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,
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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 self-tissues 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 decay-accelerating 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).
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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 µm), 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, 15, 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 one-way 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A and 2). As expected, the
mucosa of the animals subjected to mesenteric IR for 2 h displayed
signs 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).
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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 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).
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Effect of Crry-Ig administration on small intestinal eicosanoid generation
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, 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 sham-operated 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.
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). 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.
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 between 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.
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Footnotes |
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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: tshea{at}usuhs.mil
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.
Received for publication May 23, 2001. Accepted for publication September 12, 2001.
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) and villus height (micrometers;
) as described in Materials and Methods. Villus
heights of individual villi were measured using an ocular micrometer.
All measurements were obtained at x200 magnification. Each bar is the
average ± SEM with six to eight animals per group. *,
Significant difference from sham group, p < 0.05
(by ANOVA with Newman-Keuls post hoc test).
