By
From the * Immunology Research Group, Department of Medical Physiology, University of Calgary,
Calgary, Alberta T2N 4N1, Canada; Department of Comparative Medicine, University of Alabama
at Birmingham, Birmingham, Alabama 35294; § Department of Microbiology and Immunology,
Baylor College of Medicine, Houston, Texas 77030-2399; Department of Molecular and Human
Genetics, Baylor College of Medicine and Howard Hughes Medical Institute, Houston, Texas 77030;
and ¶ Department of Inflammation/Autoimmune Diseases, Hoffmann La-Roche Inc., Nutley, New
Jersey 07110-1199
In this study, we examined the relationship between the endothelial selectins (P-selectin and
E-selectin) and whether they are critical for 4-integrin-dependent leukocyte recruitment in inflamed (late phase response), cremasteric postcapillary venules. Animals were systemically
sensitized and 2 wk later challenged intrascrotally with chicken ovalbumin. Leukocyte rolling
flux, adhesion, and emigration were assessed at baseline and 4 and 8 h postantigen challenge.
There was a significant increase in leukocyte rolling flux, adhesion, and emigration in sensitized
and challenged mice at both 4 and 8 h. At 8 h, the increase in leukocyte rolling flux was ~50%
inhibitable by an anti-
4-integrin antibody, 98% inhibitable by fucoidin (a selectin-binding
carbohydrate), and 100% inhibitable by an anti-P-selectin antibody. P-selectin-deficient animals displayed no leukocyte rolling or adhesion at 8 h after challenge. However, at 8 h there
were many emigrated leukocytes in the perivascular space suggesting P-selectin-independent
rolling at an earlier time point. Indeed, at 4 h postantigen challenge in P-selectin-deficient mice,
there was increased leukocyte rolling, adhesion, and emigration. The rolling in the P-selectin-
deficient mice at 4 h was largely
4-integrin dependent. However, there was an essential E-selectin-
dependent component inasmuch as an anti-E-selectin antibody completely reversed the rolling, and in E-selectin and P-selectin double deficient mice rolling, adhesion and emigration
were completely absent. These results illustrate that P-selectin underlies all of the antigen-induced
rolling with a brief transient contribution from E-selectin in the P-selectin-deficient animals.
Finally, the antigen-induced
4-integrin-mediated leukocyte recruitment is entirely dependent
upon endothelial selectins.
The movement of leukocytes from the intravascular
compartment to the extravascular space is mediated by
three sequential events. First, circulating leukocytes in the
flowing stream of blood make initial contact with the endothelium that is manifest as a tethering and rolling motion
along the length of the venule. Second, rolling leukocytes
are activated to firmly adhere to the endothelium by various proinflammatory mediators. Once firmly adherent, the
cells are able to perform the third and final motion, emigration out of the vasculature. Each of these events are sequential inasmuch as inhibition of leukocyte tethering and
rolling inhibits further adhesion and emigration. In addition, each event is mediated by distinct adhesion molecules.
It is generally accepted that two endothelial selectins are
responsible for leukocyte tethering and rolling, P-selectin
mediating early rolling events and E-selectin overlapping with
P-selectin with time of inflammation. However, the exact
temporal relationship between the selectins remains unclear.
The integrins and members of the immunoglobulin superfamily are responsible for leukocyte adhesion and emigration (1). More recently, however, a number of reports
have suggested that the Luscinskas et al. (5) recently reported that under flow
conditions in vitro, monocytes firmly adhere via the At sites of leukocyte-endothelial cell interactions in vivo,
the shear stresses are usually much higher than 1 dyn/cm2,
ranging between 2-16 dyn/cm2, where 2 dyn/cm2 is at the
very low end of physiologic shear (7). One might predict,
therefore, that in vivo, the In this study, we developed an allergen model of inflammation, and using intravital microscopy we studied the contribution of P-selectin, E-selectin, and Mice deficient in P-selectin, E-selectin, or both P-selectin and
E-selectin were generated by gene targeting in embryonic stem
cells as previously described (9). The mutant mice used for
these experiments were from a 129/Sv × C57Bl/6 mixed background, whereas both 129/Sv × C57Bl/6 and C57Bl/6 nonmutant mice were used as wild-type controls. The results from these
two sets of control animals were not significantly different from
each other and therefore, were grouped together. Only E-selectin-P-selectin double mutant mice that did not show obvious
signs of disease were used for these studies (10). All animals used
weighed between 20-35 g.
Immunization Protocol.
To develop a model of chronic inflammation, a type I hypersensitivity reaction was elicited by systemically (intraperitoneal injection) sensitizing animals with 10 µg
of chicken ovalbumin (Sigma Chemical Co., St. Louis, MO) and
10 mg grade V AlOH (Sigma Chemical Co.) in a total volume of
0.2 ml saline. 2 wk later, animals were challenged locally (intrascrotal injection) with the sensitizing antigen. Sham sensitization
and sham challenge involved systemic and local injection of 0.2 ml
saline, respectively. The animals were prepared for intravital microscopy and leukocyte-endothelial cell interactions were examined during the late phase response, at 4 or 8 h after saline or
ovalbumin challenge.
Intravital Microscopy.
Animals were anesthetized by intraperitoneal injection of a cocktail of 10 mg/kg Xylazine (MTC Pharmaceuticals, Cambridge, Ontario, Canada) and 200 mg/kg ketamine hydrochloride (Rogar/STB Inc., Montreal, Quebec, Canada).
The left jugular vein was cannulated to administer anesthetic and
various drugs. An incision was made in the scrotal skin to expose
the left cremaster muscle, which was then carefully removed from
the associated fascia. A lengthwise incision was made on the ventral
surface of the cremaster muscle. The testicle and epididymis were
separated from the underlying muscle and reintroduced into the
abdominal cavity. The muscle was then spread out over an optically
clear viewing pedestal, and secured along the edges with 5-0 suture.
