Gut Mucosal Homeostasis and Cellular Mediators after Severe ...

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Endocrinology 148(1):354 –362 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-0883

Gut Mucosal Homeostasis and Cellular Mediators after Severe Thermal Trauma and the Effect of Insulin-Like Growth Factor-I in Combination with Insulin-Like Growth Factor Binding Protein-3 Marc G. Jeschke, Ullrich Bolder, Dai H. Chung, Rene Przkora, Ulla Mueller, James C. Thompson, Steven E. Wolf, and David N. Herndon Shriners Hospitals for Children (M.G.J., R.P., J.C.T., D.N.H.) and Department of Surgery (M.G.J., D.H.C., D.N.H.), The University Texas Medical Branch, Galveston, Texas 77550; Department of Surgery (U.B., U.M.), University of Regensburg, 93053 Regensburg, Germany; and Department of Surgery (S.E.W.), University of Texas Health Science Center, San Antonio, Texas 78229 Increased small bowel epithelial cell apoptosis and decreased cell proliferation lead to impairment of gut mucosal integrity and function after thermal injury. Impairment of gut integrity is associated with increased bacterial translocation and incidence of sepsis. The purpose of this study was to determine whether IGF-I/IGF binding protein (IGFBP)-3 can improve small bowel homeostasis after injury and by which cellular mechanisms these changes occur and to identify changes in apoptosis-related genes after burn and the effect of bile acid on small bowel epithelial cell apoptosis after burn. Rats sustained a thermal injury and received saline or the IGF-I/ IGFBP-3 complex. Serum and small intestine were taken at 1, 2, 5, and 7 d after injury and serum inflammatory cytokines and mucosal apoptosis, proliferation, villous morphology, and apoptotic and proliferative mediators were measured. Apoptosis-related gene expression and the bile acid pool were

determined in separate experiments up to 6 h after burn. Gut epithelial cell apoptosis as well as apoptosis-related genes were increased after the thermal injury, whereas bile acid secretion was significantly decreased (P < 0.05). IGF-I/ IGFBP-3 significantly improved villous height and cells per villous by decreasing small bowel epithelial cell apoptosis and increasing proliferation (P < 0.05). Decreased apoptosis was associated with decreased Fas, Fas-ligand, and TNF when compared with saline (P < 0.05). A severe thermal injury caused an up-regulation of apoptosis and apoptosis-related genes and down-regulation of bile acid secretion. IGF-I/ IGFBP-3 decreases small bowel epithelial cell apoptosis through down-regulation of the Fas pathway, which improves gut mucosal integrity after a severe thermal injury. (Endocrinology 148: 354 –362, 2007)

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year, with an estimated 330,000 deaths related to the thermal injury (3). Numerous deaths are associated with infectious complications or sepsis. Because increased bacterial translocation may be a source for the increased incidence of sepsis and infections, the gut may play a significant role during the postburn response (4). A thermal injury decreases gut mucosal weight, protein, and DNA content, which are indicative of decreased cellular mass and absorptive surface of the small bowel. Deceased cellular mass and surface is due to an increased rate of small bowel mucosal apoptosis with a relative decrease in small bowel cell proliferation (5, 6). Small bowel mucosal loss has been associated with decreased small bowel nutrient absorption, dysfunction in nutrient transportation, and increased gut permeability (2, 4, 7). Increased gut permeability can lead to increased bacterial translocation with an increased risk of infection or even sepsis and multiorgan failure (2, 4, 7–9). One approach to counter increased apoptosis and decreased proliferation with concomitant loss of mucosal protein and DNA content would be to either decrease the rate of programmed cell death or increase small bowel epithelial cell proliferation maintaining gut homeostasis and function. Growth factors are known to affect either apoptosis or proliferation (10, 11), thus leading us to the investigation of anabolic growth factors on small bowel epithelial cell homeostasis.

RESERVATION OF SMALL bowel mucosal integrity and homeostasis depends on a balance between cell proliferation and cell death (1). Cell proliferation in small bowel epithelial cells occurs by mitosis in the intestinal crypts (1). Cell death occurs in the small bowel by two distinctly different mechanisms, apoptosis and necrosis. Apoptosis, or programmed cell death, is a genetically determined energydependent process by which senescent or dysfunctional cells are removed without extrusion of the intracellular contents or inflammation (1). This is in direct contrast to necrosis, which is a passive process initiated by direct injury to the cell. Alterations in the balance between apoptosis and proliferation may lead to changes in small bowel function and integrity (2). A severe burn injury represents one of the most severe forms of trauma and occurs according to the World Health Organization in more than 2 million people worldwide per First Published Online September 28, 2006 Abbreviations: IGFBP, IGF binding protein; PCNA, proliferative cell nuclear antigen; rh, recombinant human; TBSA, total body surface area; TUNEL, terminal deoxyuridine nick end labeling. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

