The role of selectins and integrins in adenovirus vector-induced neutrophil recruitment to the liver

0014-2980/02/1212-3443$17.50+.50/0© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The role of selectins and integrins in adenovirusvector-induced neutrophil recruitment to the liver

Yang Li1, Daniel A. Muruve2, Robert G. Collins4, Samuel S. Lee1 and Paul Kubes3

1 Gastrointestinal Research Group, Department of Medicine, University of Calgary, Calgary,Canada

2 Department of Medicine, University of Calgary, Calgary, Canada3 Immunology Research Group, Department of Physiology and Biophysics, University of Calgary,

Calgary, Canada4 Baylor College of Medicine, Department of Pediatrics, Section of Leukocyte Biology, Children’s

Nutritional Research Center, Houston, USA

Adenovirus vectors for human gene therapy induce early host inflammatory responses intransduced tissues that limit gene transfer efficiency and can result in significant morbidity.The present study aimed to elucidate the cellular mechanisms underlying the acute inflam-mation induced by adenovirus vectors in the liver. Leukocyte rolling and adhesion inresponse to an intravenously administered adenovirus vector was examined by intravitalmicroscopy in mouse liver. Adenovirus vectors significantly increased leukocyte rolling andadhesion in the postsinusoidal venules within minutes of transduction. Unlike other inflam-matory states in the liver, no leukocyte retention was seen in the sinusoids in response toadenovirus vector administration. Inhibition of P-selectin, § 4-integrin, and E-selectin wasnecessary to completely block leukocyte rolling and subsequent adhesion. The administra-tion of an anti- § 4-integrin antibody alone significantly reduced leukocyte adhesion. In con-trast, adenovirus vector-induced leukocyte adhesion was unchanged in CD18-knockoutmice. Depletion of circulating neutrophils eliminated leukocyte rolling and adhesion inresponse to adenovirus vector transduction in the liver. In conclusion, adenovirus vectorsinduce rapid neutrophil-mediated inflammation in the post-sinusoidal venules by selectinsand § 4-integrin but surprisingly not by CD18.

Key words: Adenovirus vectors / Leukocyte trafficking / Liver inflammation / Selectin / Integrin

Received 29/4/02Revised 10/9/02Accepted 27/9/02

[I 23111]

The first two authors contributed equally to this work.

Abbreviation: AdGFP: Serotype 5 adenovirus vectorencoding green fluorescent protein

1 Introduction

A key pathological event during acute inflammation isthe recruitment of leukocytes to afflicted sites. Therecruitment of leukocytes involves the movement ofthese cells from the intravascular compartment to theextravascular space and is mediated by three sequentialevents. First, circulating leukocytes in the bloodstreammake initial contact with the endothelium that manifestsas tethering and rolling along the length of the venule.Second, rolling leukocytes are activated to firmly adhereto the endothelium by various pro-inflammatory media-tors. Once firmly adherent, leukocytes are able to per-form the third and final step, emigration out of the vascu-lature. The selectins (L-, P-, and E-selectin) mediate the

initial tethering and rolling of leukocytes to the vascularendothelium, whereas firm adhesion of leukocytes to theendothelial wall is mediated by interaction of leukocyteintegrins, such as CD18 ( g 2-integrin), with cell surfacemolecules such as ICAM-1 or ICAM-2 present on theendothelium [1, 2]. These interactions are followed bytransendothelial migration and chemotaxis of leukocytesto the site of inflammation [1, 2]. § 4-Integrin expressedon leukocytes is unique among the integrins in its abilityto support both leukocyte rolling and leukocyte adhe-sion, in vitro and in vivo [3, 4]. While § 4-integrin is gener-ally thought to mediate monocyte, lymphocyte andeosinophil-endothelial cell interactions, neutrophil adhe-sion via § 4-integrin occurs only in chronic or systemicinflammatory states [4, 5].

Neutrophils participate in inflammatory responses byphagocytosing and killing pathogenic organisms. Thereare a number of situations where liver injury results frominappropriate neutrophil recruitment and activation [6, 7,8]. In these states, the recruitment of neutrophils to theliver is in part dependent upon selectins and integrins.

