AJP - GI Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 286: G791-G796, 2004. First published December 23, 2003; doi:10.1152/ajpgi.00407.2003
0193-1857/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/5/G791    most recent
00407.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by von Dobschuetz, E.
Right arrow Articles by Messmer, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by von Dobschuetz, E.
Right arrow Articles by Messmer, K.

INFLAMMATION/IMMUNITY/MEDIATORS

Soluble complement receptor 1 preserves endothelial barrier function and microcirculation in postischemic pancreatitis in the rat

E. von Dobschuetz,1 O. Bleiziffer,2 S. Pahernik,2,3 M. Dellian,4 T. Hoffmann,5 and K. Messmer2

1Department of General and Visceral Surgery, Albert Ludwigs University, 79106 Freiburg; 2Institute for Surgical Research, Ludwig Maximilians University, 81366 Munich; 3Department of Urology, University of Mainz, 55101 Mainz; 4Department of Otorhinolaryngology, Ludwig Maximilians University, 81377 Munich, Germany; and 5Maria Theresia Klinik, 80336 Munich, Germany

Submitted 17 September 2003 ; accepted in final form 18 December 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Components of the activated complement cascade are considered to play a pivotal role in ischemia-reperfusion-induced organ injury. With the use of intravital epifluorescence microscopy, we investigated the effect of complement inhibition by the recombinant soluble complement receptor 1 (sCR1; TP10) on the effect of macromolecular microvascular permeability, functional capillary perfusion, and leukocyte endothelium interaction in postischemic pancreatitis. Anaesthetized Sprague-Dawley rats were subjected to 60 min of normothermic pancreatic ischemia induced by microclipping of the blood-supplying arteries of the organ. Rats who received sCR1 (15 mg/kg body wt iv; n = 7) during reperfusion showed a significant reduction of permeability (1.77 ± 1.34 x 10-8 cm/s; n = 7) of tetramethylrhodamine isothiocyanate-labeled albumin injected 90 min after the onset of reperfusion compared with vehicletreated animals (6.95 ± 1.56 x 10-8 cm/s; n = 7). At 120 min after the onset of reperfusion, the length of red blood cell-perfused capillaries (functional capillary density) was significantly improved (from 279 ± 15.7 to 330 ± 3.7 cm-1; n = 7) and the number of leukocytes adherent to postcapillary venules was significantly reduced (from 314 ± 87 to 163 ± 71 mm-2; n = 7) by sCR1 compared with vehicle treatment. Complement inhibition by sCR1 effectively ameliorates pancreatic ischemia-reperfusion-induced microcirculatory disturbances and might be considered for treatment of postischemic pancreatitis.

permeability; ischemia; reperfusion; acute pancreatitis; transplantation

ISCHEMIA-REPERFUSION IS A causative factor in the pathogenesis of acute pancreatitis after mesenteric artery embolization (20), cardiac bypass operation (5), and the early inflammatory response after pancreas transplantation (21). The stimulus of ischemia-reperfusion induces generation of oxidative stress and then apoptosis of endothelial cells, which are shown to activate the complement cascade by the classical (12) and lectin (4)-mediated pathways. Furthermore, ischemia-reperfusion in the pancreas is considered to activate and release trypsin, which is a well-known activator of the complement cascade (5). Microcirculatory pancreatic damage showing impairment of functional capillary perfusion (7), activation and adhesion of leukocytes in postcapillary venules (7), and increase of endothelial macromolecular permeability (3) are the crucial hallmarks of early reperfusion. Recombinant soluble complement receptor 1 (sCR1; TP10) has proven its therapeutic usefulness in ischemia-reperfusion injury of myocardium (31), skeletal muscle (17), and small intestine (32) through blocking of the complement cascade during the early onset of reperfusion. Furthermore, sCR1 ameliorated graft function after experimental kidney (18), lung (15), and liver transplantation (10). Because of all of these studies, reduction of microcirculatory disturbances by inhibition of leukocyte adherence and preservation of the endothelial barrier function was considered as the decisive effect of sCR1 in reperfusion injury. Although graft pancreatitis is one of the major complications of pancreas transplantation, until now there has been no reliable prevention of this early postischemic microcirculatory disturbance. The aim of this study was to investigate the effect of sCR1 on the early microcirculatory deterioration during ischemia-reperfusion-induced pancreatitis using a novel method of measuring macromolecular permeability combined with analyzed leukocyte adherence and functional capillary perfusion by means of intravital microscopic technique.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Anesthesia and monitoring. The experimental protocol of this study was approved by the local ethical governmental committee and conforms with American Physiological Society Guiding Principles in the Care and Use of Animals. Male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) weighing 180–260 g were anaesthetized with ether and pentobarbital (50 mg/kg body wt ip) after an overnight fast with free access to tap water. After tracheotomy, respiration was volume controlled (frequency, 57–65 breaths/min; tidal volume, 2–2.5 ml; FIO2, 0.25–0.40; Harvard Rodent Ventilator model 683; Harvard Apparatus, Hollistan, MA). The right carotid artery and the right jugular vein were cannulated with a polyethylene catheter (PE-50; 0.58 mm inner diameter; Portex, Hythe, UK) for continuous monitoring of mean arterial pressure (MAP) and heart rate. Rectal temperature was kept between 36.5 and 37.5°C by means of a heating pad (Fa Effenberger, Pfaffing, Germany). Adequate anesthesia was maintained by intravenous infusion of pentobarbital (12 mg·kg body wt-1·h-1) and N2O admixture (0.65–0.75) to the inspired air. Arterial blood gases, acid/base status, and blood lactate concentration were measured intermittently by a blood gas analyzer (Ciba Corning 860; Chiron Diagnostics, Fernwald, Germany) and were adjusted to the following values for baseline conditions by means of ventilator adjustment and intravenous injection of 8.4% : PO2, 100–120 mmHg; PCO2, 30–40 mmHg; pH, 7.39 ± 0.02; and base excess, 0 ± 2. During the experiment, arterial PO2 was kept >90 mmHg by adjustment of FIO2 and PCO2 was kept >45 mmHg by alteration of the breathing frequency. Hematocrit in arterial blood was measured by a Coultercounter T540 (Coulter Electronics, Hialeah, FL).