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Jeschke et al. • IGF-I Alters Gut Homeostasis

Endocrinology, January 2007, 148(1):354 –362

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IGF-I is a small polypeptide (⬃7.5 kDa) that has been shown to be mitogenic (12) and improve gut mucosal function after a thermal injury (13). A recent study in transgenic mice that overexpress IGF-I showed that IGF-I stimulated crypt cell mitosis and increased growth of the small bowel (12). In addition, IGF-I has been shown to exert antiapoptotic effects (14 –16). Despite the possible advantages of IGF-I, therapeutic use is restricted because of adverse side effects such as hypoglycemia, electrolyte imbalance, or even cardiac arrest (17, 18). The introduction of a new complex in which IGF-I is bound to its principal binding protein (IGF-I/ IGFBP-3) has been shown to be safe and efficacious in humans (19 –22). Therefore, we used this complex in the present study to administer physiologic doses of IGF-I that are safe and efficacious. The purpose of our studies was severalfold: 1) to determine apoptosis-related genes after burn and confirm previous studies; 2) to determine the role of bile acid production and composition during the postburn stress response and its effect on small bowel epithelial cell apoptosis; and 3) to test the effect of IGF-I/IGFBP-3 on small bowel homeostasis and cellular mediators after a severe thermal injury and whether IGF-I/IGFBP-3 would be an agent that could be used in the clinical setting to positively affect gut homeostasis.

were treated humanely and given pain medication, special nutrition, and fluid substitution according to human burn treatment.

Materials and Methods

All animals were pair fed according to the following protocol: rats were fed with a liquid diet, rich in vitamins, proteins, and carbohydrates (Sustacal; Mead Johnson Nutritionals, Evansville, IN) with a caloric distribution of 24% protein, 21% fat, and 55% carbohydrate, resulting in an energy intake of 1.01 cal/ml. Both groups of rats were pair fed according to the caloric intake. The feeding protocol was as follows: 25 calories on the day of burn (25 cc of food), 51 calories on the first postburn day (50 cc of food), 76 calories on the second (75 cc of food), and 101 calories from the third day after burn on. The nutritional intake was the same in all groups.

Fifty-six rats received a 60% TBSA as described above (23) and were than randomly divided into two groups to receive either saline (0.4 cc iv every 12 h, n ⫽ 28) or recombinant human (rh) IGF-I/IGFBP-3 (10 mg/kg in 0.4 cc iv, every 24 h, n ⫽ 28). To establish normal values, eight additional rats were anesthetized and killed 1, 2, 5, and 7 d after anesthesia. Killing of the animals and tissue collection followed the same protocol as described below. IGF-I/IGFBP-3 is a recombinant human complex in which IGF-I is bound to IGF-I/IGFBP-3 in a 1:1 molar ratio. The rhIGF-I/IGFBP-3 complex was provided by Celtrix Pharmaceuticals, Inc. (Santa Clara, CA) in a 1:1 molar ratio of rhIGF-I to rhIGF-I/GFBP-3. This corresponds to the naturally occurring protein complex purified by cation exchange column chromatography. Infusions were prepared from vials containing 10 mg/ml rhIGF-I/IGFBP-3 in sterile 50 mm sodium acetate and 105 mm sodium chloride buffered to pH 5.5. The complex was used in clinical studies (19 –22) and was shown to be safe and efficacious. Injection (⬃0.4 cc) was performed by slow-tail vein injection. The dose of 10 mg/kg was determined by a dose-response study in rats. Treatment with IGF-I/IGFBP-3 or saline began 30 min after burn. Rats were killed at 1, 2, 5, or 7 d after burn (n ⫽ 7 in each group at each time point), approximately 20 h after the last IGF-I/IGFBP-3 injection, and serum, small bowel, and kidney (as a positive control for apoptosis) were taken for analysis. The entire small bowel was removed intact and divided into proximal and distal halves. Preliminary experiments indicated that the proximal small bowel had a greater apoptotic response; therefore, the present study focused on changes in the proximal half of the small bowel. The proximal half of the small bowel was flushed with ice-cold saline to remove enteric debris. Two 1-cm sections of the proximal end were excised and immediately fixed overnight in 10% buffered formalin and then transferred to 80% ethanol until embedded in paraffin within 48 h of the time the animals were killed. These segments were used for the immunohistochemical measurements.

