- Email: [email protected]
IL-11 Pretreatment Reduces Cell Death after Intestinal Ischemia–Reperfusion
Journal of Surgical Research 108, 268 –272 (2002) doi:10.1006/jsre.2002.6542
IL-11 Pretreatment Reduces Cell Death after Intestinal Ischemia–Reperfusion Keith A. Kuenzler, M.D.,* Philip Y. Pearson, M.D.,* and Marshall Z. Schwartz, M.D.* ,† *Department of Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; and †Department of Surgery, A. I. duPont Hospital for Children, Wilmington, Delaware 19899 Submitted for publication May 28, 2002
Background. Intestinal ischemia–reperfusion (IR) injury results in enterocyte necrosis and apoptosis. This study was designed to evaluate the potential protective effects of interleukin-11 (IL-11) pretreatment on intestinal mucosa following IR injury. Materials and methods. Sham (n ⴝ 7) and control animals (n ⴝ 7) received 48 h of intravenous saline while treatment animals (n ⴝ 7) received IL-11 (750 g/kg/day). Sham animals then underwent laparotomy alone, while control and treatment animals underwent 35 min of mesenteric artery occlusion and 120 min of reperfusion. Midjejunum samples were obtained and serum was drawn. Fluorometric assays were performed for hexosaminidase A (HEX A) and -glucuronidase (GLUC), markers of enterocyte necrosis. Apoptosis was quantified by TUNEL and confirmed by DNA fragmentation. Transcription of Bcl-2, an antiapoptotic regulator, was assessed by multiplex RTPCR. Statistical analysis was performed using ANOVA and expressed as means ⴞ SEM. Results. In pretreated animals, HEX A and GLUC activities after IR were reduced from 570 ⴞ 54 to 426 ⴞ 47 nmol/ml/h (P < 0.05) and from 183 ⴞ 29 to 125 ⴞ 7 nmol/ml/h (P < 0.01), respectively. Pretreated animals had a reduced number of apoptotic cells per 10 crypts (79 ⴞ 11) compared with untreated rats (255 ⴞ 17) after IR injury (P < 0.01). Mucosal DNA from pretreated rats qualitatively showed less fragmentation on electrophoresis. Relative Bcl-2 band intensity was higher in pretreated animals (1.04 ⴞ 0.09) compared with controls (0.78 ⴞ 0.07) (P < 0.05). Conclusions. IL-11 pretreatment reduced crypt cell apoptosis after IR injury, possibly by upregulating Bcl-2. Treated animals also demonstrated attenuation in the release of certain lysosomal enzymes. These Presented at the Annual Meeting of the Association for Academic Surgery, Milwaukee, Wisconsin, November 15–17, 2001
0022-4804/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
data indicate that following IR injury, IL-11 improves enterocyte survival by reducing necrosis and apoptosis. © 2002 Elsevier Science (USA) Key Words: ischemia–reperfusion injury; interleukin-11; intestinal ischemia; lysosomal enzymes; programmed cell death; apoptosis; BCL-2.
Ischemia–reperfusion (IR) injury to the small intestine occurs in both the pediatric and the adult age groups. Midgut volvulus, necrotizing enterocolitis, shock states, and mesenteric emboli are only some of the entities that can produce IR injury. The mechanisms by which the intestine is damaged after IR are not well defined. Contributing factors include toxic oxygen species and the local recruitment of inflammatory leukocytes [1]. In the past, necrosis had been presumed to be the chief cause of epithelial cell death following an ischemic insult. More recent studies have indicated, however, that apoptosis is a significant and perhaps the principal contributor to cell death after IR injury [2– 4]. In contrast to cell necrosis, apoptosis is a coordinated, gene-directed process. Thus, understanding the sequence of events central to the apoptotic mechanism may potentially lead to treatments for diseases that are characterized by accelerated rates of programmed cell death [4]. Interleukin-11 (IL-11) is a multifunctional cytokine which our laboratory has shown to enhance absorptive function in animal models of small bowel adaptation [5] and intestinal IR injury [6]. Du et al. [7] previously reported that IL-11 pretreatment reduced morbidity and mortality in mice subjected to small bowel necrosis. They described both an increase in mitotic activity and a decline in enterocyte apoptosis. Additional studies have supported the ability of IL-11 to reduce apoptosis in radiation injury to the small intestine [8] and skin [9]. The goal of the current study was to evaluate
268
KUENZLER, PEARSON, AND SCHWARTZ: IL-11 REDUCES CELL DEATH
the effects of pretreatment with IL-11 in reducing mucosal cell death by necrosis and apoptosis after moderate IR injury. MATERIALS AND METHODS Animal care. All animal care and use complied with the institutional regulations set forth and approved by the Animal Care and Use Committee of the A. I. duPont Hospital for Children and the Nemours Research Programs (Wilmington, DE). Fourteen adult male Sprague–Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 275 to 300 g were used in this study. Rats were kept in individual cages and fed standard rat chow and water ad libitum. After the animals were allowed to acclimate to the environment for a period of 1 week, surgical procedures were performed using aseptic technique. General anesthesia was administered with an intramuscular injection of a combination of ketamine (70 mg/kg) and xylazine (14 mg/kg). Animal model. Each rat underwent placement of a polyethylene jugular venous catheter that was connected to a subcutaneously placed osmotic pump designed to deliver its contents at a rate of 1 l/h (Alzet osmotic minipump, Model 