Eur. J. Immunol. 2002. 32: 3443–3452 Selectins and integrins in adenovirus-induced inflammation 3443

Fig. 1. Flux of rolling neutrophils (A) and number of adherentneutrophils (B) in response to AdGFP administration in theliver postsinusoidal venules of C57BL/6 mice (wild type)over 120 min. Data are expressed as mean ± SEM of fiveanimals. *p X 0.05 compared with 0 min. †p X 0.05 comparedwith corresponding vehicle controls.

P-selectin expression is found in the portal veins andpostsinusoidal venules and mediates the neutrophil mar-gination in these vasculatures in murine endotoxin shock[9]. CD18 ( g 2-integrin) mediates neutrophil adhesion andplays an important role in the subsequent neutrophil-induced liver cell injury in endotoxin shock [8]. In contrastto the postsinusoidal venules, neutrophil recruitment inthe liver sinusoids appears to occur independent ofselectins and integrins [9, 10]. An understanding of thebiology of leukocyte recruitment into the different vascu-lar compartments of the liver is therefore essential todevelop potential for anti-adhesive therapies for hepaticinflammatory diseases.

Adenoviridae are being developed as agents for geneand cancer therapies in humans. Recombinant adenovi-rus vectors have several advantages including broad celltropism, ease of production in high titer and a capacity totransfer large amounts of DNA. However, host inflamma-tory and immune responses limit the efficacy of adenovi-rus vectors resulting in transient transgene expressionand ultimate vector loss [11, 12]. Adenovirus vectorsactivate both innate and adaptive arms of the immunesystem. Adaptive immune responses to adenovirus vec-tors occur several days following transduction and arelargely dependent on viral gene transcription [12]. On theother hand, adenovirus-induced innate responses aretriggered early and occur independent of virus transcrip-tion [13]. The liver is the major target of intravenouslyadministered adenovirus vectors [14, 15]. Thus, theinnate arm of the immune system is efficiently activatedin this organ following transduction with adenovirus vec-tors in vivo inducing the expression of numerous cyto-kines/chemokines and the recruitment of immune effec-tor cells (neutrophils, natural killer cells and monocyte-macrophages) to the liver [13]. Activation of the innatesystem leads to the rapid elimination of the deliveredadenovirus vector and the accompanying inflammationresults in acute hepatic toxicity [16]. Although manyinflammatory genes are up-regulated following adenovi-rus vector transduction of the liver, little is known regard-ing the cellular mechanisms that mediate the recruitmentof effector leukocytes to this organ in response to theseagents. Understanding the biology of the early hostinnate response to adenovirus vectors is essential toimprove the safety and effectiveness of adenovirus-mediated gene therapy. The present study aimed todefine the role of selectins and integrins in the biology ofthe innate response to adenovirus vectors in the liver.

2 Results

2.1 Adenovirus vector induced leukocyte-endothelial interactions in the liver

Adenovirus vectors induce acute inflammation in theliver; however, little is known about the biology ofleukocyte-endothelial interactions following transductionwith these agents. To directly visualize the effect of ade-novirus vector administration on leukocyte recruitmentto the liver we employed intravital microscopy in C57BL/6 mice. Under baseline condition (0 min), we observed alow basal level of rolling (Fig. 1A) but not adhering(Fig. 1B) leukocytes in postsinusoidal venules of the liver.No leukocytes were seen in liver sinusoids under basalconditions. The intravenous administration of virus vehi-cle alone did not increase basal leukocyte rolling oradhesion over 120 min of observation. Administration ofserotype 5 adenovirus vector encoding green fluores-cent protein (AdGFP; 2.5×1011 particles/animal) very rap-idly (in minutes) and significantly increased leukocyterolling in the postsinusoidal venules over 120 min com-

3444 Y. Li et al. Eur. J. Immunol. 2002. 32: 3443–3452

Fig. 2. Number of adherent leukocytes in response toAdGFP or TNF- § in the liver sinusoids of C57BL/6 mice (wildtype). Data are expressed as mean ± SEM of five animals.*p X 0.05 compared with control. †p X 0.05 compared withAdGFP.

