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A Sphingosine-1 Phosphate Agonist (FTY720) Limits Trauma/Hemorrhagic Shock–Induced Multiple Organ Dysfunction Syndrome

Bonitz, Joyce A.; Son, Julie Y.; Chandler, Benjamin; Tomaio, Jacquelyn N.; Qin, Yong; Prescott, Lauriston M.; Feketeova, Eleonora; Deitch, Edwin A.

doi: 10.1097/SHK.0000000000000227
Basic Science Aspects
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ABSTRACT Background: Trauma/hemorrhagic shock (T/HS) is one of the major consequences of battlefield injury as well as civilian trauma. FTY720 (sphingosine-1-phosphate agonist) has the capability to decrease the activity of the innate and adaptive immune systems and, at the same time, maintain endothelial cell barrier function and vascular homeostasis during stress. For this reason, we hypothesize that FTY720, as part of resuscitation therapy, would limit T/HS-induced multiple organ dysfunction syndrome in a rodent T/HS model. Methods: Rats subjected to trauma/sham shock (T/SS) or T/HS (30 mm Hg × 90 min) were administered FTY720 (1 mg/kg) post-T/HS during volume resuscitation. Lung injury (permeability to Evans blue dye), polymorphonuclear leukocyte (PMN) priming (respiratory burst activity), and red blood cell (RBC) rigidity were measured. In addition, lymph duct–cannulated rats were used to quantify the effect of FTY720 on gut injury (permeability and morphology) and the biologic activity of T/HS versus T/SS lymph on PMN-RBC and RBC deformability. Results: Trauma/hemorrhagic shock–induced increased lung permeability, PMN priming, and RBC rigidity were all abrogated by FTY720. The systemic protective effect of FTY720 was only partially at the gut level, because FTY720 did not prevent T/HS-induced gut injury (morphology or permeability); however, it did abrogate T/HS lymph-induced increased respiratory burst and RBC rigidity. Conclusions: FTY720 limited T/HS-induced multiple organ dysfunction syndrome (lung injury, red cell injury, and neutrophil priming) as well as T/HS lymph bioactivity, although it did not limit gut injury.

Department of Surgery, Rutgers New Jersey Medical School, Newark, New Jersey

Received 10 Mar 2014; first review completed 27 Mar 2014; accepted in final form 23 Jun 2014

Address reprint requests to Joyce A. Bonitz, MD, Rutgers New Jersey Medical School, Medical Science Bldg G-506, 185 S Orange Ave, Newark, NJ 07101. E-mail: alexanj2@njms.rutgers.edu.

Supported by Department of Defense Grant W81XWH-12-1-0091 (to E.A.D.) and T32 069330 (to J.A.B. and J.Y.S.)

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INTRODUCTION

Trauma/hemorrhagic shock (T/HS) is a major consequence of battlefield injury as well as civilian trauma. It has several peaks of mortality, one of which is due to the initial extent of blood loss and inability to adequately resuscitate individuals (1). The second peak is related to head injury. The third peak is related to the development of multiple organ failure, which is a consequence of the injury shock state. Our work focusing on the pathogenesis of multiple organ failure indicates that many components of the early post-T/HS–induced multiple organ dysfunction syndrome (MODS) are related to gut injury and lipid and protein factors exiting the stressed gut via the intestinal lymphatics (2–4). Because the mechanism by which these gut-derived factors appear to cause acute organ dysfunction involves the induction of an acute immune-inflammatory state, the goal of this study was to study a novel potential mechanism-based agent for preventing or limiting the development of MODS after traumatic shock. This potential therapeutic agent, FTY720 (fingolimod), is a sphingosine-1-phosphate (S1P) agonist and was chosen because of its multiple potentially protective physiologic activities. Specifically, FTY720 has the unique ability to limit the activation of the innate and adaptive immune systems after stress and injury while maintaining endothelial cell barrier function and vascular homeostasis (5). As described by Garris et al (6), S1P is a lipid second messenger that signals via five G protein–coupled receptors. This receptor signaling is associated with a wide variety of physiologic processes, such as vascular development, central nervous system homeostasis, and lymphocyte biology, particularly their recirculation and determination of T cell phenotype. Hence, FTY720 has the potential to limit tissue damage and excessive systemic inflammation and thereby reduce the development or magnitude of trauma-induced MODS. Further support for this study of FTY720 are based on preclinical studies documenting that FTY720 and/or S1P administration can attenuate ischemia-reperfusion injuries to the liver as well as other organs (7) and is protective in a model of endotoxin-induced lung injury (8). In addition, a recently published study by Hawksworth et al. (9) documented that FTY720 improved survival time and limited immune and inflammatory changes in a porcine hemorrhagic shock model. Importantly, FTY720 could be rapidly transitioned from preclinical to clinical studies, because it is currently approved for human use in the treatment of multiple sclerosis (10) and has been used safely in several phases I, II, and III studies in other patient populations, including transplant recipients (11).

