The role of {alpha}4 and LFA-1 integrins in selectin-independent monocyte and neutrophil migration to joints of rats with adjuvant arthritis

Ulrieke Birner1, Thomas B. Issekutz1,2,3, Ulrike Walter2,3 and Andrew C. Issekutz1,2,3

1 Departments of 1Pathology,
2 Pediatrics and
3 Microbiology–Immunology, Dalhousie University, Halifax, Nova Scotia B3J 3G9, Canada

Correspondence to: A. C. Issekutz, Department of Pediatrics, Division of Immunology, Rheumatology and Infectious Diseases, IWK Grace Health Centre, 5850 University Avenue, Halifax, Nova Scotia B3J 3G9, Canada


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Monocytes and neutrophils are chronically recruited to joints in rheumatoid arthritis. In the joints of rats with adjuvant arthritis, this is mediated, in part, by selectin-dependent and selectin-independent mechanisms. To define the selectin-independent mechanisms, 51Cr-labeled blood monocytes, 111In-labeled neutrophils and function blocking mAb to the selectins and integrins were utilized. Integrins contributed to the selectin-independent monocyte migration to arthritic joints with 58–70% inhibition of this recruitment by anti-{alpha}4 or anti-LFA-1 mAb, relative to selectin blockade alone. {alpha}4 plus P-selectin blockade was as effective as combined blockade of {alpha}4, P-, E- and L-selectin, mediating ~83% of the overall monocyte migration to the joints. In contrast, LFA-1 was the predominant selectin-independent mechanism for neutrophil recruitment to the joints. LFA-1 together with P-selectin had essential roles in the talar joint. In dermal inflammation in the arthritic rats, LFA-1 accounted for most (69%) of the selectin-independent monocyte migration to the chemoattractant C5adesArg (zymosan-activated serum), whereas LFA-1 and Mac-1 both contributed to selectin-independent neutrophil recruitment to C5adesArg. {alpha}4 integrin and P-selectin in concert mediated monocyte recruitment to lipopolysaccharide and IFN-{gamma} lesions (81%). Thus: (1) either {alpha}4 or LFA-1 can mediate monocyte migration to arthritic joints in the absence of selectin function and {alpha}4 together with P-selectin is particularly important; (2) LFA-1 is the predominant mechanism of selectin-independent migration of neutrophils to inflamed joints; and (3) in arthritic rats, selectin-independent migration of monocytes and neutrophils to dermal inflammation is mediated by {alpha}4 or LFA-1 or both LFA-1 and Mac-1, depending on the leukocyte type, and inflammatory stimulus.

Keywords: adhesion molecule, CD11a, CD49d, inflammation, leukocyte


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Leukocyte recruitment into inflamed tissues is dependent upon adhesive and activational events between leukocytes and vascular endothelium, which are mediated by cell adhesion molecules (CAM) and chemoattractants. The leukocyte–endothelial cell adhesion cascade, a process whereby leukocytes emigrate out of the blood into extravascular tissues, is broadly divided into three sequential but overlapping steps: (1) leukocyte tethering and rolling along endothelial venules, (2) leukocyte activation leading to arrest and firm adhesion, and (3) leukocyte transmigration across the endothelial lining (for reviews, see 1,2).

Each step in the cascade is thought to be governed by a distinct set of molecular signals. Selectin–carbohydrate interactions between leukocytes and activated endothelium mediate the initial capture and rolling of the leukocyte along post-capillary venules. Specifically, the endothelial selectins E- and P-, and the leukocyte-expressed L-selectin, interact with mucin-like glycoproteins containing fucosylated, sialyated and sulfated ogliosaccharide side chains (e.g. ESL-1, PSGL-1, CD34) (3). Activation is mediated by chemo- attractant–receptor ligations, and the resulting arrest, firm adhesion and spreading of leukocytes on endothelium are dependent upon leukocyte integrins of the ß1 (e.g., {alpha}4ß1), ß2 (e.g. {alpha}Lß2, {alpha}mß2) and ß7 (e.g. {alpha}4ß7) subfamilies interacting with endothelial ligands of the Ig superfamily (IgS; e.g. VCAM-1, ICAM-1, ICAM-2). To date, the ß2 integrins LFA-1 (CD11a/CD18; {alpha}Lß2) and Mac-1 (CD11b/CD18; {alpha}mß2) are the best characterized leukocyte integrins involved in firm adhesion (4). LFA-1 is expressed by lymphocytes, monocytes and granulocytes, whereas Mac-1 expression is largely limited to monocytes and granulocytes. ICAM-1 serves as an endothelial ligand for both LFA-1 and Mac-1, and LFA-1 can also bind to ICAM-2. Finally, transmigration of the leukocyte between the endothelial cell junctions and into the interstitial tissue is orchestrated by additional integrin–IgS CAM interactions (1).

The ß1 integrin, VLA-4 (CD49/CD29; {alpha}4ß1) is a heterodimer composed of an {alpha}4 chain (CD49d) non-covalently linked to a ß1 chain (CD29). The {alpha}4 subunit can also associate with ß7 chain to form LPAM-1 ({alpha}4ß7), a CAM important in mucosal immunity (5). Ligands for {alpha}4 include VCAM-1, expressed on cytokine-activated endothelium, MAdCAM-1, expressed on mucosal venules and an alternatively spliced connecting segment (CS1) region on fibronectin, expressed in extracellular matrices. {alpha}4 is expressed by a variety of immune-inflammatory cells (e.g. monocytes, lymphocytes, eosinophils, mast cells and also neutrophils) (6). {alpha}4 expression on neutrophils has been confirmed by several laboratories. Rat neutrophils have low constitutive levels of {alpha}4, as detected by flow cytometry using mAb specific for {alpha}4 (79). Despite low levels of expression, {alpha}4 on rat neutrophils can function to mediate adhesion to VCAM-1, MAdCAM-1 and perhaps fibronectin (e.g. as expressed on cardiac myocytes) (7,9). Although {alpha}4 is not constitutively expressed by human neutrophils, after transendothelial migration or exogenous stimulation, human neutrophils can rapidly mobilize preformed stores of {alpha}4ß1 subunits to their membrane surface (10). {alpha}4ß1-expressing human neutrophils are able to tether and stably adhere to VCAM-1, but not ICAM-1 on transfected L-cells (11).