Fig. 3. Flux of rolling neutrophils (A) and number of adherentneutrophils (B) in postsinusoidal venules of C57BL/6 mice(wild type) following AdGFP administration. Mice weretreated with anti-P-selectin or isotype control antibody10 min prior to AdGFP challenge. Data are expressed asmean ± SEM of five animals. *p X 0.05 compared with 0 min.†p X 0.05 compared with corresponding control.

pared with either baseline values (0 min) or the corre-sponding vehicle control (p X 0.01; Fig. 1A). AdGFP alsoincreased leukocyte adhesion in the postsinusoidalvenules in a time-dependent manner over the 120 minexperiment. This increase was significantly higher com-pared with either baseline (0 min) or the correspondingcontrols (Fig. 1B).

Surprisingly, no increase in leukocyte sequestration wasnoted in sinusoids following the administration of AdGFP.This was in direct contrast to the response to tumornecrosis factor-alpha (TNF- § ), which induced a signifi-cant increase in leukocyte number in the liver sinusoids(p X 0.001; Fig. 2). Since leukocyte trapping in the sinu-soids is seen in most inflammatory models studied todate [9, 10, 17], the lack of this phenomenon appears tobe unique to the inflammatory response induced by ade-novirus vectors.

2.2 The role of P-selectin in adenovirus vector-induced leukocyte-endothelial interactions inthe liver

P-selectin is known to mediate early leukocyte rolling innumerous inflammatory conditions. The administrationof anti-P-selectin antibody 10 min prior to the intrave-nous injection of 2.5×1011 particles of AdGFP signifi-cantly decreased leukocyte rolling in the postsinusoidalvenules. At 30 and 60 min, the number of rolling leuko-cytes was significantly lower than the corresponding val-ues in mice receiving isotype control antibody andAdGFP (Fig. 3A). At 90 and 120 min, leukocyte rollingbegan to increase and approach the degree of rollingseen in mice that received control antibody and AdGFP(Fig. 3A).

Pretreatment with anti-P-selectin antibody also affectedadenovirus vector-induced leukocyte adhesion. At 30and 60 min, anti-P-selectin antibody significantlyreduced AdGFP-induced leukocyte adhesion comparedto mice pretreated with isotype control IgG1 (Fig. 3B). At90 min, and coinciding with the observed increase in leu-kocyte rolling, leukocyte adhesion increased, and by120 min, was similar to the leukocyte adhesion observedin control mice (Fig. 3B).

2.3 Role of E-selectin in adenovirus vector-induced leukocyte-endothelial interactions inthe liver

Inhibition of leukocyte rolling and adhesion was onlydelayed by blocking P-selectin suggesting that othermolecules were involved in adenovirus vector-inducedleukocyte rolling. E-selectin also mediates leukocyte roll-ing but its effects are delayed compared to P-selectin[18]. To address the role of E-selectin in adenovirus


Fig. 4. Flux of rolling neutrophils (A) and number of adherentneutrophils (B) in postsinusoidal venules of E-selectin-deficient (E-selectin–/–) mice in response to AdGFP adminis-tration. Mice were treated with anti-P-selectin, both anti-P-selectin and anti- § 4-integrin, or isotype control antibodies10 min prior to AdGFP challenge. Data are expressed asmean ± SEM of five animals. *p X 0.05 compared with 0 min.†p X 0.05 compared with corresponding control.

vector-induced leukocyte rolling, experiments were per-formed in E-selectin-deficient mice. The administrationof 2.5×1011 particles of AdGFP to E-selectin-deficientmice increased leukocyte rolling in the postsinusoidalvenules similar to the effect seen in wild-type C57BL/6mice (data not shown). Pretreatment of E-selectin-deficient mice with anti-P-selectin antibody significantlyreduced leukocyte rolling following AdGFP administra-tion compared to E-selectin-deficient mice receivingIgG1 control antibody and AdGFP (Fig. 4A). The numberof rolling leukocytes at 60 and 90 min was reduced fur-ther in E-selectin-deficient mice pretreated with anti-Pselectin antibody versus wild-type mice receiving P-selectin antibody (Fig. 4A). At 120 min, the number ofrolling leukocytes once again began to increase; how-ever, the number (3.8±0.9) was considerably less than inwild-type mice receiving AdGFP and anti-P-selectin anti-body (7.4±1.0). Similar to the effect seen in wild-typemice, the administration of anti-P-selectin antibody to E-

selectin-deficient mice only delayed AdGFP-inducedleukocyte adhesion in the liver (Fig. 4B).