Animal model and experimental protocol. After transverse laparotomy, complete ischemia of the pancreas was induced by clipping the four blood-supplying arteries (left gastric artery, gastroduodenal artery, splenic artery, and caudal pancreaticoduodenal artery) to the pancreas by means of microvascular clips (closing force 70 g; Aesculap, Tuttlingen, Germany). The complete microsurgical technique was described previously (7). Sham-operated animals underwent the surgical procedure without induction of ischemia. After a stabilization period of 15 min, animals were randomly assigned to three groups: 1) sham-operated group without ischemia (sham control, n = 7); 2) 1-h ischemia group receiving 3 ml/kg body wt iv vehicle (ischemia control, n = 7); and 3) 1-h ischemia group receiving 15 mg/kg body wt iv sCR1 (ischemia sCR1, n = 7). sCR1 was further injected into three sham-operated animals (15 mg/kg body wt); they showed no significant differences compared with animals of the sham control group (data not shown). The dose (15 mg/kg body wt) of sCR1 used has proven its efficacy in other experiments in rats (10). Arterial blood samples (1 ml) were taken for assay of serum total hemolytic complement activity before induction of ischemia at the end of the experiment and were immediately replaced by aliquots of saline. Fifteen minutes after administration of the solutions, the pancreas and spleen were exteriorized on an adjustable microscope stage and covered by a thin transparent plastic membrane to prevent drying. To avoid cooling of the organs, the whole animal and the stage were covered by swabs between the microscopy measurements. MAP was continuously determined and registered on a recorder (Siemens XT Kompensograph; Siemens, Munich, Germany). Arterial blood gases from the carotid artery and hematocrit were measured at baseline conditions and 15, 60, and 120 min after administration of the solutions. The experiments were discontinued by an intravenous injection of an overdose of pentobarbital.

Drugs. Lyophilized sCR1 (TP10) was kindly provided by AVANT Immunotheraputics (Needham, MA). The vials contained mannitol (80 mg), monobasic potassium phosphate (4.36 mg), dibasic potassium phosphate (5.76 mg), and sodium chloride (5.48 mg) as buffer salt. With the use of water for injection as reconstitution fluid, a solution containing 5 mg/ml sCR1 was prepared, aliquoted into sterile syringes, and frozen at -20°C until use. Vehicle was prepared containing the same buffer salt concentration.

