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₄ Integrin-Dependent Leukocyte Recruitment Does Not Require VCAM-1 in a Chronic Model of Inflammation¹

Brent Johnston^*, Alex Chee^*, Thomas B. Issekutz, Tatiana Ugarova, Alison Fox-Robichaud^*, Michael J. Hickey^* and Paul Kubes^2,*

^* Immunology Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada; Dalhousie Inflammation Group, Departments of Pediatrics, Medicine, Immunology, and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada; and Center for Thrombosis and Vascular Biology, Lerner Research Institute, Cleveland, OH 44195

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rats immunized with Mycobacterium butyricum in Freund’s adjuvant develop a chronic vasculitis, with large increases in leukocyte rolling and adhesion in mesenteric postcapillary venules that are significantly inhibited with an ₄ integrin Ab. Using intravital microscopy to visualize chronically inflamed microvessels, we demonstrated that ₄ integrin-dependent leukocyte rolling and adhesion was inhibited with a ß₁ integrin, but not a ß₇ integrin Ab. To date, VCAM-1 has been presumed to be the primary ligand for ₄ß₁ integrin in the vasculature. However, ₄ß₁ integrin-dependent interactions were not reduced by monoclonal or polyclonal VCAM-1 Abs or a VCAM-1 antisense oligonucleotide despite increased VCAM-1 expression in the mesenteric vasculature. To ensure that the VCAM-1 Abs were functional and used at saturating concentrations, blood from Ab-treated rats was perfused over monolayers of CHO cells transfected with rat VCAM-1. Sufficient ₄ integrin or VCAM-1 Ab was present to inhibit leukocyte interactions with rat VCAM-1 by 95–100%. Under in vitro flow conditions, only mononuclear leukocytes were recruited from blood of control rats onto purified VCAM-1. However, neutrophils were also recruited onto VCAM-1 from whole blood of adjuvant-immunized animals via ₄ integrin. Another ligand for ₄ß₁ integrin is the connecting segment-1 (CS-1) region of fibronectin. An Ab to the CS-1 portion of fibronectin, which did not reduce rolling and adhesion in adjuvant arthritis animals, completely inhibited leukocyte adhesion to CS-1 under static conditions. These findings provide the first evidence that ₄ß₁ integrin-dependent leukocyte rolling and adhesion can occur in vivo via a mechanism other than VCAM-1.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leukocytes are recruited from the blood to sites of inflammation via a multistep adhesive cascade (1, 2, 3). Initially, leukocytes tether to the endothelium and engage in transient rolling interactions. Rolling leukocytes may than become activated and adhere firmly to the endothelium before they transmigrate into the tissues. The ₄ integrin appears to be unique among the integrins in its ability to support both leukocyte rolling and leukocyte adhesion, in vitro and in vivo (4, 5, 6, 7, 8). As this molecule can mediate multiple steps in the adhesion molecule cascade, it has been proposed that the ₄ integrin could potentially recruit leukocytes to sites of inflammation without a contribution from the selectins, which mediate leukocyte rolling interactions in acute models of inflammation (9, 10, 11). This proposal increases the therapeutic potential for targeting ₄ integrin-dependent recruitment pathways. Indeed, inhibition of the ₄ integrin has been shown to block both leukocyte recruitment and the associated tissue dysfunction in several models of chronic inflammation (7, 8, 12, 13, 14, 15, 16).

VCAM-1 is thought to be the primary ligand for ₄ integrin-dependent leukocyte recruitment as the ₄ integrin will bind to purified VCAM-1 protein or VCAM-1-transfected cell lines under static or laminar flow conditions in vitro (4, 5, 6, 17, 18, 19, 20). Moreover, ₄ integrin-dependent rolling and/or adhesion on cytokine-activated endothelium can be inhibited with VCAM-1 Abs (6, 17, 19, 20, 21). There is also evidence to support a role for VCAM-1 as a ligand for the ₄ß₁ integrin in vivo. Blockade of VCAM-1 has been shown to reduce leukocyte recruitment to sites of inflammation to approximately the same degree as an anti-₄ integrin Ab in response to TNF- or IL-4 (22, 23, 24). More recently, Mazo et al. (25) reported using intravital microscopy that hemopoietic progenitor cells rolled in bone marrow microvessels via the ₄ integrin/VCAM-1 pathway. Clearly, the in vitro and in vivo data support the view that ₄ß₁ integrin-dependent leukocyte rolling and adhesion can occur via VCAM-1. Based on this work and the fact that no other ligand for ₄ß₁ integrin has been reported to mediate leukocyte-endothelial cell interactions under flow conditions, it has been proposed that VCAM-1 mediates ₄ integrin-dependent leukocyte rolling and adhesion in the microcirculation even in more prolonged chronic inflammatory conditions where this contention has not been tested directly.