2.4 Role of > 4-integrin in adenovirus vector-induced leukocyte-endothelial interactions inthe liver

Adenovirus vector-induced rolling was not completelyabolished in anti-P-selectin antibody-treated, E-selectin-deficient mice. Experiments were therefore performed toexamine the role of § 4 integrin, a leukocyte-derived mol-ecule that has been shown to contribute to leukocyterolling in the liver [17]. Pretreatment of E-selectin-deficient mice with anti-P-selectin antibody and anti- § 4-integrin antibody (R1–2) completely inhibited AdGFP-induced leukocyte rolling at all time points (Fig. 4A). Fur-thermore, the complete lack of rolling seen in these ani-mals was accompanied by a complete lack of leukocyteadhesion for the duration of the experimental time periodfollowing AdGFP administration (2.5×1011 particles/ani-mal; Fig. 4B). In contrast, the administration of isotypecontrol IgG1 and IgG2b antibodies did not affect AdGFP-induced rolling and adhesion in the E-selectin-deficientmice compared to AdGFP alone.

The § 4-integrin supports rolling and adhesion both invitro and in vivo [3, 4]. The findings in the precedingexperiments led us to examine further the independentrole of § 4-integrin in adenovirus vector-induced leuko-cyte rolling and adhesion in liver. For this purpose, twofunctionally similar blocking § 4-integrin antibodies, R1–2and PS/2, were used [19, 20]. In wild-type C57BL/6mice, R1–2 pretreatment partially reduced the flux of roll-ing leukocytes in the postsinusoidal venules. This effectstarted at 30 min, and persisted to 120 min following theintravenous administration of AdGFP. The number of roll-ing leukocytes at 30, 60, 90 or 120 min was significantlylower than in mice receiving IgG2b control and AdGFP(Fig. 5A). R1–2 pretreatment also significantly decreasedAdGFP-induced leukocyte adhesion in the postsinusoi-dal venules at every time point (Fig. 5B). Pretreatment ofanimals with PS/2 antibody had the same effect onAdGFP-induced leukocyte rolling and adhesion as pre-treatment with R1–2 antibody (Fig. 5A, B).

2.5 Role of CD18 in adenovirus vector-inducedleukocyte-endothelial interactions in the liver

CD18 is known to play an important role in leukocyteadhesion to endothelium. The effect of CD18 in AdGFP-induced leukocyte rolling and adhesion was investigatedusing a blocking anti-CD18 antibody and CD18-deficientmice. Pretreatment of C57BL/6 mice with an anti-CD18


Fig. 5. Flux of rolling neutrophils (A) and number of adherentneutrophils (B) in the liver postsinusoidal venules of C57BL/6mice (wild type) following AdGFP administration. Mice weretreated with anti- § 4-integrin or isotype control antibody10 min prior to AdGFP challenge. Data are expressed asmean ± SEM of five animals. *p X 0.05 compared with 0 min.†p X 0.05 compared with corresponding control.

Fig. 6. Flux of rolling neutrophils (A) and number of adherentneutrophils (B) in postsinusoidal venules of C57BL/6 mice(CD18+/+) or CD18-deficient mice (CD18–/–) following AdGFPadministration. CD18+/+ mice were treated with anti-CD18antibody or isotype control antibody 10 min prior to AdGFPchallenge. Data are expressed as mean ± SEM of five ani-mals. *p X 0.05 compared with 0 min. †p X 0.05 comparedwith corresponding IgG1 control antibody.

antibody did not reduce AdGFP-induced leukocyte roll-ing in the postsinusoidal venules. Interestingly, the flux ofrolling leukocytes at 30, 60, 90 and 120 min was signifi-cantly higher in mice that received anti-CD18 antibodyand AdGFP than in animals that received IgG1 controland AdGFP (Fig. 6A). Although the reason for increasedrolling is not immediately apparent, the further enhancedrolling was not seen in CD18-deficient mice and sug-gests that this observation is related to the antibody perse. Anti-CD18 antibody did not prevent leukocyte adhe-sion in response to AdGFP (Fig. 6B). To probe further therole of CD18 in adenovirus vector-induced leukocyte roll-ing and adhesion, experiments were performed in CD18-deficient mice. Leukocyte rolling and adhesion inresponse to AdGFP were similar to the effects induced inwild-type C57BL/6 mice. Intravenous administration of2.5×1011 particles of AdGFP still caused a time-dependent increase in rolling and adherent leukocytes inthe postsinusoidal venules of CD18-deficient mice(Fig. 6A, B).