Ethics

Measurements

These studies were reviewed and approved by the Animal Care and Use Committee of the University Texas Medical Branch (Galveston, TX), assuring that all animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The animals were visited twice daily by the investigators and daily by the Animal Care and Use Committee to ensure that animals were not suffering or in pain. Animals

Apoptosis. We used the terminal deoxyuridine nick end labeling (TUNEL) immunohistochemical method (Apoptag; Oncogene, Baltimore, MD) to allow histologic identification of apoptotic cells in the small bowel mucosa. We followed the same protocol as previously described (6, 25, 26). Briefly, six sections of each block were obtained at 40- to 50-␮m intervals. Within each section, a blinded observer selected 10 full-length villi for counting TUNEL-positive cells. Three blinded

All three studies presented followed the same Institutional Animal Care and Use Committee, nutrition, anesthesia and analgesia, and burn protocol. Adult male Sprague Dawley rats weighing 300 –350 g (HarlanSprague Dawley, Houston, TX) were housed in wire bottom cages in a temperature-controlled room with a 12-h light, 12-h dark cycle. All animals were acclimated to their environment for 7 d. Rats received water ad libitum for the entire study period.

Burn model The burn model is very well established, inducing a 60% total body surface area (TBSA) burn, which was first described by Walker-Mason and modified by Herndon et al. (23). All animals received analgesia (Buprenex 0.1 mg/kg, im) and general anesthesia (pentobarbital 25 mg/kg, ip) before the burn. After receiving the thermal injury, rats were immediately resuscitated with ip Ringer’s lactate (60 ml/kg, ip). Analgesia was administered every 12 h or when animal was in pain.

Nutrition

Study 1: apoptosis-related genes after burn Eighteen rats were divided into the sham group (n ⫽ 9) or burn group (n ⫽ 9). The burn group received a 60% TBSA burn as described above (23). Rats were killed at 1, 3, and 6 h after burn with the small bowel removed. Samples from the proximal small bowel were used for analysis. Expression for apoptosis-related genes p21Waf-1/Cip-1, growth arrest and DNA damage inducible gene-45 (GADD-45), and GADD-153 were determined by Northern blot technique.

Measurements Apoptosis-related gene expression. Samples of whole proximal small bowel (25–30 mg) were homogenized in 1 ml ULTRASPEC RNA isolation buffer from BIOTECX (Houston, TX), using an Omni EZ Connect homogenizer 5-mm blade (Gainesville, VA). Samples were purified following ULTRASPEC RNA isolation buffer protocol (24). Samples were run on an RNA formaldehyde gel for separation and transferred onto Nytran Plus nylon transfer and immobilization membrane (Schleicher & Schuell, Keene, NH). Membranes were left overnight in hybridization solution and then hybridized with P-32-labeled cDNA for p21. The membranes were then stripped, hybridized with GADD-45 cDNA, exposed, and analyzed as described above. The process was then repeated with GADD-153 cDNA.

Study 2: effect of IGF-I/IGFBP-3 on small bowel homeostasis

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observers to treatment counted cells. All epithelial cells within the villi were counted, and apoptosis was expressed as a percentage of apoptotic cells per 100 villous epithelial cells. Values for all sections were averaged to calculate an apoptosis score for the proximal gut of each animal as previously described (25, 26). To corroborate our TUNEL findings, we performed an ELISA (Boehringer Ingelheim, Ingelheim, Germany) using proximal small bowel. Frozen scraped mucosa from each animal was thawed in ice slurry while in collection vials. Each vial had 400 ␮l incubation buffer added and was thoroughly shaken. The samples were incubated at 4 C for 30 min to allow for complete lysis of the cells. Samples were then centrifuged at 15,000 rpm (20,000 ⫻ g) for 10 min and the supernatant removed. ELISA was performed according to the manufacturer’s guidelines. The ELISA estimation of apoptosis was determined as the ratio between absorbance at 290 and 450 nm. Proliferation. Small bowel mucosal proliferation was determined by immunohistochemical staining for proliferative cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Santa Cruz, CA; SC-56, 1:50 dilution overnight at 4 C) as previously published (6, 25, 26). PCNA-positive cells (stained red-brown) were counted on six sections with at least 10 crypts from each animal. The proliferation index was calculated by determining the number of PCNA-positive cells per 100 intestinal crypt cells. Villous height and cell count. To determine mucosal atrophy, three blinded observers determined mucosal/villous height by randomly selecting 10 complete villi from each section and measuring the distance from the villous tip to the villous base and the distance from the crypt tip to the base of the crypt. The values from each villous and crypt were averaged to obtain average villous and crypt height per animal. In addition, the same 10 villi from each section cell number from the tip of the villous to the base of the villous were counted. The values from each villous were averaged to obtain the average cell number per villous per animal.