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MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass) weighing 350 to 400 g were housed under barrier-sustained conditions and kept at 25°C with 12-h light/dark cycles. They were provided with chow and water ad libitum. All animals were maintained according to the Guide for the Care and Use of Laboratory Animals. In addition, all animal protocols were approved by the New Jersey Medical School Animal Care Committee.

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Trauma/hemorrhagic shock

Rats were anesthetized with intraperitoneal pentobarbital (50 mg/kg). Using aseptic techniques, a midline laparotomy was made, and the intestines were eviscerated for 15 minutes. After this time, the intestines were placed back into the abdominal cavity, and the laparotomy incision was closed using 4-0 silk suture. The right external jugular vein was dissected out and cannulated using silastic PE-50 tubing for blood withdrawal. In the same way, the right femoral artery was cannulated using polyethylene PE-50 catheter for blood pressure monitoring. Trauma/sham shock (T/SS) animals were prepared as previously stated, but no blood was removed. For the T/HS rats, blood was withdrawn into a heparinized syringe through the jugular vein line. The mean arterial blood pressure was reduced to 30 mmHg and maintained at this level for 90 min by reinfusing or withdrawing blood as needed (12). After shock was complete, the rats received 1 mL/kg of either FTY720 (1 mg/kg in normal saline) or vehicle over 3 minutes. After this was complete, the T/HS rats were resuscitated with three times the amount of shed blood in lactated Ringer’s solution at a rate of 1 mL/min and observed for 3 h. The mean arterial pressure (MAP) returned toward a normal level within a few minutes after the rats were volume resuscitated.

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Experimental design

The primary hypothesis being tested is that FTY720, through its immunoinflammatory and vascular effects, will limit T/HS-induced systemic inflammation and MODS when administered as part of the shock-resuscitation regimen. The secondary hypothesis is that this systemic protective effect of FTY720 would be associated with a reduction in the biologic activity of mesenteric lymph and less gut injury after T/HS.

To test the hypothesis that T/HS-induced MODS is prevented by the use of FTY720, four groups of rats were studied. These include two nonshock control groups of rats subjected to T/SS receiving either vehicle or FTY720 (1 mg/kg) as well as two groups of T/HS rats, one of which received vehicle and the other FTY720. The FTY720 or vehicle was administered at the end of the shock period during the first 5 min of volume resuscitation and thus utilized a posttreatment strategy. Based on our earlier results showing that acute lung and gut injury and neutrophil activation and red blood cell (RBC) dysfunction are present as early as 3 h after the end of the resuscitation period (12), these four groups of rats were killed at this time period. Lung injury was quantified using Evans blue dye (EBD) to measure lung permeability. Gut injury was assessed morphologically based on histologic criteria. (13) In a second group of rats, gut barrier function was measured physiologically, using a fluorescent-labeled dextran intestinal permeability probe. (14) In addition, neutrophil priming, as reflected by an increased respiratory burst, was measured as were RBC deformability and function, because increased RBC rigidity after T/HS has been associated with impaired microcirculatory blood flow (15). Both samples were obtained using fresh heparinized blood. Lastly, because of the potential effects of FTY720 on lymphocyte sequestration, a complete blood count was also obtained using 25 μL of fresh heparinized blood in the Hema True blood analyzer (HESKA, Loveland, Colo).

