Complement Inhibitor, Complement Receptor 1-Related Gene/ Protein y-Ig Attenuates Intestinal Damage After the Onset of Mesenteric Ischemia/Reperfusion Injury in Mice1 Scott Rehrig,2*† Sherry D. Fleming,2‡§ Jimie Anderson,*† Joel M. Guthridge,¶ Jonathan Rakstang,¶ Charles E. McQueen,‡ V. Michael Holers,¶ George C. Tsokos,‡§ and Terez Shea-Donohue3‡ Complement receptor 1-related gene/protein y (Crry) is a murine membrane protein that regulates the activity of both classical and alternative complement pathways. We used a recombinant soluble form of Crry fused to the hinge, CH2, and CH3 domains of mouse IgG1 (Crry-Ig) to determine whether inhibition of complement activation prevents and/or reverses mesenteric ischemia/ reperfusion-induced injury in mice. Mice were subjected to 30 min of ischemia, followed by 2 h of reperfusion. Crry-Ig was administered either 5 min before or 30 min after initiation of the reperfusion phase. Pretreatment with Crry-Ig reduced local intestinal mucosal injury and decreased generation of leukotriene B4 (LTB4). When given 30 min after the beginning of the reperfusion phase, Crry-Ig resulted in a decrease in ischemia/reperfusion-induced intestinal mucosal injury comparable to that occurring when it was given 5 min before initiation of the reperfusion phase. The beneficial effect of Crry-Ig administered 30 min after the initiation of reperfusion coincided with a decrease in PGE2 generation despite the fact that it did not prevent local infiltration of neutrophils and did not have a significant effect on LTB4 production. These data suggest that complement inhibition protects animals from reperfusion-induced intestinal damage even if administered as late as 30 min into reperfusion and that the mechanism of protection is independent of neutrophil infiltration or LTB4 inhibition. The Journal of Immunology, 2001, 167: 5921–5927.
I
n many clinical situations, such as hemorrhagic shock, sepsis, multisystem organ failure, myocardial infarction, severe burns, mesenteric thrombosis/embolism, and early organ transplant rejection, tissues become temporarily ischemic, followed by restoration of blood flow (1–3). The injury sustained after an ischemic event and subsequent restoration of blood flow surpasses that expected by ischemia alone (4). Organ ischemiareperfusion (IR)4 causes the generation of numerous inflammatory mediators, such as proteolytic fragments of complement proteins, cytokines, and eicosanoids; the inappropriate expression of adhe-
*Department of Surgery, Walter Reed Army Medical Center, Washington, DC 20307; Departments of †Surgery and ‡Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; §Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; and ¶Departments of Medicine and Immunology, University of Colorado Health Sciences Center, Denver, CO 80206 Received for publication May 23, 2001. Accepted for publication September 12, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Medical Research Material Command Scientific and Technical Objective R, Complement Program, and Grant G183HO (to T.S.-D.) from Walter Reed Army Institute for Research Scientific and Technical Objective R; National Institutes of Health Grant RO-1AI31105 (to V.M.H.); and a National Research Fellowship from Walter Reed Army Institute of Research (to S.D.F.). The opinions contained herein are the private ones of the authors and are not to be construed as official policy or as reflecting the views of the Department of Defense. 2
S.R. and S.D.F. contributed equally to this article.
3
Address correspondence and reprint requests to Dr. Terez Shea-Donohue, Department of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. E-mail address:
[email protected] 4 Abbreviations used in this paper: IR, ischemia/reperfusion; cNOS, constitutive NO synthase; Crry, complement receptor 1-related gene/protein y; CVF, cobra venom factor; LTB4, leukotriene B4; MPO, myeloperoxidase; PMNs, neutrophils; s, soluble.