The exposed tissue was suffused with warm bicarbonate-buffered
saline (pH 7.4). The cremasteric microcirculation was observed
through an intravital microscope (Nikon Inc., Mississauga, Canada)
with a 25× objective lens (L25/0.35; Wetzlar E. Leitz Inc., Munich, Germany) and a 10× eyepiece. The image of the microcirculatory bed (magnification of 1400 on the video monitor) was recorded
using a video camera (Panasonic-Digital 5100; Panasonic, Osaka,
Japan) and a video recorder (NV8950, Panasonic). This preparation
has previously been used to visualize the microcirculation (12, 13).
Images of the microcirculation were recorded at 0, 15, and 30 min
and all experimental parameters were measured at these time points.
4-integrin is not only capable of
mediating leukocyte adhesion, but also initiating the rolling
interactions (4).
4-integrin to activated endothelial cells. In that study, however,
the authors illustrated that the initial tethering and rolling
interaction was entirely dependent upon selectins (L-selectin
and P-selectin). Neutralizing selectin function in that system prevented the
4-dependent adhesion regardless of the
shear stress (0.8-4.0 dynes[dyn]1/cm2). These observations
suggested that in the presence of selectins the
4-integrin
was capable of mediating only the adhesion event. In another in vitro study, however, Alon et al. (4) illustrated that
the
4-integrin on lymphocytes was capable of mediating
tethering, rolling, and firm adhesion on its ligand vascular
cell adhesion molecule 1 (VCAM-1). It should be noted,
however, that the entire sequence of events occurred at a
shear stress of <1 dyn/cm2. At higher shear stresses where
selectins can clearly tether leukocytes and support rolling,
the
4-integrin could not support tethering to VCAM-1.
4-integrin would not be likely to
tether cells to the endothelial surface. However, in vivo, there
are other features that may promote leukocyte-endothelial cell interactions and allow for
4-integrin-dependent tethering and rolling. For example, circulating red blood cells
displace larger cells (leukocytes) from the mainstream of blood
to the periphery of blood vessels, forcing them to make initial contact with vascular endothelial cells (7). It is conceivable, therefore, that in the presence of red blood cells,
4-integrins may be sufficient to recruit circulating leukocytes
independent of selectins. Indeed, introduction of red blood
cells into flow chambers increased leukocyte-endothelial cell interactions (8). Another critical difference between in
vitro and in vivo systems may be the site density of ligands for the
4-integrin. The use of various in vitro substrata
instead of microvascular endothelium may greatly underestimate the site density of ligands (e.g., VCAM-1) for
4-integrins. Therefore, it is conceivable that under inflammatory
conditions,
4-integrin ligands may be expressed in sufficient numbers to gain the capacity to mediate leukocyte
tethering, rolling, and adhesion independent of selectins.
4-integrin to leukocyte recruitment in post-allergen-treated cremaster muscle.
Moreover, this approach permitted us to examine the interrelationship between each of these three adhesion molecules with particular emphasis on establishing whether
4-integrin-mediated leukocyte recruitment was dependent upon P-selectin and/or E-selectin. Additionally, it permitted examination of the time profiles for P-selectin and
E-selectin in mediating leukocyte rolling during a late
phase allergic response.
) was calculated based on the Newtonian definition:
= 8(Vmean/Dv), and
venular wall shear stress was
× blood viscosity, where blood
viscosity was assumed to be 0.025 poise (14).
Passive Cutaneous Anaphylaxis Reaction. Serum was obtained from all OVA-sensitized animals at the end of the experiment by intracardiac puncture. Serial dilutions (1:8-1:64) of the serum samples were prepared and 200 µl of each sample was injected intradermally into the shaved backs of control, untreated mice and Sprague-Dawley rats. Serum from immunized mice elicited the same response in Sprague-Dawley rats as it did in C57Bl/6 mice. Therefore, rats were used for all subsequent passive cutaneous anaphylaxis (PCA) reactions primarily because detection of PCA was easier to record. After 72 h, animals were challenged with an intracardiac injection of a solution containing 2.5 mg Evan's blue dye and 5 mg chicken OVA in a total volume of 1.5 ml (saline). The final reaction was read 60 min later as the highest dilution that produced a distinct blue region (Evan's blue dye extravasation) at the center of the injection site (15). Sensitized animals had serum anti-OVA antibody titres of at least 1:64, whereas sham-sensitized animals had no anti-OVA antibodies. To determine if the serum-induced PCA reaction was mediated by antiOVA IgG, the serum samples were heated for 30 min at 56°C in another series of experiments. This protocol denatures IgE leaving IgG immunoglobulins intact (15). A PCA reaction was then carried out with heat-treated serum samples as described above.
Drug Administration and Experimental Protocol.
All drugs were
administered intravenously, at 5 min of the experimental protocol:
10 mg/kg of fucoidin (Sigma Chemical Co.); 75 or 150 µg/animal
of a monoclonal anti-4-integrin antibody (R1-2; PharMingen,
San Diego, CA); 20 µg/animal of a monoclonal anti-P-selectin antibody (RB40.34; PharMingen); and 100 µg/animal of a monoclonal
anti-E-selectin antibody (9A9) (12, 13, 176-19). Some animals received an isotype-matched (IgG) nonbinding control antibody.
Circulating Leukocyte Counts. At the end of each experiment, whole blood was drawn via cardiac puncture. Total leukocyte counts were performed using a hemocytometer (Bright-line; Hausser Scientific, Horsham, PA) in untreated, wild-type, and the various mutant animals.
Statistical Analysis. Data are presented as mean ± SEM. A Student's t test with bonferroni correction was used for multiple comparisons. Statistical significance was set at P <0.05.
