Antibody-induced modulation of the leukocyte CD11b integrin prevents mild but not major renal ischaemic injury

Luis C. F. Tajra1,2, Xavier Martin2, Jacqueline Margonari1, Nelly Blanc-Brunat3, Michio Ishibashi1, Geneviève Vivier3, Jean P. Steghens5, Hiroto Kawashima6, Masayuki Miyasaka6, Jean-M. Dubernard2 and Jean-P. Revillard4,

1 INSERM, Unité 281, Laboratoire de Recherche Chirurgicale, 2 Service d'Urologie et Chirurgie de la Transplantation, 3 INSERM, Laboratoire d'Immunopathologie, 4 INSERM, Unité 503, Immuno-Biologie Fondamentale et Clinique, 5 Laboratoire de Biochimie, Hôpital Edouard Herriot, Lyon, France, and 6 Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, Suita, Japan



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. CD11/CD18 ß2 integrins are involved in leukocyte adhesion to the activated endothelium, and therefore represent a possible therapeutic target in the prevention of ischaemic acute renal failure (ARF).

Methods. To assess the effect of an anti-CD11b monoclonal antibody (mAb) in ischaemic ARF, uninephrectomized Fischer rats were subjected to 45 or 60 min of warm renal ischaemia, then received 1 mg of anti-CD11b mAb 5 min before reperfusion.

Results. After 45 min of ischaemia, renal function tests at 24 and 48 h were less altered in mAb-treated than in control rats, but after 60 min of ischaemia the same level of renal insufficiency was observed in the two groups. In parallel, milder tubular necrosis and less leukocyte infiltration were observed in the treated group after 45 min of ischaemia, but no difference was seen after 60 min compared to the control group. The mAb was detected on blood neutrophils up to 48 h after infusion and a marked down-regulation of CD11b expression on neutrophil surfaces was documented by flow cytometry.

Conclusion. These results indicate that anti-CD11b mAb administered prior to reperfusion decreases moderate ischaemic ARF but fails to prevent renal injury secondary to prolonged ischaemia in this model.

Keywords: adhesion molecules; integrins; Mac-1; monoclonal antibody; rat; renal ischaemic injury



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The process of ischaemia–reperfusion (IR) results in the production of reactive oxygen species and proinflammatory mediators, including cytokines, platelet-activating factor, and other products of arachidonic acid metabolism. These mediators contribute to reperfusion-induced leukocyte accumulation in renal tissue [1,2]. Leukocytes, particularly polymorphonuclear granulocytes (PMNs), play a critical role in the development of IR-induced organ injury. Indeed, neutrophil depletion before ischaemia was reported to provide some functional and morphological protection in a model of renal ischaemia in rats [3]. Furthermore, activated PMNs introduced in post-ischaemic reperfusion of isolated perfused rat kidneys induced exacerbation of ischaemic acute renal failure (ARF), with decreased glomerular filtration rate and tubular Na reabsorption [4]. However, other studies in rabbits and rats failed to demonstrate either functional or morphological protection by depleting PMNs [5,6].

The process of leukocyte extravasation from the circulation to a site of ischaemic injury involves several sequential steps: tethering and rolling mediated by selectins, triggering by chemokines and lipidic mediators, then firm adhesion and transmigration that primarily involve integrins and their ligands [2,7,8].

Integrins are leukocyte transmembrane proteins made of {alpha} and ß subunits [8]. The common ß2-chain CD18 may associate with one of three {alpha} subunits: CD11a, CD11b, or CD11c. The integrin CD11a/CD18 (LFA-1), expressed on all leukocytes, is primarily involved in modulating T-lymphocyte adhesion and, to a lesser extent, neutrophil–endothelial interaction [9]. CD11b/CD18 (Mac-1) is the most abundant and an important integrin for neutrophil adhesion to endothelial cells [7]. CD11c/CD18, a ligand of the proteolytic fragment of complement protein C3 (iC3b) and fibrinogen, may also contribute to neutrophil adhesion [7,8]. The primary endothelial ligands for the ß2 integrins are the intercellular adhesion molecules (ICAM-1, ICAM-2, and ICAM-3). ICAM-1 (CD54) is a ligand for both CD11a/CD18 and CD11b/CD18, while ICAM-2 (CD102) binds only to CD11a/CD18 [7,8]. ICAM-1 is expressed constitutively on endothelium throughout the normal kidney, whereas the renal tubules are usually negative [10].