Copyright © 2001 by The American Association of Immunologists
sion molecules; and the activation of neutrophils (PMNs) that invade local and remote tissues (reviewed in Refs. 1, 5, and 6). Complement activation occurs through the classical, lectin, or alternative pathways and constitutes a major portion of the innate immune response. Complement fragments enhance chemotaxis, promote phagocytosis, and lead to the formation of terminal membrane attack complexes on the surface membrane of invading organisms and target cells. However, excessive activation of the complement cascade may induce tissue damage. To protect selftissues in the face of inflammation, natural inhibitors, including proteins expressed on the surface membrane of various cells, control complement activation. These inhibitors include complement receptor 1 (CR1), decay-accelerating factor and membrane cofactor protein (7, 8). Soluble human CR1 (sCR1) is a potent inhibitor of C3 activation. In humans, widely distributed CR1 binds and contributes to the inactivation of both C3b and C4b (8). In the mouse, however, CR1 is expressed on a limited number of cells. In addition, the mouse protein encoded by complement receptor-related gene y, (Crry) has a wider distribution pattern, binds both C4b and C3b, and has the same complement inhibitory activity as human CR1 (9). Therefore, Crry is a more appropriate complement C3 inhibitor for murine models and may provide greater insight into the comparable role of sCR1 in humans. Crry is a mouse membrane complement inhibitor with decayaccelerating activity for both classic and alternative pathways (9). It also possesses cofactor activity comparable to that of CR1 for the factor I-mediated cleavage of C3b and C4b (9, 10). Crry-Ig is a recombinant, soluble protein with an increased half-life due to fusion of Crry with the Fc portion of a non-complement-activating mouse IgG1 partner. Overall, Crry-Ig is more potent complement inhibitor than mouse CR1 (9, 10). 0022-1767/01/$02.00
5922 Previous studies demonstrated that complement activation plays an essential role in many models of mesenteric IR and that inhibitors of complement activation may limit IR-induced damage (3, 4, 11, 12). Previously, we showed that administration of sCR1 to rats before or during the reperfusion of intestinal tissues significantly reduced mucosal injury, PMN infiltration, and leukotriene B4 (LTB4) production (3). To allow for a better comparison with human complement inhibitors and to further examine the mechanisms of inhibition, in this study we used the Crry-Ig fusion protein to inhibit mesenteric IR-induced injury. We report that Crry-Ig effectively prevents the development of tissue damage even when administered 30 min into the reperfusion phase despite the presence of substantial numbers of biologically active PMNs.
Materials and Methods Animal preparation Adult male BALB/c mice (National Cancer Institute, Bethesda, MD) were obtained and prepared for surgery after a 7-day acclimation period. Anesthesia was induced with ketamine (16 mg/kg) and xylazine (8 mg/kg) administered i.m. All procedures were performed with the animals breathing spontaneously, and body temperature was maintained at 37°C using a water-circulating heating pad. Experiments were performed according to the principles set forth in the Guide for the Care and Use of Laboratory Animals of the Institute of Animal Resources (National Research Council; DHEW publication no. 85-23).
Crry-Ig and control Crry-Ig was made and purified as described previously (9). IgG1 mAb 994, used to control for Fc binding, was purified from supernatants by passage over protein G-Sepharose. mAb 994 does not react with mouse tissues, but has the same Fc portion as Crry-Ig. Both Crry-Ig and the control Ab appeared to be safe and nontoxic.
Experimental protocol Mice were divided into six experimental groups (n ⫽ 7–9 animals/group): 1) 30 min of ischemia, followed by 120 min of reperfusion (IR120); 2) sham laparotomy (sham); 3) IR120 pretreated with 2 mg Crry-Ig at 5 min before the start of reperfusion (T-5); 4) IR120 treated with 2 mg Crry-Ig 30 min after the start of reperfusion (T ⫹ 30); 5) IR120 pretreated with 2 mg control mouse IgG1 5 min before the start of reperfusion (Ig-5); and 6) IR120 treated with 2 mg control IgG1 30 min after the start of reperfusion (Ig ⫹ 30). Additional groups included Crry-Ig-treated, sham-operated mice and cobra venom factor (CVF)-treated sham and IR mice. Twelve units of CVF (Sigma) was administered i.p. at 24 and 18 h before laparotomy. The abdomen was entered via a midline laparotomy incision, and all animals were subjected to a 30-min equilibration period. Next, the superior mesenteric artery was identified and isolated, and a small vascular clamp (Roboz Surgical Instruments, Rockville, MD) was applied. Ischemia to the mid-jejunum was confirmed by noting a change in the color of the bowel from pink to pale gray and an absence of pulsations of the mesenteric vessels distal to the clamp. Desiccation of the intestine was prevented by covering the bowel with surgical gauze moistened with warm 0.9% normal saline. After 30 min of mesenteric ischemia, the clamp was removed under direct visualization, and the intestine was allowed to reperfuse for 120 min. Five minutes before the start of reperfusion, one group of mice in the IR group was given an i.p. dose of Crry-Ig. A separate group of IR-treated animals received an i.p. dose of control IgG1. Thirty minutes after reperfusion began, additional IR-treated animals were given Crry-Ig or IgG1 in a similar manner. The sham animals underwent the same surgical intervention, except for omission of superior mesenteric artery occlusion. The IR nontreated control group underwent identical surgical intervention without treatment with Crry-Ig or control IgG1. After clamp removal, reperfusion was confirmed by observing a change in the color of the bowel from pale gray to pink and the return of pulsatile flow to the superior mesenteric artery and its branches. Next, the midline laparotomy incisions were closed with a 6.0 prolene suture in a running fashion. All animals were monitored during the reperfusion period, and additional ketamine and xylazine were administered i.m. as needed and immediately before sacrifice. Once anesthetized, the small intestine 10 –20 cm distal to the gastroduodenal junction was harvested for histologic evaluation, eicosanoid determination (LTB4 and PGE2), and quantification of neutrophil (PMN) infiltrate. Mice were excluded from the study if they
Crry-Ig DECREASES IR INJURY failed to survive the full 120-min reperfusion period. The survival rate was not significantly different among treatment groups.