All sensitized animals used in this study had serum anti-OVA antibody titres of at least 1/64 as assessed by a PCA reaction. There was no evidence of anti-OVA antibodies in sham-sensitized animals. Approximately 10% of the heat-treated samples were also able to induce a dermal hypersensitivity response (data not shown) suggesting that at least 90% of the OVA-induced late phase responses were mediated by anti-IgE antibodies (heating inactivates IgE, but leaves IgG intact [15]).
Allergen Increases Leukocyte-Endothelial Cell Interactions in Sensitized Animals.Fig. 1 illustrates the flux of rolling leukocytes (top), leukocyte adhesion (middle), and leukocyte
emigration (bottom) in untreated animals at baseline, and animals that were sham sensitized and sham challenged (SS),
sham sensitized and OVA-challenged (SO), OVA sensitized and sham challenged (OS), and OVA sensitized and OVA challenged (OO) at 4 and 8 h after challenge. In untreated animals, the flux of rolling leukocytes was ~60
cells/min. In sham-sensitized, OVA challenged and OVAsensitized, sham-challenged animals, leukocyte rolling flux
was not different from untreated animals. In OVA-sensitized animals at 4 h after OVA challenge, however, there
was a very dramatic increase in leukocyte rolling flux compared to the respective control groups. Furthermore, after 8 h
of challenge, leukocyte rolling was even further increased to ~300 cells/min, a value above that observed after 4 h of
challenge (P <0.05). We have not seen this degree of leukocyte rolling in any of our previous models including rolling induced by histamine (20), oxidants (21), or leukotriene
C4 (22) in the rat, ischemia/reperfusion in the cat (23), or
cytokine-induced rolling in the mouse (our unpublished
observations). Fig. 1 also illustrates that there was very little
leukocyte adhesion (middle) and leukocyte emigration (bottom) in untreated and sham animals at 4 and 8 h after challenge. However, in sensitized animals exposed to allergen, there was a significant increase in both leukocyte adhesion
(middle) and emigration (bottom) after 4 and 8 h of challenge. The adhesion and emigration was not unlike values
observed after exposure to potent chemoattractants (24-
26). All leukocyte parameters were stable over the 30 min
experimental protocol. Finally, at the end of the 8-h treatment with OVA, histological analysis (hematoxylin and
eosin) revealed that at least 50% of the leukocytes were
PMN, whereas the remaining cells were eosinophils or
mononuclear cells.
Table 1 summarizes leukocyte rolling velocity, VRBC, Dv, and venular shear stress in sensitized mice at 8 h after allergen challenge. There was no difference in leukocyte rolling velocity, red blood cell velocity, or venular diameter over the course of the experiment or when compared to untreated controls. Venular shear stress increased at 15 min, but was back down to control levels by 30 min. Leukocyte rolling velocity, VRBC, Dv or shear stress did not differ in any of the experimental groups (data not shown).
|
To ensure that each animal tested had a late phase
leukocytic response, antibodies were added during the course
of the experiment. Administration of a monoclonal antibody against murine 4-integrin (75 µg/animal) reduced leukocyte rolling by 50% (Fig. 2). Nevertheless, there still remained more than twice the number of rolling cells as that
observed in any of the sham groups of animals (~60 cells/ min) suggesting a population of leukocytes that rolled independent of
4-integrin. In every case, there was a short delay (15 min) before the
4-integrin antibody blocked leukocyte rolling. Therefore, in some animals a higher dose of
the
4-integrin antibody was used (150 µg/animal). Although this concentration immediately reduced leukocyte
rolling, the reduction was identical to the magnitude of response observed with the lower concentration. Therefore, the data are pooled. Administration of an isotype-matched
(IgG) control antibody had no effect on the antigen-induced
leukocyte rolling. Neither antibody affected systemic leukocyte counts.
P-selectin Is Critical to All Leukocyte Rolling at 8 h After Allergen Challenge.
A role for P-selectin at 8 h after antigen
challenge was established in three different ways. In the first
instance, we treated animals with a selectin-binding carbohydrate, fucoidin (Fig. 3; top), which reduced the antigeninduced increase in leukocyte rolling by ~98% at 8 h after
allergen challenge. An anti-P-selectin antibody (Fig. 3: middle) completely reversed the OVA-induced increase in leukocyte rolling, suggesting that P-selectin is critical for the
leukocyte recruitment in this model. The leukocytes that
were already adherent were not affected by either the P-selectin antibody or fucoidin treatment, consistent with the view
that the firm adhesion per se was due to other adhesive
mechanisms (data not shown). In the third series of experiments, we elicited the late phase response in animals genetically deficient in P-selectin. The bottom panel of Fig. 3
illustrates that in P-selectin-deficient mice there was absolutely no leukocyte rolling at 8 h after antigen challenge.
This three-pronged approach suggests that both the 4-integrin-dependent and
4-integrin-independent leukocyte rolling was contingent upon functional P-selectin.
Leukocyte-Endothelial Interactions Do Occur in P-selectin- deficient Mice.
Despite the lack of rolling at 8 h after antigen
challenge in the P-selectin-deficient animals, there was always a very significant increase in leukocyte emigration.
