• View in gallery

    Mean ± SEM serum IL-6 activity in 4 dogs that underwent CMA occlusion (duration of ischemic phase, 60 minutes) followed by reperfusion (duration of reperfusion phase, 480 minutes; I-R group; black bars) and 4 sham-operated control dogs (control group; white bars). Data were recorded immediately before the CMA occlusion was relieved (end of ischemic phase [EOI]) and at 60, 180, and 480 minutes of reperfusion, and are expressed as the change from baseline value (measurement obtained immediately before CMA occlusion). *At this time point, value for I-R group is significantly (P < 0.05) different from that for the control group. †At this time point, value is significantly (P < 0.05) different from baseline value for this group.

  • View in gallery

    Mean ± SEM ratio of TNF-α mRNA (A) and IL-6 mRNA (B) to β-actin mRNA in jejunal tissue of 4 dogs that underwent CMA occlusion (duration of ischemic phase, 60 minutes) followed by reperfusion (duration of reperfusion phase, 480 minutes; I-R group; black bars) and 4 sham-operated control dogs (control group; white bars). See Figure 1 for key.

  • View in gallery

    Mean ± SEM ratio of TNF-α mRNA (A) and IL-6 mRNA (B) to β-actin mRNA in liver tissue of 4 dogs that underwent CMA occlusion (duration of ischemic phase, 60 minutes) followed by reperfusion (duration of reperfusion phase, 480 minutes; I-R group; black bars) and 4 sham-operated control dogs (control group; white bars). See Figure 1 for key.

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Effects of small intestinal ischemia and reperfusion on expression of tumor necrosis factor-α and interleukin-6 messenger RNAs in the jejunum, liver, and lungs of dogs

Yoshinori Nezu DVM, PhD1, Yoko Nezu DVM, PhD2, Kae Shigihara DVM3, Yasuji Harada DVM, PhD4, Takuya Yogo DVM, PhD5, Yasushi Hara DVM, PhD6, and Masahiro Tagawa DVM, PhD7
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  • 1 Division of Veterinary Surgery, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo, 180-8602 Japan.
  • | 2 Igusa Animal Hospital, 1-31-16 Igusa, Suginami-ku, Tokyo, 167-0021 Japan.
  • | 3 Veterinary Medical Teaching Hospital, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo, 180-8602 Japan.
  • | 4 Division of Veterinary Surgery, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo, 180-8602 Japan.
  • | 5 Division of Veterinary Surgery, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo, 180-8602 Japan.
  • | 6 Division of Veterinary Surgery, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo, 180-8602 Japan.
  • | 7 Division of Veterinary Surgery, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo, 180-8602 Japan.

Abstract

Objective—To determine the effects of intestinal ischemia and reperfusion on the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNAs in the jejunum, liver, and lungs of dogs.

Animals—8 healthy adult Beagles.

Procedures—In each dog, the cranial mesenteric artery was occluded for 0 (control group; n = 4) or 60 (I-R group; 4) minutes, followed by reperfusion for 480 minutes; serum TNF-α and IL-6 activities and expression levels of TNF-α and IL-6 mRNAs in jejunal, hepatic, and lung tissues were measured before and at the end of the ischemic period and at intervals during reperfusion. For each variable, values were compared between the control and I-R groups at each time point.

Results—Compared with the control group, serum IL-6 activity increased significantly after 180 minutes of reperfusion in the I-R group; also, jejunal TNF-α mRNA expression increased significantly after 60 (peak) and 180 minutes of reperfusion. In the I-R group, expressions of IL-6 mRNA in the liver and TNF-α and IL-6 mRNAs in the lungs increased significantly at 480 minutes of reperfusion, compared with the control group. Serum TNF-α activity, expression of IL-6 mRNA in the jejunum, and expression of TNF-α mRNA in the liver in the control and I-R groups did not differ.

Conclusions and Clinical Relevance—Results indicated that the liver, lungs, and jejunum contributed to the production of TNF-α and IL-6 after intestinal ischemia and reperfusion in dogs, suggesting that intestinal ischemia and reperfusion induce a systemic proinflammatory cytokine response in dogs.

Abstract

Objective—To determine the effects of intestinal ischemia and reperfusion on the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 mRNAs in the jejunum, liver, and lungs of dogs.

