Journal of Vascular Surgery
Volume 33, Issue 4 , Pages 840-846, April 2001

Recombinant bactericidal/permeability–increasing protein attenuates the systemic inflammatory response syndrome in lower limb ischemia-reperfusion injury☆☆☆★★★

Presented in part to the Surgical Research Society, Plenary Session, London, UK, Dec 3, 1999.

  • Denis W. Harkin, MD

      Affiliations

    • Department of Surgery, The Queen's University of Belfast Belfast, Northern Ireland
    • ,
  • Aires A.B. Barros D'Sa, MD

      Affiliations

    • Vascular Surgical Unit, The Royal Victoria Hospital. Belfast, Northern Ireland
    • ,
  • Magdi M.I. Yassin, MD

      Affiliations

    • Department of Surgery, The Queen's University of Belfast Belfast, Northern Ireland
    • ,
  • Margaret Hoper, PhD

      Affiliations

    • Department of Surgery, The Queen's University of Belfast Belfast, Northern Ireland
    • ,
  • M.Isla Halliday, PhD

      Affiliations

    • Department of Surgery, The Queen's University of Belfast Belfast, Northern Ireland
    • ,
  • T.George Parks, MD

      Affiliations

    • Department of Surgery, The Queen's University of Belfast Belfast, Northern Ireland
    • ,
  • F.Charles Campbell, MD

      Affiliations

    • Department of Surgery, The Queen's University of Belfast Belfast, Northern Ireland

Received 13 April 1999; accepted 14 August 2000.

Article Outline

Abstract 

Objectives: Hind limb ischemia-reperfusion (I/R) injury increases gut permeability, and resultant endotoxemia is associated with an amplified systemic inflammatory response syndrome leading to multiple organ dysfunction syndrome. We studied the potential role of recombinant bactericidal/permeability–increasing protein (rBPI 21 ), a novel antiendotoxin therapy, in modulating endotoxin-enhanced systemic inflammatory response syndrome in hind limb I/R injury. Methods: In this prospective, randomized, controlled, experimental animal study, 48 male Wistar rats, weighing 300 to 350 g, were randomized to a control group (sham) and five groups undergoing 3 hours bilateral hind limb ischemia with 2 hours reperfusion (I/R) (n = 8 per group). The control and untreated I/R groups received thaumatin, a control- protein preparation, at 2 mg/kg. Treatment groups were administered rBPI 21 intravenously at 1, 2, or 4 mg/kg body weight at the beginning of reperfusion; an additional group was administered rBPI 21 intravenously at 2 mg/kg after 1 hour of reperfusion. Plasma interleukin-6 concentration was estimated by bioassay as a measure of systemic inflammation. Plasma endotoxin concentration was determined by use of an amebocyte lysate chromogenic assay. Crossreactive immunoglobulin G and M antibodies to the highly conserved inner core region of endotoxin were measured by use of an enzyme-linked immunosorbent assay. The lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were used as markers of edema and neutrophil sequestration, respectively. Results: I/R provoked highly significant elevation in plasma interleukin-6 concentrations (1351.20 pg/mL [860.16 - 1886.40 pg/mL]) compared with controls (125.32 pg/mL [87.76-157.52 pg/mL; P < .0001]), but treatment with rBPI 21 2 mg/kg at onset of reperfusion (715.89 pg/mL [573.36-847.76 pg/mL]) significantly decreased interleukin-6 response compared with the nontreatment group ( P < .016). I/R increased plasma endotoxin concentrations significantly (21.52 pg/mL [6.20-48.23 pg/mL]), compared with control animals (0.90 pg/mL [0.00-2.30 pg/mL; P < .0001]), and treatment with rBPI 21 4 mg/kg at reperfusion significantly decreased endotoxemia (1.30 pg/mL [1.20-2.20 pg/mL]), compared with the untreated group ( P < .001). The lung tissue myeloperoxidase level was significantly increased in the untreated I/R group (208.18% [128.79%-221.81%]), compared with in controls (62.00% [40.45%-80.92%; P < .0001]), and attenuated in those treated with rBPI 21 2 mg/kg (129.54% [90.49%-145.78%; P < .05]). Data represent median and interquartile range, comparisons made with the nonparametric Mann-Whitney U test. Conclusions: These findings show that hind limb ischemia-reperfusion injury is associated with endotoxemia, elevations in plasma interleukin-6, and pulmonary leukosequestration. Treatment with rBPI 21 after ischemia reduces endotoxemia, the interleukin-6 response, and attenuates pulmonary leukosequestration in response to hind limb reperfusion injury. (J Vasc Surg 2001;33:840-6.)