To further investigate this observation, some P-selectin-
deficient animals were examined at 0 and 4 h after antigen
challenge (Fig. 4). No leukocyte rolling (top), adhesion (middle), or emigration (bottom) was noted in the P-selectin-deficient mice immediately after challenge (0 h). At 4 h after
antigen challenge, however, the P-selectin-deficient mice
displayed significant leukocyte rolling (top). Although there was some leukocyte rolling in every P-selectin-deficient
vessel examined at 4 h, there was a tremendous amount of
variability between vessels ranging from as few as 4 cells/
min to as many as 421 cells/min. On average, ~100-150
cells/min rolled in the P-selectin-deficient vessels. Associated with the increased rolling of leukocytes at this time
point, leukocyte adhesion (middle) and emigration (bottom)
were also noted. In fact, more than twice as many cells adhered at 4 h in P-selectin-deficient vessels compared to
their wild-type counterparts (Fig. 1 versus Fig. 4). At 8 h after antigen challenge, leukocyte rolling disappeared (top)
and there was very little leukocyte adhesion in the P-selectin-deficient mice compared to the wild-type controls at
the 8 h time point (Fig. 1 versus Fig. 4). However, there
was a significantly elevated number of emigrated leukocytes. In fact, the amount of emigrated cells did not differ
between the P-selectin-deficient mice and their wild-type
counterparts (Fig. 1 versus Fig. 4). It is unlikely that the
cells were emigrating from other vessels or other regions of
the microcirculation since an examination of all other vessels in the general area revealed no rolling cells at 8 h after
antigen challenge. Clearly, the only explanation is that at
~4 h after challenge, a P-selectin-independent mechanism
was transiently induced which allowed leukocytes to roll,
adhere, and emigrate into the interstitium.
Leukocyte Rolling in P-selectin-deficient Vessels Is Dependent on
As there was significant 4-integrin-dependent leukocyte rolling at 8 h after allergen challenge in
wild-type animals, we examined whether this mechanism
was responsible for leukocyte rolling at 4 h in the P-selectin-deficient vessels. Fig. 5 illustrates that when the anti-
4-antibody was administered to sensitized and challenged
P-selectin-deficient mice, the rolling dissipated by 90% to
fewer than 15 rolling cells/min. This was, however, specific to the P-selectin-deficient vessels as addition of the same anti-
4-integrin antibody 4 h after challenge in wildtype mice had no detectable effect on the flux of rolling
leukocytes. Clearly a much greater proportion of rolling
cells were dependent upon
4-integrin in P-selectin-deficient mice (>90%) at 4 h than in the wild-type mice at either 4 h (0%) or 8 h (50%, Fig. 2) after allergen challenge.
The anti-
4-antibody had no effect on leukocyte adhesion
or emigration in either wild-type animals or the P-selectin-
deficient animals at this time point (data not shown).
To determine if the other endothelial selectin, E-selectin, contributed to the 4-integrin-
dependent rolling pathway at 4 h in the P-selectin-deficient vessels, P-selectin-deficient mice were treated with an
antimurine E-selectin antibody at 4 h after antigen challenge. Fig. 6 (top) illustrates that administration of the anti-
E-selectin antibody completely reversed the antigen-induced leukocyte rolling at 4 h after challenge in the P-selectin-deficient mice. In an additional series of experiments, we sensitized and challenged E-selectin- and P-selectin-deficient
mice and examined the leukocyte recruitment profiles at 0, 4, and 8 h after challenge. Fig. 6 (bottom) also illustrates that
there was absolutely no leukocyte rolling in the E-selectin-
and P-selectin-deficient vessels at 0, 4, or 8 h after allergen
challenge. Consistent with this observation is the lack of
leukocyte adhesion and emigration at 0, 4, or 8 h of OVA
exposure (data not shown). The lack of emigration at 8 h in
the E-selectin- and P-selectin-deficient animals is consistent with no leukocyte-endothelial cell interactions throughout the 8 h time point.
For completeness, the same experiments were also carried out in E-selectin-deficient mice (Fig. 7). After 4 or 8 h
of antigen challenge in these animals, leukocyte rolling (top),
adhesion (middle), and emigration (bottom) were indistinguishable from wild-type controls.
Fig. 8 illustrates the rolling velocity profiles for wild-type
animals under control conditions and wild-type animals,
P-selectin-deficient, and E-selectin-deficient animals at 4 h
after antigen challenge. On average leukocyte rolling velocity was not different between wild-type animals and any
of the experimental groups at either 0 or 4 h. In each series
of animals, >90% of the leukocytes rolled between 30-60
µm/sec, regardless of whether they were rolling on P-selectin (A, B, and C) or E-selectin (D) with a contribution from 4-integrin (D).
Total circulating leukocyte counts were significantly elevated in P-selectin-deficient mice (13.7 ± 1.5 × 106/ml) and
E-selectin- and P-selectin-deficient mice (16.5 ± 1.4 × 106/ml) when compared to control animals (3.3 ± 0.6 × 106/ml). Leukocyte counts in the E-selectin-deficient mice
(2.9 ± 1.4 × 106/ml) were not significantly different from
control animals. All leukocyte counts were performed at the
end of each experiment. Administration of the anti-4-integrin antibody, the anti-P-selectin antibody, or the anti-
E-selectin antibody had no effect on blood leukocyte counts
in either the control animals or the P-selectin-deficient animals. All animals used were properly sensitized as assessed by anti-OVA antibody titres of at least 1:64.
The late phase reaction in response to allergens is characterized by the recruitment of numerous leukocyte populations that are thought to ultimately cause the tissue injury
associated with various pathological conditions including
asthma and various skin disorders (15, 27). It is becoming apparent that the leukocytic infiltrate is dependent upon
various adhesion molecules including the selectins and various integrins. Antibodies directed against P-selectin, E-selectin, and 4-integrin have all been reported to attenuate leukocyte infiltration into various tissues or have prevented
tissue injury (edema formation) in allergic models (19, 29,
31, 32). Although these experiments suggest an important role for each of these adhesion molecules to the recruitment
of leukocytes and ultimate tissue injury, a major limitation
of these studies is the inability to determine the actual
mechanism(s) and times at which the different adhesion molecules contribute to the multi-step recruitment process. We
report herein the development of an allergic model of
inflammation which permits us to visualize leukocyte recruitment into regions undergoing a late phase response associated with type I hypersensitivity. We define a role for P-selectin, E-selectin, and the
4-integrin in antigen-induced leukocyte recruitment, and also highlight the subtle interrelationships that exist between these adhesive molecules.