{alpha}4 integrin-dependent adhesion pathways play a role in experimental inflammation and autoimmune pathologies including allergic lung inflammation, experimental allergic encephalitis, insulitis, contact hypersensitivity (as reviewed in 6) and adjuvant arthritis (8,12,13). {alpha}4–ligand interactions contribute to arrest, firm adhesion and migration of leukocytes (6). However, an additional function for {alpha}4 is materializing since recent studies report that {alpha}4 can mediate tethering and/or rolling of leukocytes both in vitro and in vivo (1416). Thus, a multifunctional capacity for {alpha}4 to support tethering, rolling and adhesion in the adhesion cascade is emerging.

Recently, we have demonstrated that selectins mediate part of the neutrophil and monocyte recruitment to inflamed joints of rats with adjuvant arthritis (17). By employing function-blocking mAb to individual selectins alone, or in combination, it was observed that the accumulation of 111In-labeled blood neutrophils in inflamed joints partly depended on both P- and E-selectin, whereas 51Cr-labeled monocyte recruitment was primarily P-selectin dependent. However, even after blockade of P-, E- and L-selectins, significant monocyte and neutrophil migration to the arthritic joints still occurred. Thus another adhesion mechanism, independent of the selectins, may also mediate this recruitment. Whether {alpha}4 integrin is an important alternate mechanism for neutrophils and monocytes during leukocyte recruitment, when the selectins are unavailable, has not been addressed in a chronic arthritis model. However, this is an important question because leukocyte adhesion molecules including, E- and P-selectin, ICAM-1 and VCAM-1, VLA-4 and Mac-1 have all been shown to be increased in synovial tissue from patients with rheumatoid arthritis (18,19). Therefore, by utilizing function-blocking mAb against {alpha}4 in combination with mAb to individual or combined selectin members, we investigated in a rat model of chronic arthritis: (1) if {alpha}4 integrin could mediate the selectin-independent accumulation of neutrophils and monocytes to inflamed joints; (2) the relative contribution of {alpha}4 in conjunction with specific selectins to mediate recruitment of neutrophils and monocytes to arthritis; and (3) whether LFA-1, an integrin thought to participate primarily in events downstream to tethering and rolling, could mediate selectin-independent accumulation of neutrophils and monocytes in arthritic joints.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Adjuvant arthritis was induced in 6- to 8-week-old male Lewis rats (175–200g) by s.c. immunization with 0.5 mg Mycobacterium butyricum (Difco, Detroit, MI) in 0.05 ml mineral oil at two sites on each side of the base of the tail. Fifteen days later, the arthritis was scored clinically from 0 to 4 points per limb and tail, based on the severity of swelling, erythema and limitation of movement as described by Taurog et al. (20). The maximal possible score was 20. The migration studies were performed at the height of clinical arthritis, 15 days post immunization.

mAb
The mAb RMP-1 (IgG2a) reacts with and blocks the adhesive function and rolling mediated by rat P-selectin (21,22). The mAb RME-1 (IgG1) reacts with and blocks the adhesive function of rat E-selectin (23). HRL-3 (IgG; a gift from Drs D. C. Anderson Pharmacia-Upjohn Laboratories, Kalamazoo, MI and M. Miyasaka, Osaka, Japan) is a hamster anti-rat L-selectin antibody which blocks the adhesive function of rat L-selectin (24). TA-2 (IgG1) is a mouse mAb which reacts with the rat {alpha}4 integrin chain and blocks the adhesive function of VLA-4 (25, 26). TA-3 (IgG1) reacts with the {alpha} chain of LFA-1 and blocks its function (27). OX-42 (IgG2a) reacts with rat CD11b/CD18 (Mac-1; a gift from Dr D. W. Mason, Oxford, UK), and blocks adhesive function and neutrophil migration (28,29). WT.3 (IgG1) is a mouse mAb (gift from Dr M. Miyasaka, Osaka, Japan) which reacts with rat ß2 chain of the CD11/CD18 integrins blocking the adhesive function of these integrins (30). All mAb (each at 1 mg/rat, except WT.3 at 2 mg/rat) were given i.v. immediately before the rats received the radiolabeled leukocytes (see below). The doses have been previously determined to provide saturating plasma levels 5- to 10-fold higher than required for maximal adhesion blocking activity (17,31). None of the mAb treatments caused leukopenia, clearance of the radiolabeled leukocytes from the circulation or caused leukocyte sequestration in vascular beds (e.g. spleen, liver, lung) as compared to animals not receiving mAb. mAb HRL-3 was used as a F(ab')2 preparation because intact antibody can cause neutropenia (24). mAb RME-1, RMP-1, TA-2, TA3 and WT.3 were used as intact antibody in all experiments, since no significant difference in the results were observed when F(ab')2 fragments were used (17 and unpublished observations). The specific effects of the blocking mAb to the selectins has been previously shown by using non-blocking mAb to the selectins and by in vivo demonstration of their inhibitory effect (17,32).

Monocyte and neutrophil isolation and labeling
Rat blood monocytes and neutrophils were isolated by hydroxyethyl starch (Hespan; Dupont Merck, Wilmington, DE) exchange transfusion (8,12). Briefly, a 25-gauge butterfly needle was inserted into the femoral vein of an anesthetized arthritic rat and the heparinized blood gradually exchanged with 50 ml 6% hydroxyethyl starch/saline (Hespan). After recovery of 45–50 ml of the blood hydroxyethyl starch perfusate in acid–citrate–dextrose (ACD, formula A; Fenwal-Travenol, Malton, Canada) anticoagulant, erythrocytes were allowed to sediment. The leukocyte-rich plasma was harvested, leukocytes pelleted at 200 g for 10 min and resuspended in Ca2+/Mg2+-free Tyrode's solution containing 10% rat platelet-poor plasma (PPP). Leukocytes were layered onto 63% isotonic plasma–Percoll (Pharmacia Fine Chemicals, Dorval, Canada) above 74% Percoll and sedimented by centrifugation (350 g for 30 min at 22°C).

Monocytes
After centrifugation, the mononuclear layer on the top of the 63% Percoll was removed, washed and resuspended in Tyrode's/10% PPP. The osmolality was increased from 284 to 299 mOsm by addition of 9% NaCl to facilitate separation of the monocytes from the lymphocytes as previously reported (33). After a brief incubation (10 min at 37°C), the cell suspension was layered onto a second discontinuous Percoll gradient (40/55/58%). After centrifugation (350 g for 30 min at 22°C) monocytes were harvested at the 40/55% and the 55/58% interphases. Fractions with >80% monocyte purity and minimal platelet contamination were washed, resuspended at 5x107 cells/ml in Tyrode's/10% PPP and radiolabeled with 75 µCi of Na251CrO4 (Amersham, Oakville, Canada) for 30 min at 37°C. Monocytes were then washed and resuspended in Tyrode's/10% PPP for i.v. injection.