 

Hind limb ischemia-reperfusion injury (I/R) has been shown to initiate a systemic inflammatory response syndrome, 1, 2 leading to multiple organ failure, 3, 4 which carries a high mortality rate in spite of conventional intensive care support. 3, 5, 6 Acute pulmonary dysfunction has been demonstrated after experimental limb I/R injury 7, 8 and is a leading cause of morbidity and death after abdominal aortic aneurysm (AAA) surgery. 2, 6 Increased numbers of circulating activated neutrophils have been reported after limb I/R injury, 9, 10 and leukosequestration is a feature of postreperfusion lung injury. 11 The development of systemic inflammation after revascularization procedures represents a complex cascade of proinflammatory mediator, 12 cytokine production 2, 10 and leukocyte recruitment and activation. 9 Several researchers have reported that AAA repair is associated with portal and systemic endotoxemia, 1, 12, 13, 14, 15 and revascularization itself has been associated with increased gut permeability. 16, 17 Endotoxin, a lipopolysaccharide component of the cell wall of gram-negative bacteria, is a potent stimulus to cytokine generation, coagulation and complement activation, 18 and leukocyte activation. 19, 20 It has been implicated in the pathogenesis of human systemic inflammatory response syndrome 16, 21 and acute lung injury after lower limb I/R injury. 19, 21, 22, 23, 24 The gut, with its large resident bacterial population 25 is acknowledged as a major source of endogenous endotoxin in a range of septic responses to injury. 1, 22, 26 Recently, at this center it has been shown that lower limb I/R is associated with altered gut permeability and changes in villus structure. 17 This in turn was associated with an increased systemic endotoxin concentration and significantly increased levels of interleukin-6 (IL-6). 27, 28 The concentration of IL-6, a pleiotropic cytokine, is known to correlate with the degree of systemic inflammation, prognostic scores, and death. 29, 30

Bactericidal/permeability-increasing protein (BPI), an antibacterial protein isolated from human neutrophils, binds lipopolysaccharide; competitively inhibits lipopolysaccharide-induced polymorphonuclear leukocyte stimulation; alters bacterial cell wall permeability, causing cell death; and reduces many of the harmful effects of endotoxin both in vitro and in vivo. 31, 32, 33 Recent reports on recombinant amino-terminal fragments and analogues of BPI (rBPI 21 and rBPI 23 ) have demonstrated increased survival rates, reduced cytokine production, and decreased hemodynamic and metabolic derangement associated with lethal endotoxin challenge in animals 34 and human beings. 35

The purpose of this study was to determine whether treatment after ischemia with antimicrobial rBPI 21 would inhibit endotoxin-induced systemic inflammation after hind limb I/R injury. We also proposed to determine whether neutralizing endotoxin with rBPI 21 would also attenuate acute lung injury associated with I/R injury.

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Material and methods 

Animal model 

Adult male Wistar rats weighing 300 to 350 g obtained from a breeding colony were used throughout. Animals were kept under standard conditions and had free access to standard rat chow and water. Before the experiment, animals were fasted overnight but were allowed water as desired. The animals were anesthetized with subcutaneous ketamine 80 mg/kg and xylazine 8 mg/kg and allowed to breathe spontaneously. Hydration was maintained by injection of sterile 0.9% sodium chloride, 3 mL/kg per hour subcutaneously. With a heat lamp, body temperature was kept at 37°C. Bilateral hind limb ischemia was induced by application of rubber band tourniquets high around each thigh, a highly reproducible technique previously reported by our group. 17 At the end of the ischemic period, reperfusion of the limbs was achieved by releasing the tourniquets. All procedures involving animals were carried out in accordance with the regulations of the United Kingdom Animals (Scientific Procedures) Act, 1986.