In this study, we report that P-selectin is essential for all of the leukocyte rolling at 8 h after allergen challenge in sensitized tissues. This contention is based on the view that addition of a P-selectin antibody or a selectin-binding carbohydrate completely prevented leukocyte rolling at this time point. Moreover, the postcapillary venules of P-selectin- deficient mice also displayed a lack of leukocyte-endothelial cell interactions at the 8 h time point. This suggests that leukocyte recruitment at 8 h after allergen challenge is unequivocally P-selectin mediated. Although initial baseline rolling (11, 33, 34) and acute mediator-induced leukocyte rolling (20) are thought to be P-selectin-dependent, more prolonged leukocyte recruitment associated with cytokines or use of intraperitoneal thioglycollate have been thought to be E-selectin-dependent, based on the original work in the P-selectin-deficient mice (11). However, Subramaniam et al. (31) recently observed reduced leukocyte recruitment in a delayed-type hypersensitivity response (24 h) in P-selectin-deficient mice. Although the authors postulated that the late effect could have reflected an early defect, i.e., the sensitization phase, or defective infiltration in the first few hours of the response, our data clearly demonstrate that P-selectin plays a critical role as late as 8 h after antigen challenge. Therefore, the profound reduction in leukocyte recruitment into challenged tissues observed by Subramaniam et al. (31), could be partially related to P-selectin-dependent leukocyte recruitment certainly as late as 8 h, and perhaps even at 24 h after allergen challenge. Indeed, our observation that P-selectin is very important in the long-term effect of leukocyte recruitment is consistent with transcriptional regulation of P-selectin by various cytokines and perhaps reuse of previously endocytosed P-selectin (5, 35), and challenges the view that E-selectin completely replaces P-selectin with time of inflammation.
Unlike P-selectin-deficient mice, the E-selectin-deficient animals failed to show any reduction in rolling at 8 h or at a time (4 h) when its upregulation is predicted. These data are consistent with the work of Labow et al. (38), who have reported a lack of phenotype for E-selectin-deficient mice in numerous inflammatory models including a delayed-type hypersensitivity reaction. Data from E-selectin antibody studies have also failed to provide significant protection against leukocyte-mediated injury in several inflammatory models including indomethacin-induced gastric mucosal injury in the rat (39), a primate model of spontaneous chronic colitis (40), skeletal muscle ischemia/reperfusion injury (41), and immune complex-mediated nephritis in the rat (42). By contrast a handful of studies in skin and lung have revealed a role for E-selectin in leukocyte recruitment, raising the possibility that E-selectin may recruit leukocytes to only a few tissues (32, 43, 44). This latter statement must be tempered by our observation of a critical role for E-selectin in recruiting a significant number of leukocytes in P-selectin-deficient vessels during a late phase response. Nevertheless, the expression and rolling was transient inasmuch as it was never observed at the 8 h time point. Interestingly, however, an E-selectin-dependent pathway was sufficient to gather as many leukocytes into P-selectin- deficient tissue as all of the adhesion molecules recruited in wild-type tissue. This highlights the importance of being able to visualize leukocyte behavior in inflamed vessels inasmuch as histology would have derived the wrong conclusion that leukocyte influx or emigration into postantigen challenged tissues at 8 h was not dependent on P-selectin based on the results from P-selectin-deficient mice.
Interestingly, the P-selectin- and E-selectin-mediated rolling in this in vivo study did not differ in the magnitude of the rolling velocity despite observations in vitro that E-selectin and P-selectin may differ in this regard (45, 46). Rather than the type of selectin, we feel that it is more likely that the leukocyte rolling velocity is dictated by the number of binding sites between the leukocyte and endothelium and perhaps the activation state of the cells. Indeed, numerous site density studies (45, 47, 48) have revealed that high binding site density for any selectin will increase the number of rolling cells as well as decrease the rolling velocity. Additionally, we have demonstrated in vivo (16, 49) and in vitro (50, 51) that inhibiting CD18 directly, or inactivating chemotactic agents that activate CD18, will reverse the reduction in leukocyte rolling velocity. For example, P-selectin-dependent rolling velocity in vivo can be very slow in response to leukotriene C4 (22) but not histamine (20), suggesting that the mediator may also be important. It is conceivable that particular proinflammatory mediators induce the rapid synthesis of chemotactic agents including platelet-activating factor, which may then reduce leukocyte rolling velocity. Although a direct comparison between E-selectin- and P-selectin-dependent rolling velocity is impossible in our study without knowing the specific number of receptors expressed, we report that the rolling velocity did not differ when cells rolled on E-selectin in P-selectin-deficient mice or on P-selectin in E-selectin-deficient mice. A direct comparison of leukocyte rolling velocity in vitro on P-selectin versus E-selectin incorporated into lipid bilayers at similar densities suggested that cells rolled more slowly on P-selectin (45).
In this study, we clearly demonstrate a role for 4-integrin as a mediator of leukocyte rolling at 8 h (not 4 h) after
allergen challenge. This is consistent with an important role
for this adhesion molecule in the recruitment of leukocytes
in late phase reactions (18, 19, 29, 52). Moreover, the observation is also consistent with previous reports that
4-integrin is capable of supporting leukocyte rolling in vivo (6,
53). The novelty of our study was the fact that we could
examine the interplay between
4-integrin and the selectins. This revealed that the
4-integrin-mediated leukocyte
rolling was in turn entirely dependent upon endothelial selectins. This conclusion is supported by the fact that
4-integrin-mediated rolling was eliminated in animals deficient in
P-selectin or in animals treated with fucoidin or an anti-
P-selectin antibody. We also observed
4-integrin-dependent leukocyte rolling in the P-selectin-deficient animals at
4 h after antigen challenge. However, this interaction was
entirely dependent upon E-selectin inasmuch as deleting
E-selectin in the P-selectin-deficient animals i.e., by immunoneutralization or E-selectin and P-selectin deletion at
the genetic level, again eliminated the
4-integrin-dependent rolling. Therefore, this study clearly demonstrates that
4-integrin-dependent rolling will not occur independent
of the selectins in postcapillary venules of the inflamed cremaster muscle.