1003D; Alza Scientific Products, Palo Alto, CA). Rats were divided into two groups as follows: control animals (n ⫽ 7) received intravenous saline and treatment animals (n ⫽ 7) received intravenous recombinant human IL-11 (750 g/kg/day) (Genetics Institute, Cambridge, MA) dissolved in saline. After 48 h, each animal underwent a midline laparotomy and superior mesenteric artery occlusion for 35 min. After 120 min of reperfusion, serum and intestine samples were taken. Following surgery, rats were euthanized. Serum lysosomal enzyme assays. Serum was assayed for levels of -glucuronidase (GLUC) and hexosaminidase A (HEX A). GLUC and HEX A activities were quantified by fluorometric assays using 4MU-D-glucuronide and 4MU--D-NAc-glucosamine sulfate, respectively, as substrates, according to the protocol of the Thomas Jefferson University Hospital Lysosomal Diseases Testing Laboratory (Philadelphia, PA). Ten microliters of rat serum was incubated at 37°C with substrate in a final volume of 200 l for 10 min, at which point the reaction was stopped by a glycine carbonate solution (pH 10). Fluorometric readings were compared to known standards and expressed as nmol/ml/h. Multiplex semiquantitative RT-PCR to detect mucosal expression of Bcl-2. Midjejunal mucosal samples (20 cm from the ligament of Treiz) were harvested at operation and immediately stored at ⫺70°C for later RT-PCR analysis. Total RNA was extracted from mucosa using the RNEasy Kit (Qiagen, Valencia, CA) and DNase treated. RNA concentration and purity were determined by absorbency at 260 and 280 nm. One microgram of total RNA was reverse transcribed. The resultant cDNA was amplified using oligonucleotide primers for a 298-bp product in Bcl-2 (5⬘-ATGACTTCTCTCGTCGCTACCG, 3⬘-CGACCCTACGGAAACACCTTCA) with glyceraldehyde3-phosphate dehydrogenase (GAPDH) used as an internal control. The temperature profile of the polymerase chain reactions consisted of a melting step at 94°C followed by 30 cycles of 30 s at 60°C, 60 s at 65°C, 30 s at 94°C, with a final extension step of 5 min at 75°C. Independent experiments established that 30 cycles were within the linear range of the multiplex PCR assay. PCR products were separated on a 4% agarose gel and stained with ethidium bromide. Images were analyzed using EagleEye II analysis software and relative band intensities were calculated. Histologic analysis. Cross sections of jejunum 15 cm distal to duodenum were immediately fixed in formalin solution. Cut samples were then embedded in paraffin. Several sections per animal were stained with hematoxylin and eosin for light microscopic examination. Areas in each of four quadrants of the intestinal cross sections, in four separate samples per animal, were graded by a blinded, independent reviewer using the Park/Chiu scoring system of bowel
269
injury as described by Quaedackers [10]. According to this system, observations of increased damage were assigned increasing points as follows: normal mucosa (0); subepithelial space at villus tips (1); extension of subepithelial space with moderate lifting (2); massive lifting, some denuded tips (3); denuded villi, dilated capillaries (4); disintegration of lamina propria (5); crypt layer injury (6); transmucosal infarction (7); and transmural infarction (8). Each animal was assigned an average score. Immunohistochemistry (terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick-end labeling (TUNEL)) and DNA fragmentation analysis. Apoptotic crypt cells were identified using the TUNEL method using a standard laboratory detection kit (TdTFragEL; Oncogene Research Products, San Diego, CA). Samples were dewaxed, rehydrated in ethanol, and incubated with 3% hydrogen peroxide. Slides were treated with 100 l equilibration buffer for 30 min. Sections were then incubated for 1 h at 37°C with 60 l TdT enzyme, followed by incubation for 30 min with 100 l conjugate solution and detection with diaminobenzidine solution. Slides were counterstained with methyl green. TUNEL-positive cells were counted by examining crypts of approximately equal size (70 m or 30 cells around) in 10 regions/slide/animal. Results were expressed as TUNEL ⫹ cells/10 crypts. DNA fragmentation electrophoresis was performed as described by Eastman [11]. DNA was isolated from mucosal samples using the Wizard Genomic DNA purification kit (Promega, Madison, WI). Equal quantities (20 g) of DNA were loaded and run on 2% agarose gels. Gels were stained with ethidium bromide in water for 1 h, destained in distilled water overnight, and then analyzed qualitatively for fragment sizes and relative intensities. Statistical analysis. Statistical analysis was performed using ANOVA where appropriate and results were expressed as means ⫾ SEM.
RESULTS
Serum Lysosomal Enzyme Assays After 120 min of reperfusion, mean serum HEX A was significantly elevated in IR control animals (570 ⫾ 54 nmol/ml/h) compared with sham-operated animals (335 ⫾ 31 nmol/ml/h) (P ⬍ 0.01). In IL-11-pretreated animals, mean HEX A levels also significantly rose with IR injury to 425 ⫾ 47 nmol/ml/h (P ⬍ 0.05), but in these animals the HEX A values were significantly lower than in IR controls (P ⬍ 0.05) (Fig. 1). Mean serum GLUC in untreated animals was significantly higher in IR controls (183 ⫾ 29 nmol/ml/h) than
FIG. 1. Mean serum hexosaminidase A activity. IL-11-pretreated animals have significantly lower levels after 35 min of intestinal ischemia and 120 min of reperfusion. **P ⬍ 0.01, *P ⬍ 0.05.