Treatment 

All rats were randomized before the induction of anesthesia to receive treatment with either rBPI 21 , at various doses, or the control protein thaumatin (XOMA [US] LLC, Berkeley, Calif). The controls underwent 5 hours of anesthesia, and all I/R injury groups underwent 3 hours of bilateral hind limb ischemia followed by 2 hours reperfusion on release of tourniquets. The rBPI 21 was administered to three (I/R) groups (n = 8 per group) intravenously at 1, 2, or 4 mg/kg at the beginning of reperfusion. An additional (I/R) group (n = 8) was administered rBPI 21 intravenously at 2 mg/kg after 1 hour of reperfusion. The control group and the untreated I/R group (n = 8 per group) received thaumatin as a control-protein preparation at 2 mg/kg.

Blood and tissue sampling 

Blood and tissue samples were obtained after 2 hours of reperfusion. The chest wall was cleansed with chlorhexidine in spirit, a 1-cm disc of skin was excised, and a butterfly needle connected to a 10-mL syringe was then used to obtain a blood sample by direct heart puncture. Blood samples were collected in heparinized (20 U/mL blood) sterile pyrogen-free tubes, and immediately transferred on ice to be spun in a centrifuge at 200 rpm (at 4°C) for 10 minutes. Plasma was aliquoted into sterile cryotubes (Nunc; Intermed, Roskidle, Denmark) and stored at –70°C until the time of assay. Immediately after blood sampling, a midline thoracotomy was performed, and the entire right lung was excised, placed immediately in a sterile pyrogen-free cryotube (Nunc), snap-frozen in liquid nitrogen, and stored at –70°C until time of assay.

IL-6 assay 

Biologically active IL-6 was measured by use of a bioassay on the basis of the proliferation of IL-6–dependent B9 hybridoma cells (a generous gift of L. Aarden, Amsterdam, The Netherlands). Samples were serially diluted with IL-6–free growth medium and dispensed in duplicate into 96-well microtiter plates. Similarly a standard curve ranging from 0 to 500 pg/mL was generated by use of recombinant human IL-6 (British Biotechnology, Cowley, United Kingdom) and plated out in duplicate. B9 cells were washed free of IL-6 and resuspended in IL-6–free B9 growth medium. Standard cell suspension (2.5 × 10 4 /mL) 100 μL was plated into wells and incubated at 37°C for 4 days. MTT (3-[4,5-dimethylthiazol-2yl]-2,5 diphenyltetrazolium bromide) in phosphate-buffered saline solution (0.5 mg/mL) was added to each well, followed 5 hours later by sodium dodecyl sulfate 50 mL (20% in 0.01 mol/L hydrochloric acid). The plates were incubated for another 24 hours. Absorbance was then read at 570 nm, and the amount of IL-6 in each sample was computed from the standard curve. Interassay and intraassay coefficients of variation were less than 10%.

Antiendotoxin antibody assay 

The “EndoCAb” enzyme-linked immunosorbent assay, which measures antibody levels to the core glycolipid region of lipopolysaccharide, was used to measure antiendotoxin antibody levels. The microplates (G. R. Barclay, Scottish Blood Transfusion Centre, Edinburgh, United Kingdom) were previously coated with a “cocktail” of core lipopolysaccharide from rough mutants of a number of bacteria in a complex with polymyxin and then blocked with 5% bovine serum albumin. Plasma samples were diluted 1:200 by use of phosphate buffered saline solution, pH 7.4, containing 0.05% Tween (Sigma, Poole, United Kingdom), 0.5% bovine serum albumin (Sigma), and 4% polyethylene glycol (BDH Chemicals, Poole, United Kingdom). A normal plasma dilution curve was obtained by use of plasma at a range of dilution from 1:50 to 1:1600. Standards and samples were added to the plate at 100 μL per well. The plate was incubated for 1 hour at room temperature with continuous shaking. A 1:250 dilution of the antirat immunoglobulin G (IgG) peroxidase or 1:500 dilution of the antirat immunoglobulin M (IgM) peroxidase (Serotec, Oxford, United Kingdom), prepared in the above diluent, was then added to each well (100 μL) for 1 hour at room temperature with continuous shaking. After this, 100 μL 3,3,5,5,tetramethyl-benzidine substrate (Sigma) was added to each well for 10 minutes, and the reaction was stopped with sulphuric acid 2 mol/L. The plate was then read on a Thermomax kinetic reader (Molecular Devices, Crawley, United Kingdom) at an absorbance of 450 nm, and the results were calculated as a percentage of the normal range.