Animals—8 healthy adult Beagles.

Procedures—In each dog, the cranial mesenteric artery was occluded for 0 (control group; n = 4) or 60 (I-R group; 4) minutes, followed by reperfusion for 480 minutes; serum TNF-α and IL-6 activities and expression levels of TNF-α and IL-6 mRNAs in jejunal, hepatic, and lung tissues were measured before and at the end of the ischemic period and at intervals during reperfusion. For each variable, values were compared between the control and I-R groups at each time point.

Results—Compared with the control group, serum IL-6 activity increased significantly after 180 minutes of reperfusion in the I-R group; also, jejunal TNF-α mRNA expression increased significantly after 60 (peak) and 180 minutes of reperfusion. In the I-R group, expressions of IL-6 mRNA in the liver and TNF-α and IL-6 mRNAs in the lungs increased significantly at 480 minutes of reperfusion, compared with the control group. Serum TNF-α activity, expression of IL-6 mRNA in the jejunum, and expression of TNF-α mRNA in the liver in the control and I-R groups did not differ.

Conclusions and Clinical Relevance—Results indicated that the liver, lungs, and jejunum contributed to the production of TNF-α and IL-6 after intestinal ischemia and reperfusion in dogs, suggesting that intestinal ischemia and reperfusion induce a systemic proinflammatory cytokine response in dogs.

In mammals, intestinal ischemia and reperfusion can lead to a systemic inflammatory response; this response may play a pivotal role in the development of MODS because systemic inflammatory response is considered to be a risk factor of MODS.1–3 The intestinal tract has traditionally been considered the central organ in the pathogenesis of MODS in critically ill patients.1–3 The intestines are affected by ischemia in the earliest stage of various types of critical illness, including shock, trauma, and sepsis.4–6 Subsequent resuscitation results in reperfusion of the ischemic intestinal tissues. Many studies1–3 have revealed that intestinal ischemia and reperfusion may not only lead to an acute local intestinal injury but may also cause distant organ injury and MODS.

With regard to the systemic inflammatory response to intestinal ischemia and reperfusion, researchers have presumed that increases in inflammatory mediator activities, such as the activities of proinflammatory cytokines, play a central role in development of distant organ injury because those increases are common pathophysiologic processes of autodestructive systemic inflammatory responses.1,2 These mediators induce microvascular dysfunction, such as failure in the regulation of microcirculation and promotion of disseminated intravascular coagulation, via activation of endothelial cells and neutrophils.7,8 Organ dysfunction is subsequently caused by this microvascular dysfunction and the direct cytotoxic effects of activated neutrophils.

Following intestinal ischemia and reperfusion, not only the intestinal tract but also distant organs may contribute to increases in systemic amounts of proin flammatory cytokines and the spread of the inflammatory response. Although many researchers have tried to identify a cytokine-synthesizing organ involved in intestinal ischemia and reperfusion, the details of the role of a remote organ remain unclear, whereas the production and release of cytokines by the intestinal tissues have been determined.9,10 Our previous study11 in which the kinetics of TNF-α and IL-6 in portal and systemic circulations in dogs that underwent CMA occlusion were investigated revealed that the IL-6 was synthesized mainly in remote organs and not in the intestines and that the major source of circulating TNF-α might change from the intestines to remote organs over time. However, on the basis of the findings of that study,11 it was not possible to directly identify the cytokine-synthesizing organ. We assumed that the primary transport pathways of various inflammatory mediators released from the intestinal tract were from intestinal tissues through the portal vein to the liver and on into the systemic circulation and from intestinal tissues through the thoracic duct to the lungs and into the systemic circulation. In addition, the liver and lungs contain large numbers of resident macrophages, and there have been many reports on leukocyte recruitment and microvascular dysfunction in the liver12,13 and lungs14,15 following intestinal ischemia and reperfusion.

The purpose of the study reported here was to determine the effects of small intestinal ischemia and reperfusion on the expression of TNF-α and IL-6 mRNAs in the jejunum, liver, and lungs of dogs. To induce intestinal ischemia and reperfusion, dogs underwent CMA occlusion. Levels of expression of TNF-α and IL-6 genes in intestinal tissue and liver were assessed at intervals during reperfusion, and levels of expression in the lungs were assessed at the end of the experiment; those assessments were performed by use of quantitative expression analysis that was based on data obtained via real-time RT-PCR procedures.