A subsequent experiment was done testing a range of doses to determine if lower doses (0.1 mg/kg or 0.5 mg/kg) of FTY720 would be as protective as the 1.0-mg/kg dose of FTY720. The same experimental design as outlined above was carried out.

The third set of experiments tested the hypothesis that one of the major mechanisms by which FTY720 prevents/limits T/HS-induced MODS is by limiting T/HS-induced gut injury and dysfunction and consequently the production of biologically active mesenteric lymph. The same two T/HS and T/SS groups of rats, receiving vehicle or FTY720 (1 mg/kg) described above, in which mesenteric lymph duct catheters were placed, was used in this experiment. Separate aliquots of lymph were collected prior to the induction of T/HS, during the 90-min shock (sham-shock) period as well as during the first 3 h after the end of the shock or sham-shock period (postshock lymph). This 3-h postshock lymph collection period was based on our previous studies documenting that the in vivo biologic activity of T/HS lymph is maximal during the first-3-h postshock period and is relatively rapidly lost thereafter (16). After the lymph was collected, it was processed, aliquoted, and stored at −80°C until tested. Subsequently, the ability of the lymph samples (T/HS and T/SS lymph ± FTY720) to activate neutrophils from normal animals as well as to alter RBC physiology was examined by incubating the various lymph samples (T/SS ± FTY720 and T/HS ± FTY720) with whole blood from naive male rats. The number of lymph samples from each group ranged from 9 to 11. We used three normal (naive) rats as blood donors for the in vitro assay with a similar number of lymph samples from each group being tested in each animal. Physiologically relevant concentrations of lymph (5%–10% vol/vol) were tested (12,17).

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Mesenteric lymph duct cannulation

As previously reported (12), the mesenteric lymph duct was catheterized using the following protocol: a midline incision was made, and the efferent mesenteric lymphatic vessel was identified (adjacent to the superior mesenteric artery) by reflecting the loops of intestine with moist gauze swabs to the left of the animal. Next, the lymphatic duct was isolated and cannulated with silastic PE-50 tubing. This tubing was secured and brought out through a separate stab incision in the right flank. Lymph was collected on ice in sterile tubes, and the sterility of the samples was validated by culturing an aliquot of each collected sample.

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Gut permeability assay

At the end of the shock resuscitation period, the midline laparotomy incision was released. The cecum was identified and protected with moist gauze. A 12-cm segment of terminal ileum was ligated proximally and distally with 4-0 silk tie. An enterotomy was made at both ends. This segment was flushed with 0.9% saline to remove the feces. The enterotomies were excluded, and a 10-cm segment was ligated. The permeability probe, fluorescein isothiocyanate (FITC)–dextran (FD4) was prepared at a concentration of 25 mg/mL, and a total of 1 mL was carefully injected into the 10-cm segment to prevent any spillage onto surrounding bowel. The segment was protected from light under a moist gauze and aluminum foil. Blood samples of 1 mL were removed from the external jugular catheter prior to and 30 min after injection of FD4. As previously reported (14), plasma FD4 measurements were obtained by centrifuging the samples at 10,000 revolutions/min for 2 min at 4°C and compared with a standard curve of FD4 measured on FLX800 Microplate Fluorescence reader (Biotek, Shoreline, Wash) at excitation 485/20, emission 528/20, and a sensitivity of 40.

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Lung permeability assay

On completion of the FD4 protocol, animals were injected with 1 mL of 1% EBD through the external jugular catheter. Blood sample of 1 mL was removed 20 min after injection of EBD from the inferior vena cava. The animals were then killed, and a cardiopulmonectomy was performed. Modified bronchoalveolar lavage (BAL) was performed on the excised lungs using 5 mL of normal saline for each of the two washes (as previously reported 12). Two small cuts were made in the lower lobes to facilitate fluid removal. The blood samples and BAL fluid were centrifuged at 10,000 revolutions/min for 2 min at 4°C. The plasma was serially diluted to form a standard curve. The plasma and supernatant fluid were assayed with a spectrophotometer at 620 nm for dye concentration and was compared with the standard curve.