2.6 Involvement of neutrophils in adenovirusvector-induced leukocyte recruitment to theliver

Herein, we have demonstrated predominately § 4-integrin-dependent and CD18-independent leukocyteadhesion following AdGFP administration. Since neutro-phils have been shown to use CD18 for adhesion toendothelium, it remained unclear whether neutrophilswere the dominant cell type recruited in this experimen-tal model. C57BL/6 mice were treated with a neutrophil-depleting antibody 24 h prior to the administration of2.5×1011 particles of AdGFP. Antibody effectiveness wasmonitored by peripheral circulating neutrophil and totalleukocyte counts. Treatment with the anti-neutrophilantibody decreased total peripheral leukocyte numberby 40% (Fig. 7A) and completely depleted circulatingneutrophils in vehicle-treated mice by 90% (Fig. 7B). The


Fig. 7. Peripheral circulating leukocytes counts and percent-age of neutrophils in mice receiving vehicle, AdGFP alone,anti-neutrophil antibody plus vehicle, and anti-neutrophilantibody plus AdGFP. Anti-neutrophil antibody was given24 h before the experiments. Data are expressed as mean± SEM of five animals. *p X 0.05 compared with vehicle con-trol. †p X 0.05 compared with AdGFP alone.

Fig. 8. Flux of rolling neutrophils (A) and number of adherentneutrophils (B) in the postsinusoidal venule of C57BL/6 mice(wild type) following AdGFP administration. Mice weretreated with anti-neutrophil or isotype control antibody 24 hprior to AdGFP challenge. Data are expressed as mean± SEM of five animals. †p X 0.05 compared with correspond-ing control.

administration of 2.5×1011 particles of AdGFP signifi-cantly increased circulating leukocyte number and neu-trophil percentage when compared to vehicle-treatedanimals. Treatment with the anti-neutrophil antibodyblocked the elevation of circulating leukocyte numberand neutrophil percentage induced by AdGFP (Fig. 7A,B). Interestingly, a complete inhibition of circulating neu-trophils was not observed, perhaps due to mobilizationof neutrophils from bone marrow following AdGFP injec-tion.

Neutrophil-depleted mice displayed reduced leukocyterolling in the postsinusoidal venules in response toAdGFP. The flux of rolling leukocytes remained at basallevels 30, 60, 90 and 120 min following vector adminis-tration (Fig. 8A). AdGFP-induced leukocyte adhesion inthe postsinusoidal venules was also significantly dimin-ished at all time points following neutrophil depletion(Fig. 8B). These results suggest that the early leukocyterecruitment to the liver in response to adenovirus vectorsis primarily composed of neutrophils.

3 Discussion

Adenovirus vectors and adenovirus-based therapies willpotentially be used to treat a variety of genetic and non-genetic human diseases. Host inflammatory and immuneresponses limit the effective application of these agentsin humans. Our results demonstrate that adenovirus vec-tors cause rapid neutrophil-endothelial interactions inmouse liver characterized by increased rolling and adhe-sion of neutrophils in the postsinusoidal venules. P-selectin and § 4-integrins mediate early neutrophil rolling.E-selectin is also required for optimal neutrophil rolling,but as expected, the effect is delayed compared to P-selectin. Surprisingly, § 4-integrin but not CD18 mediatesneutrophil adhesion to the postsinusoidal venules inresponse to adenovirus vectors.

The acute inflammation of AdGFP-transduced liver dem-onstrated in the present study is consistent with previousfindings of innate immune activation [13, 21]. Innate


immune activation not only dramatically reduces vectorpersistence and transgene expression but also results inacute inflammation and tissue injury [16, 22]. Since ade-novirus activation of the innate immune system is dose-dependent, strategies to improve gene transfer effi-ciency by increasing vector titers only exacerbates theinflammatory response [23]. This problem has been high-lighted by the death of a patient undergoing gene ther-apy for the ornithine transcarbamylase deficiency afterintrahepatic arterial injection of an adenovirus vector car-rying a wild-type version of the defective enzyme [24].Since the liver is the major target of intravenous adminis-tered adenovirus vectors, a complete understanding ofthe inflammatory consequences in this organ is requiredto improve the effectiveness and safety of adenovirus-based therapies for humans. Our results show, for thefirst time, the molecular adhesion mechanism by whicheffector cells of the innate immune system respond toadenovirus vectors.