Materials and Methods

Animals—The study conformed to the Bioethics Guidlines of Nippon Veterinary and Life Science University. Eight healthy adult Beagles of both sexes that weighed 9.5 to 14 kg were used. Food was withheld from the dogs for 18 hours prior to the experiment, but water was supplied ad libitum.

Surgical procedure—For each dog, anesthesia was induced via IV administration of thiamylal sodiuma (25 mg/kg). After endotracheal intubation, the endotracheal tube was connected to a pressure-limited ventilatorb and the dog was mechanically ventilated (fraction of inspired oxygen, 1.0). Spontaneous respiration was completely stopped by use of an IV bolus of pancuronium bromidec (0.1 mg/kg); throughout the experimental period, additional pancuronium bromide was administered as necessary. Minute ventilation was adjusted to maintain normocarbia (arterial PCO2, 40.0 ± 5.0 mm Hg). Anesthesia was maintained via inhalation of isofluraned in 100% oxygen.

During the experimental period, each dog received a continuous IV infusion of lactated Ringer's solutione (10 mL/kg/h) and 5% glucose solution (5 mL/kg/h) as fluid replacement. Body temperature was maintained at 36.5° to 37.5°C by use of a heating mat.

In each dog, the left femoral artery was cannulated with a sterile catheter positioned in the abdominal portion of the aorta for arterial blood sample collection. The left cephalic vein was cannulated for administration of fluids and drug infusions. A midline laparotomy was performed. The CMA was identified and cleaned of surrounding tissue close to its origin from the aorta. A silastic vessel loop was positioned around the CMA for complete occlusion of the vessel. After all measurement systems were in place, the abdominal incision was closed and sutured, except for the portion necessary for the experimental procedures. The open area was covered with a sterile moist gauze pad to prevent evaporation of body fluids. After surgical preparation was completed, 100% oxygen was switched to air gas for ventilation and the fraction of inspired oxygen was reduced to 0.21. After preparation, a 60-minute period was allowed for all variables to stabilize before baseline measurements were obtained.

Experimental protocol—The 8 dogs were each allocated to 1 of 2 experimental groups. The I-R group (n = 4) underwent 60 minutes of CMA occlusion and 480 minutes of reperfusion after the occlusion was relieved. A sham operation was performed in each control dog (n = 4). To perform the ischemic phase in the I-R group, the CMA was completely occluded by application of constant tension to the silastic vessel loop positioned around the CMA. The reperfusion phase was achieved via release of the loop tension. In the control group, the abdominal surgery was performed in the same way as I-R group, but the CMA was not occluded. The open area of the abdominal incision was covered with a sterile moist gauze pad.

Arterial blood samples were collected prior to CMA occlusion (baseline); immediately before the occlusion was relieved (end of the ischemic episode); and at 60, 180, and 480 minutes after the occlusion was relieved. These blood samples were allowed to clot and were centrifuged at 4°C for 20 minutes; the serum was stored at −80°C until the cytokine assays were performed.

Samples of liver and jejunum were collected before CMA occlusion (baseline); immediately before the occlusion was relieved (end of the ischemic episode); and at 60, 180, and 480 minutes after the occlusion was relieved. At the time of each sample collection, the left lateral, left medial, right lateral, right medial, or quadrate lobe was chosen at random and then a sample of the liver tissue was collected by use of a wedge biopsy method. Only 1 sample was collected from each hepatic lobe in each dog to minimize tissue damage associated with the sampling procedure. To collect samples from the small intestine, a biopsy punchf (diameter, 10 mm) was applied to the intestinal wall of the antimesenteric part of the central portion of the jejunum. At the time of each sample collection, jejunal tissue was obtained from a site that was approximately 10 cm from the last sample site to minimize tissue damage associated with the sampling procedure.

After the last liver and jejunal tissue samples were collected, each dog was euthanatized by IV administration of an overdose of pentobarbital sodiumg and tissue samples from the lungs were immediately collected.