270
JOURNAL OF SURGICAL RESEARCH: VOL. 108, NO. 2, DECEMBER 2002
FIG. 2. Mean serum -glucuronidase activity. IL-11-pretreated animals have significantly lower levels after 35 min of intestinal ischemia and 120 min of reperfusion. **P ⬍ 0.01.
in sham-operated animals (100 ⫾ 16 nmol/ml/h) after 120 min of reperfusion (P ⬍ 0.01). IL-11-pretreated animals had GLUC values (125 ⫾ 7 nmol/ml/h) which were significantly lower than those of IR controls at 120 min (P ⬍ 0.01) and, in fact, were not statistically different from those of sham-operated animals (Fig. 2). Histologic Analysis Both IR-injured animal groups revealed mild to moderate damage in intestinal villi after 35 min of ischemia and 120 min of reperfusion. Areas of structural villus injury were not consistent and were separated by regions of preserved villus architecture. Mean histologic mucosal injury scores assigned by a blinded pathologist were not significantly different between the untreated control group (2.00) and the IL-11pretreatment group (2.14). Immunohistochemistry (TUNEL) and DNA Fragmentation Analysis IL-11-pretreated animals had significantly fewer TUNEL-positive cells per 10 crypts (79 ⫾ 11) than
FIG. 4. Mean number of TUNEL ⫹ cells per 10 crypts. IL-11pretreated animals show a significant reduction at 120 min of reperfusion. **P ⬍ 0.01.
untreated control animals (255 ⫾ 17) (P ⬍ 0.01) subjected to IR injury (Figs. 3 and 4). Electrophoresis of DNA isolated from adjacent jejunal mucosa revealed an impressive increase in DNA laddering in the IRinjured group compared to sham-operated animals (Fig. 5, lanes 1 and 2). DNA from IL-11-pretreated rats showed relatively less DNA digestion (Fig. 5, lane 3), supporting our interpretations of in situ TUNEL staining. RT-PCR Average Bcl-2 relative band intensities were significantly increased in treated animals (1.04 ⫾ 0.09) compared with control animals (0.78 ⫾ 0.07) (P ⬍ 0.05) (Fig. 6). DISCUSSION
IL-11, a multifunctional bone marrow-derived cytokine, belongs to a family of human growth factors that utilizes the gp130 signal-transducing subunit [12]. In addition to the hematopoietic activities of IL-11, recent experiments have shown numerous effects of IL-11 on
FIG. 3. Micrograph of rat jejunal crypt, stained by TUNEL method with methyl green counterstain (original magnification 400⫻). (A) Untreated rat jejunal crypt after IR and (B) IL-11-pretreated rat jejunal crypt after IR.
KUENZLER, PEARSON, AND SCHWARTZ: IL-11 REDUCES CELL DEATH
the intestine. Studies have demonstrated that IL-11 enhances small intestine mucosal mass in animal models of short bowel syndrome [5, 13, 14]. IL-11 also appears to reduce apoptosis in murine models of bowel necrosis [7] and radiation injury [8]. We have previously shown that IL-11 administered at the time of IR injury can enhance the recovery of small intestinal absorptive function in rats [6]. In the current study we sought to examine the effects of pretreatment with IL-11 on preventing two modes of enterocyte death, necrosis and apoptosis. To evaluate the effects of IR-induced necrosis, we measured the release of certain lysosomal enzymes and assessed morphologic changes in the small intestine following injury. Abe et al. were the first investigators to show that GLUC was released into the circulation following intestinal IR [15]. Subsequently, Polson et al. demonstrated that HEX levels increased in the serum following IR injury [16]. Further studies by Schwartz and colleagues demonstrated HEX increases in neonatal clinical settings [17] and in an animal model of intestinal transplantation [18]. Serum levels of these enzymes rise consistently over the first 3 h following mesenteric artery occlusion, presumably as cell membrane integrity is lost in damaged enterocytes. Thus, we believe that levels of these enzymes are proportional to the loss of cells throughout the small bowel. Tissue samples were taken following 120 min of reperfusion because previous reports note histologic evidence of severe crypt cell apoptosis in the early reperfusion period [19]. In the current study, systemic infusion of IL-11 for 48 h prior to IR injury prevented the sharp increase in both of the enzymes. We believe this reflects a protective or stabilization effect of IL-11 on the mucosal cells of the small intestine. Apoptosis is the other significant mode of enterocyte cell death following IR injury. In healthy bowel, apo-
FIG. 5. Agarose gel electrophoresis of fragmented DNA from jejunal mucosa after IR. Lane A, sham; lane B, IR; lane C, IR ⫹ IL-11.
271
FIG. 6. Effect of IL-11 pretreatment on mucosal Bcl-2 mRNA expression after 35 min of intestinal ischemia and 120 min of reperfusion. Relative band intensities after RT-PCR are shown for Bcl-2 compared with the GAPDH internal standard. *P ⬍ 0.05.
ptosis allows continuous cell renewal as enterocytes are shed from villus tips. Apoptosis in normal jejunal crypts is quite rare (less than one cell per 10 crypts), but following IR it can be markedly increased in the first few hours of reperfusion [2]. Because apoptosis is a gene-mediated cellular process, it is possible that growth factors may have a role in affecting this phenomenon. We have recently described a reduction in IR-induced crypt cell apoptosis using hepatocyte growth factor [20]. Michalsky et al. [21] have observed inhibition of apoptosis using both flow cytometric studies and “cell death” ELISAs with another growth factor, HB-EGF, in intestinal epithelial cell cultures stimulated by inflammatory mediators. Although we were not able to demonstrate a benefit in the overall villus injury score, we did demonstrate a marked decrease in apoptotic crypt cells in animals receiving IL-11 pretreatment by the TUNEL technique. The reduction in apoptotic cells was also reflected in our observation that there was less DNA fragmentation in the mucosa overall. The specific mechanisms for the apparent antiapoptotic effects of IL-11 have not yet been elucidated. We hypothesize that IL-11 may be stimulating antiapoptotic regulators such as Bcl-2, which has been recently shown to render mice resistant to IR-induced intestinal apoptosis [22]. We have demonstrated a modest increase in the transcription of Bcl-2 in our current study. We realize that these methods are semiquantitative and reflect mucosal cellular transcription without precisely localizing the responsible cells. Nevertheless, this preliminary finding may help begin to explain this particular effect of IL-11 on injured intestine. Additionally, IL-11 might be attenuating the mucosal injury that precedes apoptosis by other mechanisms such as reducing or neutralizing toxic oxygen species like superoxide, hydrogen peroxide, or hydroxyl radicals. Based on this study and previous reports regarding IL-11, we postulate that in the future it may be appropriate to pretreat patients and potentially stabilize the
272
JOURNAL OF SURGICAL RESEARCH: VOL. 108, NO. 2, DECEMBER 2002
mucosa at risk for intestinal ischemia, e.g., prior to surgery for abdominal aortic aneurysms, intestinal transplantation, or infants at risk for necrotizing enterocolitis.