Histology Small intestine specimens were promptly fixed in 10% buffered formalin phosphate, embedded in paraffin, sectioned transversely (5–7 m), and stained with Giemsa. The mucosal injury score was graded on a six-tiered scale defined by Chiu et al. (3, 13). The villus height of at least 10 villi from the same sections was measured using an ocular micrometer.
Immunohistochemistry Additional tissues were fixed at 4°C for 2 h in 4% paraformaldehyde in PBS. Sections were obtained and stained by immunohistochemistry. Nonspecific Ab binding sites were blocked by incubation with a solution of 20% rat serum in PBS for 30 min. After washing in PBS, the tissues were incubated with isotype control Ab or FITC-conjugated rat anti-mouse GR-1 (rat IgG1) mAb to identify PMN (BD PharMingen, San Diego, CA) for 1 h at room temperature. After washing, the slides were mounted using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). A blinded observer examined the slides by fluorescent microscopy using a Leica DM RX/A fluorescent microscope (Leica Microsystems, Atlanta, GA) with SPOT diagnostic computer software (Sterling Heights, MI) and counted fluorescent cells per field. Additional sections were stained for NADPH diaphorase activity, a specific marker for constitutive NO synthase (cNOS) activity in the enteric nerves, using a modification of the method described by Shuttleworth et al. (14 –16).
Eicosanoid determination The ex vivo generation of eicosanoids in small intestine tissue was determined using a previously validated method (17). Briefly, sections of minced fresh mid-jejunum were washed and resuspended in 37°C oxygenated Tyrode’s buffer (Sigma, St. Louis, MO). After tissues were incubated for 20 min at 37°C, supernatants and tissue were collected and stored at ⫺80°C until assayed. The concentrations of LTB4 and PGE2 were determined using an enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). The LTB4 assay has ⬍0.01% cross-reactivity with LTC4, LTD4, LTE4, and LTF4. The tissue protein content was determined using the bicinchoninic acid assay (Pierce, Rockford, IL) adapted for use with microtiter plates. BSA was used as the standard. Eicosanoid generation was expressed per milligram tissue protein per 20 min.
Myeloperoxidase (MPO) assay Supernatants generated for the eicosanoid assays were also used to determine MPO activity by measuring oxidation of 3,3⬘,5,5⬘-tetramethylbenzidene (18). Supernatants were incubated with equal volumes of 3,3⬘,5,5⬘-tetramethylbenzidene peroxidase substrate (Kirkegaard & Perry, Gaithersburg, MD) for 45 min. The reaction was stopped by the addition of 0.18 M sulfuric acid, and the OD (A450) was determined. The concentration of MPO was determined using HRP (Sigma) as a standard and was plotted as picograms of myeloperoxidase activity per milligram of tissue.
Statistical analysis All data are presented as the mean ⫾ SEM. Data were compared by oneway ANOVA with post hoc analysis using Newman-Keuls test (Graph Pad Software, Philadelphia, PA). The difference between groups was considered significant at p ⬍ 0.05.