Endotoxin 

Endotoxin concentration was determined by use of a quantitative Limulus amebocyte lysate chromogenic assay (Quadratech, Epsom, United Kingdom). The detection limits of the assay were between 0.1 to 100 pg/mL, interassay coefficient of variation was less than 10%, and intra-assay coefficient of variation was less than 10%.

Measurement of lung tissue myeloperoxidase 

The specimen (200-400 mg) was minced in a beaker containing 1 mL of hexadecyltrimethylammonium bromide buffer on ice, transferred to a test tube, and homogenized with a Polytron homogenizer (Phillip Harris International, Ashby de la Zouch, Leicestershire, United Kingdom) (3 times for 30 seconds each, on ice) to determine myeloperoxidase activity in tissue. After homogenization the homogenizer was rinsed twice with 1 mL of HTAB. The pooled homogenate and washes were sonicated for 10 seconds, freeze-thawed thrice, and spun in a centrifuge at 40,000 g for 15 minutes. The supernatant was assayed for myeloperoxidase activity. Myeloperoxidase activity was measured spectrophotometrically: 0.1 mL of supernatant was combined with 2.9 mL of 50 mmol/L phosphate buffer, pH 6.0, containing 0.167 mg/mL O -dianisidine hydrochloride and 0.0005% hydrogen peroxide. The change in absorbance at 460 nm was measured with a Beckmann DU-2 spectrophotometer (Beckmann Instruments, Inc, Cedar Grove, NJ). One unit of myeloperoxidase activity is defined as that degrading 1 μmol of peroxide per minute at 25°C.

Lung tissue wet-to-dry weight ratios 

Wet-to-dry ratios of lung tissue samples (200-400 mg) were calculated. Each specimen was blotted dry, weighed, and then placed in a vacuum freeze dryer at –70°C for 48 hours. Specimens were then reweighed, and the wet-to-dry tissue weight ratio was calculated.

Statistical analysis 

Results are expressed as the median (interquartile range). A nonparametric one-way analysis of variance (Kruskal-Wallis H) was used to detect differences between groups, and statistical comparisons were made by use of the Mann-Whitney U test. A P value of .05 or less was considered to indicate statistical significance. Analysis was performed with the software package SPSS version 9.0 for Windows (SPSS Inc, Chicago, Ill).

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Results 

Plasma IL-6 

Limb I/R increased plasma IL-6 concentrations significantly (1352.20 pg/mL [860.16-1886.40 pg/mL]) compared with control animals (125.32 pg/mL [87.76-157.52 pg/mL]; P < .0001). Treatment with rBPI 21 intravenously reduced the IL-6 response to bilateral hind limb I/R significantly in all groups ( P < .05); this significance was greatest when rBPI 21 2 mg/kg was given at reperfusion (715.89 pg/mL [573.36-847.76 pg/mL]) compared with the untreated group (1352.20 pg/mL [860.16-1886.40 pg/mL]; P < .016) (Fig 1 ) .

  • View full-size image.
  • Fig. 1. 

    Effect of bilateral lower I/R injury on plasma IL-6 concentration with comparison to control group and groups treated with rBPI 21 . Data represent median (interquartile range). * P < .0001 versus control group, † P < .002 versus control group, ‡ P < .05 versus I/R injury group. IRI , I/R injury.