10.
Quaedackers, J. S. L. T., Beuk, R. J., Bennet, A., et al. An evaluation of methods for grading histologic injury following ischemia/reperfusion of the small bowel. Transplant. Proc. 32: 1307, 2000.
11.
Eastman, A. Assays for DNA fragmentation, endonucleases, and intracellular pH and Ca 2⫹ associated with apoptosis. In L. M. Schwartz and B. Osbourne (Eds.), Methods in Cell Biology. San Diego: Academic Press, 1995.
12.
Du, X. X., and Williams, D. A. Interleukin-11: Review of molecular, cell biology, and clinical use. Blood 89: 3897, 1997.
13.
Liu, Q., Du, X. X., Schindel, D. T., et al. Trophic effects of interleukin-11 in rats with experimental short bowel syndrome. J. Pediatr. Surg. 31: 1047, 1996.
14.
Fiore, N. F., Ledniczky, G., Liu, Q., et al. Comparison of interleukin-11 and epidermal growth factor on residual small intestine after massive small bowel resection. J. Pediatr. Surg. 33: 24, 1998.
15.
Abe, H., Carballo, J., Appert, H. E., et al. The release and fate of the intestinal lysosomal enzymes after acute ischemic injury of the intestine. Surg. Gynecol. Oncol. 135: 581, 1972.
16.
Polson, H., Mowat, C., and Himal, H. S. Experimental and clinical studies of mesenteric infarction. Surg. Gynecol. Oncol. 153: 360, 1981.
17.
Lobe, T. E., Schwartz, M. Z., Richardson, C. J., et al. Hexosaminidase: A marker for intestinal gangrene in necrotizing enterocolitis. J. Pediatr. Surg. 18: 449, 1983.
18.
Maeda, K., Schwartz, M. Z., Bamberger, M. H., et al. A possible serum marker for rejection after small bowel transplantation. Am. J. Surg. 153: 68, 1987.
19.
Shah, K. A., Shurey, S., and Green, C. J. Apoptosis after intestinal ischemia–reperfusion injury. Transplantation 64: 1393, 1997.
20.
Kuenzler, K. A., Pearson, P. Y., and Schwartz, M. Z. Hepatoctye growth factor pretreatment reduces apoptosis and mucosal damage following intestinal ischemia–reperfusion. J. Pediatr. Surg. 37: 1093, 2002.
21.
Michalsky, M. P., Kuhn, A., Mehta, V., et al. Heparin-binding EGF-like growth factor decreases apoptosis in intestinal epithelial cells in vitro. J. Pediatr. Surg. 36: 1130, 2001.
22.
Coopersmith, C. M., O’Donnell, D., and Gordon, J. I. Bcl-2 inhibits ischemia–reperfusion-induced apoptosis in the intestinal epithelium of mice. Am. J. Physiol. 276: G677, 1999.
ACKNOWLEDGMENTS The authors thank Juan P. Palazzo, M.D., of the Department of Pathology, Thomas Jefferson University Hospital, for his teaching and help with histologic interpretation, and David A. Wenger, Ph.D., of the Thomas Jefferson University Hospital Lysosomal Diseases Testing Laboratory, for his assistance with the enzyme assays.
REFERENCES 1. 2.
3.
4. 5.
6.
7.
8.
9.
Schoenberg, M. H., and Beger, H. G. Reperfusion injury after intestinal ischemia. Crit. Care Med. 21: 1376, 1993. Shah, K. A., Shurey, S., and Green, C. J. Characterization of apoptosis in intestinal ischaemia–reperfusion injury—A light and electron microscopic study. Int. J. Exp. Pathol. 78: 355, 1997. Noda, T., Iwakiri, R., Fujimoto, K., et al. Programmed cell death induced by ischemia–reperfusion in rat intestinal mucosa. Am. J. Physiol. 274: G270, 1998. Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456, 1995. Alavi, K., Prasad, R., Lundgren, K., et al. Interleukin-11 enhances small intestinal absorptive function and mucosal mass after intestinal adaptation. J. Pediatr. Surg. 35: 371, 2000. Kuenzler, K. A., Pearson, P. Y., and Schwartz, M. Z. Interleukin-11 enhances intestinal absorptive function after ischemia–reperfusion injury. J. Pediatr. Surg. 37: 457, 2002. Du, X. X., Liu, Q., Zhixiang, Y., et al. Protective effects of interleukin-11 in a murine model of ischemic bowel necrosis. Am. J. Physiol. 272: G545, 1997. Orazi, A., Du, X. X., Yang, Z., et al. Interleukin-11 prevents apoptosis and accelerates recovery of small intestinal mucosa in mice treated with combined chemotherapy and radiation. Lab. Invest. 75: 33, 1996. Scordi, I. A., Nassiri, M., Hanly, A. J., et al. Interleukin-11 reduces apoptosis in UVB-irradiated skin. Dermatology 199: 296, 1999.