Results Although both pathways of complement activation have been suspected to participate in the IR-induced tissue damage, previous murine studies have used complement inhibitors that block both pathways in humans, but whose enzymatic activities were not clearly defined in the mouse. We planned experiments to investigate the ability of the Crry-Ig fusion protein, which has been shown to inhibit both classical and alternative mouse complement activation pathways (10), to prevent intestinal damage after mesenteric IR. Mice were subjected to IR or sham operation with or without Crry-Ig treatment and the extent of the mucosal damage was determined. The intestinal mucosa of the sham-operated animals remained normal as indicated by an injury score of 0.6 ⫾ 0.1 and normal villi height (Figs. 1A and 2). As expected, the mucosa of the animals subjected to mesenteric IR for 2 h displayed signs
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FIGURE 1. Effect of Crry-Ig on IR-induced mucosal injury. Formalinfixed intestinal sections were stained with Giemsa. A, Sham treatment; B, 30-min ischemia and 2-h reperfusion (IR); C, treatment with 2 mg IgG1 5 min before beginning reperfusion; D, treatment with 2 mg IgG1 30 min after beginning reperfusion; E, treatment with 2 mg Crry-Ig 5 min before beginning reperfusion; F, treatment with 2 mg Crry-Ig 30 min after beginning reperfusion; G, CVF treatment followed by sham; H, CVF treatment followed by IR. Original magnification, ⫻200. Data are representative of six individual experiments.
of significant damage with a mean score of 3.6 ⫾ 0.2 (Figs. 1 and 2). Macroscopically, the intestines of the animals subjected to IR appeared swollen and edematous with areas of red streaks. Microscopically, the observed changes ranged from shortened and vacuolated villi to complete destruction of normal mucosal architecture with frank hemorrhage (Figs. 1B and 2). Treatment of animals with IgG1, a control biological reagent for the Crry-Ig, 5 min before (Fig. 1C) or 30 min after (Fig. 1D) the initiation of reperfusion did not prevent the development of mucosal injury (mean injury scores, 3.6 ⫾ 0.3 and 3.5 ⫾ 0.4, respectively). In contrast, animals pretreated with Crry-Ig 5 min before the initiation of reperfusion significantly preserved the macro- and microscopic appearance of the intestinal mucosa (mean injury score, 1.2 ⫾ 0.2; Fig. 2). In animals treated with Crry-Ig, the changes in intestinal architecture
ranged from normal villi to the development of subepithelial Grugenhagen’s space (injury score, 2) at the apex of the villus (Figs. 1E and 2). The intestinal villus height remained normal. Surprisingly, treatment with Crry-Ig 30 min into the reperfusion period (Figs. 1F and 2) was equally or more effective (mean score, 0.70 ⫾ 0.2) in protecting against mucosal injury as administration of Crry-Ig 5 min before the initiation of reperfusion. Moreover, treatment with Crry-Ig either before or during reperfusion preserved the intestinal villus height (Fig. 2). To verify that complement was a critical factor in the development of mucosal injury after IR we subjected animals to mesenteric IR after treatment with CVF, which is known to completely consume complement. CVF alone had no effect on sham-operated rats (Fig. 1G) and protected against IR-induced mucosal injury (Fig. 1H).
5924
FIGURE 2. Crry-Ig protects intestinal mucosa from IR-induced injury. Giemsa-stained intestinal sections from each treatment group were scored for mucosal injury (0 – 6; 䡺) and villus height (micrometers; f) as described in Materials and Methods. Villus heights of individual villi were measured using an ocular micrometer. All measurements were obtained at ⫻200 magnification. Each bar is the average ⫾ SEM with six to eight animals per group. ⴱ, Significant difference from sham group, p ⬍ 0.05 (by ANOVA with Newman-Keuls post hoc test).
Crry-Ig DECREASES IR INJURY
FIGURE 4. Treatment with Crry-Ig before, but not after, reperfusion prevents PMN infiltration. Intestinal sections from sham (A), IR-treated (B), Crry-Ig-treated 5 min before reperfusion (C), or Crry-Ig-treated 30 min into reperfusion (D) animals were stained for GR-1 mAb as described in Materials and Methods. Original magnification, ⫻200. Data are representative of four individual experiments.