Bioassay for serum TNF-α activity—Serum TNFA activity was determined by use of a cytotoxicity bioassay involving WEHI 164 clone 13 murine fibrosarcoma cells.h The cytotoxicity bioassay was conducted according to the method of Eskandari et al16 with a minor modification. In brief, RPMI 1640 mediumi was used to establish 100-μL volumes of serial 2-fold dilutions of serum samples or recombinant human TNF-α,j which were added to 96-well flat-bottomed tissue culture plates.k Then, 5 × 104 WEHI 164 cells in 100-μL volumes of RPMI 1640 medium containing 1 Mg of actinomycin Dl/mL were added to each well and incubated at 37°C in 5% CO2 for 20 hours. After incubation, cytotoxicity was measured colorimetrically by use of tetrazolium salt. In brief, 50 μL of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) solutionl (1 mg/mL; XTT in RPMI 1640 containing 20 ML of 0.383 mg of phenazine methosulfatel/mL) was added to each well. After an additional 4 hours of incubation at 37°C in 5% CO, color development was measured at 450 nm by use of a microplate reader.m Tumor necrosis factor-α activity was determined from the standard curve derived from recombinant human TNF-α. All determinations were performed in triplicate.

Bioassay for serum IL-6 activity—Serum IL-6 activity was determined by use of a proliferative bioassay involving the IL-6–dependent murine hybridoma cell line 7TD1.n The proliferative bioassay was conducted according to the method of Van Snick et al17 with a minor modification. In brief, RPMI 1640 mediumi was used to establish 100-μL volumes of serial 2-fold dilutions of serum samples or recombinant human IL-6,i which were added to 96-well flat-bottomed tissue culture plates. Then, 5 × 103 7TD1 cells in 100-μL volumes of RPMI 1640 were added to each well and incubated at 37°C in 5% CO2 for 68 hours. After incubation, proliferation was measured colorimetrically by use of tetrazolium salt. Interleukin-6 activity was determined from the standard curve derived from recombinant human IL-6. All determinations were performed in triplicate.

Quantitative RT-PCR assay—Tissue samples collected from the lungs, liver, and jejunum were placed in a tissue storage reagent.o The tissues were incubated at 4°C for approximately 12 hours and then stored at −80°C before RNA isolation. Total RNA was extracted by use of a kitp according to the manufacturer's instructions. The isolated RNA was suspended in ribonuclease-free water,j quantified by use of a spectrophotometer,q and adjusted to a concentration of approximately 0.02 μg/ML.

Complementary DNA was synthesized from RNA by use of an RT method.r The RT reaction was performed in 50-μL samples containing 10mM Tris-HCl (pH, 8.3); 50mM KCl; 2.5mM MgCl2; 10mM each of 2′-deoxyadenosine 5′-triphosphate, 2′-deoxyguanosine 5′-triphosphate, 2′-deoxythymidine 5′-triphosphate, and 2′-deoxycytidine 5′-triphosphate; ribonuclease inhibitor (2.5 U/μL); murine leukemia virus RT (2.5 U/μL); random hexamers (2.5 U/μL); and 7.5 mL of total RNA. The thermal cycling conditions were as follows: 15 minutes at 42°C, 5 minutes at 99°C, and 5 minutes at 5°C. The RT reactions were performed with a thermal cycler.s

Quantitative real-time RT-PCR assay—To evaluate the level of expression of the target genes, a quantitative RT-PCR assay was performed with a real-time technique by use of a sequence detector.t The dog-specific detection probes and primers were generated according to procedures reported by Aihara et al18 (Appendix). Dog-specific detection probesu were labeled with a reporter fluorescent dye, FAM (6-carboxyfluorescein), on the 5′ nucleotide and with a quenching fluorescent dye, TAMRA (6-carboxy-tetramethyl-rhodamine), on the 3′ nucleotide. The amplification reaction (total volume, 50 μL) contained diethyl pyrocarbonate water (13.5 μL), master mixv (25 μL), sense and antisense primersw (200 nmol/L [2.25 μL] each), each of the detection probes (100 nmol/L [2 μL] each), and cDNA (5 mL). The thermal cycling conditions were as follows: 40 cycles of 20 seconds at 94°C for denaturation and 1 minute at 60°C for annealing and extension.