In this study we have tested this hypothesis using a model of chronic vasculitis induced by immunizing rats with Mycobacterium butyricum in Freund’s mineral oil adjuvant (7, 8). Within 12 days of immunization, leukocyte rolling and adhesion within mesenteric postcapillary venules are increased 10-fold over the levels observed in untreated control animals. In these animals, >50% of the leukocyte rolling and 75–80% of the leukocyte adhesion are consistently inhibited by an Ab against the ₄ integrin (7, 8). Our first objective was to ascertain which ß subunit was responsible for the rolling and adhesion (i.e., the ₄ß₁ integrin or the ₄ß₇ integrin). Next we used a systematic approach to determine whether VCAM-1 is the functional ligand for the ₄ integrin in this model of inflammation. Polyclonal and mAbs against VCAM-1 and an antisense oligonucleotide that blocks VCAM-1 expression were evaluated for their ability to inhibit the ₄ integrin-dependent leukocyte recruitment in vivo. The third objective was to design a system to confirm that the concentrations of VCAM-1 Ab were sufficient to inhibit VCAM-1-dependent interactions. This was accomplished by perfusing blood from Ab-treated rats through a laminar flow chamber over monolayers of CHO cells transfected with rat VCAM-1. Finally, we examined whether VCAM-1 was actually up-regulated in this chronic vasculitis model. The results revealed for the first time that leukocyte recruitment to the endothelial surface in this chronic model of inflammation was dependent on the ₄ß₁ integrin, but the ligand was not VCAM-1. This system also very unexpectedly revealed that neutrophils from adjuvant-immunized rats, but not those from their nonimmunized counterparts, exhibited recruitment onto VCAM-1 under flow conditions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adjuvant immunization

Under light anesthetic (diethyl ether, BDH, Toronto, Canada), male Sprague Dawley rats (160–220 g) were injected s.c. at the base of the tail with a solution of heat-killed M. butyricum (Difco, Detroit, MI) in Freund’s mineral oil adjuvant (Difco; 0.75 mg of M. butyricum in 0.1 ml of adjuvant). This protocol has been used as a model of arthritis in other laboratories and was described in detail previously (14, 15, 26). Joint swelling became apparent 10–12 days after immunization, and the rats were used in this study at this time point. Previous experiments using intravital microscopy revealed a tremendous increase in leukocyte trafficking through mesenteric postcapillary venules 4–20 days after immunization (7, 8, 26).

Intravital microscopy

Rats were maintained on a purified laboratory diet and fasted for 18–24 h before surgery. Animals were anesthetized with an i.p. injection of sodium pentobarbitol (55 mg/kg body weight). The right jugular vein was cannulated to maintain anesthesia, and the right carotid artery was cannulated to measure systemic arterial blood pressure (model P23XL pressure transducer, Viggo-Spectramed, Oxnard, CA; model 7 physiologic recorder, Grass Instruments, Quincy, MA). Following laparotomy, rats were placed in a supine position on an adjustable Plexiglass microscope stage, and a segment of the midjejunum was exteriorized and prepared for intravital microscopy as previously described (7, 8, 9, 10, 11, 26).

The mesenteric preparation was observed through an intravital microscope (Optiphot-2, Nikon, Mississauga, Canada) with a x25 objective lens (Wetzlar L25/0.35, Leitz, Munich, Germany) and a x10 eyepiece. A video camera (model 5100 HS, Panasonic, Osaka, Japan) mounted on the microscope projected the image onto a color monitor (model PVM 2030, Sony, Tokyo, Japan), and the images were recorded using a videocassette recorder (model AG-1790, Panasonic) for subsequent playback analysis. The final magnification of the image on the monitor was x1800. Single unbranched mesenteric venules (30–50 µm in diameter) were selected for study. The same section of venule was observed throughout the experiment to control for variations between different regions. Venular diameter was measured on-line using a video caliper (Microcirculation Research Institute, Texas A & M University, College Station, TX). Centerline RBC velocity was also measured on-line using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A & M University).