Bcl-2 was determined by following the same protocol, with primary antibody anti-Bcl-2 (Santa Cruz; sc-492, 1:100) and secondary antibody (Santa Cruz; sc-2004, 1:200). Bcl-2 concentration was determined by grading positive reactions as described above. Caspase-3 followed the same protocol except pretreatment with trypsin for 35 min at room temperature. Instead of BSA we used goat serum. The primary antibody was anticaspase-3 (R&D Systems, Minneapolis, MN; AF 835, 1:150), and the secondary antibody (Santa Cruz; sc-2004, 1:200). Caspase-3 concentration was determined by counting the whole villus. Three blinded observers counted following the same standardized protocol. Fifteen subsequent villi were counted. Values given are the positive cells in percentage of the entire villous. Caspase-7 followed the same immunohistochemical protocol, except pretreatment with pepsin for 7 min. Primary antibody used was anticaspase-7 (Santa Cruz; sc-8512, 1:50) and the second antibody used (Santa Cruz; sc-2768, 1:200). Caspase-7 concentration was determined by counting positive cells per total cells of the villus. Three blinded observers counted following the same standardized protocol.

Study 3: bile acid pool and composition after burn Five rats received a 60% TBSA burn as described above (23). Five animals were given anesthesia and analgesia but were not burned; these rats served as sham control animals. All animals underwent laparotomy under general anesthesia and analgesia, and the bile duct was prepared and punctured. Bile was collected from anesthetized rats in preweighed tubes chilled on ice. Total bile acid secretion in biliary fistula rats was measured by the 3␣-hydroxysteroid dehydrogenase assay (28). Bile acid secretion is reported in three consecutive 10-min intervals while the animal was under general anesthesia and analgesia. The composition of the endogenous bile acid pool was investigated by HPLC (Beckmann

Serum TNF, IL-1, and IL-6. TNF levels were determined with a rat specific ELISA (Endogen, Woburn, MA). IL-1␤ levels were determined using ELISA (Biosource International, Camarillo, CA). Serum levels of IL-6 were determined by bioassay using B9 cells (mouse hybridoma line) in their log phase of growth and treated with increasing concentrations of serum. Cell proliferation in response to serum addition was measured spectrophotometrically as previously described (27). Serum IGF-I concentration. We determined human and endogenous rat IGF-I concentrations in the serum using a human IGF-I RIA (Nichols Institute Diagnostics, San Juan Capistrano, CA) or a rat IGF-I RIA (Diagnostic System Laboratories, Webster, TX). By measuring both human and rat IGF-I, total IGF-I levels could be determined. Fas, Fas-ligand, Bax, Bcl-2, and caspase-3 and -9 expressions. Fas concentration in the small bowel was measured by antibodies against Fas using standard immunohistochemical techniques. The primary antibody antiFas was diluted with BSA 1% to 1:100 (Santa Cruz Biotechnology), applied to the samples, and then incubated at 4 C overnight. After another washing the secondary antibody Dako E 0466 (Santa Cruz; 1:200) was incubated for 1 h at 37 C followed by another washing. The samples were then incubated with diaminobenzidine for 15 min at 37 C. The samples were thoroughly washed and hematoxylin was applied for the counterstaining. Fas concentration was determined by grading the samples from 0 to 2 (0 ⫽ no staining and 2 ⫽ maximal staining). Three observes blinded to treatment counted each sample at six different sites for Fas-positive cells. Fas-ligand concentration was determined following a similar protocol as described for Fas. The first antibody was an anti-Fas-ligand (Santa Cruz; sc-834, 1:100) and the second antibody was Dako E 0466 (Santa Cruz; sc-2004, 1:200). Fas-ligand concentration was determined by grading the samples as described above (Fas). Bax concentration was determined following the same protocol as above. The first antibody used was an anti-Bax antibody (Oncogene; PC66, 1:50) and the secondary antibody was from Santa Cruz (sc-2004 in a dilution of 1:200). Bax was determined by counting positive cells per defined fields as described above.

FIG. 1. A, Gene expression for p21waf-1/cip-1, GADD-45, and GADD153 in whole proximal small bowel RNA extracts 1, 3, and 6 h after burn. *, Significant difference to normal expression, P ⬍ 0.05. B, RNA hybridization blot for individual proximal small bowel samples from sham burned rats and 40% TBSA burned rats 1 h after injury. 18S was used as a loading control.



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Inc., Palo Alto, CA) using an isocratic elution at 0.75 ml/min and an octadecylsilane column (RP C-18) (29).