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Intestinal villous injury

After the rats were killed, a segment of the terminal ileum was excised and fixed in 10% buffered formalin for morphologic analysis as previously described (18). Briefly, once processed, semithin sections (2–4 μm) were cut and stained with toluidine blue. Five random fields with 100 to 250 villi from each animal were analyzed in a blinded fashion using light microscopy at ×100 magnification. The overall incidence of villous damage was expressed as a percentage where the number of injured villi was divided by the total number of villi counted.

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Determination of neutrophil respiratory burst

To assess the in vivo effects of T/HS on polymorphonuclear leukocyte (PMN) priming, heparinized blood samples were obtained from each animal before shock/sham shock and 3 h after the end of shock/sham shock for the measurement of neutrophil respiratory burst activity. As previously described (17), the samples were treated with RBC lysis buffer (1 mL Lyse Buffer; Sigma, St Louis, Mo) and incubated for 15 min at room temperature to remove the RBCs. Next, they were centrifuged at 25°C, washed twice with Hanks balanced salt solution, and the supernatant was discarded. The resulting pellet was then suspended in Hanks balanced salt solution at a concentration of 4 × 106 cells/mL. Dihydrorhodamine (15 ng/mL) was added to 100 μL of the PMN sample and warmed to 37°C. Phorbol myristic acid (0.4 μmol/L) was then added to stimulate the cells for 15 min at 37°C. The PMN respiratory burst was measured by flow cytometry, where the neutrophils were identified by forward- and side-scatter analysis. The data are expressed as the mean fluorescence index. To assess the effects of the various lymph samples on PMN priming in vitro, PMNs isolated from the whole blood of naive rats were incubated with rat T/SS or T/HS lymph (10% vol/vol) for 5 minutes. Following this incubation period, PMA-induced respiratory burst was measured as described above.

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Determination of RBC deformability

Because RBC dysfunction has been shown to impair microvascular blood flow (15), we assessed the in vivo effects of T/HS on RBC deformability, by analyzing heparinized whole blood that was collected before shock/sham shock and 3 h after shock/sham shock. Red blood cell deformability was determined with a laser optical rotational cell analyzer (LORCA; RR Mechatronics, East Providence, RI) as previously reported (15). An aliquot of RBCs (15 μL) containing approximately 30 million cells was suspended in 1 mL of 5% polyvinylpyrrolidone (molecular weight 360,000; Sigma) in phosphate-buffered saline at a final viscosity of 30 mPa. After gently mixing for 15 min at room temperature to ensure complete oxygenation of the hemoglobin, cell deformability was determined at 37°C. Cell deformability was assessed by calculating the elongation index at shear stresses ranging from 0.3 to 30 Pa. From the shear-stress elongation curve created previously, the data were analyzed using the Lineweaver-Burk analysis to determine the stress required for the erythrocytes to reach 50% of their maximal elongation (kinetic energy interactive [KEI]). An elevation in KEI reflects a decrease in RBC deformability. To assess the effects of the various lymph samples on RBC deformability in vitro, the whole blood of naive rats was incubated with rat T/SS or T/HS lymph (10% vol/vol) for 3 h. Following this incubation period, RBC deformability was measured as described above.

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RBC–endothelial cell adhesion assay

To assess the effects of the various lymph samples on RBC adhesion to the endothelium, whole blood of naive rats was incubated with rat T/SS or T/HS lymph (10% vol/vol) for 3 h. Following this incubation period, RBC adhesion to an endothelial cell monolayer was measured as follows. Briefly, human umbilical vein endothelial cells (HUVECs; LONZA, Walkersville, Md) were seeded onto 35-mm tissue culture plates and used 48 h after seeding when the endothelial cells had reached confluence. Then, RBC (60 μL of whole blood) samples incubated with the T/HS or T/SS lymph collected from FTY720 or vehicle-treated rats were added to the tissue culture plates containing the HUVECs monolayers together with 2 mL of essential basal media (LONZA). The plates were gently swirled, then incubated for 5 min at room temperature and washed three times with 2 mL of essential basal media following which the supernatant was discarded and the plates were placed in an inverted position for 10 s. Red blood cell adhesion was quantified by optical microscopy at ×40 magnification (ZEISS microscope; Carl Zeiss AG, Thornwood, NY). In this assay, the number of adherent RBC in three randomly selected ×40-magnification40 fields was counted in a blinded fashion.