Effect of Crry-Ig administration on small intestinal inflammation The precise contribution of neutrophils to IR-induced mucosal damage is controversial. Hernandez et al. (19) concluded that neutrophil infiltration in the intestine was responsible for mucosal damage in response to IR injury. However, others found that neutrophils were not required for IR-induced damage (20). To determine the extent of neutrophil infiltration into the intestinal tissue in this mouse model, we stained sections of the small intestine with the granulocyte-specific marker, GR-1. Sham-treated animals with normal mucosal architecture had few neutrophils in the villi (Figs. 3 and 4A). Surprisingly, tissue taken from animals subjected to IR after 2-h reperfusion also had few PMNs in intestinal villi (Figs. 3 and 4B). However, compared with tissues from sham-treated animals, the villus height was very low in the IR-treated group (Fig. 2). Both of these facts correlate with the denuded villi and lamina propria exuding from the villi seen in the Giemsa-stained sections
FIGURE 3. Administration of Crry-Ig at 30 min into the reperfusion period allows PMN infiltration and activity. PMN in intestinal tissue sections were stained with FITC-labeled GR-1 mAb, and the number of fluorescent PMN per high power field was counted from three independent experiments. MPO activity per milligram of tissue was determined as described in Materials and Methods. ⴱ, Significant difference from sham group, p ⬍ 0.05; †, significant difference from IR group, p ⬍ 0.05 (by ANOVA with Newman-Keuls post hoc test).
in this group (Figs. 1 and 4B). This suggests that infiltrating PMN located in the villi tips at earlier time points were extruded into the intestinal lumen with the sloughed epithelium by 2 h postreperfusion. Pretreatment with control Ig did not alter IR-induced PMN infiltration (data not shown). Crry-Ig administered 5 min before the beginning of the reperfusion also prevented PMN infiltration with an average of 8.5 ⫾ 3 PMN/high power field (Figs. 3 and 4C). In addition, there was no significant decrease in villus height of either Crry-Ig-treated group. Interestingly, treatment with Crry-Ig 30 min into the reperfusion period did not prevent the infiltration of PMN into the intestinal tissue despite the fact that the tissue displayed normal microscopic appearance (Fig. 4D). The average PMN per high power field in these tissues was 40.7 ⫾ 5 (Fig. 3). To confirm the surprisingly low number of PMN in the IR group and the high number in the Crry T ⫹ 30 group, we determined MPO activity. At 2 h after beginning reperfusion, MPO was not significantly different in tissues from sham- or IR-treated animals. In addition, the protection afforded to animals treated with Crry at
FIGURE 5. Crry-Ig prevents LTB4 production by intestinal tissues. Eicosanoid production by tissue sections from each treatment group was determined as described in Materials and Methods. Each bar is the average ⫾ SEM, with n ⫽ 3–5 animals/group. ⴱ, Significant difference from sham group, p ⬍ 0.05; †, significant difference from IR group, p ⬍ 0.05 (by ANOVA with Newman-Keuls post hoc test).
The Journal of Immunology
FIGURE 6. Crry-Ig prevents PGE2 production by intestinal tissues. Ex vivo eicosanoid production by tissue sections from each treatment group was determined as described in Materials and Methods. Each bar is the average ⫾ SEM with four to six animals per group. ⴱ, Significant difference from sham group, p ⬍ 0.05 (by ANOVA with the NewmanKeuls post hoc test).
5 min before reperfusion included normal MPO activity. In contrast, when Crry was administered at 30 min after beginning reperfusion, MPO activity was significantly increased. Thus, Crry protects the mucosa from injury despite the fact that it does not prevent PMN infiltration or activity when administered well into the reperfusion period.
5925 such as LTB4 and PGE2. We first measured the levels of the PMN chemotactic factor, LTB4, in all groups of treated and control animals. LTB4 generation was low in the small intestine of shamoperated mice and was significantly elevated in response to IR. In contrast, LTB4 production was low in mice treated with Crry-Ig 5 min before the initiation of the reperfusion, (Fig. 5). The delayed administration of Crry-Ig 30 min into the reperfusion, however, only partially blunted the IR-induced increase in LTB4 generation. PGE2 generation is elevated in response to a number of physiological and pathophysiological stimuli. Similar to LTB4, PGE2 production was increased significantly in response to IR in both vehicle- and Ig-treated groups (Fig. 6). Treatment with Crry-Ig either 5 min before or 30 min into the reperfusion phase significantly decreased the levels of PGE2 (Fig. 6). These findings show that Crry-Ig has a divergent effect on the production of the two eicosanoids. Effect of Crry-Ig on cNOS activity One measure of oxidative stress within the tissue is the production of cNOS by the enteric nerves. Using NADPH diaphorase staining of whole mounts of small intestine, the intensity of which is correlated with cNOS activity, we showed that IR decreased the intensity of staining and the number of nerve cells stained compared with those in sham-operated animals (Fig. 7, A and B). This decrease was similar after treatment with Ig control Ab given before reperfusion or 30 min into the reperfusion period (Fig. 7, E and F). In contrast, administration of Crry-Ig at either time point preserved the staining intensity and the number of cells stained (Fig. 7, C and D).