The number of rolling and adherent leukocytes was determined off-line during video playback analysis. Leukocytes were considered adherent to the venular endothelium if they remained stationary for a period of time equal to or exceeding 30 s. Adhesion is expressed as cells per 100 µm of venule length. Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of erythrocytes within a given vessel. The flux of rolling leukocytes was determined as the number of white blood cells that rolled past a fixed point in the venule during a 1-min interval using frame-by-frame video analysis.

Experimental protocols

Leukocyte trafficking was examined in mesenteric postcapillary venules of naive and M. butyricum-immunized animals (12 days postimmunization). Anti-adhesion molecule therapies were administered following two baseline recordings (at the 20 min point) so that each animal could serve as its own control for effects of treatments on leukocyte rolling flux and adhesion. In some experiments animals were treated with a polyclonal rabbit serum against rat VCAM-1 (0.5 ml/rat i.v.), a mAb against rat VCAM-1 (3 mg/kg i.v.; 5F10) (22, 23), a mAb against the rat ₄ integrin (4 mg/kg i.v.; TA-2) (7), a mAb against the rat ß₇ integrin (4 mg/kg i.v.; TA-6), or a mAb against the mouse ß₁ integrin (2 mg/kg i.v.; HMß1-1, PharMingen Canada, Mississauga, Canada). In preliminary experiments the ß₁ integrin Ab was found to block the ß₁ integrin, but induced ß₂ integrin (CD18)-dependent adhesion. Therefore, experiments were performed using ß₁ integrin Ab plus a blocking ß₂ integrin mAb (2 mg/kg i.v.; WT-3). On its own, WT-3 blocks chemokine-induced adhesion in normal animals (27), but does not reduce adhesion in mesenteric venules of adjuvant-immunized rats (7, 8) or enhance the effect of the ₄ integrin or ß₇ integrin mAbs (data not shown). In another experiment a polyclonal Ab directed against the connecting segment-1 (CS-1)³ portion of fibronectin (0.1 and 0.3 ml/rat) was given to immunized rats. All these Abs were used at concentrations that were previously reported to inhibit leukocyte recruitment. Nevertheless, to confirm that suprasaturating concentrations of Ab were used, and that the concentration of Ab was sufficient to block binding to the targeted ligand (VCAM-1 or CS-1), whole blood flow chamber assays were used as described below.

In other experiments rats were treated with a phosphorothioate antisense oligodeoxynucleotide (ISIS 18155, a gift from Dr. C. F. Bennett, ISIS Pharmaceuticals, Carlsbad, CA), which blocks translation of rat and mouse VCAM-1 mRNA (C. F. Bennett, unpublished observation). The sequence of ISIS 18155 is 5'-CGCGACCATCTTCACAGGCA-3'. ISIS 18155, or a scrambled control oligonucleotide, ISIS 18154, was given at 50 mg/kg i.v. 16–24 h before intravital microscopy. Antisense oligonucleotides given in this manner were found to be effective in murine and rat models of inflammation (28, 29). The dose of antisense in this study prevented significant VCAM-1 expression.

Whole blood flow chamber assay

CHO cells stably transfected with rat VCAM-1 (a gift from Dr. R. R. Lobb, Biogen, Cambridge, MA) were grown to confluence (2 days) on glass coverslips (Fisher Scientific, Ottawa, Canada) using Ham’s F-12 medium (Life Technologies, Grand island, NY) with 10% FBS (Life Technologies). In other experiments glass coverslips were coated with 5 µg/ml soluble recombinant human VCAM-1 (a gift from Dr. R. R. Lobb, Biogen) and incubated for 2 h at 37°C. It has been shown previously that rat ₄ integrins will bind to human VCAM-1 (30, 31). Coverslips were then incubated with 1% BSA (Sigma, St. Louis, MO) for an additional 2 h (37°C) to block nonspecific leukocyte interactions with glass. Coverslips were assembled into parallel plate flow chambers (2, 32) and washed with HBSS (Life Technologies) before experiments. Conditions were maintained at 37°C using a warm air cabinet surrounding the flow chamber.