Neutrophils
Neutrophils were recovered at the interphase between 63 and 74% Percoll and were > 95% pure. Neutrophils were washed, resuspended in Tyrode's/10% PPP at 108 cells/ml and labeled with 1.5 µCi/107 cells of 111In-oxine (Amersham) for 10 min at room temperature. Labeled neutrophils were then washed and resuspended in Tyrode's/10% PPP for i.v. injection. Each rat received 3–6x106 51Cr-labeled monocytes carrying 1–1.5x105 c.p.m. together with 5x106 neutrophils carrying 3–7x105 c.p.m. Viability of the labeled cells was confirmed by the active accumulation of the labeled cells in the joints and in acute dermal inflammatory reactions. The label was >90% retained on the cells in the circulation after i.v. injection as has been previously reported (34). Monocytes and neutrophils purified and labeled by these techniques do not show an appreciable increase in Mac-1 expression (12,33) or shedding of L-selectin as compared to blood monocytes and neutrophils (17).

Dermal inflammatory reactions
Acute inflammatory reactions were induced in the arthritic rats by intradermal injection of inflammatory stimuli (0.05 ml) in duplicate sites on the back as described (8). Stimuli included zymosan-activated serum (ZAS 50%), a source of the chemotactic factor C5adesArg, generated as described (8), 1 ng Escherichia coli 0111 endotoxin [lipopolysaccharide (LPS); List Biologicals, Campbell, CA], 300 U recombinant rat IFN-{gamma} (gift from P van der Meide, TNO Primate Center, The Netherlands) or diluent (RPMI 1640/0.5% HSA) as a negative control. Inflammatory agents were injected i.d. at the time of the injection of mAb and radiolabeled neutrophils.

Quantification of monocyte and neutrophil accumulation
After injection of the mAb, radiolabeled neutrophils and monocytes, and dermal inflammatory stimuli, neutrophils and monocytes were allowed to migrate for 2 h, and then the animals were euthanized. As previously described (8), 2 ml of blood was collected into ACD, 12 mm punch skin biopsies of the injected dermal sites were taken, and samples of tissues were collected for determination of 51Cr and 111In content. Tissues containing the carpal joints of the forepaws and the talar joints of the hindpaws were obtained by sectioning above and below the joint. Radioactive cell accumulation in spleen, liver, lung, lymph nodes, blood and plasma was also monitored. Neutrophil and monocyte accumulation in the tissues are expressed as c.p.m./106 c.p.m. injected.

Statistical analysis
After confirming normality (Kolmogorov–Smirnov test), data were analyzed by ANOVA followed by post hoc Bonferroni comparisons. There were fewer animals with inflamed carpal than talar joints. Therefore, statistical analysis in some groups for carpal arthritis, where indicated, was not performed.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effect of {alpha}4 or LFA-1 blockade on selectin-independent monocyte migration to rat arthritis.
As previously observed (17), 51Cr-labeled monocytes migrated rapidly (within 2 h) to the talar joint of the hindlimb and the carpal joint of the forepaw in rats with adjuvant arthritis. The mean monocyte accumulation in the talar joint, the most severely affected joint in this model, was 23,125 c.p.m. (Fig. 1Go left panel) and in the carpal joint was 5957 c.p.m. (Fig. 1Go right panel). Administration (i.v.) of anti-E- and P-selectin mAb inhibited the accumulation of monocytes to the talar joint by 58% and to the carpal joint by 48%, as previously observed (17). Blocking L-selectin in addition to E- and P- selectin did not further inhibit the migration to either of the joints (Fig. 1Go).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1. The effect of {alpha}4 or LFA-1 blockade on the selectin-independent monocyte migration to the talar (left panel) and carpal (right panel) joints of arthritic rats. Fifteen days post-immunization with M. butyricum, rats with polyarticular arthritis were injected i.v. with PBS (arthritic controls) or with function-blocking mAb to the selectins (white bars) and integrins (solid bars), alone or in combination as indicated. Immediately afterwards, 51Cr-labeled monocytes were injected i.v., and after a 2 h migration period the animals were euthanized and 51Cr monocyte accumulation in the tissues was determined as described in Methods. Values for background monocyte accumulation in the joints of naive (non-arthritic) but mAb-treated (anti-{alpha}4 plus anti-E- and P-selectin or anti-LFA-1 plus anti-E- and P-selectin) animals were subtracted from the groups (mean talar = 1143 c.p.m. and mean carpal = 467 c.p.m.). There were fewer animals with inflamed carpal than talar joints. Therefore, valid statistical analysis on carpal joints after anti-E-, P- and L- selectin plus {alpha}4 or CD18 mAb treatment was not performed. Values are the means + SEM of three to 25 animals in each group except for anti-{alpha}4 + E- + P- + L-selectin group where n = 2 for carpal joints. *P < 0.05; #P < 0.01; §P < 0.001 compared to treatment with the anti-selectin mAb treated group in each subset of three.

 
In order to investigate whether {alpha}4 or LFA-1 integrins could mediate the remaining (42–52%) monocyte accumulation in the joints, separate groups of rats were injected i.v. with blocking mAb to E- and P-selectin plus mAb to {alpha}4 or LFA-1 integrin. As shown in Fig. 1Go, blockade of {alpha}4 integrin, in addition to E- and P-selectin blockade, significantly decreased monocyte accumulation to both the talar (P < 0.001) and carpal (P < 0.001) joints by 58 and 70% respectively relative to animals treated only with mAb to E- plus P-selectin. Compared to untreated arthritic rats, combined E- and P-selectin and {alpha}4 integrin blockade resulted in an inhibition of monocyte accumulation to the talar and carpal joints by 83 and 84% respectively. Adding mAb to L-selectin to mAb to E- and P-selectin plus {alpha}4 integrin did not further inhibit monocyte accumulation to either the talar (84%) or carpal (80%) joints.

It was of interest to know whether integrins involved in monocyte migration, aside from {alpha}4, could mediate the selectin-independent monocyte accumulation. mAb to LFA-1 integrin combined with E- and P-selectin resulted in significant inhibition of monocyte accumulation in both the talar (67%; P < 0.01) and carpal (60%; P < 0.05) joints relative to E- plus P-selectin blockade alone. Compared to untreated arthritic rats, combined E- and P-selectin and LFA-1 integrin blockade inhibited monocyte accumulation at the talar and carpal joints by 86 and 79% respectively (Fig. 1Go). Blocking the CD18 (ß2) chain and thus the function of LFA-1, Mac-1 and p150/95, in combination with blockade of E-, P- and L-selectin resulted in a similar overall inhibition (80% talar and 72% carpal) as observed with mAb to only LFA-1 plus E- and P-selectin.