Study 2: effect of IGF-I/IGFBP-3 on small bowel homeostasis

Statistics

Apoptosis. All animals survived the thermal injury and treatment. The apoptotic index increased in both groups after thermal injury and stayed increased until 5 d after burn when compared with normal small bowel apoptosis (Fig. 2A). When apoptosis was measured using a different technique quantifying exposed histones by ELISA, there was also an increase detected at 24, 48, and 120 h after burn (data not shown). Changes in both these measures corroborate an increase in apoptosis after burn injury. There was no evidence of necrosis in any of the sections. Thermally injured rats receiving IGF-I/ IGFBP-3 had significantly decreased apoptotic rates in the proximal small bowel at 5 and 7 d after burn when compared with thermally injured rats receiving saline (P ⬍ 0.05; Fig. 2A).

Statistical comparisons were made by ANOVA with post hoc Bonferroni’s correction or Student’s t test where appropriate. Data are expressed as means ⫾ sd in tables and as means ⫾ sem in figures. Significance was accepted at P ⬍ 0.05.

Results Study 1: apoptosis-related genes postburn

To assess early changes in cell cycle-related gene expression associated with increased gut epithelial apoptosis, we analyzed whole mucosal RNA extracts for expression of p21WAF-1, GADD-45, and GADD-153. At 1, 3, and 6 h after burn, we found that expression for all three genes significantly increased, compared with sham rats at 1 h (P ⬍ 0.05; Fig. 1A). Expression of GADD-45 remained elevated at 3 h and approached control levels at 6 h after burn. The expression of p21WAF-1 remained significantly increased at 3 and 6 h after burn (P ⬍ 0.05; Fig. 1, A and B).

Proliferation

Small bowel epithelial cell proliferation decreased after the thermal injury beginning postburn d 2 until postburn d 7, compared with normal. IGF-I/IGFBP-3 significantly in-

FIG. 2. A, Percentage of apoptotic cells measured by TUNEL assay 1, 2, 5, and 7 d after burn. *, Significant difference between IGF-I/IGFBP-3 and saline, P ⬍ 0.05. Data presented as means ⫾ SEM with n ⫽ 7 (normal small bowel epithelial cell apoptosis: 0.14 ⫾ 0.02%). B, Percent of proliferating cells measured by PCNA. *, Significant difference between IGF-I/IGFBP-3 vs. saline, P ⬍ 0.05. Data presented as means ⫾ SEM with n ⫽ 7 (normal small bowel epithelial crypt cell proliferation: 55 ⫾ 4%). C, Villous height measured in micrometers with a ⫻40 magnification. *, Significant difference between IGF-I/IGFBP-3 vs. saline, P ⬍ 0.05. Data presented as means ⫾ SEM with n ⫽ 7 (normal small bowel villous height: 71 ⫾ 1 ␮m). D, Cell number per villous counted through ⫻40 magnification. *, Significant difference between IGF-I/IGFBP-3 vs. saline, P ⬍ 0.05. Data presented as means ⫾ SEM with n ⫽ 7 (normal small bowel cell number: 435 ⫾ 9).

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creased mucosal cell proliferation at 2, 5, and 7 d after burn, compared with burn saline, and prevented the burn-induced decrease in small bowel epithelial cell proliferation (P ⬍ 0.05; Fig. 2B). Villous height and cell count. Villous height decreased after the severe trauma. Increased proliferation with IGF-I/IGFBP-3 administration was associated with improved small bowel villous morphology. Rats receiving IGF-I/IGFBP-3 had significantly higher villi with an increased cell number when compared with rats receiving saline at d 2, 5, and 7 after burn (P ⬍ 0.05; Fig. 2, C and D). Serum TNF, IL-1, and IL-6

Serum TNF increased in both groups, the saline and IGF-I/ IGFBP-3 group, immediately after burn. IGF-I/IGFBP-3 significantly decreased serum TNF at d 1 after burn, compared with saline (P ⬍ 0.05; Fig. 3A). Similarly, IL-1␤ was increased after severe injury. IGF-I/IGFBP-3 significantly decreased serum IL-1␤ on postburn d 1 and 2 when compared with saline (P ⬍ 0.05; Fig. 3B). Serum IL-6 also increased after burn, but there were no significant differences between saline and IGF-I/IGFBP-3 for serum IL-6 (Fig. 3C). Serum IGF-I concentration

Immediately after the trauma, serum IGF-I decreased in all groups. Despite a moderate increase, IGF-I levels remained decreased over the study period (Table 1). rhIGF-I/IGFBP-3 significantly increased human IGF-I levels after the burn injury (P ⬍ 0.05; Table 1). Increased human IGF-I levels along with decreased rat IGF-I would imply that the human IGFI/IGFBP-3 competed with the rat IGF-I/IGFBP-3 for binding to the acid-labeled subunit. Total IGF-I was, however, significantly increased in the rhIGF-I/IGFBP-3 group throughout the entire study (P ⬍ 0.05; Table 1). Fas, Fas-ligand, Bax, Bcl-2, and caspase-3, and -9 expression