At first glance it may be difficult to explain how two different adhesion molecules (a selectin and 4-integrin) might
support the same event i.e., leukocyte rolling. However,
there is a growing body of evidence that leukocyte rolling
per se can be divided into at least two mechanistically separate components; the initial capturing of the leukocyte is referred to as tethering and the second event is thought to be
rolling (1). To date, this has been demonstrated for L-selectin and E-selectin in flow chambers in vitro i.e., the capturing or tethering of leukocytes to E-selectin was dependent
on L-selectin (47). However, if L-selectin-deficient cells
were permitted to settle on E-selectin thereby bypassing the initial tethering event and flow in the chamber was resumed, these cells could then roll at very high shear rate.
We propose the same mechanism for the selectins and the
4-integrin. We would predict that the selectins tether or
capture leukocytes because (a)
4-integrin is far less effective at tethering leukocytes to its ligand VCAM-1 (0.7 dyn/cm2) than selectins are to their ligands (>1 dyn/cm2),
and (b)
4-integrin can support rolling at even very high
shear forces despite its inability to capture at these shear
conditions (4). As the shear stress was much higher than 1 dyn/cm2 in the inflamed cremasteric postcapillary venules,
the view that it was the selectins rather than
4-integrin
that tethered the cells to the endothelium permitting the
integrin to then mediate rolling is supported. Although the
possibility cannot be excluded that selectins and
4-integrin
work cooperatively to tether and then allow leukocytes to
roll, our data are conclusive that selectins are essential for
4-dependent rolling in the late phase reaction.
In conclusion, we illustrate that endothelial selectins, primarily P-selectin, are essential for 4-dependent leukocyte
rolling in chronically inflamed venules in vivo. In P-selectin-
deficient mice, E-selectin is induced transiently to recruit leukocytes. Nevertheless, a critical role for P-selectin resumes
at 8 h. This study unequivocally illustrates that selectins are
critical for the initiation of events leading to leukocyte recruitment regardless of the induction of subsequent rolling
via other routes, including the
4-integrin pathway. Clearly,
despite the importance of
4-integrin in asthma and associated pathologies, targeting the selectins may still be an efficient means of controlling leukocyte infiltration during a
late phase reaction.
Address correspondence to Dr. Paul Kubes, Immunology Research Group, Department of Medical Physiology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1 Canada.
Received for publication 18 September 1996 and in revised form 12 December 1996.
1Abbreviations used in this paper: Dv, venular diameter; dyn, dynes; PCA, passive cutaneous anaphylaxis; VCAM-1, vascular cell adhesion molecule 1.We would like to thank Dr. Ivan Richards from The Upjohn Company for help in developing the model.
This work was supported by grants from the Medical Research Council (MRC HT 11537) of Canada and the National Institutes of Health (AI-32177, GM-15483, HL-42550). P. Kubes is an AHFMR and Medical Research Council scholar. S. Kanwar is supported by an Alberta Heritage Foundation for Medical Research studentship.
1. | Springer, T.A.. 1994. Traffic signals of lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell. 76: 301-314 [Medline]. |
2. | Zimmerman, G.A., S.M. Prescott, and T.M. McIntyre. 1992. Endothelial cell interactions with granulocytes: tethering and signaling molecules. Immunol. Today. 13: 93-99 [Medline]. |
3. |
Albelda, S.M.,
C.W. Smith, and
P.A. Ward.
1994.
Adhesion
molecules and inflammatory injury.
FASEB J.
8:
504-512
|
4. | Alon, R., P.D. Kassner, M.W. Carr, E.B. Finger, M.E. Hemler, and T.A. Springer. 1995. The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J. Cell Biol. 128: 1243-1253 [Abstract]. |
5. | Luscinskas, F.W., H. Ding, P. Tan, D. Cumming, T.F. Tedder, and M.E. Gerritson. 1996. L- and P-selectin, but not CD49d (VLA-4) integrins, mediate monocyte initial attachment to TNF-alpha-activated vascular endothelium under flow in vitro. J. Immunol. 156: 326-335 . |
6. | Johnston, B., T.B. Issekutz, and P. Kubes. 1996. The alpha4-integrin supports leukocyte rolling and adhesion in chronically inflamed post-capillary venules in vivo. J. Exp. Med. 183: 1995-2006 [Abstract]. |
7. | Schmid-Schonbein, G.W., S. Usami, R. Skalak, and S. Chien. 1980. The interaction of leukocytes and erythrocytes in capillary and postcapillary vessels. Microvasc. Res. 19: 45-70 [Medline]. |
8. | Melder, R.J., L.L. Munn, S. Yamada, C. Ohkubo, and R.K. Jain. 1995. Selectin- and integrin-mediated T-lymphocyte rolling and arrest on TNF-alpha-activated endothelium: augmentation by erythrocytes. Biophys. J. 69: 2131-2138 [Abstract]. |
9. | Bullard, D.C., L. Qin, I. Lorenzo, W.M. Quinlin, N.A. Doyle, R. Bosse, D. Vestweber, C.M. Doerschuk, and A.L. Beaudet. 1995. P-selectin/ICAM-1 double mutant mice: acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli. J. Clin. Invest. 95: 1782-1788 [Medline]. |
10. | Bullard, D.C., E.J. Kunkel, H. Kubo, M.J. Hicks, I. Lorenzo, N.A. Doyle, C.M. Dorschuk, K. Ley, and A.L. Beaudet. 1996. Infectious susceptibility and severe deficiency of leukocyte rolling and recruitment in E-selectin and P-selectin double mutant mice. J. Exp. Med. 183: 2329-2336 [Abstract]. |
11. | Mayadas, T.N., R.C. Johnson, H. Rayburn, R.O. Hynes, and D.D. Wagner. 1993. Leukocyte rolling and extravasation are severely compromised in P-selectin-deficient mice. Cell. 74: 541-554 [Medline]. |
12. | Ley, K., D.C. Bullard, M.L. Arbones, R. Bosse, D. Vestweber, T.F. Tedder, and A.L. Beaudet. 1995. Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J. Exp. Med. 181: 669-675 [Abstract]. |
13. | Kunkel, E.J., U. Jung, D.C. Bullard, K.E. Norman, B.A. Wolitzky, D. Vestweber, A.L. Beaudet, and K. Ley. 1996. Absence of trauma-induced leukocyte rolling in mice deficient in both P-selectin and intercellular adhesion molecule 1. J. Exp. Med. 183: 57-65 [Abstract]. |
14. | Lipowsky, H.H., S. Usami, and S. Chien. 1980. In vivo measurements of "apparent viscosity" and microvessel hematocrit in the mesentry of the cat. Microvasc. Res. 19: 297-319 [Medline]. |
15. | Saloga, J., H. Renz, G. Lack, K.L. Bradley, J.L. Greenstein, G. Larsen, and E.W. Gelfand. 1993. Development and transfer of immediate cutaneous hypersensitivity in mice exposed to aerosolized antigen. J. Clin. Invest. 91: 133-140 [Medline]. |