Intravital microscopy and quantification of microvascular parameters. Intravital fluorescence microscopy of the pancreas was performed by using a modified Leitz-Orthoplan microscope (Leitz, Wetzlar, Germany) with a mercury lamp (100 W; HBO; Leitz) attached to a Ploemo-Pak illuminator with I2/3 (excitation 450–490 nm, emission >515 nm, used for permeability and leukocyte-adherence) and N2 [excitation 530–560 nm, emission >580 nm, used for functional capillary density (FCD)] filterblocks (Leitz) for epi-illumination. Microvascular macromolecular permeability was measured by using the following setup. To enable subtraction of tissue autofluorescence, a randomly selected capillary field (region of interest) of the exocrine pancreas was acquired by using a light immersion objective (L x 32/0.4; Leitz) and a silicon-intensified target video camera (model C2400-08; Hamamatsu, Herrsching, Germany). The picture was recorded on videotape (video recorder: AG-Panasonic, Munich, Germany) for offline evaluation. Rhodamin-labeled BSA (40 mg/kg body wt; Molecular Probes, Leiden, The Netherlands) was intravenously injected, and the identical capillary field was recorded during the first 6 min after injection every 30 s and up to 20 min every 60 s. Fluorescence illumination was reduced to 2 s per time point to avoid photoxic effects and bleaching. Recorded pictures were digitized by an image analysis system (model IBAS 2000; Kontron, Eching, Germany), and the fluorescence intensity of the capillary field was measured offline after subtraction of tissue autofluorescence obtained before the injection by densitometric measurement. Effective macro-molecular permeability of albumin was calculated as described previously (29) using the following formula of Yuan et al. (34): P = (1 - HTm) VVes/S (1/Im0 dIm/dt + 1/k), where P is the vascular permeability based on Im0, which represents the average fluorescence intensity (Im) of the capillary area recorded immediately after the vessels were filled with tracer molecules. In separate experiments (n = 3), the time constant (k) of plasma clearance of rhodamin-labeled BSA was quantified by collecting arterial blood samples at t = 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, and 60 min after injection and fitting these data on an exponential function. Vessel volume (Vves) pro-to-surface area (S ratio) in a pancreatic capillary field was analyzed in three animals by measuring the length of capillaries and the diameter with an CAMAs image analyzing system (developed by Dr. Zeintl, Heidelberg, Germany) (9). The dynamic microhematocrit (HTm) of the pancreatic capillary field was measured in a separate experiment by the method described by Sarelius and Duling (22) using the following formula: HTm = (FRBC/LF x MCV x 100)/[VRBC x (D/2)2].

Briefly, rat erythrocytes were labeled with fluoresceinisothiocyanate (Sigma, St Louis, MO). The erythrocytes were injected into the animal, and red blood cell flux (FRBC) of labeled cells, the average cell velocity (VRBC), and diameter of the vessel (D) were measured in the pancreatic capillary fields. Labeled cell fraction (LF) of the whole erythrocyte volume was determined by using a fluorescence-activated cell sorter analyzer (Becton Dickinson, Heidelberg, Germany). Mean corpuscular volume (MCV) was calculated from a full blood cell count (Coulter Electronics, Hialeah, FL). FCD and leukocyte adherence were measured by the following setup: 0.2 ml of 2.5% BSA labeled with the fluorochrome fluorescein isothiocyanate (Sigma) for contrast enhancement of microvessels and 0.1 ml 0.2% rhodamin 6G (497 mol wt; Sigma) for in vivo staining of cytochrome c-containing cells (leukocytes) was injected intravenously into the right jugular vein before the measurement time point. A saltwater immersion objective (SW x25/0.6; Leitz) allowed magnification of approximately x800. Observations were recorded by means of a chargecoupled device (CCD) video camera (model FK 6990; Cohu, Prospective Measurements, San Diego, CA) and stored on videotape (video recorder; AG-Panasonic) for offline evaluation. Quantitative assessment of the microcirculation included determination of the FCD and the number of adherent leukocytes in the postcapillary venules. These parameters were measured at 120 min after the start of reperfusion. FCD is defined as the length of red blood cell perfused capillaries (cm) per observation area (cm2) (16). The FCD was determined by analysis of the videotapes using CAMAs image analyzing system (9). Ten randomly selected regions of interest (400 x 300 µm) of the pancreas were evaluated. For quantification of leukocyte-endothelial interaction, at least three postcapillary venules (<40 µm diameter and <150 µm length) per animal were recorded for 30 s. Adherent leukocytes were defined as cells remaining stationary on the surface of the endothelium for the whole observation time of 30 s. The surface area of the vessel segments was calculated based on diameter measurement, assuming a cylindrical geometry of the vessels. Adherent leukocytes are given as cells per endothelial surface (mm-2).