To assess the relative contribution of {alpha}4 or LFA-1 integrin in conjunction with either E- or P-selectin in the accumulation of monocytes to the joints, separate groups of animals were treated with mAb to: (1) P-selectin ± {alpha}4, (2) E-selectin ± {alpha}4, (3) P-selectin ± LFA-1, (4) E-selectin ± LFA-1, or for comparison (5) {alpha}4 alone or (6) LFA-1 alone. Administration of mAb to {alpha}4 partially inhibited monocyte accumulation in the talar joint (33%) but did not alter migration to the carpal joint. Anti-LFA-1 treatment alone inhibited monocyte migration to both the talar (52%) and carpal (57%) joints. P-selectin blockade alone inhibited monocyte migration to the joints by ~50%, whereas mAb to E-selectin alone had no effect on monocyte migration to either joint, as previously reported (17). Monocyte accumulation in the joints during E-selectin blockade, in combination with either P-selectin, or {alpha}4 or LFA-1 blockade was comparable to accumulation during blockade of P-selectin, {alpha}4 or LFA-1 individually, suggesting that under these conditions E-selectin is functionally redundant (Fig. 1a and bGo). In the talar joint (Fig. 1Go left panel), blocking either {alpha}4 or LFA-1, in addition to P-selectin, resulted in a significant further inhibition (P < 0.001; both) as compared to blocking P-selectin alone. In contrast, blockade of Mac-1 in conjunction with P-selectin had no effect on migration compared to P-selectin blockade alone (percent inhibition anti-Mac-1 + P-selectin = 44%, n = 5; anti-P-selectin = 44%, n = 11; data not shown). In the carpal joint (Fig. 1Go right panel), blocking {alpha}4 and P-selectin, but not LFA-1 and P-selectin, inhibited monocyte accumulation, relative to P-selectin blockade alone.

Effect of {alpha}4 or LFA-1 blockade on selectin-independent neutrophil migration to rat arthritis
During the 2 h migration period, the mean 111In-labeled neutrophil accumulation in the talar joint (Fig. 2Go left panel) was 20,590 c.p.m. and in the carpal joint (Fig. 2Go right panel) this was 4662, comparable to values in a previous study (17). Treatment with anti-E- plus P-selectin mAb inhibited neutrophil migration to the talar joint by 61% and to the carpal joint by 43%. Blocking L-selectin in addition to E- plus P-selectin did not further inhibit the migration to either of the joints (Fig. 2a and bGo).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2. The effect of {alpha}4 or LFA-1 blockade on the selectin-independent neutrophil migration to the talar (left panel) and carpal (right panel) joints of arthritic rats. Fifteen days post-immunization with M. butyricum, rats with polyarticular arthritis were injected i.v. with PBS (arthritic controls) or with function-blocking mAb to the selectins (white bars) and integrins (solid bars), alone or in combination as indicated. Immediately afterwards, 111In-labeled neutrophils were injected i.v., and after a 2 h migration period the animals were euthanized and 111In-labeled neutrophil accumulation in the tissues was determined. Values for background monocyte accumulation in the joints of naive (non-arthritic) but mAb-treated (anti-{alpha}4 plus anti-E- and P-selectin or anti-LFA-1 plus anti-E- and P-selectin) animals were subtracted from the groups (mean talar = 978 c.p.m. and mean carpal = 365 c.p.m.). Values are the means + SEM of three to 25 animals in each group except for anti-{alpha}4 + E- + P- + L-selectin group where n = 2 for carpal joints). *P < 0.05; #P < 0.01; §P < 0.001 compared to treatment with the anti-selectin mAb treated group in each subset of three.

 
As illustrated in Fig. 2Go, the remaining (40%) neutrophil accumulation in the talar joint was not further inhibited by blocking {alpha}4 integrin and E- and P-selectins together (P > 0.05), whereas the same mAb treatment significantly inhibited neutrophil migration by 53% to the carpal joint (P < 0.05), compared to E- plus P-selectin blockade. Blocking L-selectin in addition to anti-{alpha}4 and E- and P-selectin did not further inhibit neutrophil accumulation to the carpal joint, and had a minimal effect in the talar joint (73% inhibition anti-{alpha}4 + E- + P-selectin versus 78% inhibition anti-{alpha}4 + E- + P- + L- selectin).

Blockade of LFA-1 and E- and P-selectin simultaneously resulted in a significant inhibition of neutrophil accumulation in both the talar (63%; P < 0.01) and carpal (68%; P < 0.05) joints relative to E- plus P-selectin blockade. Compared to untreated arthritic rats, this treatment inhibited neutrophil migration to the talar and carpal joints by 86 and 82% respectively. mAb to CD18 in combination with E-, P- and L-selectin resulted in comparable overall inhibition of neutrophil migration to the talar (93%) and carpal (86%) joints as combined anti-LFA-1, E- and P-selectin treatment.

As with monocytes, the relative contribution of {alpha}4 or LFA-1 integrin in conjunction with either E- or P-selectin to recruitment of neutrophils was assessed. Administration of {alpha}4 mAb alone did not decrease neutrophil accumulation in either the talar or carpal joints, as has been previously reported (8). Anti-LFA-1 treatment alone partially inhibited the accumulation of neutrophils at the talar (46%) and carpal (24%) joints. mAb to P-selectin alone inhibited neutrophil migration to the joints by 26–40% but anti- E-selectin mAb alone had no effect, as previously reported (17) (Fig. 2Go). A significant further decrease in the accumulation of neutrophils in the talar joint was observed after blocking {alpha}4 plus P-selectin (P < 0.05) or LFA-1 plus P-selectin (P < 0.001) as compared to blocking P-selectin alone (Fig. 2Go left panel). In contrast, blocking Mac-1 in addition to P-selectin had no effect on neutrophil migration, compared to P-selectin blockade alone (% inhibition anti-Mac-1 + P-selectin = 28%, n = 5; anti-P-selectin = 26%, n = 11; data not shown). Blockade of {alpha}4 or LFA-1 (or Mac-1, not shown) plus P-selectin did not further decrease the migration of neutrophils to the carpal joint, as compared to P-selectin blockade alone (Fig. 2Go right panel).