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Flow cytometry assay for CD36-positive RBC

Since our recent work has documented that increased adhesion of T/HS RBC to the endothelium is mediated to a large extent by increased expression of CD36 (19), the ability of the various lymph samples to increase RBC CD36 expression was quantified. In this assay, whole-blood samples from naive male rats that had been previously incubated with the lymph samples were stained for 30 min with CD36 FITC (Invitrogen, Camarillo, Calif) or an isotype control antibody (Invitrogen). Following which they were resuspended in 400 μL of 1 × phosphate-buffered saline and analyzed by a fluorescence-activated cell sorter (FACS-Calibur; Becton-Dickinson, Franklin Lakes, NJ). A 488-nm argon laser beam was used for excitation. A two-parameter dot-plot of forward light scatter and side light scatter was set up to include only RBCs and to exclude white blood cells and lymphocytes. Green fluorescence of gated 10,000 RBCs was then measured using linear amplification. The percentage of CD36 positive FITC RBCs was derived by Cell Quest software.

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Statistical analysis

Analysis of variance with post hoc Turkey-Kramer multiple-comparisons test was used for comparison between multiple groups, whereas Student t tests were used for comparison between two groups. Results are expressed as mean ± SD. P < 0.05 was considered statistically significant.

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RESULTS

The first major set of key observations was that post-T/HS treatment with FTY720 protected against T/HS-induced (a) acute lung injury, (b) neutrophil priming, and (c) RBC dysfunction (Table 1). Specifically, the administration of FTY70 given during the post-T/HS resuscitation period completely prevented the threefold increase in T/HS-induced lung permeability. Furthermore, although some degree of increased neutrophil priming was observed in the T/SS groups (Table 1), indicating that the laparotomy and instrumentation led to some neutrophil activation, the magnitude of neutrophil priming was significantly increased in the T/HS plus vehicle group and abrogated by FTY720. However, neutrophil priming remained increased in the T/HS plus FTY720 group as compared with the T/SS group. Like the response observed for neutrophil priming, FTY720 significantly limited T/HS-induced RBC rigidification, but its protective effect was not complete (Table 1).

Table 1

Table 1

Having shown that FTY720, at a dose of 1 mg/kg, was effective at limiting T/HS-induced organ and cellular injury, a dose-range study of FTY720 was performed using two lower doses. The results of this study showed that the beneficial effects of FTY720 observed with the 1-mg/kg dose also existed when lower doses (0.5 and 0.1 mg/kg) were tested (Figs. 1 and 2). Specifically, all of the doses of FTY720 tested were similarly effective in limiting T/HS-induced lung injury, neutrophil priming, and RBC dysfunction. A dose-range study was also done with T/SS rats, but these values were not different from each other or the T/SS vehicle group.