Effect of Crry-Ig administration on small intestinal eicosanoid generation
Discussion
We considered that the observed discrepancy between the presence of PMNs and the lack of microscopic tissue damage might be linked to changes in the production of inflammatory eicosanoids,
We present evidence that inhibition of complement activation with Crry-Ig effectively decreased mesenteric IR-induced mucosal damage. The involvement of complement in tissue IR-induced injury is
FIGURE 7. Crry-Ig restores NADPH diapherase activity. Intestinal sections from sham (A), IR-treated (B), control Ig-treated 5 min before reperfusion (C), control Ig-treated 30 min into reperfusion (D), Crry-Ig-treated 5 min before reperfusion (E), or Crry-Ig-treated 30 min into reperfusion (F) animals were stained for NADPH diaphorase activity as described in Materials and Methods. Original magnification, ⫻200. Data are representative of at least three individual experiments.
5926 well established, yet the precise mechanism remains unknown. Our study introduces two new findings regarding IR-induced intestinal pathology. First, we showed that delaying treatment with Crry-Ig until 30 min into the reperfusion period continued to provide mucosal protection despite the presence of infiltrating PMNs. Second, we demonstrated a disassociation of the activity of two major enzymatic pathways activated by IR, the cyclo-oxygenase production of PGE2 and the 5-lipoxygenase production of LTB4. Complement plays a central role in regulating many aspects of the innate and acquired immune response. Inhibition of complement activation to limit organ injury is inherently connected with unwanted side effects on the immune response, and obviously a competent immune system is needed in all clinical situations associated with mesenteric ischemia to prevent infections and/or sepsis. Therefore, consideration of the duration of the inhibition of complement activation and compartmentalization of the tissues where complement is inhibited are important. Crry has been shown to inhibit complement activation in animal models of nephrotoxic tissue-induced glomerular inflammation (9, 21) and in animal models of autoimmunity (22). In the present study Crry-Ig limits IR-induced intestinal injury. It is proposed that naturally occurring IgM Abs, which are present in all normal animals, bind to the surface membrane of cells injured by hypoxia during the ischemic phase and subsequently activate complement (23, 24). Such a model of injury requires the deposition of both Ig and complement products in the affected tissues. In this study we were unable to demonstrate the presence of Ig or C3 after 2-h reperfusion (data not shown), indicating that deposition of these products probably occurs earlier in the reperfusion period. Is there a key factor in the development of intestinal mucosal damage following mesenteric IR? Traditionally, PMN infiltration has been given a significant priority (19, 25). Tissue damage obviously can generate chemoattractants such as LTB4 that enhance PMN infiltration. In addition, tissue damage can augment the expression of adhesion molecules on the surface of endothelial cells that may trap PMNs circulating through the damaged tissue during the reperfusion phase. Inhibition of complement activation in rats treated with human sCR1 prevented tissue damage and infiltration of PMNs (3). In contrast, in the current study and in a study by Simpson et al. (20), damage was prevented despite PMN infiltration. It is possible that complement activation and tissue damage associated with infiltration of PMN occur within the first 30 min of the reperfusion period. The subsequent maintenance of PMN activation provides a continuous source of substances deleterious to the surrounding tissue. This conclusion is supported by the fact that administration of Crry-Ig 30 min after initiation of the reperfusion phase prevented tissue damage, but not the entry of PMNs. However, interruption of complement activation at this point still blocks the ability of PMNs to injure tissue. In the rat model of IR-induced injury, PGE2 and LTB4 are usually generated under the same conditions and play a role in edema and inflammation. LTB4 is a product of the 5-lipoxygenase enzyme located primarily in inflammatory cells including PMN. It is a potent chemotactic substance and is considered to be proinflammatory. PGE2 is a product of the cyclo-oxygenase pathway located in a wide variety of cells and is released during inflammation, but has many anti-inflammatory actions. Treatment with Crry-Ig 30 min into the reperfusion phase significantly reduced the tissue damage and PGE2 generation, but not the generation of LTB4. These experiments suggest the LTB4 production may account for the infiltration of PMNs in this group. Therefore, the mechanism by which complement activation inhibition with the soluble membrane complement regulatory protein Crry causes dissociation be-