In this study, AdGFP stimulated an immediate inflamma-tory response manifested by neutrophil rolling and adhe-sion in the postsinusoidal venules. It is known that P-selectin is constitutively synthesized in endothelial cellsand packaged in secretory granules (Weibel-Palade bod-ies). Stimuli such as thrombin, histamine, hydrogen per-oxide, and other secretagogues rapidly induce translo-cation of P-selectin to the cell surface during fusion ofgranular membranes with the plasma membranes [25,26]. Our data show that P-selectin is essential for thevery early neutrophil rolling in the postsinusoidal venulesimmediately after AdGFP administration. Although previ-ous studies [9, 10, 17] have demonstrated that anti-P-selectin therapy can prevent leukocyte rolling in postsi-nusoidal venules, these same approaches did notreduce sequestration in sinusoids. The fact that AdGFPonly induced leukocytes in postsinusoidal vessels isencouraging in terms of therapeutically blocking leuko-cyte recruitment.

Our data show that E-selectin is responsible for delayedneutrophil rolling induced by adenovirus. Unlike P-selectin, E-selectin requires de novo protein synthesisfollowing stimulation. The delayed role of E-selectin inadenovirus-induced neutrophil rolling is consistent withprevious work examining E-selectin inducibility inresponse to TNF- § , LPS and IL-1 [27]. Althoughadenovirus-induced responses in E-selectin-deficientmice are not different from responses in wild-type mice,this is entirely consistent with previous work demonstrat-ing the requirement to inhibit P-selectin to uncover theoverlapping role of E-selectin [28]. Labow et al. foundthat in two models of inflammation, thioglycollate-induced peritonitis and delayed-type hypersensitivity inthe skin, E-selectin-deficient mice displayed no signifi-

cant change in trafficking of neutrophils. While neutrophilaccumulation at early times was dependent on P-selectin, neutrophil accumulation at later time points wasblocked by an anti-P-selectin antibody only in E-selectin-deficient mice [28]. Our present data confirmthat P-selectin mediates the initial neutrophil rolling, andboth P-selectin and E-selectin are involved in thedelayed neutrophil rolling induced by adenovirus vectorsin the postsinusoidal venules.

At first glance it is somewhat surprising that P-selectinand E-selectin could inhibit 90–95% of neutrophil rolling,and yet neutrophil adhesion was only affected modestly.However, this observation is consistent with previousfindings that selectin-dependent rolling must be inhibitedby at least 90% to significantly affect adhesion [29]. Fur-thermore, the inhibition of selectins in slower flowingvessels had absolutely no effect on leukocyte adhesion[29]. Since the liver blood flow is quite low, bypassingselectins or a greater reliance on integrins for adhesion isquite likely. In this study, we found that anti- § 4-integrinplus anti-P-selectin antibodies administered to E-selectin-deficient mice completely abolishes both neu-trophil rolling and adhesion elicited by AdGFP in thepostsinusoidal venules. These data demonstrate that § 4-integrin accounts for the remaining selectin-independentrolling. When § 4-integrin alone was blocked, only 50% ofthe leukocyte rolling was inhibited but a dramaticdecrease in adhesion was seen. These data suggest that§ 4-integrin is important in primarily mediating the leuko-

cyte adhesion caused by AdGFP in the postsinusoidalvenules. Clearly, § 4-integrin contributed to both leuko-cyte rolling as well as leukocyte adhesion, a unique char-acteristic attributed to § 4-integrin [3, 4]. The rapidity of§ 4-integrin-mediated rolling and adhesion is consistent

with the expression of its ligand vascular adhesionmolecule-1 (VCAM-1). Essani and co-workers found thatin livers of normal control mice, only a very weak expres-sion of VCAM-1 was observed in sinusoidal lining cells;however, VCAM-1 was moderately expressed in postsi-nusoidal blood vessels [30]. Furthermore, they alsofound that increase in VCAM-1 expression on sinusoidallining cells could be seen only at 4 h after endotoxinadministration [30]. Our results of predominant leukocyterolling and adhesion in postsinusoidal venules within 2 hafter AdGFP administration are comparable to their find-ings.