Whole blood was isolated from normal and adjuvant-immunized rats by cardiac puncture, treated with heparin (30 U/ml) to prevent clotting, and maintained at 37°C in a water bath. Some animals had been treated with Abs against VCAM-1 or the ₄ integrin before blood isolation (see above). A syringe pump (model 22, Harvard Apparatus, South Natick, MA) was used to draw blood across VCAM-1-coated coverslips or over CHO cells expressing rat VCAM-1 at a defined shear force of 10 dyn/cm². Whole blood was perfused over coverslips for 5 min, followed by perfusion with HBSS as previously described (32). Within 20 s of perfusion with HBSS, the RBC cleared and leukocytes could be observed interacting with the surface of the coverslip. Leukocyte interactions were examined using an inverted phase contrast microscope (Axioskop, Carl Zeiss Canada, Don Mills, Canada) with a x10 objective. Images were projected onto a video monitor using a CCD camera and were videotaped for subsequent analysis. Four different fields of view were each recorded for 15 s. The numbers of rolling and adherent leukocytes were determined. Rolling leukocytes could be distinguished from those in the bulk flow by phase contrast and their reduced velocity. In these experiments leukocytes were defined as adherent if they remained stationary for a period 10 s (33).

To distinguish the types of leukocytes that were interacting with VCAM-1, the coverslips were gently removed from the flow chamber and stained using a Wright-Giemsa staining kit. This procedure resulted in minimal loss of interacting leukocytes as determined by comparing the number of leukocytes on the coverslip before removal from the chamber with that after the staining procedure (32).

Flow cytometry

Leukocytes were isolated from citric acid, sodium citrate, and dextrose (ACD) anticoagulated rat blood by a 45-min sedimentation over 4% dextran (250,000 m.w.; Spectrum, Gardena, CA) followed by hypotonic lysis of contaminating RBC. Cells were diluted to 1 x 10⁶/tube and incubated with an Ab against the ₄ integrin (TA-2; 1 µg/tube) at room temperature for 30 min. Cells were washed and stained with a FITC-labeled goat anti-mouse IgG (Becton Dickinson, Mississauga, Canada) for 30 min. After washing, the level of ₄ integrin expression was measured using a FACScan flow cytometer (Becton Dickinson). Neutrophils and lymphocytes were differentiated based on forward and side scatter characteristics.

Radioiodination of mAbs for VCAM-1 expression

The binding (5F10) and nonbinding (P-32) mAbs were labeled with ¹²⁵I and ¹³¹I (DuPont-NEN, Boston, MA), respectively, using the Iodogen method previously described (34). Untreated M. butyricum-immunized rats, and M. butyricum-immunized rats treated with ISIS 18155 as previously described were anesthetized, and the carotid artery and jugular vein were cannulated. A mixture of ¹²⁵I-labeled 5F10 and a fixed dose of ¹³¹I-labeled nonbinding mAb was administered through the jugular vein catheter. Exactly 5 min after injection of the mAb mixture a blood sample was obtained from the carotid artery. Immediately thereafter, the animals were heparinized (40 U of sodium heparin) and rapidly exsanguinated by perfusion of bicarbonate-buffered saline through the jugular venous catheter with simultaneous blood withdrawal through the carotid arterial catheter. This was followed by perfusion of 60 ml of buffered saline through the carotid artery after severing the inferior vena cava at the thoracic level. The mesentery was harvested and weighed.

The method of calculating VCAM-1 expression has been described previously (34). Briefly, the ¹²⁵I binding and ¹³¹I nonbinding mAb activities in the mesentery and in 100-µl samples of cell-free plasma were measured. A 3-µl aliquot of the radiolabeled mAb mixture was assayed to determine the total injected activity of each labeled mAb. The accumulated activity of each mAb in the mesentery was expressed as a percentage of the injected activity per gram of tissue. VCAM-1 expression was calculated by subtracting the accumulated activity per gram of tissue of the nonbinding mAb (¹³¹I-labeled P-23) from the activity of the binding VCAM-1 (¹²⁵I-labeled 5F10). This value, expressed as a percentage of the injected dose per gram of tissue, was converted to nanograms of mAb per gram of tissue by multiplying the above value by the total injected binding mAb.

Statistical analysis

Data are presented as the mean ± SEM. Means were compared using Student’s t test with Bonferroni’s correction for multiple comparisons where appropriate. Statistical significance was set at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ₄ß₁ integrin mediates leukocyte recruitment in vivo