Immediately after burn, Fas-ligand increased in small bowel epithelial cells and remained increased over the study period. IGF-I/IGFBP-3 significantly decreased Fas-ligand concentration in small bowel epithelial cells at d 1 after burn when compared with saline controls (P ⬍ 0.05; Fig. 4A). Fas concentration was also increased in small bowel epithelial cells immediately after burn but then decreased over time. IGF-I/IGFBP-3 significantly decreased Fas concentration on d 1, 2, and 5 when compared with saline (P ⬍ 0.05; Fig. 4B). Bax expression in the small bowel increased immediately after burn and increased also toward postburn d 5 and 7. IGF-I/IGFBP-3 had no effect on Bax expression during the study period (Fig. 4C). Bcl-2 concentration decreased in all groups after the thermal injury. rhIGF-I/IGFBP-3 had no effect on Bcl-2 concentration in the small bowel (Fig. 4D). Caspases-3 and -7 increased immediately after burn and showed a mild decrease over time. IGF-I/IGFBP-3 significantly decreased caspases-3 at d 1, 5, and 7, compared with saline (P ⬍ 0.05; Fig. 4E). IGF-I/IGFBP-3 also decreased caspase-7 at the first postburn day, which was due to large se of the mean not significant (Fig. 4F).

FIG. 3. A, IGF-I/IGFBP-3 significantly decreased serum TNF at d 1 after burn (normal serum TNF: ⬍ 2 pg/ml). B, IGF-I/IGFBP-3 significantly decreased serum IL-1␤ on d 1 and 2 when compared with saline (normal serum IL-1␤: ⬍ 2 pg/ml). C, Serum IL-6 also increased after burn, but there were no significant differences between saline and IGF-I/IGFBP-3 for serum IL-6 (normal IL-6: ⬍ 10 pg/ml). *, Significant difference between IGF-I/IGFBP-3 and saline, P ⬍ 0.05.

Study 3: bile acid pool and composition after burn

Thermally injured animals had a significantly lower bile acid secretion in comparison with controls (P ⬍ 0.05; Fig. 5). Control animals had a higher bile volume and a higher bile acid concentration than burned rats. At any given time during the biliary diversion studies, the bile acid output was significantly greater in control animals, although the output decreased with every interval measured consistent with a diminished recirculation from the intestine (Fig. 5).



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TABLE 1. Serum human (h) IGF-I, rat IGF-I, and total IGF-I concentrations Days after burn 1

Saline hIGF-I (␮g/ml) Rat IGF-I (␮/ml) Total IGF-I (␮g/ml) rhIGF-I/IGFBP-3 hIGF-I (␮g/ml) Rat IGF-I (␮g/ml) Total IGF-I (␮g/ml)

2

5

7

3.4 ⫾ 0.3 10.4 ⫾ 1.5 13.8 ⫾ 1.5

3.6 ⫾ 0.3 12.7 ⫾ 1.1 16.3 ⫾ 1.5

3.2 ⫾ 0.2 12.6 ⫾ 1.5 15.8 ⫾ 1.7

3.1 ⫾ 0.3 13.2 ⫾ 1.2 16.3 ⫾ 1.5

35.4 ⫾ 11.1a 5.4 ⫾ 1.8a 40.8 ⫾ 12.1a

44.4 ⫾ 17.6a 8.1 ⫾ 3.0a 52.5 ⫾ 20.5a

39.4 ⫾ 15.0a 7.1 ⫾ 2.2a 46.5 ⫾ 17.1a

35.4 ⫾ 7.5a 7.6 ⫾ 3.8a 43 ⫾ 11.1a

Data presented as means ⫾ SD. Normal levels for hIGF-I are: 3–5 ␮g/ml; rat IGF-I: 19 –25 ␮g/ml; and thus, total IGF-I: 22–30 ␮g/ml. Total IGF-I, Rat IGF-I plus hIGF-I. a There is a significant difference between rhIGF-I/IGFBP-3 and saline, P ⬍ 0.05.