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Because FTY720 has vasoactive properties, and this could have accounted for part of its protective effects, we measured the postshock hemodynamic response. In addition, to confirm standardization of the groups, we measured the volume of blood required to be withdrawn to induce and maintain the shock state. There were no differences in the volume of blood removed between the T/HS animals receiving vehicle and the three T/HS groups receiving different doses of FTY720 (Fig. 1A). On the other hand, the average MAP postresuscitation was significantly higher in the rats treated with 1 mg/kg of FTY720 compared with the vehicle group. Yet, no difference was seen between the 0.5-mg/kg and the 0.1-mg/kg FTY720 groups compared with the vehicle group or the 1-mg/kg FTY720 group. (Fig. 1B) A second way in which FTY720 could have abrogated T/HS-induced lung injury was through its ability to increase leukocyte sequestration, thereby decreasing circulating leukocyte counts (5, 20). Thus, the pre- and post-T/HS (T/SS) neutrophil and lymphocyte counts were quantified (Table 2). For clarity, only the data obtained for the 1.0-mg/kg dose of FTY720 is shown, because similar results were observed in rats receiving the 0.1- and the 0.5-mg/kg doses of FTY720. None of the pre-T/HS PMN values differed between the T/SS and T/HS groups. Although the post-T/HS (T/SS) PMN counts were lower than the pre-T/HS (T/SS) PMN counts, this decrease was not statistically significant. However, there was a significant decrease when the postshock PMN T/SS + vehicle and T/SS + FTY groups were compared with the T/HS + FTY group. Similarly, the post-T/HS lymphocyte counts had significantly decreased in all groups. However, although the post-T/HS lymphocyte counts were reduced, no statistical differences were observed between the various T/HS and T/SS groups, suggesting that FTY720’s protective effects were not mediated to any significant extent by lymphocyte sequestration.

Table 2

Table 2

Because early post-T/HS–induced lung injury, neutrophil priming, and RBC changes have been mechanistically tied to the generation of biologically active lymph exiting the stressed gut, we next tested the hypothesis that FTY720 would limit the biologic effects of T/HS lymph. This was done by incubating whole blood from naive healthy male rats with lymph (10% vol/vol) collected from T/SS or T/HS rats that had received vehicle or FTY720 at a dose of 1 mg/kg. As shown in Figure 3A, T/HS lymph decreased the deformability of RBCs as reflected in an elevated KEI. This T/HS lymph effect on RBC deformability was prevented by the administration of FTY720, and the KEI values of the naive RBCs incubated with T/HS lymph from the rats receiving FTY720 were similar to those of the RBCs incubated with T/SS lymph samples (Fig. 3A). This ability of T/HS lymph to decrease RBC deformability was also associated with increased adherence to a HUVEC endothelial monolayer (Fig. 3B) and surface expression of CD36 (Fig. 3C). The T/HS lymph from the FTY720 rats reduced but did not totally prevent these effects on RBC adhesion and CD36 expression (Fig. 3). Likewise, the T/HS lymph samples primed normal neutrophils to a greater degree than the T/SS lymph samples (Fig. 3D). Treatment with FTY720 significantly reduced the ability of T/HS lymph to prime PMNs; however, FTY720 was not fully protective (Fig. 3D). There were no differences between the sham values in the dose-range experiments comparing lung injury, neutrophil activation, or RBC deformability (Fig. 1C and Fig. 2). These results suggest that part of FTY720’s systemic protective effects could be mediated through abrogation of the production of toxic intestinal lymph.

Fig. 3

Fig. 3

It is important to stress that the intestinal lymph samples were sterile and were tested after all of the cells had been removed by centrifugation. Cell-free lymph was tested based on our earlier studies showing that lymph’s bioactivities were not related to the cellular components of lymph, but were due to humoral factors contained in lymph (21). However, because FTY720 has been reported to reduce lymphocyte counts and a large number of lymphocytes exit the gut through the lymphatics, it is possible that FTY720 would reduce the lymphocyte count of intestinal lymph. Thus, pre-T/HS (T/SS) and post-T/HS (T/SS) lymphocyte counts were quantified in mesenteric lymph (Table 3). The lymphocyte count decreased from the pre-T/HS or T/SS levels in all the groups except the T/SS + vehicle group, indicating that both the administration of FTY720 as well as actual T/HS was associated with a decrease in the lymphocyte count of the lymph samples. Thus, consistent with the potential of FTY720 to sequester lymphocytes in lymphoid organs, the lymphocyte count in the T/SS rats receiving FTY720 was significantly reduced as compared with the T/SS rats receiving vehicle.