Assay of serum total hemolytic C activity. Blood was withdrawn through the arterial catheter and centrifuged (3,000 rpm, 4°C, 10 min) to obtain serum. The samples were then stored at -70°C until assayed. For an assay, 5 µl of serum were added to wells placed in agarose gel containing standardized sheep erythrocytes sensitized with hemolysin (kit RC001; The Binding Site, San Diego, CA). Plates were incubated for 18 h at 4°C and then for 1 h at 37°C. Areas of the zones of hemolysis around each well (radial immunodiffusion) were measured by imaging these zones with a CCD camera after scanning of the images. Their relative optical densities were calculated by using software-based evaluation for analyzing electrophoretic gels. These values were converted to total hemolytic C activity (CH100) units (one unit being the amount of C that lyses 100% of the erythrocytes) by interpolation from calibration curves plotted using the manufacturer's standard, diluted from neat to 1:32 (minimum sensitivity, 32 CH100 units).

Statistical analysis. All data are presented as means ± SD. Data analyses were performed by using a statistical software package (Sigma Stat 2.0; Jandel Scientific Software, San Rafael, CA). Data comparison between the groups were tested by Mann-Whitney U-test followed by a Bonferroni-Holm correction. Within each group, Friedman repeated-measures ANOVA on ranks followed by Dunnett's method was performed. P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Macrohemodynamics and general data. There was no significant difference of MAP in sham operated animals during the whole observation time (Fig. 1). In both ischemia groups, MAP dropped significantly compared with baseline values and sham-operated animals at the early onset of reperfusion. In the sCR1-treated group, this drop of MAP could not be reversed completely and was more severe compared with initial control values during the whole reperfusion time. Whereas there were no significant changes in lactate values (Table 1) in the sCR1-treated and sham-operated animals, lactate values were significantly increased in the vehicle-treated ischemia group 120 min after reperfusion. Hematocrit and negative base excess values were significantly higher 120 min after reperfusion in both ischemic groups compared with sham-operated animals.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1. Mean arterial pressure before (-60 min) and after (up to 120 min) reperfusion in animals with sham operation ({bullet}), 60-min pancreatic ischemia and 3 ml/kg vehicle infusion ({circ}), and 60-min pancreatic ischemia and 15 mg/kg recombinant soluble complement receptor 1 (sCR1) infusion ({blacktriangledown}). Values are means ± SD; *P < 0.05 vs. sham; #P < 0.05 vs. -60 min.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Lactate, hematocrit, and acid-base balance during the experiment

 

Microvascular permeability. At 90 min after reperfusion, vehicle-treated animals showed a significant increase (6.95 ± 1.56 x 10-8 cm/s; n = 7, P < 0.01) of rhodamin-labeled albumin permeability compared with the sham group (3.18 ± 0.7 x 10-8 cm/s; n = 7) (Fig. 2). sCR1 treatment resulted in significant (P < 0.01) reduction of permeability (1.77 ± 1.34 x 10-8 cm/s; n = 7) when compared with the vehicletreated group. There was no significant difference of micro-vascular permeability between sham-operated and sCR1-treated animals undergoing ischemia.



View larger version (8K):
[in this window]
[in a new window]
  		Fig. 2. Microvascular permeability of rhodamin-labeled albumin 90 min after the onset of reperfusion. Values are means ± SD. #P < 0.01 vs. sham and ischemia + sCR1. There were no significant differences between sCR1 and sham.

 

FCD. FCD (Fig. 3) was reduced to 279 ± 15.7 cm-1 (n = 7, P < 0.01) and 330 ± 3.7 cm-1 (n = 7, P < 0.01) in vehicle- and sCR1-treated ischemia groups, respectively, compared with sham-operated animals (393 ± 8.9 cm-1; n = 7). Treatment with sCR1 was associated with improved FCD (P < 0.01) compared with the vehicle-treated group.



View larger version (8K):
[in this window]
[in a new window]
  		Fig. 3. Functional capillary density in exocrine pancreas 120 min after reperfusion. Values are means ± SD; #P < 0.01 vs. sham and ischemia + sCR1; *P < 0.01 vs. sham.

 

Leukocyte-endothelium interaction. Normothermic ischemia-reperfusion of the pancreas resulted in a significant (314 ± 87 mm-2, n = 7) increase of leukocyte adherence in postcapillary pancreatic venules (P < 0.01) compared with the shamoperated group (128 ± 47 mm-2; n = 7) (Fig. 4). Intravenous sCR1 treatment during reperfusion reduced this increase to values (163 ± 71 mm-2; n = 7) not significantly different from values of sham-operated animals.



View larger version (8K):
[in this window]
[in a new window]
  		Fig. 4. Number of adherent leukocytes in postcapillary venules 120 min after reperfusion. Values are means ± SD; #P < 0.05 vs. sham and ischemia + sCR1 treatment.