In Fig. 1 (A and B) the data reveal that on day 12 250–300 cells rolled/min and 30–40 cells adhered/100 µm of venule length in rats immunized with M. butyricum 12 days earlier. These values are at least 10-fold higher than those in control untreated animals (data not shown). Treatment with an ₄ integrin mAb (4 mg/kg i.v.; TA-2) significantly reduced both the leukocyte rolling flux (55%) and leukocyte firm adhesion (75%) observed in mesenteric postcapillary venules of adjuvant-immunized rats. Previous work has demonstrated that the remaining 50% of leukocyte rolling is L-selectin dependent (7). Treatment with an Ab against the ß₁ integrin (2 mg/kg i.v.; HMß1-1) caused a similar reduction in leukocyte rolling flux (Fig. 1). The inset in Fig. 1 (top) demonstrates the temporal kinetics of ß₁ integrin Ab inhibition; leukocyte rolling was 300 cells/min in adjuvant-treated rats and decreased to <150 cells/min following ß₁ integrin Ab administration. As we were unable to find a ß₁ integrin Ab that did not activate CD18 on rat leukocytes, we inhibited the CD18-dependent adhesion when testing the role of ß₁ integrin. It should be noted that the mAb to CD18 did not block adhesion on its own (Fig. 1). In the presence of a CD18 mAb (2 mg/kg i.v.; WT-3), the ß₁ integrin mAb reduced adhesion by about 55%. In direct contrast, an Ab against the ß₇ integrin (4 mg/kg i.v.; TA-6) did not reduce leukocyte rolling or adhesion alone (Fig. 1) or in the presence of a CD18 Ab (data not shown). These data suggest that the ₄ integrin-dependent leukocyte recruitment in adjuvant-immunized rats is dependent on very late activation Ag-4 (₄ß₁) rather than ₄ß₇ integrin.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Role of the 4ß1 and 4ß7 integrins in the leukocyte rolling (A) and adhesion (B) in mesenteric postcapillary venules of M. butyricum-immunized (day 12) rats. After a baseline control period of 20 min, rats were treated with Abs against 4 integrin (TA-2; 4 mg/kg i.v.; n = 5), ß1 integrin (HMß1-1; 2 mg/kg i.v.; n = 3), ß7 integrin (TA-6; 4 mg/kg i.v.; n = 4), and/or CD18 (WT-3; 2 mg/kg i.v.; n = 5). The administration protocol is illustrated in the inset; the administration of HMß1-1 at 20 min significantly reduced leukocyte rolling flux at later time points. Time points after Ab administration were compared with the initial baseline time points within each animal. *, p < 0.05 compared with time zero; , p < 0.05 compared with untreated day 12 immunized animals.

VCAM-1 does not mediate ₄ integrin-dependent leukocyte recruitment in vivo

As VCAM-1 is the major ligand for very late activation Ag-4 in vitro (4, 5, 6, 17, 18, 19), animals were treated with a function-blocking mAb against rat VCAM-1 (3 mg/kg i.v.; 5F10). Surprisingly, this treatment did not reduce leukocyte rolling or adhesion (Fig. 2). As multiple VCAM-1 epitopes have been shown to mediate ₄ integrin/VCAM-1 interactions (17, 18, 19, 35), a polyclonal serum against rat VCAM-1 (0.5 ml/rat) was also tested. As with the mAb, this treatment had no effect on leukocyte rolling or adhesion (Fig. 2). It should be noted that in the same animals in which inhibition of VCAM-1 had no effect on leukocyte rolling in the microvasculature of an adjuvant-immunized rat, the Ab directed against ₄ integrin was again able to inhibit the rolling of >150 cells/min (not shown). In additional experiments, VCAM-1 inhibition with an antisense oligonucleotide failed to reduce leukocyte trafficking in vivo (Fig. 2).

View larger version (23K): [in this window] [in a new window]   FIGURE 2. The role of VCAM-1 in leukocyte rolling (A) and adhesion (B) in mesenteric postcapillary venules of M. butyricum-immunized (day 12) rats. After a baseline control period of 20 min, rats were treated with an mAb against VCAM-1 (5F10; 3 mg/kg i.v.; n = 4), or a polyclonal serum against rat VCAM-1 (0.5 ml/rat i.v.; n = 4). Other rats were pretreated 16–24 h before the experiment with a phosphorothioate antisense oligonucleotide against VCAM-1 (ISIS 18155; 50 mg/kg i.v.; n = 4) or a scrambled control oligonucleotide (ISIS 18154; 50 mg/kg i.v.; n = 4). The administration protocol is illustrated in the inset. The administration of 5F10 at 20 min did not alter leukocyte rolling flux at later time points. Time points after Ab administration were compared with the initial baseline time points within each animal. *, p < 0.05 compared with time zero; , p < 0.05 compared with untreated day 12 immunized animals.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Leukocyte recruitment onto rat VCAM-1 under flow conditions in vitro. Whole blood from adjuvant-immunized rats was perfused over monolayers of CHO cells transfected with rat VCAM-1. Some animals had been pretreated with mAbs against the 4 integrin (TA-2; 4 mg/kg i.v.; n = 2) or VCAM-1 (5F10; 3 mg/kg i.v.; n = 3), or a polyclonal serum against rat VCAM-1 (0.5 ml/rat i.v.; n = 3). Total leukocyte interactions (rolling and adhesion) were quantitated after 5 min of perfusion. *, p < 0.05 compared with untreated day 12 immunized animals.