Several studies with different experimental models of IR organ injury suggest that blockade of leukocyte–endothelial cell interactions by interference with the binding of integrins to their ligands may be beneficial. However, controversial results about efficiency of anti-adhesion molecules mAbs in IR organ injury have been reported. Antibodies to CD11b were reported to prevent IR injury of myocardium [11], liver [12], and brain [13], whereas in other studies anti-CD11b was inefficient in preventing these injuries in the myocardium [14]. No results have been reported in the literature using this mAb alone against IR injury in kidney. Here we report that a CD11b mAb of the IgA isotype, although effective in moderate ischaemia, did not reduce ARF in an in vivo model of severe renal IR injury in the rat, despite the fact that the antibody induced a profound and long-lasting down-regulation of surface expression of the CD11b/CD18 molecule on neutrophils during treatment.



   Subjects and methods
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 Subjects and methods
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Production of monoclonal antibodies
Hybridoma cell lines producing mouse IgA anti-rat CD11b (WT.5) were provided by M. Miyasaka of the Tokyo Metropolitan Institute of Medical Science, Japan. WT.5 was produced as previously described [15]. Briefly, 2x106 hybridoma cells in exponential growth phase were injected into the peritoneal cavity of pristane-primed, 6-week-old male nude mice. About 5 days later, the antibody-containing ascites were collected, centrifuged, and immediately frozen at -80°C prior to use. Specificity of WT.5 for the CD11b ß2 integrins had been previously determined by immunoprecipitation analysis of peritoneal exudate neutrophil lysates showing the 140–145/95–100 kDa {alpha}/ß heterodimer that corresponds to CD11b/CD18 [15]. WT.5 stains rat polymorphonuclear leukocytes but not lymphocytes.

Animal and surgical procedure
Male inbred specific pathogen-free (SPF) Fischer rats weighing 280–320 g were given humane care in compliance with French regulations, as well as with the ‘Principles of Laboratory Animal Care’ (NIH publication no. 86–23, revised 1985). Animals were anaesthetized with ether. The abdomen was prepared with Betadine®. The abdominal cavity was exposed via a midline incision. The left renal pedicle was either occluded with a non-traumatic vascular clamp for 45 or 60 min, or simply exposed. Temperature at the surface of the exposed kidney ranged from 30 to 31°C. Before closure, a right nephrectomy was performed and the left kidney was inspected for reperfusion. Blood samples (0.5 ml) were obtained from the vena cava and WT.5 was administered via the same vein. The abdomen was closed in two layers with 5–0 Dexon suture. Rats were returned to their cage and allowed ad libitum access to food and water.

Experimental protocol
The rats were divided into three groups according to the time of renal ischaemia: group 1 (45 min), group 2 (60 min), and sham (rats operated without occlusion of the renal pedicle).

In all treated animals, mAb was injected intravenously 5 min before reperfusion. One group of animals without ischaemia was treated with 5 mg WT.5/rat to analyse the eventual toxicity of WT.5.

Blood samples for BUN and creatinine determination were obtained from the vena cava at the time of surgical procedure and from the femoral vein at days 1 and 2. Creatinine and BUN levels were measured by the Jaffé method, taking into consideration the sample blank (twin-mode) and kinetic UV test respectively. The animals were killed at day 7, blood samples were taken, and kidneys were removed for histological study.

For pharmacokinetic studies four rats were injected with 1 mg WT.5 and peripheral blood was collected before injection and at 0.5, 1, 6, 24 and 48 h after injection for immunofluorescence analysis and measurement of residual free antibody levels. Two additional rats were used for plasma WT.5 determination.