Systemic endotoxemia 

Limb I/R increased plasma endotoxin concentrations significantly (21.52 pg/mL [6.20-48.23 pg/mL]) compared with control animals (0.90 [0.00-2.30 pg/mL]; P < .0001). Treatment with rBPI 21 intravenously reduced the endotoxin response to bilateral hind limb I/R significantly when rBPI 21 4 mg/kg was given at reperfusion (1.30 pg/mL [1.20-2.20 pg/mL]) compared with the untreated group ( P < .001) (Table).

Systemic endotoxemia
Group (n = 8)Indexes of systemic endotoxemia
Endotoxin (pg/mL)EndoCAb IgG (% control)EndoCAb IgM (% control)
Control0.9 (0.0-2.3)——
I/R21.5 (6.2-48.2) *37.9 (18.9-55.5) †62.3 (53.3-70.8) †
I/R + rBPI 21 (1 mg/kg)2.5 (2.0-3.20) ‡24.9 (14.7-35.3) †89.5 (72.6-131.6)
I/R + rBPI 21 (2 mg/kg)2.5 (2.0-5.4) ‡47.3 (16.9-288.9)82.7 (62.9-230.7)
I/R + rBPI 21 (4 mg/kg)1.3 (1.2-2.2) §44.7 (28.7-126.3)106.5 (51.4-127.1)
I/R + rBPI 21 (2 mg/kg) at 1 h into reperfusion3.5 (2.7-4.0) ‡63.3 (23.3-158.7)135.0 (83-150.7)
*P < .0001 versus control group. †P < .005 versus control group. ‡P < .05 versus I/R injury group. §P < .001 versus I/R injury group.

The effect of bilateral lower I/R injury on plasma EndoCAb IgG concentration with comparison to control group and groups treated with rBPI 21 . Results expressed as percentage of pooled control plasma (control = 100%). Data represent median (interquartile range).

A significant decrease in plasma concentrations (percent of control value) of cross-reactive IgG antibodies to core glycolipid (endotoxin) was observed after 3 hours of bilateral hind limb ischemia followed by 2 hours of reperfusion in the untreated group (37.85% [18.90%-55.53%]; P < .002). No significant consumption compared with controls was noted in the I/R groups treated with rBPI 21 at reperfusion with 2 mg/kg (47.30% [16.85%-288.88%]) and 4 mg/kg (44.70% [28.73%-126.25%]) and at 1 hour into reperfusion at 2 mg/kg (63.30% [23.30%-158.70%]). There was a significant decrease in plasma concentrations of cross-reactive IgM antibodies to core glycolipid (endotoxin) in the I/R group compared with control ( P < .005). There was no significant change in plasma concentrations of antiendotoxin antibody IgM in any rBPI 21 treated group compared with control (Table ).

Lung tissue myeloperoxidase concentration 

Limb I/R significantly increased lung tissue myeloperoxidase concentration (208.18 [128.79-221.81]) absorbance units per gram dry tissue, compared with the control group (62.00 [40.45-80.92]; P < .0001). Treatment with 2 mg/kg rBPI 21 at reperfusion significantly decreased lung tissue myeloperoxidase concentration (129.54 [90.49-145.78]) compared with the untreated group (208.18 [128.79-221.81]; P < .050) (Fig 2 ) .

  • View full-size image.
  • Fig. 2. 

    Effect of bilateral lower I/R injury on lung tissue myeloperoxidase concentration with comparison to control group and groups treated with rBPI 21 . Data represent median (interquartile range). * P < .0001 versus control group, † P < .002 versus control group, ‡ P < .05 versus I/R injury group. IRI , I/R injury.

Lung wet-to-dry tissue weight 

Limb I/R increased lung tissue wet-to-dry weight ratio (6.08 [5.15-7.89]) compared with the control group, (4.63 [3.59-5.17]; P < .007. Treatment with 2 mg/kg rBPI 21 at reperfusion significantly decreased the lung tissue wet-to-dry tissue weight (4.94 [3.65-5.26]) compared with untreated I/R injury (6.08 [5.15-7.89]; P < .04); however, this was still significantly higher than in the control group (4.63 [3.59-5.17]; P < .02) (Fig 3 ) .