To exclude a role for CD18 in adenovirus-induced leuko-cyte recruitment, we also performed experiments inCD18-deficient mice. Our data suggest that § 4-integrinbut not CD18 is involved in the leukocyte recruitmentinduced by AdGFP. This adhesion profile would infer thatmononuclear cells rather than neutrophils were the leu-kocytes involved. However, our previous histological


assessments [13] and the neutrophil depletion studiesherein clearly show that neutrophils were the predomi-nate infiltrating cells. Although rare, there is a precedentfor § 4-integrin-dependent neutrophil recruitment inrodent models of inflammation. First, § 4-integrin wasfound to be able to mediate CD18-independent neutro-phil recruitment in endotoxin- or KC-induced lunginflammation [31, 32]. Second, § 4-integrin expressionwas observed on resting and activated mouse neutro-phils [33]. The novelty of our work is that AdGFP causedneutrophil adhesion in the postsinusoidal venulesthrough an exclusive and rapid § 4-integrin-dependent,but CD18-independent pathway not reported previously.Whether this is a direct effect of AdGFP on leukocytes oron endothelium remains to be determined.

Our data demonstrate that adenovirus vectors inducerapid neutrophil recruitment restricted to the postsinu-soidal venules of the liver. Although overt liver injury isnot seen until 16–24 h post-administration, our data pro-vide insight to the early mechanism of liver injury inducedby adenovirus vectors. Anti-adhesion therapy canreduce early leukocyte recruitment into the liver inresponse to adenovirus vectors but a combination of P-selectin, E-selectin and § 4-integrin inhibition will at aminimum be required. Tandem § 4-integrin, P-selectinand E-selectin inhibition does not affect leukocyterecruitment into the liver in other models of inflammationdue to sequestration of leukocytes in sinusoids [9, 10,17]. Therefore, the lack of recruitment of leukocytes intosinusoids in this model system bodes well for anti-adhesion therapy. The early modulation of leukocyterecruitment to the liver provides a strategy to improve thesafety and effectiveness of adenovirus-based therapiesfor humans.

4 Materials and methods

4.1 Animals

Male C57BL/6 mice (Charles River Laboratories, Quebec,Canada) were used throughout the study. Mice deficient inE-selectin and CD18 integrin were originally generated bygene targeting in embryonic stem cells as previouslydescribed [34, 35], then backcrossed on to C57BL/6 for atleast seven generations. These mice were housed in specificpathogen-free facilities. All animals used weighed 22–30 g.Animal protocols were approved by the University of CalgaryAnimal Care Committee and met the Canadian Guidelinesfor Animal Research.

4.2 Intravital microscopy in the mouse liver

Mice were anesthetized with a mixture of 200 mg/kg keta-mine (Rogar/STB Inc., Montreal, Quebec, Canada) and

10 mg/kg xylazine (MTC Pharmaceuticals, Cambridge,Ontario, Canada) injected intraperitoneally (i.p.). The rightjugular vein was cannulated to maintain anesthesia and forinjection of the adenovirus vector and the monoclonal anti-bodies. The abdomen was opened via a midline incision, theskin and peritoneum were removed close to the costal mar-gin, and the hepatoform ligament was carefully releasedfrom the gallbladder. Mice were then placed in a left supineposition on a Plexiglas form (Universal Plastic, Calgary, Can-ada) and the left lobe of the liver was gently placed onto thestage. The liver was covered with Saran Wrap (Dow Brands,Ontario, Canada) to hold the organ in position and the intes-tines were kept close to the abdomen and covered withsaline-soaked gauze. Animals were kept warm with an infra-red heat lamp.