Light microscopy and immunohistology
Kidneys were divided into three parts and fixed with Bouin's solution, Carnoy's solution, or preserved in liquid nitrogen. Specimens for light microscopy examination were fixed with Bouin's solution, then embedded in paraffin. Sections were stained with periodic acid–Schiff reagent, with H&E and Masson's trichrome. Indirect immunoperoxidase technique was also applied. Primary antibodies were as follows: ED1 murine monoclonal antibody against rat macrophages 1 : 250 (IgG1, ascites; Serotec Ltd, Oxford, UK) and RP1, anti-granulocyte-macrophages mouse mAb 1 : 200 (IgG1, ascites, a gift from M. Miyasaka, Japan). The second antibody was a biotinylated horse anti-mouse antiserum (H+L) 1 : 100 (Biosys Laboratory, Compiegne, France). ED1 was used on Carnoy-fixed, paraffin-embedded sections. RP1 was utilized for tissues snap-frozen in liquid nitrogen. Immunostaining was performed using the avidin–biotin–peroxidase complex technique [16]. Histological sections were evaluated in a ‘blinded’ fashion. Ischaemic injury was evaluated on five independent criteria: intensity and extension of tubular necrosis and tubular dilatation, number of leukocytes, ED1+ (macrophages) and RP1+ (macrophages, granulocytes). Scores from – (normal kidney) to +++ (maximal alteration) were used for each criteria independently. Data are presented as mean score value in each group (Table 1Go).


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Table 1. Histological pattern in the different groups

 

Flow cytometry studies
To determine the level of binding of WT.5 mAb to neutrophils and the presence of free mAb in the plasma of treated rats, control cells, i.e. blood cells before injection, were incubated in vitro with a saturating concentration of WT.5 mAb (50 µl of 1 mg/ml) or with the plasma samples obtained at different times (0, 0.5, 1, 6, 24 and 48 h) from treated animals, then with goat anti-mouse FITC-conjugated antibody. Blood samples taken after intravenous injection of WT.5 mAb were incubated for 30 min at 4°C with or without WT.5, then in the presence of goat anti-mouse FITC-conjugated antibody, and washed in phosphate-buffered saline/1% bovine serum albumin (PBS/BSA). After red cells lysis, the remaining cells, including PMNs, were fixed in PBS/azide/1% formaldehyde and stored at 4°C. Cells were analysed using a FACSCAN flow cytometer (Becton Dickinson, Mountain View, CA, USA) using the Lysys II software. Results were expressed as median fluorescence intensity in arbitrary units.

Statistics
Creatinine and BUN values are expressed as the mean±SEM. Differences among mean values were evaluated by analysis of variance, i.e. one-way Friedman test. Kruskal–Wallis test was used to compare non-parametric data between groups. Scores of histological lesions were evaluated by analysis of variance (ANOVA) followed by protected least significance difference Fischer's test. P<0.05 was considered significant.



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 Results
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Renal function
After 45 min of ischaemia, plasma creatinine of control animals increased more than twofold over baseline levels at 24 h (P<0.05). It was still higher than baseline at 48 h (P<0.05) but close to normal at day 7 (Figure 1Go). Administration of anti-CD11b decreased post-ischaemic renal dysfunction: after 24 h of ischaemia, mean creatinine was 107±5.3 vs 139±8.5 µmol/l in the control group (P<0.05). Creatinine values were 88.5±2.3 and 111.1±9.7 µmol/l (P<0.05) 48 h after ischaemia in the anti-CD11b and control group respectively.



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Fig. 1. Effect of anti-CD11b mAb on the course of ARF.

 
After 60 min of ischaemia, plasma creatinine of control animals increased more than threefold over baseline levels at 24 h (P<0.05). Administration of anti-CD11b did not decrease post-ischaemic renal dysfunction (Figure 1Go): after 24 h of ischaemia, mean creatinine was 189±22 vs 163.6±14 µmol/1 in the control group (P=n.s.). Creatinine values were 161±35 and 143.9±28 µmol/l 48 h after ischaemia in the anti-CD11b and control group respectively (P=n.s.).

In all anti-CD11b mAb-treated groups, mean creatinine at 24 and 48 h of reperfusion was still higher than in baseline or sham-operated animals (P<0.05). At 7 days no significant differences were observed between the groups. BUN values paralleled those of creatinine with a significant decrease (P<0.05) after 45 min of ischaemia but without significant difference after 60 min of ischaemia regarding all groups (Figure 1Go). Injection of WT.5 at 5 mg/rat did not result in any clinical adverse effect or significant change in renal function.