Table 3

Table 3

To assess the potential protective effect of FTY720 on T/HS-induced gut injury, we measured gut permeability using the permeability probe FD4, where FD4 is placed in the lumen of the gut, and 30 min later, plasma samples are collected and the blood FD4 concentration is measured. Morphologic studies of the intestinal villi were also performed to complement the physiologic FD4 studies. We found that, in contrast to the protective effects of FTY720 on T/HS lymph bioactivities, the FTY720 did not prevent T/HS-induced increases in gut permeability or limit the extent of morphologic gut injury (Fig. 4).

Fig. 4

Fig. 4

Because previous studies document that mesenteric lymph diversion or lymph duct ligation limits T/HS-induced lung injury, PMN priming, and RBC injury (2, 19), we also measured these variables in the FTY720- and vehicle-treated groups that had systemic lymph diversion via the implanted catheters to compare the protective effects of FTY720 to that of lymph diversion. This experiment is based on the concept that the injured gut produces toxic lymph, which enters the systemic circulation and can lead to end organ failure. If the toxic lymph is prevented from reaching systemic circulation (lymph catheter), end organ failure is prevented. (13) As predicted, both the T/HS-vehicle and T/HS-FTY720 groups did not manifest acute lung injury when lymph was prevented from entering the systemic circulation (Fig. 5A). Likewise, there was no difference in the postshock neutrophil respiratory burst priming or RBC deformability between the groups. However, the manipulations associated with the laparotomy, lymph collection, and other instrumentation were associated with increased PMN priming and decreased RBC deformability of the postshock values compared with the animals’ preshock values (Fig. 5). As in the T/HS and T/SS groups that did not undergo lymph diversion, the post-T/SS and T/HS blood lymphocyte counts decreased similarly among the groups, whereas the neutrophil counts did not decrease (Table 4).

Table 4

Table 4

Fig. 5

Fig. 5

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DISCUSSION

The major observation of this study is that FTY720 administered postshock as part of the fluid resuscitation therapy is able to abrogate T/HS-induced acute lung injury, neutrophil priming, and RBC dysfunction, as well as abrogate the deleterious biologic activity of mesenteric lymph. These results are encouraging because postinjury therapy with FTY720 was able to prevent lung injury and neutrophil priming, which are two of the major consequences of trauma-hemorrhage that have been associated with increased morbidity and mortality (22, 23). This failure of FTY720 to completely abrogate the effects of T/HS on RBC function and neutrophil priming shows that its protective effects were not complete. Two possibilities are that this is the result of involvement of other pathways, or higher drug doses may have been necessary. A limitation of this study is that neither of these possibilities were investigated. Another potential limitation to this work is that we did not perform a mortality study to investigate whether FTY720 prevents mortality as well as morbidity. However, the fact that the protective effect of FTY720 was apparent over a range of doses and at levels as low as 0.1 mg/kg indicates that the drug is effective at doses not that much higher than those that have been safely administered to patients, which range from a total dose of 0.5 to 1.25 mg (24).

Because of its broad spectrum of biologic activities, there are several potential mechanisms by which FTY720 could have limited T/HS-induced acute lung injury (5). Specifically, FTY720 has been shown to reduce lymphocyte cell trafficking, induce lymphopenia, limit immune and inflammatory cell activation, maintain vascular homeostasis, and limit tissue injury in several ischemia-reperfusion models (5, 7, 8). These biologic activities of FTY720 appear to overlap with the processes involved in the pathogenesis of acute lung injury. That is, dysregulation of vasomotor tone, immune-inflammatory cell trafficking and activation, and gut injury have been documented to play a role in several models of acute lung injury (2, 25), whereas RBC dysfunction has been shown to impair microvascular blood flow (15, 26). Thus, we measured the effect of FTY720 on the number of circulating leukocytes, the amount of withdrawn blood required to induce shock, the postshock hemodynamic response and the magnitude of gut injury, the biologic activity of mesenteric lymph, neutrophil priming, and a panel of RBC parameters.