 

Serum CH100. Total hemolytic complement activity given as the percentage of baseline values was significantly reduced in the vehicle-treated animals undergoing ischemia compared with the sham-operated group (Fig. 5). SCR1 treatment resulted in less reduction of hemolytic activity without significant difference compared with sham-operated and vehicletreated animals.



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5. Total hemolytic serum complement activity given in %baseline values. Values are means ± SD; #P < 0.01 vs. sham.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides evidence that complement activation plays a pivotal role in ischemia-reperfusion-induced pancreatitis. With the use of a method for measurement of macromolecular permeability in pancreatic tissue by intravital epifluorescence microscopy, we could show the prevention of increased microvascular permeability and edema formation by administration of sCR1 during the early reperfusion time. Furthermore, the activation of leukocyte-endothelium interaction as well as the impairment of functional capillary perfusion was significantly reduced by intravenous injection of sCR1 during the onset of reperfusion.

The recombinant soluble form of CR1 (TP10), has proven to block the complement pathway being a cofactor of degradation of classical and alternative C3 convertase and enabling degradation of C3b to the inactive form iC3b. SCR1 is considered to block alternative and classical complement pathway on a very early level. Next to its therapeutic effectiveness in other models of ischemia-reperfusion injury as in liver (10), heart (31), intestine (32), kidney, and lung (15), its efficacy on the postischemic pancreas was unknown. Complement activation by ischemia-reperfusion is partly dependent on the generation of hydroxyl radicals as shown by the prevention of radical generation in an intestine model of experimental ischemia-reperfusion injury (25). Furthermore, it has been shown that complement may mediate ischemia-reperfusion-induced tissue damage by nitration and attenuation of superoxide dismutase (13). In contrast to other organs, the pancreas reacts on ischemia-reperfusion stimulus with circulatory liberation of digestive enzymes like trypsin (11), which are considered to be additional potent complement-activating factors (19). It is quite controversial whether all components of the activated complement system are proinflammatory noxes in the reaction of the pancreas. A hyperexpressing C5a mouse had less inflammatory tissue damage after induction of acute pancreatitis, leading to the conclusion that this single complement might be protective by an anti-inflammatory effect (2). This might be because complex single components of the complement system have their specific tasks and differences. Furthermore, application of sCR1 to the cerulein model of acute pancreatitis failed to ameliorate pancreatic edema or lung damage (30). Both cited studies used a pancreatitis model involving cerulein as an inflammatory stimulus to the pancreas, which is very much different from the ischemia-reperfusion damage our results show. In postischemic pancreatitis, blocking the complement system on a very early step of the cascade resulted in an amelioration of pancreatic microcirculatory reperfusion damage.

Edema formation is one of the most obvious signs of microcirculatory derangement during acute pancreatitis. According to the Starling hypothesis, fluid transport across the endothelial barrier is driven by the hydrostatic and oncotic pressures inside and outside of the microvessel wall (23). An increased permeability of the microvascular wall for albumin results in a decreased intravascular oncotic pressure. Due to this decreased intravasal oncotic force, fluid can easily be extravasated, resulting in edema of the organ, hemoconcentration, and fluid loss from systemic circulation that triggers development of hypovolemic shock state. Furthermore, the increased hydrostatic pressure of the tissue could further compromise microvascular perfusion and hinder oxygen diffusion, due to the increased space between microvessels. Although newer data from ex vivo and mathematical experiments suggest that convective flux across a capillary barrier is independent from different variables, like the local protein concentration underneath the endothelial glycocalix (8), the Starling model is probably the most suitable for explanation and determination of edema formation in the in vivo experiment. Methods so far used in estimation of edema formation, capillary leakage, and macromolecular permeability, e.g., Evans blue extravasation and organ wet/dry ratio, are invasive and thus difficult to apply together with quantitative microvascular analysis. They provide no specific information of the specific role of albumin, which is probably the most important oncotic component of the plasma. By applying a method developed for intravital microscopy of tumor microcirculation (29), we could show that activation of the complement system during the early onset of reperfusion is a crucial event of the initiation of the edema formation in postischemic pancreatitis.