Crry-Ig DECREASES IR INJURY tween the production of the two eicosanoids is interesting, albeit unknown. The enteric nerves of the intestine are essential to maintain normal function. The production of low levels of NO by the cNOS in nerves is a primary factor controlling smooth muscle function within the gut. In this study we assessed cNOS activity in enteric nerves as an index of the degree of oxidative stress. Previous studies showed that IR decreased cNOS activity, an effect that was attenuated by maintaining the availability of the substrate L-arginine (16). Our mouse model shows a similar decrease in NOS activity after IR. Administration of Crry-Ig at either time point restores this activity. These data suggest that Crry-Ig limits the oxidative stress during the reperfusion period despite the increase in PMN infiltration and activity, again suggesting a direct effect on the neutrophil. Complement activation results in the release of anaphylotoxins that may play a role in damage to remote organs. In the rat model, IR-induced systemic damage occurs at later time points (4 h after induction). The ability to delay the administration of Crry-Ig until well into the reperfusion period suggests that Crry-Ig may also be a useful inhibitor to control systemic damage as well. In addition, administration of Crry-Ig before reperfusion prevents PMN infiltration, suggesting that the peripheral PMN are not activated. Thus, it is possible that Crry-Ig may limit the systemic PMN production of free radical oxygen molecules (H2O2), thereby limiting the role of PMN in the systemic inflammatory response syndrome after major surgery or trauma. When Crry-Ig is administrated 30 min after beginning the reperfusion period, there is decreased mucosal injury, suggesting that it may be important in reversing tissue damage established during the ischemic and early reperfusion phases. In conclusion, we have shown in a mouse model of mesenteric IR that treatment with soluble Crry-Ig administered as late as 30 min into the reperfusion phase provides significant preservation of tissue morphology and preservation of cNOS expression. Our studies have demonstrated a dichotomous effect of Crry-Ig-mediated complement inhibition in the production of PGE2 and LTB4. Finally, our results show that mere infiltration of the intestinal tissue with PMNs does not herald tissue damage. From the clinical point of view, our data suggest that inhibitors of complement activation can be used late into the reperfusion phase with significant capability to attenuate tissue damage.
References 1. Kilgore, K. S., R. F. Todd, and B. R. Lucchesi. 1999. Reperfusion injury. In Inflammation: Basic Principles and Clinical Correlates. J. I. Gallin and R. Snyderman, eds. Lippincott Williams & Wilkins, Philadelphia, p. 1047. 2. Collard, C. D., R. Lekowski, J. E. Jordan, A. Agah, and G. L. Stahl. 1999. Complement activation following oxidative stress. Mol. Immunol. 36:941. 3. Eror, A. T., A. Stojadinovic, B. W. Starnes, S. C. Makrides, G. C. Tsokos, and T. Shea-Donohue. 1999. Antiinflammatory effects of soluble complement receptor type 1 promote rapid recovery of ischemia/reperfusion injury in rat small intestine. Clin. Immunol. 90:266. 4. Austen, W. G., C. Kyriakides, J. Favuzza, Y. Wang, L. Kobzik, F. D. Moore, and H. B. Hechtman. 1999. Intestinal ischemia-reperfusion injury is mediated by the membrane attack complex. Surgery 126:343. 5. Schoenberg, M. H., and H. G. Beger. 1993. Reperfusion injury after intestinal ischemia. Crit. Care Med. 21:1376. 6. Holers, V. M. 2000. Complement. In Current Molecular Medicine: Principles of Molecular Rheumatology. G. C. Tsokos, ed. Humana Press, Totowa, p. 145. 7. Makrides, S. C. 1998. Therapeutic inhibition of the complement system. Pharmacol. Rev. 50:59. 8. Makrides, S. C. 2000. Complement inhibitors. In Current Molecular Medicine: Principles of Molecular Rheumatology. G. C. Tsokos, ed. Humana Press, Totowa, p. 465. 9. Quigg, R. J., Y. Kozono, D. Berthiaume, A. Lim, D. J. Salant, A. Weinfeld, P. Griffin, E. Kremmer, and V. M. Holers. 1998. Blockade of antibody-induced glomerulonephritis with Crry-Ig, a soluble murine complement inhibitor. J. Immunol. 160:4553. 10. Kim, Y. U., T. Kinoshita, H. Molina, D. Hourcade, T. Seya, L. M. Wagner, and V. M. Holers. 1995. Mouse complement regulatory protein Crry/p65 uses the
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11.