Effect of {alpha}4 or LFA-1 blockade on selectin-independent monocyte and neutrophil migration to dermal inflammation in arthritic rats
It was of interest to determine the role of {alpha}4 and LFA-1 in the accumulation of monocytes and neutrophils to a different vascular bed, such as the skin, since CAM utilized by leukocytes can vary according to tissue type and inflammatory stimulus (1,2). Labeled monocytes and neutrophils migrate rapidly to dermal sites injected with ZAS (containing C5adesArg) and endotoxin (LPS). IFN- {gamma}, on the other hand, induces monocyte but not neutrophil recruitment (33). Blockade of P-selectin partly (29–60%) inhibited neutrophil and monocyte accumulation at dermal inflammation. When E-, P- and L-selectins were simultaneously blocked, monocyte and neutrophil accumulation was only partially inhibited, especially in ZAS- and LPS-induced inflammation [Fig. 3Go and as previously reported (17)]. These results indicate that monocyte and neutrophil migration to dermal inflammation has both a selectin-dependent and a selectin-independent component.



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 3. The effect of {alpha}4 or LFA-1 blockade on the selectin-independent monocyte and neutrophil migration to dermal inflammation in rats with arthritis. Immediately following the i.v. injections of the anti-selectin and anti-integrin mAb as indicated and the i.v. injection of 51Cr-labeled monocytes and 111In-labeled neutrophils, cutaneous inflammatory reactions were induced in the same rats as in Figs 1 and 2GoGo by i.d. injections of 50 µl of 50% ZAS (top panels), 1 ng endotoxin (LPS) (middle panels), 300 U IFN-{gamma} (bottom panel) or diluent alone on the back in duplicate sites. The labeled cells were allowed to accumulate in the skin sites during the same time they were migrating to the joints (2 h migration period). [51Cr]monocyte and [111In]neutrophil accumulation in the tissues was determined. Background values of diluent-injected sites were subtracted from the stimuli in each animal (mean = 169 ± 18.2 c.p.m.) individually, since each animal served as its own control. Values are mean + SEM of three to 24 animals per group. *P < 0.05; #P < 0.01; §P < 0.001 compared to treatment with the anti-selectin mAb treated group in each subset of three.

 
To evaluate the involvement of {alpha}4 or LFA-1 integrin in selectin-independent migration of monocytes and neutrophils to acute dermal inflammation, the same animals used in Figs 1 and 2GoGo were injected i.d. with ZAS, LPS, IFN-{gamma} or diluent control and radiolabeled monocyte and neutrophil accumulation in the sites was quantitated. As illustrated in Fig. 3Go(top panels), after E- and P-selectin blockade, selectin-independent mechanisms accounted for 43–52% of monocyte and neutrophil recruitment to ZAS lesions. mAb to {alpha}4, E- and P-selectin in combination did not further inhibit the migration of either monocytes or neutrophils to skin sites injected with ZAS. However, blocking LFA-1 in addition to E- and P-selectin, significantly inhibited the ZAS-induced monocyte (P < 0.01) and neutrophil (P < 0.05) accumulation, as compared to E-plus P-selectin blockade. Overall this inhibition was 87% for monocytes and 71% for neutrophils, as compared to untreated controls. Blockade of CD18 in combination with E-, P- and L-selectin did not further inhibit monocyte migration to ZAS as compared to LFA-1, E- plus P-selectin blockade. In contrast, blocking CD18 in combination with E-, P- and L-selectin virtually abolished the neutrophil accumulation (99%).

Figure 3Go (middle panels) shows that combined E- and P- or E-, P and L-selectin blockade only partially inhibited LPS-induced migration of monocytes (~72%) and neutrophils (~58%) to dermal lesions, although monocyte migration to IFN-{gamma} was largely suppressed (Fig. 3Go bottom panel). Additional blockade of LFA-1 with the selectins did not significantly inhibit further the accumulation for either cell type. However, combined CD18, E-, P- and L-blockade inhibited monocyte accumulation overall by 91%, while virtually abolishing neutrophil accumulation (99%) to LPS-induced dermal inflammatory reactions. This is in accordance with a similar degree of inhibition of neutrophil migration to dermal inflammation by anti-CD18 treatment alone (8).

In contrast to the lack of effect of LFA-1 blockade on selectin-independent migration to LPS or IFN-{gamma}, {alpha}4 blockade significantly inhibited monocyte migration to IFN-{gamma} inflammation when only P-selectin was blocked (P < 0.01). This effect was also marginally significant (P = 0.05) at LPS lesions. However, with inhibition of the other selectins (E- ± L-selectin), {alpha}4 blockade had less of an effect, not reaching significance, suggesting that {alpha}4 was not an important mechanism for selectin-independent migration to these reactions.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In human and experimental arthritis, the associated joint pathology and injury is due, in part, to the chronic recruitment of monocytes and neutrophils into the joint and periarticular tissues. The recruitment of these leukocytes, at least in some vascular beds, is believed to require initial capture, tethering and rolling of the cell in venules, mediated predominantly by the selectins (L-, P- and E-selectins) (1,2). However, leukocyte accumulation in joints of rats with adjuvant arthritis appears to be mediated by both selectin-dependent (17) and selectin-independent mechanisms. Despite selectin blockade (E- and P- or E-, P- and L-selectin) with specific function blocking mAb there was appreciable monocyte and neutrophil accumulation in inflamed joints (40–60% of control).

{alpha}4 integrin mediated a large part of the recruitment of monocytes in the absence of functional selectins, since blocking {alpha}4 integrin significantly inhibited monocyte accumulation in both the talar and carpal joints by 60–70% relative to selectin (E- and P- or E-, P- and L- selectin) blockade alone. This resulted in ~83% decrease overall in monocyte migration to these joints (Fig. 1Go). Blockade of P-selectin and {alpha}4 integrin together inhibited monocyte migration to the joints to the same extent (~81%) as blocking all of the selectins and {alpha}4 integrin (~82%). Thus, E- and L-selectin do not appear essential for monocyte recruitment to the joints. Our findings of the relatively important role of {alpha}4 and P-selectin for monocyte recruitment to joints is similar to the results with eosinophils by Patel et al. (35). They observed that P-selectin and {alpha}4 integrin mediated tethering and adhesion of eosinophils on IL-4-activated endothelium at physiological shear in vitro. Both eosinophils and monocytes express high levels of {alpha}4 integrin, which may explain why this integrin makes a significant contribution to the recruitment of these leukocytes (7).

Neutrophils express considerably less {alpha}4 integrin than monocytes (7,8). Nevertheless, {alpha}4 integrin also contributed to neutrophil accumulation in the inflamed joints (Fig. 2Go) in the presence of blockade of one or more of the selectins, but to a lesser and more variable degree than observed for monocytes (Fig. 1Go). Although {alpha}4 integrin contributed a small degree to P-selectin-independent neutrophil migration to the talar joint (Fig. 2Go), this was not significant in the carpal joint unless E-selectin was also blocked. These observations suggest that {alpha}4 in conjunction with P-, E- and L-selectin, depending on the joint, contribute to neutrophil migration to arthritis. However, other mechanisms are probably also involved since overall inhibition of neutrophil accumulation was only ~75% after selectin plus {alpha}4 blockade (Fig. 2Go).