Renal morphology and leukocyte infiltration
By light microscopy, the control group presented moderate to severe (2+ to 3+) widespread necrosis of the renal tubules, most prominent after 60 min of ischaemia. These lesions were observed in the outer medullary stripe but also as patchy involvement of the cortical tubular segments. Important dilatation of tubules was observed especially in the proximal tubules (Table 1Go). Several hyaline and epithelial cellular casts were observed in the tubular lumen. Isolated mitoses were seen in tubular epithelium. Histological damage was mild (1+) in anti-CD11b-treated kidneys after 45 min of ischaemia (Table 1Go). Slight tubular dilatation was observed and tubular necrosis was significantly less important than the control group (P<0.05). After 60 min of ischaemia, no significant difference was observed regarding renal morphology between CD11b-treated and control groups (Table 1Go). No necrosis or mitotic forms were seen in sham-operated kidneys and kidneys with highest dose (5 mg) of monoclonal antibody.

Severe leukocyte accumulation in the renal interstitium was observed by light microscopy in the treated and control groups after 60 min of ischaemia (Table 1Go). There was an evident tendency toward decreased leukocyte infiltration in the interstitium in the CD11b-treated rats after 45 min of ischaemia but this difference did not reach statistical significance compared to the control group. (Table 1Go). Finally, fewer ED1- and RP1-positive cells were observed in the treated group after 45 min of ischaemia, but not in group two (60 min).

Flow cytometry analysis
Addition of the conjugate to blood samples from treated rats revealed the exclusive coating of neutrophils, with decreasing intensity from 30 min to 48 h (Figure 2Go). When WT.5 plus conjugate were added on the same cells, the median fluorescence intensity (that parallels the number of surface CD11b epitopes) dropped markedly at 30 min or after 1 h and increased at 48 h but did not reach pre-infusion levels (Figure 2Go). Free WT.5 mAb was demonstrable in rat plasma diluted at 1 : 2 and 1 : 10 up to 6 h but not thereafter (Table 2Go). Median fluorescence intensities of rat plasma were equivalent to the intensity of control cells incubated in vitro with 5 µg/ml of WT.5 mAb as determined from the titration curve (median fluorescence intensities of WT.5 over a range of dilutions).



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Fig. 2. Presence of WT.5 mAb on blood neutrophils and down-modulation of CD11b. (a) Whole blood from a normal rat was mixed with WT.5 (50 µl) and mAb fixation was revealed by addition of FITC-GAMIg. All blood elements (with the exception of lysed red cells) are displayed according to their light-scattering properties at small angles (FSC) and 90° (SSC). The window R1 contains all the PMNs, which represent the only fluorescent cells in this experiment. (b) In the upper histograms, fluorescence intensity after FITC-GAMIg staining of PMNs collected from rat 1, before treatment (control), and at time 30 min, 60 min, 6 h, 24 h, and 48 h after i.v. injection of WT.5 mAb (1 mg/rat). Decreasing fluorescence intensity is observed along the time course. In the histograms below, a saturating concentration of WT.5 mAb (50 µl) was added in vitro to evaluate the remaining free antibody binding sites. Cell surface expression of CD11b on PMNs decreased 1 h (rats 1 and 2) or 30 min after injection of mAb. Open histograms indicate control cells and solid histograms indicate cells that were stained with anti-CD11b mAb. (c) Median fluorescence intensities of PMNs from four rats treated with WT.5.

 

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Table 2. Determination of free WT.5 mAb in rat plasma*

 



   Discussion
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 Subjects and methods
 Results
 Discussion
 References
 
ARF induced by IR lesions contributes to delayed graft function after renal transplantation from cadaveric donors. Its prevention is an important issue in clinical kidney transplantation. Our experimental model in the rat was designed to mimic the conditions of transplantation with injection of mAb after rather than before the warm ischaemia period, and to provide preclinical evaluation of treatments that may modulate IR injury of the transplant. The choice of CD11b antibody relied on the reported capacity of such mAb to decrease IR injury of myocardium [11], liver [12], brain [13], and intestine [17] in various experimental models, whereas their possible effect on renal IR lesions had not previously been investigated to our knowledge. Rabb et al. [18] demonstrated that the association of anti-CD11a with anti-CD11b promotes functional and morphological protection in a model of ischaemic ARF in rats. The ß2 integrin Mac-1 is a major adhesion molecule of polymorphonuclear neutrophils and its interaction with its ligand (CD 54) on endothelial cells is a critical event in IR lesions allowing firm adhesion and inducing reciprocal activation signals and generation of proinflammatory mediators by the two cell types [7,8].