IL-11 Pretreatment Reduces Cell Death after Intestinal Ischemia–Reperfusion Keith A. Kuenzler, M.D.,* Philip Y. Pearson, M.D.,* and Marshall Z. Schwartz, M.D.* ,† *Department of Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; and †Department of Surgery, A. I. duPont Hospital for Children, Wilmington, Delaware 19899 Submitted for publication May 28, 2002
Background. Intestinal ischemia–reperfusion (IR) injury results in enterocyte necrosis and apoptosis. This study was designed to evaluate the potential protective effects of interleukin-11 (IL-11) pretreatment on intestinal mucosa following IR injury. Materials and methods. Sham (n ⴝ 7) and control animals (n ⴝ 7) received 48 h of intravenous saline while treatment animals (n ⴝ 7) received IL-11 (750 g/kg/day). Sham animals then underwent laparotomy alone, while control and treatment animals underwent 35 min of mesenteric artery occlusion and 120 min of reperfusion. Midjejunum samples were obtained and serum was drawn. Fluorometric assays were performed for hexosaminidase A (HEX A) and -glucuronidase (GLUC), markers of enterocyte necrosis. Apoptosis was quantified by TUNEL and confirmed by DNA fragmentation. Transcription of Bcl-2, an antiapoptotic regulator, was assessed by multiplex RTPCR. Statistical analysis was performed using ANOVA and expressed as means ⴞ SEM. Results. In pretreated animals, HEX A and GLUC activities after IR were reduced from 570 ⴞ 54 to 426 ⴞ 47 nmol/ml/h (P < 0.05) and from 183 ⴞ 29 to 125 ⴞ 7 nmol/ml/h (P < 0.01), respectively. Pretreated animals had a reduced number of apoptotic cells per 10 crypts (79 ⴞ 11) compared with untreated rats (255 ⴞ 17) after IR injury (P < 0.01). Mucosal DNA from pretreated rats qualitatively showed less fragmentation on electrophoresis. Relative Bcl-2 band intensity was higher in pretreated animals (1.04 ⴞ 0.09) compared with controls (0.78 ⴞ 0.07) (P < 0.05). Conclusions. IL-11 pretreatment reduced crypt cell apoptosis after IR injury, possibly by upregulating Bcl-2. Treated animals also demonstrated attenuation in the release of certain lysosomal enzymes. These Presented at the Annual Meeting of the Association for Academic Surgery, Milwaukee, Wisconsin, November 15–17, 2001
0022-4804/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
data indicate that following IR injury, IL-11 improves enterocyte survival by reducing necrosis and apoptosis. © 2002 Elsevier Science (USA) Key Words: ischemia–reperfusion injury; interleukin-11; intestinal ischemia; lysosomal enzymes; programmed cell death; apoptosis; BCL-2.
Ischemia–reperfusion (IR) injury to the small intestine occurs in both the pediatric and the adult age groups. Midgut volvulus, necrotizing enterocolitis, shock states, and mesenteric emboli are only some of the entities that can produce IR injury. The mechanisms by which the intestine is damaged after IR are not well defined. Contributing factors include toxic oxygen species and the local recruitment of inflammatory leukocytes [1]. In the past, necrosis had been presumed to be the chief cause of epithelial cell death following an ischemic insult. More recent studies have indicated, however, that apoptosis is a significant and perhaps the principal contributor to cell death after IR injury [2– 4]. In contrast to cell necrosis, apoptosis is a coordinated, gene-directed process. Thus, understanding the sequence of events central to the apoptotic mechanism may potentially lead to treatments for diseases that are characterized by accelerated rates of programmed cell death [4]. Interleukin-11 (IL-11) is a multifunctional cytokine which our laboratory has shown to enhance absorptive function in animal models of small bowel adaptation [5] and intestinal IR injury [6]. Du et al. [7] previously reported that IL-11 pretreatment reduced morbidity and mortality in mice subjected to small bowel necrosis. They described both an increase in mitotic activity and a decline in enterocyte apoptosis. Additional studies have supported the ability of IL-11 to reduce apoptosis in radiation injury to the small intestine [8] and skin [9]. The goal of the current study was to evaluate
268
KUENZLER, PEARSON, AND SCHWARTZ: IL-11 REDUCES CELL DEATH
the effects of pretreatment with IL-11 in reducing mucosal cell death by necrosis and apoptosis after moderate IR injury. MATERIALS AND METHODS Animal care. All animal care and use complied with the institutional regulations set forth and approved by the Animal Care and Use Committee of the A. I. duPont Hospital for Children and the Nemours Research Programs (Wilmington, DE). Fourteen adult male Sprague–Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 275 to 300 g were used in this study. Rats were kept in individual cages and fed standard rat chow and water ad libitum. After the animals were allowed to acclimate to the environment for a period of 1 week, surgical procedures were performed using aseptic technique. General anesthesia was administered with an intramuscular injection of a combination of ketamine (70 mg/kg) and xylazine (14 mg/kg). Animal model. Each rat underwent placement of a polyethylene jugular venous catheter that was connected to a subcutaneously placed osmotic pump designed to deliver its contents at a rate of 1 l/h (Alzet osmotic minipump, Model 1003D; Alza Scientific Products, Palo Alto, CA). Rats were divided into two groups as follows: control animals (n ⫽ 7) received intravenous saline and treatment animals (n ⫽ 7) received intravenous recombinant human IL-11 (750 g/kg/day) (Genetics Institute, Cambridge, MA) dissolved in saline. After 48 h, each animal underwent a midline laparotomy and superior mesenteric artery occlusion for 35 min. After 120 min of reperfusion, serum and intestine samples were taken. Following surgery, rats were euthanized. Serum lysosomal enzyme assays. Serum was assayed for levels of -glucuronidase (GLUC) and hexosaminidase A (HEX A). GLUC and HEX A activities were quantified by fluorometric assays using 4MU-D-glucuronide and 4MU--D-NAc-glucosamine sulfate, respectively, as substrates, according to the protocol of the Thomas Jefferson University Hospital Lysosomal Diseases Testing Laboratory (Philadelphia, PA). Ten microliters of rat serum was incubated at 37°C with substrate in a final volume of 200 l for 10 min, at which point the reaction was stopped by a glycine carbonate solution (pH 10). Fluorometric readings were compared to known standards and expressed as nmol/ml/h. Multiplex semiquantitative RT-PCR to detect mucosal expression of Bcl-2. Midjejunal mucosal samples (20 cm from the ligament of Treiz) were harvested at operation and immediately stored at ⫺70°C for later RT-PCR analysis. Total RNA was extracted from mucosa using the RNEasy Kit (Qiagen, Valencia, CA) and DNase treated. RNA concentration and purity were determined by absorbency at 260 and 280 nm. One microgram of total RNA was reverse transcribed. The resultant cDNA was amplified using oligonucleotide primers for a 298-bp product in Bcl-2 (5⬘-ATGACTTCTCTCGTCGCTACCG, 3⬘-CGACCCTACGGAAACACCTTCA) with glyceraldehyde3-phosphate dehydrogenase (GAPDH) used as an internal control. The temperature profile of the polymerase chain reactions consisted of a melting step at 94°C followed by 30 cycles of 30 s at 60°C, 60 s at 65°C, 30 s at 94°C, with a final extension step of 5 min at 75°C. Independent experiments established that 30 cycles were within the linear range of the multiplex PCR assay. PCR products were separated on a 4% agarose gel and stained with ethidium bromide. Images were analyzed using EagleEye II analysis software and relative band intensities were calculated. Histologic analysis. Cross sections of jejunum 15 cm distal to duodenum were immediately fixed in formalin solution. Cut samples were then embedded in paraffin. Several sections per animal were stained with hematoxylin and eosin for light microscopic examination. Areas in each of four quadrants of the intestinal cross sections, in four separate samples per animal, were graded by a blinded, independent reviewer using the Park/Chiu scoring system of bowel
269
injury as described by Quaedackers [10]. According to this system, observations of increased damage were assigned increasing points as follows: normal mucosa (0); subepithelial space at villus tips (1); extension of subepithelial space with moderate lifting (2); massive lifting, some denuded tips (3); denuded villi, dilated capillaries (4); disintegration of lamina propria (5); crypt layer injury (6); transmucosal infarction (7); and transmural infarction (8). Each animal was assigned an average score. Immunohistochemistry (terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick-end labeling (TUNEL)) and DNA fragmentation analysis. Apoptotic crypt cells were identified using the TUNEL method using a standard laboratory detection kit (TdTFragEL; Oncogene Research Products, San Diego, CA). Samples were dewaxed, rehydrated in ethanol, and incubated with 3% hydrogen peroxide. Slides were treated with 100 l equilibration buffer for 30 min. Sections were then incubated for 1 h at 37°C with 60 l TdT enzyme, followed by incubation for 30 min with 100 l conjugate solution and detection with diaminobenzidine solution. Slides were counterstained with methyl green. TUNEL-positive cells were counted by examining crypts of approximately equal size (70 m or 30 cells around) in 10 regions/slide/animal. Results were expressed as TUNEL ⫹ cells/10 crypts. DNA fragmentation electrophoresis was performed as described by Eastman [11]. DNA was isolated from mucosal samples using the Wizard Genomic DNA purification kit (Promega, Madison, WI). Equal quantities (20 g) of DNA were loaded and run on 2% agarose gels. Gels were stained with ethidium bromide in water for 1 h, destained in distilled water overnight, and then analyzed qualitatively for fragment sizes and relative intensities. Statistical analysis. Statistical analysis was performed using ANOVA where appropriate and results were expressed as means ⫾ SEM.
RESULTS
Serum Lysosomal Enzyme Assays After 120 min of reperfusion, mean serum HEX A was significantly elevated in IR control animals (570 ⫾ 54 nmol/ml/h) compared with sham-operated animals (335 ⫾ 31 nmol/ml/h) (P ⬍ 0.01). In IL-11-pretreated animals, mean HEX A levels also significantly rose with IR injury to 425 ⫾ 47 nmol/ml/h (P ⬍ 0.05), but in these animals the HEX A values were significantly lower than in IR controls (P ⬍ 0.05) (Fig. 1). Mean serum GLUC in untreated animals was significantly higher in IR controls (183 ⫾ 29 nmol/ml/h) than
FIG. 1. Mean serum hexosaminidase A activity. IL-11-pretreated animals have significantly lower levels after 35 min of intestinal ischemia and 120 min of reperfusion. **P ⬍ 0.01, *P ⬍ 0.05.