  • View full-size image.
  • Fig. 3. 

    Effect of bilateral lower I/R injury on lung tissue wet-to-dry weight ratio with comparison to control group and groups treated with rBPI 21 . Data represent median (interquartile range). * P < .007 versus control group, † P < .05 versus control group, ‡ P < .04 versus I/R injury group. IRI , I/R injury.

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Discussion 

Elective or emergency vascular surgical reperfusion of ischemic lower limbs may be successful technically but still carries a high morbidity and mortality rate in an increasingly elderly patient population. 3, 4, 5 Although optimal surgical technique and critical care has improved outcomes after operations for AAA, the rupture mortality rate is still 30% to 70%. 3, 36 At least 20% of these deaths are attributable to multiple organ failure, 3, 6 the leading cause of death in surgical intensive care units. 37 Our own group 17 and others 16, 38 have shown that lower limb I/R is associated with remote gut mucosal injury and increased permeability. We have previously reported that increased levels of endotoxin, a lipopolysaccharide component of gram-negative bacterial cell wall, is associated with elevations in systemic IL-6 concentrations and high mortality rates in an experimental model of hind limb I/R injury. 27 Increased endotoxin levels have also been reported in human beings after AAA repair. 21 More recently, endotoxemia has been shown to predict death after thoracoabdominal aortic aneurysm repair. 19 In our study the modulation of the IL-6 response and remote acute lung injury by rBPI 21 , an antibacterial and antiendotoxin agent, suggests that gut-derived endotoxin may well amplify the septic response to lower limb I/R injury.

Previous work on this model of bilateral hind limb I/R at this center has demonstrated remote gut injury, systemic endotoxemia, and elevated systemic IL-6. 27, 28 Endotoxins are responsible for metabolic derangement, vascular endothelial damage, and cardiovascular changes resulting in hypotension and shock. 18, 39, 40 These also stimulate cytokine production, including significant release of IL-6. 41 Svoboda et al 29 and Calandra et al 42 have shown a close correlation between IL-6 levels and injury severity score and survival after trauma and sepsis, respectively. In this study rBPI 21 has been used to significantly diminish the IL-6 response to lower limb I/R. We have previously reported elevations in tumor necrosis factor–alpha early in the reperfusion period in a survival model of rat I/R injury. 27 Tumor necrosis factor, an early proinflammatory mediator, partially controls the release of IL-6 43 and has been implicated in the development of acute lung injury. 44 We measured IL-6 in this study because it indicates global inflammation and correlates with outcome. 1, 42, 45 IL-6 has also been associated with the activation of circulating neutrophils, 46 delaying apoptosis, 47 and therefore prolonging functional longevity and potential tissue injury. Endotoxin, a potent stimulant of IL-6 release, 41 is neutralized by rBPI 21 , but the drug also directly inhibits polymorphonuclear stimulation by endotoxin. 32, 33 The reduced IL-6 response may also be representative of a general decrease in inflammation and cytokine production.

Reperfusion of postischemic tissue initiates a systemic inflammatory response syndrome characterized by production of proinflammatory mediator production, neutrophil and monocyte activation, and hemodynamic and metabolic derangement. 2, 9, 30 I/R of lower limb is also associated with an acute lung injury or adult respiratory distress syndrome, 48 characterized by increased microvascular permeability and polymorphonuclear infiltration. 7, 9 We have demonstrated in this model that I/R is associated with acute pulmonary edema and polymorphonuclear leukocyte infiltration and that it can be partially attenuated by use of rBPI 21 . Endotoxin is a potent stimulus to neutrophil activation. Therefore the beneficial effects of rBPI 21 may be due to its role in promoting direct clearance of endotoxin and indirect attenuation of phagocytic cell activation in response to endotoxin. It may also be a reflection of a generalized diminished systemic inflammatory response syndrome in these animals in which we have shown a highly significant decrease in plasma IL-6. The lungs in the untreated I/R group showed a dense polymorphonuclear infiltrate on histologic assessment.