In the present study, we could observe an increase of the functional capillary perfusion during the reperfusion by sCR1 treatment. Although we found a significant difference between both ischemic groups compared with the control group, FCD is not significantly different at the end of the experiment when microcirculatory data are measured. Therefore, in our opinion, the existing tendencies of differences of MAP during reperfusion cannot explain the differences seen in FCD and micro-vascular permeability after 90 and 120 min. Capillary "noreflow" is a crucial hallmark of microcirculatory dearrangement of the pancreatic ischemia-reperfusion damage (7, 27). Because erythrocyte-perfused capillaries are very important for the oxygen supply to the tissue, this parameter gives valuable information on tissue viability. By measuring pancreatic tissue oxygen pressure in patients during the early reperfusion period after pancreas transplantation, it could be shown that the increase of oxygen pressure during reperfusion correlates negatively with the highest concentration of C-reactive protein (1). This gives an indirect proof that diminished oxygen supply during reperfusion is a very important factor for the development of the early reperfusion injury.

Leukocyte-endothelium interaction is a crucial event in ischemia-reperfusion-induced pancreatitis. Complement activation results in formation of proinflammatory components like C3a, C5a, iC3b, and the membrane attack complex C5b-9. These components are associated with promotion of leukocyte activation via upregulation of CD18/11b (33), ICAM-1 (26), and selectin adhesion molecules (14). Activated leukocytes can further release proteases like cathepsin G and neutrophil elastase, which are considered to have proinflammatory potential. Using the same animal model, we have previously shown that inhibition of such proteases by a recombinant serpin could effectively reduce the postischemic capillary perfusion deficit (28), suggesting the active role of leukocytes in postischemic pancreatitis. It has been shown that the development of pancreatic injury in severe pancreatitis is associated with the increased endothelial expression of ICAM-1 (24). With the use of serum obtained from rats after induction of acute necrotizing pancreatitis, Hartwig et al. (6) have shown that trypsin-generated complement activation participates in the upregulation of the integrin MAC-1 on neutrophils, which, in turn, is known to enable the firm adhesion of phagocytes to endothelium by binding to ICAM-1. The addition of sCR1 reduced this upregulation of MAC-1. Our experiments showed the reduction of leukocyte adhesion in postcapillary venules of the pancreas by the injection of sCR1 after normothermic ischemia and reperfusion. Therefore, complement activation may well account for the adhesion of activated leukocytes in postcapillary venules, probably by an ICAM-1-dependent mechanism in postischemic pancreatitis.

Total hemolytic complement activity was reduced after ischemia-reperfusion of the pancreas in vehicle-treated animals, which might be a sign of complement activation in the pathophysiological setup of ischemia-reperfusion-induced pancreatitis. sCR1 treatment resulted in a reduction of microcirculatory reperfusion damage. Hemolytic complement activity was still reduced after sCR1 treatment compared with baseline values, which is similar to the data obtained after kidney transplantation and sCR1 treatment in rats (18). Unfortunately, the cited paper shows no data of complement activity in the transplantation group not receiving sCR1, making the interpretation and comparison of the significant reduction of hemolytic activity in the vehicle-treated control group very difficult. The measured parameter might thus resemble, on the one hand, increased consumption of complement components by postischemic pancreatitis and, on the other hand, a sustained blocking of the complement system by sCR1, which would be expected to be more pronounced during the first 30 min after sCR1 injection.

In conclusion, the data presented are the first proof that blocking the complement system by sCR1 in the early reperfusion period is an effective approach for preservation of microcirculatory disturbances such as increased macromolecular permeability, capillary perfusion failure, and leukocyte adherence. Thus sCR1 might become a therapeutic option for prevention of reperfusion injury in the clinical setting of pancreas transplantation.