Data were analyzed according to the manufacturer's instructions by use of the standard curve method. Briefly, 4 serial dilutions of a standard sample in which expression of target genes had already been determined were analyzed and used to construct a standard curve. On the basis of the standard curve, the mRNA concentration of each sample was calculated by its threshold cycle value. The threshold cycle values corresponded to the cycle number at which the fluorescent emission monitored in real time reached the threshold, which was set at a value 10 SDs greater than the mean of baseline emissions calculated from cycles 5 to 15. The expression levels of target genes were evaluated by the ratio of the number of target mRNA to β-actin mRNA because total RNA concentrations from each sample could be normalized with greater certainty on the basis of the quantity of β-actin mRNA. A liver tissue sample collected from a dog in which lipopolysaccharidex (2 mg/kg, IV bolus) had been infused was used as the standard sample. All determinations were performed in triplicate, and the mean value of the triplicate runs was used for each determination.

Figure 1—
Figure 1—

Mean ± SEM serum IL-6 activity in 4 dogs that underwent CMA occlusion (duration of ischemic phase, 60 minutes) followed by reperfusion (duration of reperfusion phase, 480 minutes; I-R group; black bars) and 4 sham-operated control dogs (control group; white bars). Data were recorded immediately before the CMA occlusion was relieved (end of ischemic phase [EOI]) and at 60, 180, and 480 minutes of reperfusion, and are expressed as the change from baseline value (measurement obtained immediately before CMA occlusion). *At this time point, value for I-R group is significantly (P < 0.05) different from that for the control group. †At this time point, value is significantly (P < 0.05) different from baseline value for this group.

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.512

Statistical analysis—All data are expressed as mean ± SEM. Serum TNF-α and IL-6 activity data were assessed in terms of changes from the baseline values at each measurement point. The TNF-α and IL-6 mRNA data in the jejunum and liver were each assessed in terms of ratios of the measurement at each time point to the baseline value. A Mann-Whitney U test was used to detect significant differences between the 2 experimental groups. To detect significant differences between the baseline value and the value at each time point within a group, a Friedman test was used; a multiple comparison was performed by use of the Fisher test, when a null hypothesis was rejected. For all comparisons, a value of P < 0.05 was regarded as significant.

Results

Serum TNF-α activity—In the I-R and control groups, serum TNF-α activity did not change significantly from baseline during the course of the experimental period. Values ranged from 3.6 to 4.4 U/mL in the I-R group and from 3.7 to 6.5 U/mL in the control group.

Figure 2—
Figure 2—

Mean ± SEM ratio of TNF-α mRNA (A) and IL-6 mRNA (B) to β-actin mRNA in jejunal tissue of 4 dogs that underwent CMA occlusion (duration of ischemic phase, 60 minutes) followed by reperfusion (duration of reperfusion phase, 480 minutes; I-R group; black bars) and 4 sham-operated control dogs (control group; white bars). See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.512

Serum IL-6 activity—At 180 minutes of reperfusion in the I-R group, serum IL-6 activity was 231.1 ± 62.0 U/mL, which was significantly (P = 0.021) different from the value for the control group (13.1 ± 14.9 U/ mL). In the I-R group, a peak serum IL-6 activity value of 1,222.2 ± 699.7 U/mL was detected at the end of the experimental period (at 480 minutes of reperfusion); at this time point, the value was significantly (P = 0.021) different from the control group value and also significantly (P = 0.015) different from the baseline value for the I-R group (Figure 1). In the control group, there were no significant changes from baseline in serum IL-6 activity during the entire experimental period.

Ratio of TNF-α or IL-6 mRNA to β-actin mRNA in the jejunum—Compared with findings in the control group, the ratio of TNF-α mRNA to β-actin mRNA in the jejunum in the I-R group dogs increased significantly during reperfusion. A peak increase (P = 0.043) was detected at 60 minutes of reperfusion (45.12 ± 39.36), and the value remained significantly (P = 0.042) increased until 180 minutes of reperfusion (5.81 ± 1.92; Figure 2). In the control group, there were no significant changes in the ratio of TNF-α mRNA to β-actin mRNA in the jejunum during the entire experimental period. With regard to the ratio of IL-6 mRNA to β-actin mRNA in the jejunum, there were no significant changes in either group during the entire experimental period.