Muricholates, hyocholates, and cholates presented the main bile acid species in both groups of animals. Small but significant differences were detected with chenodeoxycholate and deoxycholate (Table 2). Discussion

Cell death in the small bowel occurs by two distinct modes of cell death, apoptosis or necrosis (1, 2, 7). Apoptosis of gut epithelial cells is characterized by cell shrinkage, DNA fragmentation (laddering), membrane blabbing, and phagocytosis of the apoptotic cell fragments by neighboring cells or extrusion into the lumen of the bowel without inflammation. This is in contrast to necrosis, which involves cellular swelling, random DNA fragmentation (laddering), lysosomal activation, membrane breakdown, and extrusion of cellular contents into the interstitium. There are two established methods to identify apoptosis, both of which are criticized (30). The TUNEL method, which has been used in the present study, labels the broken ends of DNA fragments. However, DNA fragmentation occurs also during necrosis. Thus, the TUNEL assay does not differentiate between apoptosis and necrosis. However, because necrosis is associated in vivo with histological signs of inflammation, whereas apoptosis is not, the two processes can be differentiated on histological sections. We found no evidence for necrosis in the small bowel villi in the present study. To corroborate the apoptotic measurements of TUNEL and ELISA with apoptotic gene expression, we showed an up-regulation of apoptotic-related genes in the small bowel mucosa early during the postburn response, indicating that TUNEL was a valid method to detect small bowel epithelial cell apoptosis in the present study. Severe cutaneous burn has been shown to increase small bowel mucosal epithelial cell apoptosis, which is associated with decreased gut mucosal weight and height, protein content and DNA content, and the small bowel absorptive surface (5, 6). To counter increased gut apoptosis and hence improve gut homeostasis by administration of the mitogenic growth factor, IGF-I was the aim of the present study. IGF-I plays a major role in the regulation of somatic and organ growth and has been shown to exert paracrine, autocrine, and endocrine effects (31). Recent evidence suggests that IGF-I can stimulate the growth of the small bowel mucosa (32). Ohneda et al. (12) demonstrated that long-term excess of IGF-I in a transgenic mouse model resulted in improved cell

balance along with increased mucosal DNA and protein content. Here we show that IGF-I in combination with its principal binding protein 3 (IGF-I/IGFBP-3) stimulated small bowel epithelial cell proliferation along with increased villous height, crypt depth and cell number after a severe thermal injury. In addition, IGF-I/IGFBP-3 significantly decreased programed cell death of small bowel epithelial cells. The antiapoptotic effect of IGF-I has been shown in other cells, such as hematopoietic, prostatic stromal cells, and rabbit blastocysts (14 –16). These studies suggested that IGF-I may act as a survival factor by increasing mitosis and inhibiting apoptosis (14 –16). IGF-I/IGFBP-3 significantly decreased Fas-ligand, Fas, TNF, and IL-1␤, suggesting that IGF-I/IGFBP-3 affects the Fas pathway. The antiapoptotic effect is most likely only through the Fas-Fas ligand-TNF pathway because Bax and Bcl-2 were not affected with IGFI/IGFBP-3 administration. A decreased Fas pathway led to a decreased concentration of the executive protein caspase-3. However, it could also be possible that IGF-I reduced the initial inflammatory cytokine response, and it was the reduced TNF and IL-1 levels that resulted in decreased apoptosis. The measures of caspases and apoptotic markers consecutively reflected the different levels of cytokine-induced apoptosis. The antiapoptotic and promitogenic effects of IGF-I/ IGFBP-3 make this molecule an interesting therapeutic agent for critically or severely injured patients because an imbalance in gut homeostasis has been delineated in several studies as a determining factor for morbidity and mortality. The apoptotic process has also been shown to play a pivotal role in the pathogenesis of the intestinal barrier dysfunction (2, 7). Increased small bowel epithelial apoptosis leads to an increased bidirectional permeability of the intestinal barrier (2, 7), with reduced uptake of intraluminal nutrients (33), increased permeability to macromolecules (34, 35), and most importantly an increase in permeability for bacterial translocation (13, 36). Translocation of enteric bacteria, toxins, and gut-derived factors carried in the mesenteric lymph can lead to sepsis, multiorgan failure, and increased mortality (2, 4, 7–9). Thus, the maintenance of the gut-barrier function is of major importance for survival after burn trauma (4, 7). The mechanisms whereby a severe injury, such as a cutaneous burn, induces programmed cell death in gut epithelium are not defined. Several studies suggest that hypoperfusion and ischemia-reperfusion of the gut, and the