Fig. 4 demonstrates that there was a very small constitutive amount of VCAM-1 in the mesentery of animals that were not immunized with M. butyricum. By contrast, VCAM-1 expression was increased >10-fold in this vascular bed in rats immunized 12 days earlier with M. butyricum. This increase was 50% of the response seen with optimal levels of LPS administration (1.88 ng of mAb/g), suggesting a significant amount of VCAM-1 expression in the mesentery of adjuvant-immunized rats. Fig. 4 also demonstrates that ISIS 18155 antisense inhibited VCAM-1 synthesis, as VCAM-1 expression was reduced by >60% in the mesentery of adjuvant-immunized rats.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. VCAM-1 expression in mesenteric vessels of healthy nonimmunized rats (n = 4) and rats immunized with M. butyricum 12 days (n = 5) before measurement of adhesion molecule expression. Also shown is VCAM-1 expression in rats immunized with M. butyricum receiving ISIS 18155 (n = 6) antisense 16–24 h before measurement of VCAM-1 expression. *, p < 0.05 compared with untreated healthy control rats.

In vitro whole blood experiments were also used to examine the populations of leukocytes attaching to VCAM-1. In Fig. 5A it can be seen that equivalent numbers of leukocytes from naive and adjuvant-immunized rats were recruited onto VCAM-1. However, when leukocytes on the coverslips were stained with a Wright-Giemsa staining kit for identification of cell types, differences in recruited cell types were observed between control and adjuvant-immunized animals (Fig. 5B). Neutrophil and monocyte recruitment from blood of adjuvant-immunized rats were significantly increased compared with those in naive control rats. In contrast, the number of lymphocytes recruited from adjuvant-immunized rats was significantly reduced compared with that in control animals. The recruitment of neutrophils was confirmed using an esterase staining kit (data not shown). To determine whether these differences in recruitment were due to the large increase in the number of neutrophils and monocytes in the blood of adjuvant-immunized rats (Table I), the recruited cell populations were normalized for differences in circulating numbers of leukocytes. Fig. 5C shows that the increased neutrophil recruitment and reduced lymphocyte recruitment in blood from adjuvant-immunized rats were still apparent despite normalization for circulating numbers of cells, while the increased recruitment of monocytes was entirely accounted for by the increase in circulating monocyte number. These results suggest that there may be differences in the activation state and/or adhesion molecule expression of circulating neutrophils and lymphocytes in adjuvant-immunized rats.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Differential leukocyte recruitment onto purified VCAM-1 under flow conditions in vitro. Whole blood from naive and adjuvant-immunized rats was perfused over coverslips coated with purified recombinant VCAM-1. Total leukocyte interactions (rolling and adhesion) were quantitated after 5 min of perfusion (A). Flow chambers were dismantled, and coverslips were stained with a Wright-Giemsa staining kit to determine leukocyte differentials (B). Leukocyte differentials were corrected for differences in leukocyte distribution (C) as shown in Table I. *, p < 0.05 compared with naive control rats.

₄ integrin expression on neutrophils is not altered in adjuvant-immunized rats

Flow cytometry was used to examine expression of the ₄ integrin on leukocytes from control and adjuvant-immunized rats. As reported previously (31, 36), ₄ integrin was detected on the surface of circulating rat neutrophils isolated from untreated control animals (Fig. 6). However, neutrophils from adjuvant-immunized rats did not exhibit an increase in the expression of ₄ integrin compared with that in control animals (Fig. 6). The lymphocyte results were more equivocal, as a significant proportion of lymphocytes in adjuvant-immunized animals expressed a similar level of ₄ integrin as nonimmunized rats, but a significant proportion of lymphocytes expressed less ₄ integrin. This may account for the reduced lymphocyte recruitment onto VCAM-1 in vitro.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Expression of 4 integrin on leukocytes from naive and adjuvant-immunized rats. Mean fluorescence with 4 integrin mAb (TA-2) was increased in naive (dotted line) and adjuvant-immunized (thick line) rats. There was no difference in the binding of an 4 integrin Ab to neutrophils and only a small reduction of binding to lymphocytes from naive or adjuvant-immunized rats. Plots are representative of five separate experiments.