Previous work at this center has shown that isolated hind limb I/R leads to systemic endotoxemia. 28 This was associated with consumption of endogenous antibodies (IgG and IgM) directed against inner core glycolipid of endotoxin (EndoCAb). These findings are in agreement with those of other workers 37 who have correlated low EndoCAb levels with high endotoxin concentrations in patients with sepsis. We have again demonstrated endotoxemia after hind limb I/R. The prevention of detectable systemic endotoxemia in the rBPI 21 -treated groups may reflect increased clearance of endotoxin; rBPI 21 has been shown to increase phagocytic cell clearance of endotoxin in vitro and in vivo. 17, 31, 34 There is also decreased systemic inflammation (IL-6) in the rBPI 21 -treated group, which in turn may help prevent further increased gut permeability and consequent endotoxin translocation. In our present study we witnessed significant consumption of cross-reactive IgG and IgM antibodies to core glycolipid (endotoxin) in the untreated I/R group, an outcome averted in the groups treated with rBPI 21 . The latter may represent a reduction in circulating endotoxin binding with rBPI 21 or more rapid phagocytosis of endotoxin bound to rBPI 21 .

Sepsis syndrome caused by bacteremia or endotoxemia remains a leading cause of morbidity and death, in spite of antibiotics and intensive care support. 37 The experimental finding that antiendotoxin immunotherapy with polyclonal or monoclonal antibodies reduced mortality rates in a research setting gave rise to initial optimism. 49, 50 However the clinical efficacy and endotoxin binding properties of these antibodies are now in doubt. 50, 51 BPI, an antibacterial protein isolated from human neutrophils, 32, 33 has been found to bind endotoxin and inhibit endotoxin-mediated neutrophil and monocyte activation. BPI (and its N -terminal fragments and analogues) has certain advantages over these antibodies, namely, that it is neither serotype specific nor inhibited by the O-specific side chain of lipopolysaccharide. 31, 33, 52 It therefore is not rendered ineffective in overwhelming infection by heterogenous organisms. Our control protein thaumatin, an intensely sweet protein isolated from the African plant Thaumacoccus danielli, has a molecular weight and an isoelectric point similar to that of rBPI 21 but does not bind to lipopolysaccharide or affect its ability to induce cytokine release in vitro. 53 The short plasma half-life of rBPI 21 , 54 means that the timing of drug delivery to limit endotoxin-induced inflammatory activation may be crucial. In further studies a continuous intravenous infusion during early reperfusion may overcome rapid rBPI 21 plasma clearance. There is a growing body of evidence supporting a role for gut hyperpermeability 13, 16 and translocation of endotoxin 15 in the propagation of the inflammatory response to AAA surgery. 1, 21 If gut endotoxin converts a recoverable inflammatory response to a lethal endotoxemia in a certain subset of patients, then rBPI 21 may represent an important advance in the treatment of these patients.

In conclusion, this study clearly shows that rBPI 21 can significantly reduce the IL-6 response to lower limb I/R and attenuation of the neutrophil-mediated acute lung injury. Although further studies on the mechanism of action, optimal dose, and time of delivery are required, these results suggest that rBPI 21 may well represent a useful adjuvant therapeutic tool in preventing or attenuating the septic response to ischemia-reperfusion injury.

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Acknowledgements 

We are indebted to Xoma Corporation (US) LLC, Berkeley, Calif, for generous provision of rBPI 21 .

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References 

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☆ Competition of interest: nil.

☆☆ Supported by The Royal Hospitals Research Trust and The Wellcome Trust.

★ Reprint requests: Mr D. W. Harkin, Department of Surgery, The Queen's University of Belfast, Institute of Clinical Sciences, Grosvenor Road, Belfast, Northern Ireland BT12 6BJ.

★★ Published online Feb 23, 2001.

PII: S0741-5214(01)93895-9

doi:10.1067/mva.2001.111992

Journal of Vascular Surgery
Volume 33, Issue 4 , Pages 840-846, April 2001

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