16. |
Gaboury, J.P.,
B. Johnston,
X.-F. Niu, and
P. Kubes.
1995.
Mechanisms underlying acute mast cell-induced leukocyte
rolling and adhesion in vivo.
J. Immunol.
154:
804-813
|
17. | Bosse, R., and D. Vestweber. 1994. Only simultaneous blocking of the L- and P-selectin completely inhibits neutrophil migration into mouse peritoneum. Eur. J. Immunol. 24: 3019-3024 [Medline]. |
18. |
Ferguson, T.A., and
T.S. Kupper.
1993.
Antigen-independent processes in antigen-specific immunity. A role for alpha
4 integrin.
J. Immunol.
150:
1172-1182
|
19. | Chisholm, P.L., C.A. Williams, and R.R. Lobb. 1993. Monoclonal antibodies to the integrin alpha-4 subunit inhibit the murine contact hypersensitivity response. Eur. J. Immunol. 23: 682-688 [Medline]. |
20. |
Kubes, P., and
S. Kanwar.
1994.
Histamine induces leukocyte rolling in post-capillary venules: a P-selectin-mediated
event.
J. Immunol.
152:
3570-3577
|
21. |
Johnston, B.,
S. Kanwar, and
P. Kubes.
1996.
Hydrogen peroxide induces leukocyte rolling: modulation by endogenous
antioxidant mechanisms including nitric oxide.
Am. J. Physiol.
271:
H614-H621
|
22. |
Kanwar, S.,
B. Johnston, and
P. Kubes.
1995.
Leukotriene
C4/D4 induces P-selectin and sialyl Lewis(x)-dependent alterations in leukocyte kinetics in vivo.
Circ. Res.
77:
879-887
|
23. | Kanwar, S., and P. Kubes. 1994. Ischemia/reperfusion-induced granulocyte influx is a multistep process mediated by mast cells. Microcirculation. 1: 175-182 [Medline]. |
24. | McCafferty, D.-M., P. Kubes, and J.L. Wallace. 1993. Inhibition of platelet-activating factor-induced leukocyte adhesion in vivo by a leumedin. Eur. J. Pharmacol. 232: 169-172 [Medline]. |
25. |
Wallace, J.L.,
D.-M. McCafferty,
D.N. Granger, and
P. Kubes.
1993.
Leukocyte adherence and mucosal injury induced by NSAIDs: role of endothelial adhesion molecules.
Am. J. Physiol.
265:
G993-G998
|
26. |
Gaboury, J.,
D.C. Anderson, and
P. Kubes.
1994.
Molecular
mechanisms involved in superoxide-induced leukocyte endothelia cell interactions in vivo.
Am. J. Physiol.
266:
H637-H642
|
27. | Atkins, P., G.R. Green, and B. Zwieman. 1993. Histologic studies of human skin test responses to ragweed, compound 48/80, and histamine. J. Allergy Clin. Immunol. 51: 263-273 . |
28. |
Iwamoto, I.,
S. Tomoe,
H. Tomioka, and
S. Yoshida.
1993.
Leukotriene B4 mediates substance P-induced granulocyte
infiltration into mouse skin.
J. Immunol.
151:
2116-2123
|
29. | Abraham, W.M., M.W. Sielczak, A. Ahmed, A. Cortes, I.T. Lauredo, J. Kim, B. Pepinsky, C.D. Benjamin, D.R. Leone, R.R. Lobb, et al . 1994. Alpha 4-integrins mediate antigeninduced late bronchial responses and prolonged airway hyperresponsiveness in sheep. J. Clin. Invest. 93: 776-787 [Medline]. |
30. | Wershil, B.K., Z.S. Wang, J.R. Gordon, and S.J. Galli. 1991. Recruitment of neutrophils during IgE-dependent cutaneous late phase reactions in the mouse is mast cell-dependent. J. Clin. Invest. 87: 446-453 [Medline]. |
31. | Subramaniam, M., S. Saffaripour, S.R. Watson, T.N. Mayadas, R.O. Hynes, and D.D. Wagner. 1995. Reduced recruitment of inflammatory cells in a contact hypersensitivity response in P-selectin-deficient mice. J. Exp. Med. 181: 2277-2282 [Abstract]. |
32. | Gundel, R.H., C.D. Wegner, C.A. Torcellini, C.C. Clarke, N. Haynes, R.R. Rothlein, C.W. Smith, and L.G. Letts. 1991. Endothelial leukocyte adhesion molecule-1 mediates antigen-induced acute airway inflammation and late-phase airway obstruction in monkeys. J. Clin. Invest. 88: 1407-1411 [Medline]. |
33. | Dore, M., R.J. Korthuis, D.N. Granger, M.L. Entman, and C.W. Smith. 1993. P-selectin mediates spontaneous leukocyte rolling in vivo. Blood. 82: 1308-1316 [Abstract]. |
34. |
Nolte, D.,
P. Schmid,
U. Jager,
A. Botzlar,
F. Roesken,
R. Hecht,
E. Uhl,
K. Messmer, and
D. Vestweber.
1994.
