascular endothelium. As a result, PMNs are able to accumulate at the inflammatory site. In the present study, significant decreases in the expression of CD11b and CD18 on PMNs were detected after MPSS treatment, which suggested that high-dose MPSS treatment can suppress the adhesive ability of PMNs. Similar results of induced functional suppression of PMNs by glucocorticoids and its analogues were also detected in studies conducted by other researchers; the PMN functional variables under the suppressive influence of corticosteroids were PMN migration behavior,19 expression of molecular adhesion,20 and recruitment or interaction with vascular endothelial cells.21 In those studies, various types of corticosteroids, such as glucocorticoids, prednisolone, dexamethasone, and methyl-prednisolone, had inhibitory effects on PMN functions. However, these types of drugs are not necessarily constant in terms of their PMN-suppressive effects. For example, in 1 study22 in cattle, dexamethasone treatment did not cause any inhibitory effects on PMN functions. Although there is variation in the duration of activity and modes of action for those corticosteroids (which makes it impossible to compare the aforementioned studies in parallel), it is also reasonable to assume that the treatment dosage may be an important factor affecting the corticosteroid impact on immune cells. In fact, some studies23,24 on PMN functions have revealed that functional changes in PMNs were influenced by corticosteroids in a dose-dependent manner. Analysis of results of the study reported here, in conjunction with results of other studies, leads to the conclusion that additional studies are warranted to more clearly describe the association of dose and type of glucocorticoid with PMN functions, especially for MPSS treatment in which use of high doses is relatively common in clinical settings.
Chemotaxis is dependent on a chemoattractive gradient.25 Once there is adhesion of PMNs to the vascular endothelium during this process, transendothelial migration begins, with the PMNs going through a transitional phase in which there is polymerization of cytoplasmic actin that increases cytoskeletal plasticity. It has been suggested26,27 that the essential force involved in this transformation is polymerization and cofilin-dependent depolymerization of actin.26,27
In the present study, we measured the expression of F-actin on PMNs after MPSS treatment as an index of migration ability. To detect F-actin and polymerized forms of actin during endocytosis, phalloidine staining typically is used.13,28,29 However, analysis of the data from the study reported here did not reveal significant differences in F-actin expression on PMNs. In another study,30 increased F-actin content was detected in PMNs after activation by a synthetic peptide. In contrast, PMNs were not activated in our study outside of the MPSS-treatment period. Although the lack of F-actin polymerization in the present study may have reflected a true lack of any major alterations in endocytotic function, it could also have represented an insufficient ability of the method used to detect such changes.31 In the present study, the migration capability of PMNs was not altered by high-dose MPSS treatment in the absence of inflammatory stimuli.
In contrast, a significant decrease was detected for the phagocytotic ability of PMNs after MPSS treatment. Analysis of our data revealed that there was a decrease in the complement receptor (CD11b-CD18) on the membranes of the PMNs. This result was paradoxical, considering that commonly held belief states that actin polymerization is essential for induction of PMN phagocytosis. The major action of complement receptors is to bind to microbes, which in turn is a signal to initiate phagocytosis.32–34 Therefore, the decrease in PMN expression of the receptor in the study reported here indicates that MPSS treatment has a negative influence on PMN phagocytosis. Results of another study28 on PMNs indicated that there are lower amounts of F-actin polymerization during phagocytosis than during migration. Thus, these results may partly explain the decrease in the phagocytotic function that was detected without any substantial changes in intracellular F-actin configuration in the present study.
In response to microbe contact, PMNs go through morphological and metabolic changes, which are referred to as the respiratory burst. This phenomenon, in essence, results in instantaneous production of ROS.15 The neutrophil NADPH oxidase specifically associated with PMNs catalyzes a reaction that leads to the generation of superoxide radicals. These radicals are then rapidly metabolized to other ROS. Activation of protein kinase C is responsible for phosphorylation of NADPH oxidase, which generates the superoxide anions that carry the electron transport chain. As a result, superoxide anions and other active metabolites are released from the plasma membrane. This entire process contributes to the host defense mechanism. In the study reported here, we used a method that reacted ROS with surrounding atoms to cause the ROS to change into electrically excited states that would emit chemiluminescence. Because the respiratory burst is located downstream of cascading inflammatory responses that originate from complement receptors, agents (such as zymosan) that stimulate complement receptors can enhance the chemiluminescence of PMNs. This increase in sensitivity makes it possible to detect the altered magnitude of the respiratory burst.35,36 In the present study, a significant decrease in chemiluminescence was detected on day 7 after completion of the MPSS treatment, which indicated that the MPSS treatment suppressed the ability of PMNs to have an oxidative burst. Other studies23,37 have also indicated inhibitory effects of similar drugs (such as glucocorticoids, prednisolone, dexamethasone, and methylprednisolone) on the ability of PMNs to produce superoxide anions. Results of our study were similar to those in the aforementioned studies.23,37 However, this effect was not persistent in our study because the variables returned to the baseline values on day 14 after MPSS treatment.
Overall, analysis of the results of our study suggests that high-dose MPSS treatment can induce suppression in some of the major PMN functions for at least 7 days after the cessation of treatment. There are numerous in vitro studies in which immunosuppressive effects of MPSS have been described, and the present study provided results for the in vivo effects of MPSS in dogs receiving a clinically relevant high-dose MPSS regimen that is primarily recommended for the treatment of certain critical conditions. Regarding the functional alternation of PMNs in association with corticosteroids, studies23,24 have revealed that these drugs could induce dose-dependent changes in PMN functions. Hence, considered in combination with results of the study reported here, it appears that additional studies may be beneficial to elucidate the effects of high-dose MPSS treatment on PMN functions in an attempt to maximize the medicinal effects of this drug and also to evaluate other treatment regimens for clinical use. Considering general post-traumatic and postoperative neurologic conditions, potentially negative effects such as immunosuppression may be a reason for reconsidering the use of MPSS. However, because the medicinal benefits and usefulness of MPSS have been determined for critical medical conditions, elimination or reduction in the use of MPSS is unreasonable without any attempts to ameliorate adverse effects. A better understanding of the nature of MPSS-induced functional suppression in PMNs could be a step toward maximizing the potential of MPSS as a treatment. Future studies are warranted to further evaluate detailed functions of PMNs and the impact of MPSS on PMNs.
ABBREVIATIONS
| HBSS | Hank's balanced salt solution |
| MFI | Mean fluorescent intensity |
| MPSS | Methylprednisolone sodium succinate |
| PMN | Polymorphonuclear neutrophilic leukocyte |
| ROS | Reactive oxygen species |
Solumedrol, Pfizer, NY.
Sigma-Aldrich Co, St Louis, Mo.
Dextran T-500, Wako Pure Chemical Industries, Osaka, Japan.
Ficoll-hypaque, Sigma-Aldrich Co, St Louis, Mo.
Gibco, Grand Island, NY.
Fetal bovine serum, Sanko Junyaku, Ibaragi, Japan.
AbD Serotec, Oxford, England.
Expo 2000 software, Beckman Coulter Inc, Fullerton, Calif.
Fluoresbrite carboxy YG 1.0 micron microspheres, Polysciences Inc, Warrington, Pa.
Epics flow cytometer, Beckman Coulter Inc, Fullerton, Calif.
Phalloidin-fluorescein isothiocyanate, Sigma-Aldrich Co, St Louis, Mo.
Zymosan A, Sigma-Aldrich Co, St Louis, Mo.
Luminol, Sigma-Aldrich Co, St Louis, Mo.
Horseradish peroxidase, Sigma-Aldrich Co, St Louis, Mo.
Dynex Technologies, Chantilly, Va.
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