Figure 3—
Figure 3—

Mean ± SEM ratio of TNF-α mRNA (A) and IL-6 mRNA (B) to β-actin mRNA in liver tissue of 4 dogs that underwent CMA occlusion (duration of ischemic phase, 60 minutes) followed by reperfusion (duration of reperfusion phase, 480 minutes; I-R group; black bars) and 4 sham-operated control dogs (control group; white bars). See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.512

Ratio of TNF-α or IL-6 mRNA to β-actin mRNA in the liver—During the experimental period, the ratio of TNF-α mRNA to β-actin mRNA in liver tissue did not change significantly in either the I-R or control group (Figure 3).

At 480 minutes of reperfusion in the I-R group, the ratio of IL-6 mRNA to β-actin mRNA in the liver was increased significantly (P = 0.021), compared with the control group value (6.13 ± 2.87 vs 0.32 ± 0.09; Figure 3). In the control group, there were no significant changes in the ratio of IL-6 mRNA to β-actin mRNA in the liver during the entire experimental period.

Ratio of TNF-α or IL-6 mRNA to β-actin mRNA in the lungs—At 480 minutes of reperfusion, the ratio of TNF-α mRNA to β-actin mRNA in the lungs of dogs in the I-R group was significantly (P = 0.021) higher than the value in the lungs of dogs in the control group (0.403 ± 0.027 vs 0.168 ± 0.050). At that time point, the ratio of IL-6 mRNA to β-actin mRNA in the lungs of dogs in the I-R group was significantly (P = 0.021) higher than the value of dogs in the control group (0.045 ± 0.025 vs 0.002 ± 0.000).

Discussion

In the present study, the levels of expression of TNF-α and IL-6 genes in the jejunum and liver of dogs with experimentally induced intestinal ischemia and reperfusion were assessed over time by use of quantitative expression analysis that was based on real-time RTPCR techniques. Similarly, levels of expression of those genes in the lungs of the study dogs were assessed at the end of the experimental period. The intent was to determine which organs were responsible for synthesis of these proinflammatory cytokines. The results of our study indicated that in addition to the intestinal tract (the ischemic organ), remote organs such as the liver and lungs contribute to the production of proinflammatory cytokines after intestinal ischemia and reperfusion in dogs.

In the dogs of the present study, the expression of TNF-α mRNA in the jejunum was increased significantly after reperfusion, but not during the phase of ischemia, and reached a peak increase early in the reperfusion phase (ie, at 60 minutes of reperfusion). These findings are direct evidence that production of TNF-α at the genetic transcription level in the intestinal tract occurred after reperfusion but not during the ischemic phase.

Several possible mechanisms of TNF-α synthesis in intestinal tissues during ischemia and reperfusion have been considered. First, translocation of bacteria or their products (eg, endotoxin trapped in the lamina propria by local phagocytic cells) can initiate a local inflammatory response and induce or potentiate cytokine production by the intestine-associated lymphatic tissue.19,20 Second, bacterial components, such as peptidoglycan,21 bacterial peptide,22 flagellin,23 and bacterial DNA,24 can stimulate cytokine synthesis directly via toll-like receptors. Third, oxygen-derived free radicals that are generated following ischemia and reperfusion can activate nuclear factors (eg, nuclear factor-Kβ), which bind to the enhancer sequences of the cytokine genes.25

Despite the increased TNF-α synthesis in the jejunum in the present study, the increase in TNF-α activity in arterial blood was not confirmed. In several studies,11,20,26 serum TNF-α activity in portal veins was significantly high, compared with the value in systemic circulation, after ischemia and reperfusion of the intestine. Thus, the roles of TNF-α generated by the intestinal tract may be to affect local intestinal tissue and to enhance the spread of the inflammatory response at a comparatively short range via portal veins.