Leukocyte rolling in venules of striated muscle and skin is mediated by P-selectin, not by L-selectin.
Am. J. Physiol.
267:
H1637-H1642
|
35. |
Luscinskas, F.W.,
H. Ding, and
A.H. Lichtman.
1995.
P-selectin and vascular cell adhesion molecule 1 mediate rolling and
arrest, respectively, of CD4+ T lymphocytes on tumor necrosis factor ![]() |
36. | Gotsch, U., U. Jager, M. Dominis, and D. Vestweber. 1994. Expression of P-selectin on endothelial cells is upregulated by LPS and TNF-alpha in vivo. Cell Adhes. Commun. 2: 7-14 [Medline]. |
37. | Bischoff, J., and C. Brasel. 1995. Regulation of P-selectin by tumor necrosis factor-alpha. Biochem. Biophys. Res. Commun. 210: 174-180 [Medline]. |
38. | Labow, M.A., C.R. Norton, J.M. Rumberger, K.M. Lombard-Gillooly, D.J. Shuster, J. Hubbard, R. Bertko, P.A. Knaack, R.W. Terry, M.L. Harbison, et al . 1995. Characterization of E-selectin-deficient mice: demonstration of overlapping function of endothelial selectins. Immunity. 1: 709-720 . |
39. |
Wallace, J.L.,
W. McKnight,
M. Miyasaka,
T. Tamatani,
J. Paulson,
D.C. Anderson,
D.N. Granger, and
P. Kubes.
1993.
Role of endothelial adhesion molecules in NSAID-induced
gastric mucosal injury.
Am. J. Physiol.
265:
G993-G998
|
40. | Podolsky, D.K., R. Lobb, N. King, C.D. Benjamin, B. Pepinsky, P. Sehgal, and M. de Beaumont. 1993. Attenuation of colitis in the cotton-top tamarin by anti-alpha-4 monoclonal antibody. J. Clin. Invest. 92: 372-380 [Medline]. |
41. | Seekamp, A., G.O. Till, M.S. Mulligan, J.C. Paulson, D.C. Anderson, M. Miyasaka, and P.A. Ward. 1994. Role of selectins in local and remote tissue injury following ischemia and reperfusion. Am. J. Pathol. 144: 592-598 [Abstract]. |
42. | Mulligan, M.S., K.J. Johnson, R.F. Todd, T.B. Issekutz, M. Miyasaka, T. Tamatani, C.W. Smith, D.C. Anderson, and P.A. Ward. 1993. Requirements for leukocyte adhesion molecules in nephrotoxic nephritis. J. Clin. Invest. 91: 577-587 [Medline]. |
43. | Mulligan, M.S., J. Varani, M.K. Dame, C.L. Lane, C.W. Smith, D.C. Anderson, and P.A. Ward. 1991. Role of endothelial-leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats. J. Clin. Invest. 88: 1396-1406 [Medline]. |
44. | Binns, R.M., A. Whyte, S.T. Licence, A.H. Harrison, Y.T.M. Tsang, D.O. Haskard, and M.K. Robinson. 1996. The role of E-selectin in lymphocyte and polymorphonuclear cell recruitment into cutaneous delayed hypersensitivity reactions in sensitized pigs. J. Immunol. 157: 4094-4099 [Abstract]. |
45. | Patel, K.D., K.L. Moore, M.U. Nollert, and R.P. McEver. 1995. Neutrophils use both shared and distinct mechanisms to adhere to selectins under static and flow conditions. J. Clin. Invest. 96: 1887-1896 [Medline]. |
46. |
Lawrence, M.B., and
T.A. Springer.
1993.
Neutrophils roll
on E-selectin.
J. Immunol.
151:
6338-6346
|
47. | Lawrence, M.B., D.F. Bainton, and T.A. Springer. 1994. Neutrophil tethering to and rolling on E-selectin are separable by requirement for L-selectin. Immunity. 1: 137-145 [Medline]. |
48. | Lawrence, M.B., and T.A. Springer. 1991. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell. 65: 859-873 [Medline]. |
49. |
Kubes, P.,
G. Ibbotson,
J.M. Russell,
J.L. Wallace, and
D.N. Granger.
1990.
Role of platelet-activating factor in ischemia/
reperfusion-induced leukocyte adherence.
Am. J. Physiol.
259:
G300-G305
|
50. | Lawrence, M.B., C.W. Smith, S.G. Eskin, and L.V. McIntire. 1990. Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium. Blood. 75: 227-237 [Abstract]. |
51. | Abbassi, O., T.K. Kishimoto, L.V. McIntire, D.C. Anderson, and C.W. Smith. 1993. E-selectin supports neutrophil rolling in vitro under conditions of flow. J. Clin. Invest. 92: 2719-2730 [Medline]. |
52. | Nakajima, H., H. Sano, T. Nishimura, S. Yoshida, and I. Iwamoto. 1994. Role of vascular cell adhesion molecule 1/very late activation antigen 4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue. J. Exp. Med. 179: 1145-1154 [Abstract]. |
53. |
Sriramarao, P.,
U.H. Von Andrian,
E.C. Butcher,
M.A. Bourdon, and
D.H. Broide.
1994.
L-selectin and very late
antigen-4 integrin promote eosinophil rolling at physiological
shear rates in vivo.
J. Immunol.
153:
4238-4246
|