270
JOURNAL OF SURGICAL RESEARCH: VOL. 108, NO. 2, DECEMBER 2002
FIG. 2. Mean serum -glucuronidase activity. IL-11-pretreated animals have significantly lower levels after 35 min of intestinal ischemia and 120 min of reperfusion. **P ⬍ 0.01.
in sham-operated animals (100 ⫾ 16 nmol/ml/h) after 120 min of reperfusion (P ⬍ 0.01). IL-11-pretreated animals had GLUC values (125 ⫾ 7 nmol/ml/h) which were significantly lower than those of IR controls at 120 min (P ⬍ 0.01) and, in fact, were not statistically different from those of sham-operated animals (Fig. 2). Histologic Analysis Both IR-injured animal groups revealed mild to moderate damage in intestinal villi after 35 min of ischemia and 120 min of reperfusion. Areas of structural villus injury were not consistent and were separated by regions of preserved villus architecture. Mean histologic mucosal injury scores assigned by a blinded pathologist were not significantly different between the untreated control group (2.00) and the IL-11pretreatment group (2.14). Immunohistochemistry (TUNEL) and DNA Fragmentation Analysis IL-11-pretreated animals had significantly fewer TUNEL-positive cells per 10 crypts (79 ⫾ 11) than
FIG. 4. Mean number of TUNEL ⫹ cells per 10 crypts. IL-11pretreated animals show a significant reduction at 120 min of reperfusion. **P ⬍ 0.01.
untreated control animals (255 ⫾ 17) (P ⬍ 0.01) subjected to IR injury (Figs. 3 and 4). Electrophoresis of DNA isolated from adjacent jejunal mucosa revealed an impressive increase in DNA laddering in the IRinjured group compared to sham-operated animals (Fig. 5, lanes 1 and 2). DNA from IL-11-pretreated rats showed relatively less DNA digestion (Fig. 5, lane 3), supporting our interpretations of in situ TUNEL staining. RT-PCR Average Bcl-2 relative band intensities were significantly increased in treated animals (1.04 ⫾ 0.09) compared with control animals (0.78 ⫾ 0.07) (P ⬍ 0.05) (Fig. 6). DISCUSSION
IL-11, a multifunctional bone marrow-derived cytokine, belongs to a family of human growth factors that utilizes the gp130 signal-transducing subunit [12]. In addition to the hematopoietic activities of IL-11, recent experiments have shown numerous effects of IL-11 on
FIG. 3. Micrograph of rat jejunal crypt, stained by TUNEL method with methyl green counterstain (original magnification 400⫻). (A) Untreated rat jejunal crypt after IR and (B) IL-11-pretreated rat jejunal crypt after IR.
KUENZLER, PEARSON, AND SCHWARTZ: IL-11 REDUCES CELL DEATH
the intestine. Studies have demonstrated that IL-11 enhances small intestine mucosal mass in animal models of short bowel syndrome [5, 13, 14]. IL-11 also appears to reduce apoptosis in murine models of bowel necrosis [7] and radiation injury [8]. We have previously shown that IL-11 administered at the time of IR injury can enhance the recovery of small intestinal absorptive function in rats [6]. In the current study we sought to examine the effects of pretreatment with IL-11 on preventing two modes of enterocyte death, necrosis and apoptosis. To evaluate the effects of IR-induced necrosis, we measured the release of certain lysosomal enzymes and assessed morphologic changes in the small intestine following injury. Abe et al. were the first investigators to show that GLUC was released into the circulation following intestinal IR [15]. Subsequently, Polson et al. demonstrated that HEX levels increased in the serum following IR injury [16]. Further studies by Schwartz and colleagues demonstrated HEX increases in neonatal clinical settings [17] and in an animal model of intestinal transplantation [18]. Serum levels of these enzymes rise consistently over the first 3 h following mesenteric artery occlusion, presumably as cell membrane integrity is lost in damaged enterocytes. Thus, we believe that levels of these enzymes are proportional to the loss of cells throughout the small bowel. Tissue samples were taken following 120 min of reperfusion because previous reports note histologic evidence of severe crypt cell apoptosis in the early reperfusion period [19]. In the current study, systemic infusion of IL-11 for 48 h prior to IR injury prevented the sharp increase in both of the enzymes. We believe this reflects a protective or stabilization effect of IL-11 on the mucosal cells of the small intestine. Apoptosis is the other significant mode of enterocyte cell death following IR injury. In healthy bowel, apo-
FIG. 5. Agarose gel electrophoresis of fragmented DNA from jejunal mucosa after IR. Lane A, sham; lane B, IR; lane C, IR ⫹ IL-11.
271
FIG. 6. Effect of IL-11 pretreatment on mucosal Bcl-2 mRNA expression after 35 min of intestinal ischemia and 120 min of reperfusion. Relative band intensities after RT-PCR are shown for Bcl-2 compared with the GAPDH internal standard. *P ⬍ 0.05.
ptosis allows continuous cell renewal as enterocytes are shed from villus tips. Apoptosis in normal jejunal crypts is quite rare (less than one cell per 10 crypts), but following IR it can be markedly increased in the first few hours of reperfusion [2]. Because apoptosis is a gene-mediated cellular process, it is possible that growth factors may have a role in affecting this phenomenon. We have recently described a reduction in IR-induced crypt cell apoptosis using hepatocyte growth factor [20]. Michalsky et al. [21] have observed inhibition of apoptosis using both flow cytometric studies and “cell death” ELISAs with another growth factor, HB-EGF, in intestinal epithelial cell cultures stimulated by inflammatory mediators. Although we were not able to demonstrate a benefit in the overall villus injury score, we did demonstrate a marked decrease in apoptotic crypt cells in animals receiving IL-11 pretreatment by the TUNEL technique. The reduction in apoptotic cells was also reflected in our observation that there was less DNA fragmentation in the mucosa overall. The specific mechanisms for the apparent antiapoptotic effects of IL-11 have not yet been elucidated. We hypothesize that IL-11 may be stimulating antiapoptotic regulators such as Bcl-2, which has been recently shown to render mice resistant to IR-induced intestinal apoptosis [22]. We have demonstrated a modest increase in the transcription of Bcl-2 in our current study. We realize that these methods are semiquantitative and reflect mucosal cellular transcription without precisely localizing the responsible cells. Nevertheless, this preliminary finding may help begin to explain this particular effect of IL-11 on injured intestine. Additionally, IL-11 might be attenuating the mucosal injury that precedes apoptosis by other mechanisms such as reducing or neutralizing toxic oxygen species like superoxide, hydrogen peroxide, or hydroxyl radicals. Based on this study and previous reports regarding IL-11, we postulate that in the future it may be appropriate to pretreat patients and potentially stabilize the
272
JOURNAL OF SURGICAL RESEARCH: VOL. 108, NO. 2, DECEMBER 2002
mucosa at risk for intestinal ischemia, e.g., prior to surgery for abdominal aortic aneurysms, intestinal transplantation, or infants at risk for necrotizing enterocolitis.