Although low activities of TNF-α in tissues play a beneficial role in the host immune defense, the high level of local activity in the intestinal tissues during ischemia and reperfusion generates excessive tissue damage such as mucosal barrier failure.9,27 Additionally, data from an in vitro study of Caco2 enterocyte cell monolayers by Diebel et al28 indicated that exposure to both hypoxiareoxygenation and bacteria increased Caco2 apoptosis in a delayed fashion and suggested that TNF-α plays a pivotal role in the onset of apoptosis. Thus, enhanced production of TNF-α in the intestinal tissues induces apoptosis of intestinal mucosa, which may result in aggravation of an intestinal barrier dysfunction at a delayed phase of reperfusion injury. A clinical approach to suppression of TNF-α synthesis in the intestines is important because intestinal barrier dysfunction can lead to or potentiate the systemic inflammatory response via bacterial translocation and thereby contribute to the development of MODS.

In both the I-R and control groups in the present study, there were no significant changes in the expression of IL-6 mRNA in the jejunum during the entire experimental period, whereas serum IL-6 activity increased significantly in the I-R group, compared with findings in the control group. On the basis of these results, it appears that the remote organs, not the intestinal tissues, are the main source of circulatory IL-6 in dogs. This finding corresponded with results of our previous study11 involving dogs that underwent CMA occlusion; in that earlier study, the magnitude of the IL-6 response in the portal circulation was almost equivalent to that in the systemic circulation, which suggested that IL-6 was generated mainly in the remote organs and not in the intestinal tissues.

It is likely that the liver contributes to the expansion of an inflammatory response at the systemic level after intestinal ischemia and reperfusion because the liver contains a large amount of the resident macrophages (Kupffer cells) and is affected by various humoral mediators or bacteria derived directly from the intestinal tract via portal veins. In the present study, the expression of IL-6 mRNA in the liver was increased significantly at 480 minutes of reperfusion in the I-R group, which indicated that the liver synthesized IL-6 and contributed to systemic inflammatory response following intestinal ischemia and reperfusion. However, there were no significant changes in the expression of TNF-α mRNA in the liver during the entire experimental period in either the I-R or control group, which indicated that the liver did not synthesize TNF-α in response to intestinal ischemia and reperfusion.

It is well established that high circulating amounts of IL-6 positively correlate with septic shock and with the mortality rate in several inflammatory diseases in humans.29–31 In IL-6 knock-out mice that underwent splanchnic artery occlusion, a deficiency of IL-6 attenuated the expression of adhesion molecules and neutrophil-mediated tissue injury, leading to a reduction in the mortality rate32; this finding indicates the importance of IL-6 in the development of MODS that accompanies intestinal ischemia and reperfusion. In contrast, recent data suggest that IL-6 is a key mediator of the hepatoprotective effect in direct hepatic ischemia-reperfusion injury33,34 and that exogenous IL-6 inhibits acute inflammatory responses and prevents ischemia and reperfusion injury after intestinal transplantation.35 Therefore, further investigation appears to be needed to determine whether IL-6 synthesis in the liver following intestinal ischemia and reperfusion exerts an organprotective effect or a MODS-development effect after intestinal ischemia and reperfusion.

Remarkably little has been reported regarding the expression of hepatic TNF-α following intestinal ischemia and reperfusion, to our knowledge. In rats that underwent CMA occlusion, Rahat et al36 found that intestinal ischemia and reperfusion were not associated with an increase in TNF-α mRNA expression in the liver; that finding corresponded with our observation in the present study. However, other researchers detected an obvious increase in TNF-α mRNA expression in liver tissue following intestinal ischemia and reperfusion in mice.37 This contrast in study findings may be a result of differences in species examined or differences in the duration of ischemia and reperfusion. Additionally, hepatic Kupffer cells, which constantly receive and biologically filter bacteria and various mediators of enteric origin, may have different reactivity (hyporesponsiveness), compared with that of other macrophages.38 Therefore, the experimental procedure used in the present study may have provided comparatively weak stimulation of the hyporesponsive hepatic Kupffer cells, and the liver may become a TNF-α production organ in response to stronger stimulation.

It is assumed that, in addition to the liver, the lungs play a role in systemic inflammatory responses after intestinal ischemia and reperfusion because the lungs are affected by various humoral mediators or bacteria derived from the intestinal tract via lymphatic vessels. In the present study, the expressions of TNF-α and IL-6 mRNAs in the lungs were significantly higher in the I-R group, compared with values in the control group, at 480 minutes of reperfusion; thus, the lungs synthesized TNF-α and IL-6 and contributed to the expansion of inflammatory cytokine response following intestinal ischemia and reperfusion.