   ACKNOWLEDGMENTS
 
We thank Dr. C. Pettey, AVANT Immunotherapeutics, Needham, MA, for generously providing sCR1.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. von Dobschuetz, Dept. of General and Visceral Surgery, Albert Ludwigs Univ., Hugstetter Str. 55, 79106 Freiburg, Germany (E-mail: ernst{at}von-dobschuetz.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Benz S, Bergt S, Obermaier R, Wiessner R, Pfeffer F, Schareck W, and Hopt UT. Impairment of microcirculation in the early reperfusion period predicts the degree of graft pancreatitis in clinical pancreas transplantation. Transplantation 71: 759-763, 2001.[CrossRef][Web of Science][Medline]
  2. Bhatia M, Saluja AK, Singh VP, Frossard JL, Lee HS, Bhagat L, Gerard C, and Steer ML. Complement factor C5a exerts an anti-inflammatory effect in acute pancreatitis and associated lung injury. Am J Physiol Gastrointest Liver Physiol 280: G974-G978, 2001.[Abstract/Free Full Text]
  3. Bockman DE. Microvasculature of the pancreas: relation to acute pancreatitis. Int J Pancreatol 12: 11-21, 1992.[Web of Science][Medline]
  4. Collard CD, Vakeva A, Morrissey MA, Agah A, Rollins SA, Reenstra WR, Buras JA, Meri S, and Stahl GL. Complement activation after oxidative stress: role of the lectin complement pathway. Am J Pathol 156: 1549-1556, 2000.[Abstract/Free Full Text]
  5. Fernandez del Castillo C, Harringer W, Warshaw AL, Vlahakes GJ, Koski G, Zaslavsky AM, and Rattner DW. Risk factors for pancreatic cellular injury after cardiopulmonary bypass. N Engl J Med 325: 382-387, 1991.[Abstract]
  6. Hartwig W, Jimenez RE, Fernandez-del Castillo C, Kelliher A, Jones R, and Warshaw AL Expression of the adhesion molecules Mac-1 and L-selectin on neutrophils in acute pancreatitis is protease- and complement-dependent. Ann Surg 233: 371-378, 2001.[CrossRef][Web of Science][Medline]
  7. Hoffmann TF, Leiderer R, Waldner H, Arbogast S, and Messmer K. Ischemia-reperfusion of the pancreas: a new in vivo model for acute pancreatitis in rats. Res Exp Med (Berl) 195: 125-144, 1995.
  8. Hu X and Weinbaum S. A new view of Starling's hypothesis at the microstructural level. Microvasc Res 58: 281-304, 1999.[CrossRef][Web of Science][Medline]
  9. Klyscz T, Jünger M, Jung F, and Zeintl H. Cap image–a new kind of computer-assisted video image analysis system for dynamic capillary microscopy. Biomed Tech (Berl) 42: 168-175, 1997.
  10. Lehmann TG, Koeppel TA, Kirschfink M, Gebhard MM, Herfarth C, Otto G, and Post S. Complement inhibition by soluble complement receptor type 1 improves microcirculation after rat liver transplantation. Transplantation 66: 717-722, 1998.[CrossRef][Web of Science][Medline]
  11. Mithöfer K, Fernandez del Castillo C, Frick TW, Foitzik T, Bassi DG, Lewandrowski KB, Rattner DW, and Warshaw AL. Increased intrapancreatic trypsinogen activation in ischemia-induced experimental pancreatitis. Ann Surg 221: 364-371, 1995.[Web of Science][Medline]
  12. Mold C and Morris CA. Complement activation by apoptotic endothelial cells following hypoxia/reoxygenation. Immunology 102: 359-364, 2001.[CrossRef][Web of Science][Medline]
  13. Montalto MC, Hart ML, Jordan JE, Wada K, and Stahl GL. Role for complement in mediating intestinal nitric oxide synthase-2 and superoxide dismutase expression. Am J Physiol Gastrointest Liver Physiol 285: G197-G206, 2003.[Abstract/Free Full Text]
  14. Mulligan MS, Schmid E, Till GO, Hugli TE, Friedl HP, Roth RA, and Ward PA. C5a-dependent up-regulation in vivo of lung vascular P-selectin. J Immunol 158: 1857-1861, 1997.[Abstract]
  15. Naka Y, Marsh HC, Scesney SM, Oz MC, and Pinsky DJ. Complement activation as a cause for primary graft failure in an isogeneic rat model of hypothermic lung preservation and transplantation. Transplantation 64: 1248-1255, 1997.[CrossRef][Web of Science][Medline]
  16. Nolte D, Zeintl H, Steinbauer M, Pickelmann S, and Messmer K. Functional capillary density: an indicator of tissue perfusion? Int J Microcirc Clin Exp 15: 244-249, 1995.[Web of Science][Medline]
  17. Pemberton M, Anderson G, Vetvicka V, Justus DE, and Ross GD. Microvascular effects of complement blockade with soluble recombinant CR1 on ischemia/reperfusion injury of skeletal muscle. J Immunol 150: 5104-5113, 1993.[Abstract]