12.
13. 14.
15.
16.
17.
specific mechanisms of both human decay-accelerating factor and membrane cofactor protein. J. Exp. Med. 181:151. Dong, J., J. R. Pratt, R. A. G. Smith, I. Dodd, and S. H. Sacks. 1999. Strategies for targeting complement inhibitors in ischaemia/reperfusion injury. Mol. Immunol. 36:957. Williams, J. P., T. T. V. Pechet, M. R. Weiser, R. Reid, L. Kobzik, F. D. Moore, M. C. Carroll, and H. B. Hechtman. 1999. Intestinal reperfusion injury is mediated by IgM and complement. J. Appl. Physiol. 86:938. Chiu, C.-J., A. H. McArdle, R. Brown, H. J. Scott, and F. N. Gurd. 1970. Intestinal mucosal lesion in low-flow states. Arch. Surg. 101:478. Bossone, C., J. M. Hosseini, V. Pineiro-Carrero, and T. Shea-Donohue. 2001. Alterations in spontaneous contractions in vitro after repeated inflammation of rat distal colon. Am. J. Physiol. 280:G949. Shuttleworth, C. W., C. Xue, S. M. Ward, J. de Vente, and K. M. Sanders. 1993. Immunohistochemical localization of 3⬘,5⬘-cyclic guanosine monophosphate in the canine proximal colon: responses to nitric oxide and electrical stimulation of enteric inhibitory neurons. Neuroscience 56:513. Ward, D. T., S. A. Lawson, C. M. Gallagher, W. C. Conner, and T. Shea-Donohue. 2000. Sustained nitric oxide production via L-Arginine administration ameliorates effects of intestinal ischemia-reperfusion. J. Surg. Res. 89:13. Sjogren, R. W., C. Colleton, and T. Shea-Donohue. 1994. Intestinal myoelectric response in two different models of acute enteric inflammation. Am. J. Physiol. 267:G329.
5927 18. Hines, I. N., H. Harada, S. Bharwani, K. P. Pavlick, J. M. Hoffman, and M. B. Grisham. 2001. Enhance post-ischemic liver injury in iNOS-deficient mice: a cautionary note. Biochem. Biophys. Res. Commun. 284:972. 19. Hernandez, L. A., M. B. Grisham, B. Twohig, K. E. Arfors, J. M. Harlan, and D. N. Granger. 1987. Role of neutrophils in ischemia-reperfusion-induced microvascular injury. Am. J. Physiol. 253:H699. 20. Simpson, R., R. Alon, L. Kobzik, C. R. Valeri, D. Shepro, and H. B. Hechtman. 1993. Neutrophil and nonneutrophil-mediated injury in intestinal ischemia-reperfusion. Ann. Surg. 218:444. 21. Quigg, R. J., C. He, A. Lim, D. Berthiaume, J. J. Alexander, D. Kraus, and V. M. Holers. 1998. Transgenic mice overexpressing the complement inhibitor Crry as a soluble protein are protected from antibody-induced glomerular injury. J. Exp. Med. 188:1321. 22. Davoust, N., S. Nataf, R. Reiman, V. M. Holers, I. L. Campbell, and S. R. Barnum. 1999. Central nervous system-targeted expression of the complement inhibitor sCrry prevents experimental allergic encephalomyelitis. J. Immunol. 163:6551. 23. Zhang, M., M. Xu, J. Austen, J. Hermingoda, W. Caine, L. Kobzik, H. B. Hechtman, F. D. Moore, and M. C. Carroll. 2001. Natural IgM mediated ischemia/reperfusion injury. Clin. Immunol. 99:143. 24. Fleming, S. D., S. T. Rehrig, J. M. Guthridge, T. Shea-Donohue, G. C. Tsokos, and V. M. Holers. 2000. Role of CR2- and CD19-regulated natural antibodies in intestinal ischemia reperfusion-induced injury. Immunopharmacology 49:22. 25. Kubes, P., J. Hunter, and D. N. Granger. 1992. Ischemia/reperfusion-induced feline intestinal dysfunction: importance of granulocyte recruitment. Gastroenterology 103:807.