Recent studies have demonstrated that {alpha}4 integrin is able to mediate leukocyte tethering and/or rolling under in vitro and in vivo conditions (1416). Our model does not allow us to directly observe whether the selectin-independent leukocyte accumulation we detect is via an {alpha}4-dependent rolling and/or adhesion process in the joint. However, our results do demonstrate that {alpha}4 integrin can mediate monocyte and some degree of neutrophil accumulation in the absence of selectin function, since blocking this integrin in conjunction with all of the selectins results in greater inhibition than selectin blockade alone (Figs 1 and 2GoGo). This may be of relevance to the situation in rheumatoid arthritis in which the {alpha}4ß1–VCAM-1 ligand receptor pair is known to be up-regulated in joint tissues where it may mediate tethering, rolling, arrest, adhesion, spreading and transmigration, depending on VCAM-1 density and leukocyte activating stimulus (36).

Figures 1 and 2GoGo illustrate that the ß2 integrin, LFA-1, can also mediate selectin-independent recruitment of both monocytes and neutrophils since blocking LFA-1 in addition to E- and P- or E-, P- and L-selectin inhibited both monocyte and neutrophil recruitment to the inflamed joints significantly more than blocking the selectins alone. In both the talar and carpal joints, LFA-1 contributed to selectin independent monocyte migration to an extent comparable with the {alpha}4 integrin mechanism with ~83% of the migration being mediated by E- and P- selectin plus LFA-1 or {alpha}4 integrin. In contrast, neutrophils appear to be more dependent on LFA-1 than {alpha}4 integrin for selectin-independent migration to the joints. This was most clear in the talar joint, the most severely affected joint in this model (20), where LFA-1, in conjunction with E- and P- selectin, mediated ~86% of the neutrophil accumulation (Fig. 2Go). Selectin-independent neutrophil migration was not further inhibited by mAb to CD18, indicating that LFA-1 is the predominant ß2 integrin involved. P-selectin and LFA-1 appear to be essential mechanisms for mediating monocyte and neutrophil migration to the talar joint, since blocking these adhesion molecules resulted in comparable inhibition to blocking both E- and P-selectin together with LFA-1. LFA-1 appears to have a unique role among ß2 integrins in contributing to selectin-independent recruitment, since blockade of Mac-1 did not inhibit the accumulation of either cell type to the joints to any greater extent than blocking P-selectin alone (data not shown). This is despite the fact that the same mAb to Mac-1 (OX-42) and LFA-1 (TA-3) were shown by us previously to inhibit neutrophil and monocyte migration to arthritic joints and skin, migration which is effectively mediated by either LFA-1 or Mac-1 (8,12,28). In the carpal joint the role of LFA-1 in selectin-independent monocyte and neutrophil recruitment was observed only when E-selectin as well as P-selectin function were blocked, suggesting that in some joints, E-selectin can substitute for a generally predominant P-selectin role. It needs to be emphasized that most of the residual neutrophil and especially monocyte migration after selectin (E-, P- and L-selectin) and LFA-1 or {alpha}4 integrin blockade is mediated by the functionally remaining integrin, since combined LFA-1 and {alpha}4 blockade nearly eliminates (75–95%) recruitment of these leukocytes, even without blocking the selectins, as shown previously (8,12).

The selectin-independent monocyte and neutrophil recruitment to inflammation in another vascular bed, i.e. the skin, was also observed in response to the chemoattractant C5adesArg in ZAS, the cytokine inducer LPS and the cytokine IFN-{gamma} (Fig. 3Go). LFA-1 appears to mediate most of the selectin-independent monocyte accumulation to ZAS, since blocking LFA-1 in combination with E- and P-selectin inhibited monocyte recruitment to a significantly greater extent (87%) than blocking only the selectins, E- and P- ± L-selectin (57%, Fig. 3Go top left panel). Furthermore, when all the ß2 integrins were blocked with anti-CD18 mAb along with blockade of all the selectins, monocyte recruitment was comparably inhibited (92%), suggesting that Mac-1 and p150/95 do not contribute significantly to this monocyte migration. LFA-1 also appears to mediate a part of the selectin-independent neutrophil migration to ZAS. However, neutrophil selectin-independent migration is mediated not only by LFA-1, but also other ß2 integrins, since anti-CD18 mAb blocked essentially all (99%) the migration (Fig. 3Go top right panel). Most likely Mac-1 is an important alternate to LFA-1 in mediating this selectin-independent neutrophil recruitment, because LFA-1 and Mac-1 jointly mediate 80–90% of neutrophil recruitment to dermal inflammation in normal as well as arthritic rats (8,28).

LPS and IFN-{gamma} both activate endothelium to express adhesion molecules, including P- and E-selectin, ICAM-1 and VCAM-1, and to produce chemokines and cytokines (1,2). Our results suggest that monocyte migration to these stimuli may be primarily dependent on {alpha}4 integrin functioning in concert with P-selectin (Fig. 3Go middle panels), as was observed in the joints. On the other hand, neither {alpha}4 nor LFA-1 were required for selectin-independent migration of neutrophils to LPS-induced dermal inflammation (Fig. 3Go middle right panel). Rather, neutrophil accumulation appears to require both LFA-1 and Mac-1 function, since blocking the common ß2 (CD18) chain in addition to blocking the selectins, resulted in >99% inhibition. These results are in accordance with previous observations of the dual importance of LFA-1 and Mac-1 to monocyte and neutrophil recruitment to dermal inflammation (8,12), and highlight the differences in mechanism with different leukocytes and with inflammatory stimuli acting primarily on the leukocyte (e.g. C5adesArg in ZAS) or on the endothelium and connective tissue cells (e.g. LPS and IFN-{gamma}).