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FIG. 4. A, IGF-I/IGFBP-3 significantly decreased Fas ligand concentration in small bowel epithelial cells at d 1 after burn when compared with saline controls (normal Fas ligand: 0 positive cells). B, IGF-I/IGFBP-3 decreased Fas concentration in the small bowel on d 1, 2, and 5, which was significant on d 1 and 5 when compared with saline (normal Fas: 0.2 ⫾ 0.05 positive cells). C, IGF-I/IGFBP-3 had no effect on Bax concentration in the small bowel (normal Bax: 0.05 ⫾ 0.01 positive cells). D, rhIGF-I/IGFBP-3 had no effect on Bcl-2 concentration in the small bowel (normal Bcl-2: 2 ⫾ 0.3 positive cells). E, IGF-I/IGFBP-3 significantly decreased caspase-3 at d 1, 5, and 7, compared with saline (normal caspase-3: 0.2 ⫾ 0.1 positive cells). F, IGF-I/IGFBP-3 also decreased caspase-7 at the first postburn day, which was due to large SE of the mean not significant (normal caspase-7: 0.4 ⫾ 0.2 positive cells). *, Significant difference between IGF-I/IGFBP-3 vs. saline controls, P ⬍ 0.05.

release of proinflammatory cytokines are associated to promote apoptosis of the small bowel mucosa (2, 7–9, 37, 38). We showed in the present study, as it has been shown in many other studies, that a burn injury increases proinflammatory

cytokine concentration, making cytokine signaling a possible mode for the induction of apoptosis. Another interesting hypothesis is that bile acids may cause apoptosis. In the present study, we found that biliary secretion of bile acids



361

apoptosis. Some investigators have shown that apoptosis occurs primarily at the villous tip (45), whereas others identified the apoptotic process in both areas (46, 47). We identified the apoptotic process in the crypt and villus, suggesting a responsive element to the signal in undifferentiated and differentiated mucosal epithelial cells. In conclusion, burn increases programmed cell death of small bowel epithelial cells with a concomitant loss of mucosal weight and protein content. IGF-I/IGFBP-3 increased small bowel epithelial cell proliferation and decreased epithelial cell apoptosis by modulating the Fas-Fas ligand-TNF pathway and decreasing proinflammatory cytokines. We suggest that IGF-I/IGFBP-3 attenuates enteral apoptosis in burn and trauma victims and thus may improve gut mucosal integrity. Acknowledgments FIG. 5. Bile acid output of thermally injured animals in comparison with control rats. Bile was collected over consecutive 10-min periods. When comparing the corresponding periods, bile acid secretion was significantly reduced in the burned group. *, Significant difference between burn vs. control, P ⬍ 0.001; **, significant difference between burn vs. control, P ⬍ 0.01.

was markedly diminished in thermally injured animals. Although there have not been any reports on biliary secretion after thermal injury, our findings are in line with many studies investigating bile secretion after application of endotoxin or acute phase mediators (39). Because these mediators have been shown to be increased in our model, it is likely that the diminished bile secretion is the result of increased serum levels of proinflammatory mediators such as TNF or IL-1, resulting in the down-regulation of hepatocyte transport proteins (40). Recently it was shown that bile acids increase intestinal cell migration, thereby regulating mucosal integrity (41). In rats oral administration of bile salts was correlated with a reduced bacterial translocation (42). In high concentrations (⬎0.5 mmol/liter), bile acids were shown to contribute to apoptosis (43) and cytotoxicity (44). Given the down-regulation of biliary secretion in thermally injured animals, a significant factor regulating crypt cell migration might reduce intestinal integrity and facilitate the translocation of toxins and bacteria. However, this process is most likely due to the lack of a trophic factor because the bile acid concentrations in our in vivo model are too low to induce apoptosis or cytotoxicity. This also holds true considering the observed alterations in the bile acid pool of thermally injured animals as the ratio of protective hydrophilic remains similar to that of toxic hydrophobic bile acids. There is also discussion about the predominant site of TABLE 2. Bile acid composition in control and burned animals Bile acid composition (%) n

Control 5 Burn 5 P value

Muricholates and hyocholates

Cholic

83.8 ⫾ 4.1 87.4 ⫾ 1.3 NS

14.3 ⫾ 3.5 8.9 ⫾ 1.2 NS

Data presented as means ⫾

SD.

Chenodeoxycholic Deoxycholic

1.3 ⫾ 0.4 3.3 ⫾ 0.4 0.02

0.7 ⫾ 0.2 0.2 ⫾ 0.1 0.05

Received July 5, 2006. Accepted September 20, 2006. Address all correspondence and requests for reprints to: Marc G. Jeschke, M.D., Ph.D., Shriners Hospitals for Children, 815 Market Street, Galveston, Texas 77550. E-mail: [email protected]. This work was supported by the Shriners North America Grant 8010 and Celtrix Pharmaceuticals. The study was further supported by the Deutsche Forschungsgemeinschaft (German Research Council) Je 233/ 6-1. Celtrix Pharmaceuticals kindly provided the IGF-I/IGFBP-3 complex used for this study. Disclosures: The authors have nothing to disclose. The authors declare that they have no financial interest in Celtrix Pharmaceuticals.

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