In the I-R group in our study, there were no significant changes in serum TNF-α activity during the experimental period, whereas expression of TNF-α mRNA in the lungs was significantly higher, compared with the value in control group. On the basis of these results, it appears that the TNF-α produced by the lungs acts locally rather than at the systemic level. Following intestinal ischemia and reperfusion in rats, the lungs undergo acute microvascular injury, characterized by neutrophil sequestration and increased microvascular permeability.39,40 Additionally, other researchers have reported41,42 that TNF-α is an important mediator of the expression of adhesion molecules, such as intercellular adhesion molecule-1, CD18, and CD11b, in leukocytes and the pulmonary endothelial surface after intestinal ischemia and reperfusion; these adhesion molecules induce leukocyte-endothelial adhesion, resulting in neutrophil sequestration and microvascular leakage in the lungs.

Several investigators have reported29–31 that a high level of systemic IL-6 activity is positively correlated with severity of septic shock and other inflammatory diseases. In contrast, Farivar et al43 recently determined that IL-6 reduces endothelial disruption and neutrophil sequestration in the lungs of rats that underwent direct lung ischemia-reperfusion injury, which resulted in improved oxygenation. However, the participation of IL-6 in acute lung injury is now controversial; therefore, it is unclear whether IL-6 synthesis in the lungs as well as the liver after intestinal ischemia and reperfusion has an organ-protective effect or a MODS-development effect in dogs.

Results of the present study in dogs indicated that the liver and lungs contribute to the synthesis of TNF-α and IL-6 after intestinal ischemia and reperfusion as well as the jejunum (part of the ischemic organ). This finding suggested that intestinal ischemia and reperfusion induce a systemic proinflammatory cytokine response in dogs and that not only the intestinal tract but also remote organs are involved in this cytokine response.

ABBREVIATIONS

MODS

Multiple organ dysfunction syndrome

TNF

Tumor necrosis factor

IL

Interleukin

CMA

Cranial mesenteric artery

RT

Reverse transcriptase

a.

Citosol, Kyorin Pharmaceutical, Tokyo, Japan.

b.

A.D.S. 1000, Shin-ei Industries Inc, Saitama, Japan.

c.

Mioblock injection, Sankyo, Tokyo, Japan.

d.

Rhodia, Nagase Medicals, Osaka, Japan.

e.

Solulact, Terumo, Tokyo, Japan.

f.

Kai sterile disposable biopsy punch, Kai Industries, Gifu, Japan.

g.

Nembutal injection, Dainihon, Osaka, Japan.

h.

American Type Culture Collection, Manassas, Va.

i.

Gibco BRL, Life Technologies Inc, Gaithersburg, Md.

j.

Boehringer Mannheim GmbH, Mannheim, Germany.

k.

Microtest tissue culture plate, Becton-Dickinson, Franklin Lakes, NJ.

l.

Sigma Chemical Co, St Louis, Mo.

m.

Spectra Rainbow Thermo A-5002, Wako, Osaka, Japan.

n.

Riken Cell Bank, Ibaraki, Japan.

o.

RNAlater, Takara-bio, Tokyo, Japan.

p.

RNeasy kit, Qiagen KK, Tokyo, Japan.

q.

Ultraspec 3000pro, Amersham Biosciences Corp, Piscataway, NJ.

r.

RNA PCR kit, Perkin-Elmer Applied Biosystems, Fremont, Calif.

s.

GeneAmp PCR System 9600, Perkin-Elmer Applied Biosystems, Fremont, Calif.

t.

7700 Sequence Detection System, Perkin-Elmer Applied Biosystems, Fremont, Calif.

u.

Perkin-Elmer Applied Biosystems, Fremont, Calif.

v.

TaqMan Universal Master Mix, Perkin-Elmer Applied Biosystems, Fremont, Calif.

w.

Sigma Aldrich Co, St Louis, Mo.

x.

Escherichia coli O55:B5, Sigma Chemical Co, St Louis, Mo.

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Contributor Notes

Address correspondence to Dr. Yoshinori Nezu.