The role of LFA-1 in mediating the selectin-independent accumulation of monocytes and neutrophils in arthritic joints and skin inflammation was unexpected. LFA-1 is believed to mediate events downstream from selectin function (i.e. firm adhesion and migration), although this paradigm has not been rigorously tested in the setting of chronic inflammation. Pertinent to our observation may be the report by Gadboury and Kubes (37) that CD11/CD18 mechanisms can mediate rolling of leukocytes in both unstimulated and PAF-stimulated rat mesenteric venules in vivo during reduced but physiological flow. Moreover, antibody to CD18 can attenuate rolling at reduced shear in vitro (38). Taken together with our current results, these observations raise the possibility that LFA-1 can mediate a significant degree of leukocyte recruitment, perhaps by functioning in capture and rolling of the leukocyte, under certain conditions. For example, in chronic inflammation, blood vessels dilate and hydrodynamic shear forces are estimated to be reduced by 40–70% (37). There is also usually thrombocytosis and marked leukophilia, most notably of neutrophils. As a result, leukocytes are displaced from the axial flow and marginate toward the vessel wall, which can increase the probability of leukocyte–endothelial interactions and of non-selectin-adhesive interactions being formed. These conditions can also facilitate homotypic (leukocyte–leukocyte) and heterotypic (leukocyte–platelet) interactions (39,40). Similarly, it is possible that in this environment of reduced shear and of high concentrations of leukocytes, platelets, and leukocyte priming and activating mediators (possibly with leukocyte integrins already in high affinity states), tethering/ rolling via selectins and {alpha}4 integrin may not be essential. This may explain why both neutrophil and monocyte migration to dermal inflammation in arthritic rats (with systemic inflammation) is less selectin-dependent than in normal rats, in response to the same stimuli (17,41). Similarly, in other systemic inflammatory conditions such as in models of lung injury or glomerulitis, integrins rather than selectins appear to mediate most of the leukocyte accumulation (reviewed in 42).

Our data highlight and emphasize the complexity involved in leukocyte recruitment during chronic inflammation to arthritic joints and to dermal inflammation. Optimal monocyte and neutrophil recruitment in both tissues requires selectins, especially P-selectin. However, monocytes can also utilize either {alpha}4 or LFA-1 integrins for migration in the absence of selectins. On the other hand, neutrophils utilize primarily LFA-1, to migrate to chronically inflamed joints in the absence of selectin function. Further to this, two important observations emerge: (1) there is a particular role for P-selectin in concert with {alpha}4 integrin for monocyte migration in vivo, and (2) there is a cooperative role for P- and E-selectin along with LFA-1 in neutrophil migration, especially to arthritic joints. Further definition of the essential leukocyte recruiting adhesive interactions during chronic inflammation in various tissues will be required for developing strategies to regulate this chronic process.


    Acknowledgments
 
The authors are grateful for the excellent technical assistance of Ms C. Jordan and Mr D. Rowter. This work was supported by grant 89036 from the Arthritis Society of Canada. U. B. is recipient of a Graduate student scholarship award from the IWK Grace Health Center Research Program.


    Abbreviations
 
ACD acid–citrate–dextrose
CAM cell adhesion molecule
LPS lipopolysaccharide
PPP platelet-poor plasma
ZAS zymosan-activated serum