CS-1 is not the ligand for ₄ integrin

In a final series of experiments, we examined the possibility that the CS-1 portion of fibronectin might be the ligand for the ₄ integrin. This peptide has been detected on the luminal surface of activated endothelium and mediates binding of monocytes in static adhesion assays (37). However, an Ab directed against the CS-1 portion of fibronectin had no effect on leukocyte recruitment when used in vivo at 0.1 or 0.3 ml/rat (Fig. 7, A and B). When whole blood from adjuvant-immunized animals was perfused over a fibronectin-40 fragment containing the CS-1 region, no interactions were noted, suggesting that leukocytes cannot interact with CS-1 under flow conditions (data not shown). However, many leukocytes adhered to the CS-1-containing fragment when the perfusion was stopped, and leukocytes were allowed to settle (Fig. 7C). Leukocytes from an animal treated with the anti-CS-1 Ab displayed no interactions with fibronectin-40 under these static conditions (Fig. 7C), suggesting that ample Ab was used in vivo.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. The effects of an Ab against the CS-1 portion of fibronectin on leukocyte rolling (A) and adhesion (B) in vivo and adhesion of leukocytes to fibronectin-40 fragment in vitro (C). Whole blood from treated and untreated animals was perfused into a flow chamber containing the CS-1 portion of fibronectin and then stopped for 3 min before flow was resumed. Due to limitations of the amount of Ab and identical results for two different concentrations, the data are grouped.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

VCAM-1 is thought to be the primary ligand for ₄ß₁ integrin. Although a number of other ligands support ₄ß₁ integrin-dependent leukocyte adhesion, to our knowledge no other ligand has ever been proposed to mediate ₄ß₁ integrin-dependent leukocyte rolling. However, in this study leukocyte recruitment (rolling and adhesion) could not be reduced by neutralizing VCAM-1 using three separate approaches, suggesting an alternate adhesive ligand for the ₄ integrin in mesenteric venules of adjuvant-immunized rats despite increased VCAM-1 expression in this specific condition. Although it is possible that this observation is particular only to this model of adjuvant-induced vasculitis, another group has recently observed that ₄ß₁ integrin-dependent IL-4-induced leukocyte recruitment into the rat pleural cavity could not be reduced with a VCAM-1 Ab (S. Nourshargh, Imperial College School of Medicine, unpublished observation). One could argue that rat leukocytes simply do not interact with VCAM-1 under flow conditions, but our own in vitro data clearly demonstrate that rat leukocytes interacted with both rat and human VCAM-1 in the presence of shear forces. A novel ₄ integrin ligand may also be present in other species, as Vonderheide and Springer found that human lymphocyte interactions with IL-4-activated human endothelium could be inhibited to a greater extent with Abs against ₄ integrin than with Abs against VCAM-1 (17).

This is the first report of ₄ß₁ integrin-dependent leukocyte rolling and adhesion with venular endothelium that could not be inhibited at least partly by VCAM-1 Abs. One could argue that the Abs used do not block the function of rat VCAM-1 or were used in insufficient quantities. However, the VCAM-1 mAb used in this study was able to block TNF-- and IL-4-induced eosinophil recruitment in rat skin in previous investigations (22, 23), while the polyclonal anti-VCAM-1 serum was shown to block streptococcal cell wall-induced monoarthritis in the rat (45). Furthermore, we used these Abs in vivo at concentrations that blocked subsequent leukocyte recruitment from isolated blood onto monolayers of CHO cells stably transfected with rat VCAM-1. This suggests that saturating levels of Ab were present in each animal, and that the Abs were functional. These data reveal the presence of a VCAM-1-independent ligand that can support both ₄ß₁ integrin-dependent rolling and adhesion in mesenteric venules of adjuvant-immunized rats.

Although we have established the presence of a novel ligand for ₄ß₁ integrin in a chronic inflammatory process, the identity of this ligand was not revealed. Treatment of human aortic endothelial cells with minimally modified, low density lipoprotein has been shown to induce ₄ integrin-dependent monocyte binding to endothelium that was mediated by the CS-1 domain of fibronectin rather than VCAM-1 (37). Moreover, the mRNA for alternatively spliced CS-1 fibronectin isoform has been shown to be present on blood vessel endothelium in the synovium of patients with rheumatoid arthritis (46). Although plasma fibronectin has been shown not to mediate ₄ integrin-dependent interactions under physiological flow conditions (5), the CS-1 region may be cryptic and not accessible in native fibronectin. However, in this study we show that leukocytes were unable to tether under flow conditions to the CS-1 sequence of fibronectin. Moreover, an Ab that was able to block leukocyte adhesion to CS-1 under static conditions had no effect in our adjuvant-immunized rats, suggesting that CS-1 was not a ligand for ₄ß₁ integrin-dependent leukocyte rolling and adhesion in our model of chronic vasculitis.