After the liver was isolated and placed under the intravitalmicroscope, a separate postsinusoidal venule and eight toten draining sinusoids were located. A microscope (AxiovertS100; Zeiss, Germany) with a ×40 objective lens (Plan-Neofluar; Zeiss) and a ×10 eyepiece was used to observethe microcirculation events of the liver. A video camera(NC—70; DAGE MTI, USA) was mounted on the microscopeand projected the image onto the monitor and the imageswere recorded for playback analysis using a videocassetterecorder (SLV-975HF; Sony, Japan). The numbers of rollingand adherent leukocytes were determined offline duringvideo playback analysis. Leukocytes were consideredadherent to the venular endothelium if they remained sta-tionary for 30 s. Rolling leukocytes were defined as thosemoving at a velocity less than that of erythrocytes within agiven vessel. Flux of rolling leukocytes was counted as theleukocyte number rolled along a 100 ? m distance in 1 min.

4.3 Adenovirus vector

The E1-deleted, E3-defective AdGFP (Quantum, Montreal,Canada) under the control of the cytomegalovirus promoterwas propagated in 293 cells as previously described [36].Virus vehicle (10 mM Tris pH 8.0, 1 mM MgCl2, 10% glyc-erol) was used for control.

AdGFP concentration was determined by measuring theoptical density at 260 nm [37]. Vectors were screened forreplication-competent adenovirus by plaque assay on HeLacells and remained consistently X 1:1010 particles. In thepresent study, 2.5×1011 particles/animal of AdGFP wasgiven to mice in a volume of 100 ? l/animal through injectioninto the jugular vein.

4.4 Experimental protocol

Immediately after finding an appropriate vessel the imagewas recorded for 5 min. This served as a baseline assess-ment. After AdGFP was injected via jugular vein, recordingswere taken at 30 min interval for 2 h. Virus vehicle (100 ? l/animal) was used as the control.


To study the role of selectins, integrins, and the involvementof neutrophils in this model of adenovirus vector-inducedleukocyte recruitment, we administered 20 ? g/animal of amonoclonal anti-P-selectin antibody (RB40.34; IgG1; Phar-Mingen), 75 ? g/animal of a monoclonal anti- § 4-integrin anti-body (R1–2; IgG2b; PharMingen), 200 ? g/animal of a mono-clonal anti- § 4-integrin antibody (PS/2; IgG2b; Merck, USA),or 30 ? g/animal of a monoclonal anti-CD18 antibody(GAME-46; IgG1; PharMingen) intravenously 10 min beforeAdGFP challenge in C57BL/6 mice. Mice deficient in CD18or E-selectin were directly challenged with AdGFP. To studythe additive role of adhesion molecules in this response,mice deficient in E-selectin were treated with anti-P-selectinantibody or the combination of anti-P-selectin and anti- § 4-integrin (R1–2) antibodies at the dose described above. Toidentify the involvement of neutrophils in the adenoviralresponse, neutrophil depletion experiments were per-formed. C57BL/6 mice were treated by i.p. injection of150 ? g/animal of an anti-neutrophil antibody (RB6–8C5;IgG2b) as described previously [38] 24 h prior to AdGFPadministration. Neutropenia was confirmed by peripheralblood smear. IgG1 or IgG2b antibodies were used as isotypecontrols.

4.5 Circulating leukocyte counts

At the end of experiments, whole blood was drawn via car-diac puncture. Total leukocyte counts were performed usinga hemocytometer (Bright-line; Hausser Scientific, Horsham,PA). Neutrophil number was counted by blood smear.

4.6 Statistical analysis

The data are presented as the mean ± SEM. ANOVA andStudent’s t-test with Bonferroni’s correction were used formultiple comparisons. Statistical significance was set atp X 0.05.

Acknowledgements: Dr. Kubes is an Alberta HeritageFoundation for Medical Research (AHFMR) scientist and aCanada Research Chair. Dr. Li is supported by a CanadianAssociation of Gastroenterology (CAG) Fellowship. Dr.Muruve is a Canadian Institutes of Health Research (CIHR)Scholar and AHFMR Clinical Investigator. Dr. Lee is anAHFMR Senior Scholar. This study was funded by researchoperating grants from the Canadian Liver Foundation, Cana-dian Institutes of Health Research (CIHR), and a CIHR GroupGrant.

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Correspondence: Paul Kubes, Immunology ResearchGroup, Department of Physiology and Biophysics, Univer-sity of Calgary, 3330 Hospital Drive NW, Calgary, Alberta,T2N 4N1, CanadaFax: +1-403-283-1267e-mail: pkubes — ucalgary.ca


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The role of selectins and integrins in adenovirus vector-induced neutrophil recruitment to the liver