    Notes
 
Transmitting editor: Z. Ovary

Received 4 August 1999, accepted 12 October 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Springer, T. A. 1995. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. Annu. Rev. Physiol. 57:827.[ISI][Medline]
  2. Ley, K. 1996. Molecular mechanisms of leukocyte recruitment in the inflammatory process. Cardiovasc. Res. 32:733.[ISI][Medline]
  3. McEver, R. P., Moore, K. L. and Cummings, R. D. 1995. Leukocyte trafficking mediated by selectin–carbohydrate interactions. J. Biol. Chem. 270:11025.[Abstract/Free Full Text]
  4. Gahmberg, C. G., Tolvanen, M. and Kotovuori, P. 1997. Leukocyte adhesion—structure and function of human leukocyte beta2-integrins and their cellular ligands. Eur. J. Biochem. 245:215.[Abstract]
  5. Hamann, A., Andrew, D. P., Jablonski-Westrich, D., Holzmann, B. and Butcher, E. C. 1994. Role of alpha 4-integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152:3282.[Abstract/Free Full Text]
  6. Lobb, R. R. and Hemler, M. E. 1994. The pathophysiologic role of alpha 4 integrins in vivo. J. Clin. Invest. 94:1722.[ISI][Medline]
  7. Davenpeck, K. L., Sterbinsky, S. A. and Bochner, B. S. 1998. Rat neutrophils express alpha4 and beta1 integrins and bind to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Blood. 91:2341.[Abstract/Free Full Text]
  8. Issekutz, T. B., Miyasaka, M. and Issekutz, A. C. 1996. Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation. J. Exp. Med. 183:2175.[Abstract]
  9. Reinhardt, P. H., Ward, C. A., Giles, W. R. and Kubes, P. 1997. Emigrated rat neutrophils adhere to cardiac myocytes via alpha 4 integrin. Circ. Res. 81:196.[Abstract/Free Full Text]
  10. Kubes, P., Niu, X. F., Smith, C. W., Kehrli, M. E., Jr, Reinhardt, P. H. and Woodman, R. C. 1995. A novel beta 1-dependent adhesion pathway on neutrophils: a mechanism invoked by dihydrocytochalasin B or endothelial transmigration. FASEB J. 9:1103.[Abstract/Free Full Text]
  11. Reinhardt, P. H., Elliott, J. F. and Kubes, P. 1997. Neutrophils can adhere via alpha4beta1-integrin under flow conditions. Blood 89:3837.[Abstract/Free Full Text]
  12. Issekutz, A. C. and Issekutz, T. B. 1995. Monocyte migration to arthritis in the rat utilizes both CD11/CD18 and very late activation antigen 4 integrin mechanisms. J. Exp. Med. 181:1197.[Abstract]
  13. Barbadillo, C., G-Arroyo, A., Salas, C., Mulero, J., Sanchez-Madrid, F. and Andreu, J. L. 1995. Anti-integrin immunotherapy in rheumatoid arthritis: protective effect of anti-alpha 4 antibody in adjuvant arthritis. Semin. Immunopathol. 16:427.[ISI][Medline]
  14. Alon, R., Kassner, P. D., Carr, M. W., Finger, E. B., Hemler, M. E. and Springer, T. A. 1995. The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J. Cell Biol. 128:1243.[Abstract]
  15. Berlin, C., Bargatze, R. F., Campbell, J. J., von Andrian, U. H., Szabo, M. C., Hasslen, S. R., Nelson, R. D., Berg, E. L., Erlandsen, S. L. and Butcher, E. C. 1995. Alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[ISI][Medline]
  16. Johnston, B., Issekutz, T. B. and Kubes, P. 1996. The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo. J. Exp. Med. 183:1995.[Abstract]
  17. Walter, U. M. and Issekutz, A. C. 1997. The role of E- and P-selectin in neutrophil and monocyte migration in adjuvant-induced arthritis in the rat. Eur. J. Immunol. 27:1498.[ISI][Medline]
  18. Tak, P. P., Thurkow, E. W., Daha, M. R., Kluin, P. M., Smeets, T. J., Meinders, A. E. and Breedveld, F. C. 1995. Expression of adhesion molecules in early rheumatoid synovial tissue. Clin. Immunol. Immunopathol. 77:236.[ISI][Medline]
  19. Grober, J. S., Bowen, B. L., Ebling, H., Athey, B., Thompson, C. B., Fox, D. A. and Stoolman, L. M. 1993. Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions. J. Clin. Invest. 91:2609.[ISI][Medline]
  20. Taurog, J. D., Argentieri, D. C. and McReynolds, R. A. 1988. Adjuvant arthritis. Methods Enzymol. 162:339.[ISI][Medline]
  21. Walter, U. M., Ayer, L. M., Wolitzky, B. A., Wagner, D. D., Hynes, R. O., Manning, A. M. and Issekutz, A. C. 1997. Characterization of a novel adhesion function blocking monoclonal antibody to rat/mouse P-selectin generated in the P-selectin-deficient mouse. Hybridoma 16:249.[ISI][Medline]
  22. Johnston, B., Walter, U. M., Issekutz, A. C., Issekutz, T. B., Anderson, D. C. and Kubes, P. 1997. Differential roles of selectins and the alpha4-integrin in acute, subacute, and chronic leukocyte recruitment in vivo. J. Immunol. 159:4514.[Abstract]
  23. Walter, U. M., Ayer, L. M., Manning, A. M., Frenette, P. S., Wagner, D. D., Hynes, R. O., Wolitzky, B. A. and Issekutz, A. C. 1997. Generation and characterization of a novel adhesion function blocking monoclonal antibody recognizing both rat and mouse E-selectin. Hybridoma 16:355.[ISI][Medline]
  24. Tamatani, T., Kuida, K., Watanabe, T., Koike, S. and Miyasaka, M. 1993. Molecular mechanisms underlying lymphocyte recirculation. III. Characterization of the LECAM-1 (L-selectin)-dependent adhesion pathway in rats. J. Immunol. 150:1735.[Abstract/Free Full Text]
  25. Issekutz, T. B. and Wykretowicz, A. 1991. Effect of a new monoclonal antibody, TA-2, that inhibits lymphocyte adherence to cytokine stimulated endothelium in the rat. J. Immunol. 147:109.[Abstract/Free Full Text]
  26. Issekutz, T. B. 1991. Inhibition of in vivo lymphocyte migration to inflammation and homing to lymphoid tissues by the TA-2 monoclonal antibody. A likely role for VLA-4 in vivo. J. Immunol. 147:4178.[Abstract/Free Full Text]
  27. Issekutz, T. B. 1992. Inhibition of lymphocyte endothelial adhesion and in vivo lymphocyte migration to cutaneous inflammation by TA-3, a new monoclonal antibody to rat LFA-1. J. Immunol. 149:3394.[Abstract/Free Full Text]
  28. Issekutz, A. C. and Issekutz, T. B. 1992. The contribution of LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) to the in vivo migration of polymorphonuclear leucocytes to inflammatory reactions in the rat. Immunology 76:655.[ISI][Medline]
  29. Robinson, A. P., White, T. M. and Mason, D. W. 1986. Macrophage heterogeneity in the rat as delineated by two monoclonal antibodies MRC OX-41 and MRC OX-42, the latter recognizing complement receptor type 3. Immunology 57:239.[ISI][Medline]
  30. Tamatani, T., Kotani, M., Tanaka, T. and Miyasaka, M. 1991. Molecular mechanisms underlying lymphocyte recirculation. II. Differential regulation of LFA-1 in the interaction between lymphocytes and high endothelial cells. Eur. J. Immunol. 21:855.[ISI][Medline]
  31. Issekutz, A. C., Ayer, L., Miyasaka, M. and Issekutz, T. B. 1996. Treatment of established adjuvant arthritis in rats with monoclonal antibody to CD18 and very late activation antigen-4 integrins suppresses neutrophil and T-lymphocyte migration to the joints and improves clinical disease. Immunology 88:569.[ISI][Medline]
  32. Birner, U., Issekutz, T. B. and Issekutz, A. C. 1999. The role of selectins in VLA-4 and CD18-independent neutrophil migration to joints of rats with adjuvant arthritis. Eur. J. Immunol. 29:1094.[ISI][Medline]
  33. Issekutz, A. C. and Issekutz, T. B. 1993. Quantitation and kinetics of blood monocyte migration to acute inflammatory reactions, and IL-1 alpha, tumor necrosis factor-alpha, and IFN-gamma. J. Immunol. 151:2105.[Abstract/Free Full Text]
  34. Issekutz, A. C. and Issekutz, T. B. 1991. Quantitation and kinetics of polymorphonuclear leukocyte and lymphocyte accumulation in joints during adjuvant arthritis in the rat. Lab. Invest. 64:656.[ISI][Medline]
  35. Patel, K. D. 1998. Eosinophil tethering to interleukin-4-activated endothelial cells requires both P-selectin and vascular cell adhesion molecule-1. Blood. 92:3904.[Abstract/Free Full Text]
  36. Lalor, P. F., Clements, J. M., Pigott, R., Humphries, M. J., Spragg, J. H. and Nash, G. B. 1997. Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1. Eur. J. Immunol. 27:1422.[ISI][Medline]
  37. Gaboury, J. P. and Kubes, P. 1994. Reductions in physiologic shear rates lead to CD11/CD18-dependent, selectin-independent leukocyte rolling in vivo. Blood 83:345.[Abstract/Free Full Text]
  38. Lawrence, M. B., Smith, C. W., Eskin, S. G. and McIntire, L. V. 1990. Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium. Blood 75:227.[Abstract]
  39. Bargatze, R. F., Kurk, S., Butcher, E. C. and Jutila, M. A. 1994. Neutrophils roll on adherent neutrophils bound to cytokine-induced endothelial cells via L-selectin on the rolling cells. J. Exp. Med. 180:1785.[Abstract]
  40. Lalor, P. and Nash, G. B. 1995. Adhesion of flowing leucocytes to immobilized platelets. Br. J. Haematol. 89:725.[ISI][Medline]
  41. Walter, U. M. and Issekutz, A. C. 1997. Role of E- and P-selectin in migration of monocytes and polymorphonuclear leucocytes to cytokine and chemoattractant-induced cutaneous inflammation in the rat. Immunology 92:290.[ISI][Medline]
  42. Albelda, S. M., Smith, C. W. and Ward, P. A. 1994. Adhesion molecules and inflammatory injury. FASEB J. 8:504.[Abstract/Free Full Text]