There are a number of other potential ligands that have been shown to bind ₄ integrin in static adhesion assays. In addition to fibronectin, the matrix protein osteopontin can interact with the ₄ integrin (47). In another study the ₄ integrin could bind homotypically to the ₄ integrin chain via a conserved LDV motif (48). Another possibility is that MAdCAM-1 could be the ligand for ₄ integrin if the integrin is in a high affinity state (as appeared to be the case for neutrophils in our in vitro studies). An activated conformation may allow ₄ß₁ integrin interactions with MAdCAM-1 in our model. In addition, ₄ integrin has been shown to bind to denatured proteins such as albumin (49), suggesting that the binding specificity for ₄ß₁ integrin is considerably broader than previously realized. Because proteins can be excessively denatured during chronic inflammation, and endothelium expresses many novel molecules, including ß₁ integrins (50), any of the aforementioned molecules could serve as ligands for the VCAM-1-independent, ₄ß₁ integrin-dependent leukocyte recruitment. Alternatively, there may be a unique, as yet unidentified, ligand for the ₄ integrin up-regulated in adjuvant-immunized rats. Our data suggest that one or more of these proteins is a dominant ligand for ₄ integrin in this model of inflammation.

An unexpected finding in this study is that neutrophils from adjuvant-immunized rats had a novel capacity to adhere to VCAM-1 under flow conditions in vitro, whereas neutrophils from their nonimmunized counterparts completely lacked the ability to bind to VCAM-1. It has been shown previously that rat neutrophils express low levels of ₄ integrin under basal situations (31, 36). However, this level of ₄ integrin did not mediate leukocyte trafficking under control conditions or during very acute inflammation in vivo (7, 8). Moreover, the data would suggest that there is no detectable up-regulation of the number of ₄ integrins on the surface of neutrophils in adjuvant-immunized rats. However, there is a dramatic increase in the VCAM-1 binding affinity of the neutrophils, suggesting either increased affinity or more optimal localization of ₄ integrin on the surface of neutrophils. Although it is tempting to conclude that this may not be relevant to human neutrophils, which do not normally express surface ₄ integrin, previous work from our laboratory has shown that maximal pharmacological stimulation of human neutrophils could induce sufficient ₄ integrin on the surface of neutrophils to support binding to VCAM-1 under both static and flow conditions in vitro (47, 51). The data in the present study would suggest that neutrophil ₄ integrin can serve as an adhesion molecule in chronic inflammatory conditions. Whether a similar event occurs in chronic inflammatory disease in humans awaits investigation.

In conclusion, our study demonstrates for the first time that ₄ß₁ integrin-dependent leukocyte rolling and adhesion to endothelium were not dependent upon the commonly associated ligand VCAM-1 or the CS-1 sequence of fibronectin. Although these data are obtained from one chronic model of inflammation, they raise some important questions about the importance of VCAM-1 in other chronic inflammatory diseases. This is underscored by our observation that increased VCAM-1 expression did not necessarily contribute to leukocyte recruitment in this model of inflammation. Our data, however, do not diminish the potential importance of VCAM-1 in certain inflammatory and noninflammatory conditions (13, 16, 24, 45). Finally, our data also reveal the ability of neutrophils from chronically inflamed animals to interact with ligands for ₄ integrin and raise the possibility that these cells may use this molecule when adhering to microvessels in chronic inflammation in vivo. The presence of an alternate ₄ integrin ligand in chronic inflammation may have important implications in the development of therapeutic agents for inflammatory diseases.

	Footnotes

 
¹ This work was supported by grants from the Alberta Heritage Foundation for Medical Research (AHFMR) and the Medical Research Council of Canada (MRC). P.K. is an AHFMR and MRC Scientist. B.J. is the recipient of an AHFMR studentship.

² Address correspondence and reprint requests to Dr. Paul Kubes, Immunology Research Group, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail address:

³ Abbreviations used in this paper: CS-1, connecting segment-1; MAdCAM-1, mucosal addressin cell adhesion molecule-1.

Received for publication November 17, 1999. Accepted for publication January 7, 2000.

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