  18. Pratt JR, Hibbs MJ, Laver AJ, Smith RA, and Sacks SH. Effects of complement inhibition with soluble complement receptor-1 on vascular injury and inflammation during renal allograft rejection in the rat. Am J Pathol 149: 2055-2066, 1996.[Abstract]
  19. Roxvall L, Bengtson A, Sennerby L, and Heideman M. Activation of the complement cascade by trypsin. Biol Chem Hoppe Seyler 372: 273-278, 1991.[Web of Science][Medline]
  20. Sakorafas GH, Tsiotos GG, Bower TC, and Sarr MG. Ischemic necrotizing pancreatitis–two case reports and review of the literature. Int J Pancreatol 24: 117-121, 1998.[Web of Science][Medline]
  21. Sakorafas GH, Tsiotos GG, and Sarr MG. Ischemia/reperfusion-induced pancreatitis. Dig Surg 17: 3-14, 2000.[CrossRef][Web of Science][Medline]
  22. Sarelius IH and Duling BR. Direct measurement of microvessel hematocrit, red cell flux, velocity, and transit time. Am J Physiol Heart Circ Physiol 243: H1018-H1026, 1982.[Abstract/Free Full Text]
  23. Starling EH. On the absorption of fluids from the convective tissue spaces. J Physiol 19: 312-326, 1896.
  24. Telek G, Ducroc R, Scoazec JY, Pasquier C, Feldmann G, and Roze C. Differential upregulation of cellular adhesion molecules at the sites of oxidative stress in experimental acute pancreatitis. J Surg Res 96: 56-67, 2001.[CrossRef][Web of Science][Medline]
  25. Turnage RH, Magee JC, Guice KS, Myers SI, and Oldham KT. Complement activation by the hydroxyl radical during intestinal reperfusion. Shock 2: 445-450, 1994.[Web of Science][Medline]
  26. Vaporciyan AA, Mulligan MS, Warren JS, Barton PA, Miyasaka M, and Ward PA. Up-regulation of lung vascular ICAM-1 in rats is complement dependent. J Immunol 155: 1442-1449, 1995.[Abstract]
  27. Von Dobschuetz E, Hoffmann T, Engelschalk C, and Messmer K. Effect of diaspirin cross-linked hemoglobin on normal and postischemic microcirculation of the rat pancreas. Am J Physiol Gastrointest Liver Physiol 276: G1507-G1514, 1999.[Abstract/Free Full Text]
  28. Von Dobschuetz E, Hoffmann T, and Messmer K. Inhibition of neutrophil proteinases by recombinant serpin Lex032 reduces capillary noreflow in ischemia/reperfusion-induced acute pancreatitis. J Pharmacol Exp Ther 290: 782-788, 1999.[Abstract/Free Full Text]
  29. Von Dobschuetz E, Pahernik SA, Hoffmann T, Kiefmann R, Heckel K, Messmer K, Mueller-Hoecker J, and Dellian M. Dynamic intravital fluorescence microscopy–a novel method for the assessment of microvascular permeability in acute pancreatitis. Microvasc Res 67: 55-63, 2004.[CrossRef][Web of Science][Medline]
  30. Weiser MR, Gibbs SA, Moore FD, and Hechtman HB. Complement inhibition by soluble complement receptor type 1 fails to moderate cerulein-induced pancreatitis in the rat. Int J Pancreatol 19: 129-134, 1996.[Web of Science][Medline]
  31. Weisman HF, Bartow T, Leppo MK, Marsh HCJ, Carson GR, Concino MF, Boyle MP, Roux KH, Weisfeldt ML, and Fearon DT. Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis. Science 249: 146-151, 1990.[Abstract/Free Full Text]
  32. Williams JP, Pechet TT, Weiser MR, Reid R, Kobzik L, Moore FDJ, Carroll MC, and Hechtman HB. Intestinal reperfusion injury is mediated by IgM and complement. J Appl Physiol 86: 938-942, 1999.[Abstract/Free Full Text]
  33. Witthaut R, Farhood A, Smith CW, and Jaeschke H. Complement and tumor necrosis factor-{alpha} contribute to Mac-1 (CD11b/CD18) up-regulation and systemic neutrophil activation during endotoxemia in vivo. J Leukoc Biol 55: 105-111, 1994.[Abstract]
  34. Yuan F, Leunig M, Berk DA, and Jain RK. Microvascular permeability of albumin, vascular surface area, and vascular volume measured in human adenocarcinoma LS174T using dorsal chamber in SCID mice. Microvasc Res 45: 269-289, 1993.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
W. Hartwig, M. Klafs, M. Kirschfink, T. Hackert, L. Schneider, M.-M. Gebhard, M. W. Buchler, and J. Werner
Interaction of complement and leukocytes in severe acute pancreatitis: potential for therapeutic intervention.
Am J Physiol Gastrointest Liver Physiol, November 1, 2006; 291(5): G844 - G850.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
286/5/G791    most recent
00407.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (9)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by von Dobschuetz, E.
Right arrow Articles by Messmer, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by von Dobschuetz, E.
Right arrow Articles by Messmer, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.