10.
Quaedackers, J. S. L. T., Beuk, R. J., Bennet, A., et al. An evaluation of methods for grading histologic injury following ischemia/reperfusion of the small bowel. Transplant. Proc. 32: 1307, 2000.
11.
Eastman, A. Assays for DNA fragmentation, endonucleases, and intracellular pH and Ca 2⫹ associated with apoptosis. In L. M. Schwartz and B. Osbourne (Eds.), Methods in Cell Biology. San Diego: Academic Press, 1995.
12.
Du, X. X., and Williams, D. A. Interleukin-11: Review of molecular, cell biology, and clinical use. Blood 89: 3897, 1997.
13.
Liu, Q., Du, X. X., Schindel, D. T., et al. Trophic effects of interleukin-11 in rats with experimental short bowel syndrome. J. Pediatr. Surg. 31: 1047, 1996.
14.
Fiore, N. F., Ledniczky, G., Liu, Q., et al. Comparison of interleukin-11 and epidermal growth factor on residual small intestine after massive small bowel resection. J. Pediatr. Surg. 33: 24, 1998.
15.
Abe, H., Carballo, J., Appert, H. E., et al. The release and fate of the intestinal lysosomal enzymes after acute ischemic injury of the intestine. Surg. Gynecol. Oncol. 135: 581, 1972.
16.
Polson, H., Mowat, C., and Himal, H. S. Experimental and clinical studies of mesenteric infarction. Surg. Gynecol. Oncol. 153: 360, 1981.
17.
Lobe, T. E., Schwartz, M. Z., Richardson, C. J., et al. Hexosaminidase: A marker for intestinal gangrene in necrotizing enterocolitis. J. Pediatr. Surg. 18: 449, 1983.
18.
Maeda, K., Schwartz, M. Z., Bamberger, M. H., et al. A possible serum marker for rejection after small bowel transplantation. Am. J. Surg. 153: 68, 1987.
19.
Shah, K. A., Shurey, S., and Green, C. J. Apoptosis after intestinal ischemia–reperfusion injury. Transplantation 64: 1393, 1997.
20.
Kuenzler, K. A., Pearson, P. Y., and Schwartz, M. Z. Hepatoctye growth factor pretreatment reduces apoptosis and mucosal damage following intestinal ischemia–reperfusion. J. Pediatr. Surg. 37: 1093, 2002.
21.
Michalsky, M. P., Kuhn, A., Mehta, V., et al. Heparin-binding EGF-like growth factor decreases apoptosis in intestinal epithelial cells in vitro. J. Pediatr. Surg. 36: 1130, 2001.
22.
Coopersmith, C. M., O’Donnell, D., and Gordon, J. I. Bcl-2 inhibits ischemia–reperfusion-induced apoptosis in the intestinal epithelium of mice. Am. J. Physiol. 276: G677, 1999.
ACKNOWLEDGMENTS The authors thank Juan P. Palazzo, M.D., of the Department of Pathology, Thomas Jefferson University Hospital, for his teaching and help with histologic interpretation, and David A. Wenger, Ph.D., of the Thomas Jefferson University Hospital Lysosomal Diseases Testing Laboratory, for his assistance with the enzyme assays.
REFERENCES 1. 2.
3.
4. 5.
6.
7.
8.
9.
Schoenberg, M. H., and Beger, H. G. Reperfusion injury after intestinal ischemia. Crit. Care Med. 21: 1376, 1993. Shah, K. A., Shurey, S., and Green, C. J. Characterization of apoptosis in intestinal ischaemia–reperfusion injury—A light and electron microscopic study. Int. J. Exp. Pathol. 78: 355, 1997. Noda, T., Iwakiri, R., Fujimoto, K., et al. Programmed cell death induced by ischemia–reperfusion in rat intestinal mucosa. Am. J. Physiol. 274: G270, 1998. Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456, 1995. Alavi, K., Prasad, R., Lundgren, K., et al. Interleukin-11 enhances small intestinal absorptive function and mucosal mass after intestinal adaptation. J. Pediatr. Surg. 35: 371, 2000. Kuenzler, K. A., Pearson, P. Y., and Schwartz, M. Z. Interleukin-11 enhances intestinal absorptive function after ischemia–reperfusion injury. J. Pediatr. Surg. 37: 457, 2002. Du, X. X., Liu, Q., Zhixiang, Y., et al. Protective effects of interleukin-11 in a murine model of ischemic bowel necrosis. Am. J. Physiol. 272: G545, 1997. Orazi, A., Du, X. X., Yang, Z., et al. Interleukin-11 prevents apoptosis and accelerates recovery of small intestinal mucosa in mice treated with combined chemotherapy and radiation. Lab. Invest. 75: 33, 1996. Scordi, I. A., Nassiri, M., Hanly, A. J., et al. Interleukin-11 reduces apoptosis in UVB-irradiated skin. Dermatology 199: 296, 1999.