The circulating lymphocyte counts as well as the lymphocyte count in the intestinal lymph samples were measured, because there is increasing evidence that lymphocytes are rapidly activated in an alloantigen-independent manner in conditions associated with an ischemia-reperfusion injury (27, 28). In addition, recent studies have shown that activated T lymphocytes participate in ischemia-reperfusion–induced tissue injury as well as neutrophils (7, 29). In fact, there is evidence that activated T cells may even be involved in coordinating the innate neutrophil response following ischemia-reperfusion (30). Although the administration of FTY720 was associated with a decrease in circulating blood lymphocytes, this decrease was similar to the vehicle-treated animals, suggesting that post-T/HS lymphocyte sequestration or lymphopenia is likely not involved in the protective effect of FTY720. However, FTY720 may have produced alterations in lymphocyte subpopulations, which could have had protective effects. Because lymphocyte subpopulations measurements were not made, this possibility cannot be excluded. Better preservation of postshock blood pressure could have been one mechanism by which FTY720 was beneficial based on the observation that the postshock MAP was better preserved in the T/HS group receiving 1 mg/kg of FTY720 compared with the vehicle group. On the other hand, the 0.1- and 0.5-mg/kg doses of FTY720, which were also systemically protective, did not show better preservation of postshock MAP. Thus, although FTY720 at 1 mg/kg preserved the postshock MAP, suggesting that its systemic protective effects were associated with an improved postshock hemodynamic response, the fact that the other doses of FTY720 did not improve the postshock MAP yet still limited organ injury and systemic neutrophil activation suggests that the hemodynamic effects of FTY720 cannot fully explain its systemic protective effects.

The fact that FTY720 treatment limited neutrophil priming is of potential clinical relevance because primed neutrophils have been tightly associated with the development of acute lung injury in both preclinical and clinical studies (23, 31). This suggests that one of the potential mechanisms by which FTY720 may have exerted its beneficial effects was through its ability to limit neutrophil activation. Red blood cell changes induced by T/HS were measured based on an increasing number of studies documenting that RBC rigidification and increased RBC adhesion to the endothelium can directly impair microvascular blood flow and hence lead to organ injury (15, 26). Because FTY720 limited T/HS-induced RBC rigidification, adhesion to the endothelium, and upregulation of surface CD36 expression, a second mechanism by which FTY720 could have exerted its protective effect is by limiting these T/HS-induced RBC changes. This possibility is supported by the fact that RBCs contain S1P receptors and are one of the principle cells involved in S1P regulation (5). Because activated neutrophils have been documented to bind to the endothelium and cause a capillary leak syndrome as well as contribute to the no-reflow phenomenon after shock (23, 31, 32), it is possible that both RBCs and neutrophils could have contributed to post-T/HS microvascular dysfunction and thereby potentiated acute lung injury. More work will be required to better understand these observations. Nonetheless, these studies complement the work by Hawksworth et al. (9) showing that FTY720 improves survival time in swine and found that this beneficial effect of FTY720 was associated with central lymphocyte sequestration.

Our result that FTY720 did not protect the gut from T/HS-induced injury, although it abrogated the biologic activity of the mesenteric lymph, was surprising and unexpected based on our previous work showing that loss of lymph bioactivity was consistently associated with abrogation of gut injury in this model system (2). We have no explanation for this observation, although one possibility could be that FTY720 blocked the process of toxic lymph production by the injured gut. Another possibility is that the differences between the gut and lung responses to FTY720 reflect differences in S1P receptors, because S1P-1 and S1P-2 receptors dominate in the gut (33), whereas S1P-1, S1P-2, and S1P-3 receptors dominate in the lung (34).

In summary, the posttreatment administration of FTY720 successfully prevented T/HS-induced lung injury, neutrophil priming, and RBC phenotypic and functional changes. Thus, FTY720 shows promise as a potential therapeutic agent based on the current work, the work of Hawksworth et al. (9) in a swine hemorrhagic shock model, and a number of preclinical studies documenting that FTY720 successfully limited ischemia-reperfusion mediated organ injury. The potential clinical importance of these encouraging preclinical studies is strengthened by the safety profile of FTY720 and its FDA approval for use in a number of clinical conditions.

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Keywords:

Acute lung injury; neutrophil activation; red blood cell rigidity; lymph

© 2014 by the Shock Society