Two degrees of renal IR injury were studied corresponding to mild (45 min) and severe (60 min) lesions. More prolonged periods of ischaemia induced a high degree of mortality (not shown). Both protocols induced significant renal insufficiency at 24 and 48 h, that was reversible within a week, but major tubulointerstitial lesions were still present at day 7. The results show that CD11b mAb injected before reperfusion reduced functional alterations and tubulointerstitial lesions induced by 45 min of ischaemia, with a significant decrease of tubular necrosis. Conversely, the same treatment was ineffective in rats submitted to a more prolonged period of ischaemia (60 min). All possible technical artefacts that could have accounted for this difference were carefully excluded. It therefore appears that the CD11b mAb WT.5 displays definite but limited capacity to prevent renal IR injury, depending on the severity of the lesions.

The efficacy of mAb therapy is related to several factors, depending on the mechanism of action that is primarily involved in the experimental model. Some mAbs may merely act as soluble ligands that compete with the natural ligand to prevent signalling interactions with cell surface molecules. This mechanism is restricted to mAbs that recognize epitopes identical to or closely associated with the ligand binding site. Blocking a particular epitope does not necessarily neutralize the functional activity of the protein to which the antibody is directed. Alternatively, the mAb may interfere with receptor signalling or even induce the modulation (transient down-regulation of surface expression) of the antigen [19,20]. Our study demonstrates that WT.5 down-regulates surface expression of CD11b, with a maximum 6 h after infusion (Figure 2Go). Modulation of surface CD11b was still important 48 h after WT.5 mAb infusion, that is far beyond the life-span of neutrophils in peripheral blood and beyond complete disappearance of free WT.5 from the rat plasma (Table 2Go). This suggests that the antibody may have bound to neutrophil progenitors in the bone marrow so that neutrophils that reached the peripheral blood after WT.5 mAb disappearance from the plasma compartment were still coated with low amounts of the mAb and expressed decreased surface levels of CD11b/CD18. These data strongly support the view that maximal pharmacological activity of the mAb has been achieved in our experiments, and that dose escalation or repeated injections are unlikely to increase treatment efficacy.

One possible drawback in the use of antibodies directed against integrins or their ligands is the massive antigen–antibody interaction that occurs in the circulation (involving both leukocytes and endothelial cells) which may result in complement activation and systemic inflammatory response if large amounts of complement-fixing antibodies (e.g. IgM, IgG2) are injected. The lack of any measurable side effect upon injection of WT.5 at 15 mg/kg (5 mg per rat) suggests that antibodies of the IgA class may be more suitable for this application than complement-fixing antibodies.

The limited efficiency of a single CD11b antibody that induces profound and long lasting down-modulation of surface expression of the CD11b/CD18 ß2 integrin on blood neutrophils suggests that a single molecular target may not be appropriate for the prevention of severe ARF from IR injury. For instance, the ß2 integrin CD11a/CD18, which also binds to ICAM-1 (CD54) [7.8], although expressed in lower density on neutrophil membrane, may represent an important therapeutic target, since it displays greater binding affinity than CD11b to CD54 [21]. Indeed anti-LFA-1 and anti-ICAM-1 mAbs were shown to prevent ischaemic renal injury in animal models [22] and anti-LFA-1 antibody has demonstrated encouraging results in human renal transplantation [23]. Besides, a more extensive intervention on leukocyte–endothelium interaction should include mAbs against VLA-4/VCAM-1, as these mAbs have been shown to inhibit inflammatory cell infiltration in vivo [24,25]. The association of mAbs against several adhesion molecules may increase efficacy by synergic effects, as has been demonstrated in experimental models [22,26].

In conclusion, administration of an IgA mAb alone directed against the CD11b integrin reduced the functional damage and ischaemic injury after moderate kidney ischaemia. Prevention of ARF after IR by inhibition of adhesion-dependent, leukocyte-mediated inflammatory injury will probably require the association of mAbs against different adhesion molecules and pharmacological blockage of inflammatory mediators to improve results.



   Acknowledgments
 
This work was carried out at INSERM, Unite 281, Laboratoire de Recherche Chirurgicale, Hôpital Edouard Herriot, Lyon, France.



   Notes
 
Correspondence and offprint requests to: Professor J. P. Revillard, INSERM, Unite 503, Immuno-Biologie Fondamentale et Clinique Hôpital Edouard Herriot, Place d'Arsonval, F-69437 Lyon Cedex 3, France. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 14. 9.99
Revision received 6. 6.00.