© 1995 American Heart Association, Inc.
Articles |
Leukotriene C4/D4 Induces P-Selectin and Sialyl Lewisx–Dependent Alterations in Leukocyte Kinetics In Vivo
From the Immunology Research Group, University of Calgary (Canada).
Correspondence to Dr Paul Kubes, Department of Medical Physiology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
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Abstract |
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Abstract The objective of this study was to assess the effect of leukotriene C4 (LTC4) on the flux of rolling leukocytes, leukocyte rolling velocity, and leukocyte adhesion in postcapillary venules in vivo and to study the underlying molecular mechanisms involved. LTC4 (20 nmol/L) induced a rapid and significant increase in leukocyte rolling flux that was inhibitable by an anti–P-selectin antibody and soluble sialyl Lewisx (sLex). LTC4 also induced a significant reduction in leukocyte rolling velocity, an event that was independent of P-selectin but entirely dependent on sLex. This LTC4-induced reduction in leukocyte rolling velocity was independent of any hemodynamic alterations. Another P-selectin effector, histamine, did not affect leukocyte rolling velocity even at >5000 times the concentration of LTC4. Treatment with an anti–L-selectin antibody had no effect on the LTC4-induced increase in leukocyte rolling or reduction in rolling velocity. Inhibition of LTC4 bioconversion to LTD4 by pretreatment with L-serine (100 µmol/L) prevented the LTC4-induced increase in leukocyte rolling flux and the LTC4-induced reduction in leukocyte rolling velocity. A subtle, yet significant, increase in leukocyte adhesion was also observed with LTC4. Pretreatment with a platelet-activating factor receptor antagonist returned the LTC4-induced leukocyte rolling velocity to baseline levels. The addition of a very low concentration of platelet-activating factor (1 nmol/L) induced significant leukocyte adhesion in the presence of LTC4 but not histamine. This study demonstrates that LTC4, via bioconversion to leukotriene D4, induces a P-selectin–dependent and sLex-dependent increase in leukocyte rolling flux and a P-selectin–independent but sLex-dependent reduction in leukocyte rolling velocity, a parameter that may play an essential role in subsequent leukocyte adhesion.
Key Words: leukotriene C4 • leukocyte rolling • sialyl Lewisx • P-selectin
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Introduction |
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Leukotriene C4 (LTC4), a cysteinyl leukotriene, is a biologically active lipid derived from the lipoxygenase-catalyzed metabolism of arachidonic acid.1 It is a potent proinflammatory agent and has been implicated in a wide range of inflammatory conditions in a variety of organs.1 2 3 4 5 Increased levels of LTC4 are found in plasma, pulmonary edema fluid,6 7 and bronchoalveolar and nasal lavage fluid in patients suffering from asthma,8 allergic rhinitis,9 cystic fibrosis,10 and adult respiratory distress syndrome.11 LTC4 and its metabolic products have also been reported in the synovial fluid of patients with rheumatoid arthritis,12 the small intestinal mucosa of children with celiac disease,13 and psoriatic skin lesions.14 In an experimental model, Rainsford15 has recently reported increased LTC4 levels in the efferent gastric circulation of pigs after indomethacin administration and postulated a role for LTC4 in nonsteroidal anti-inflammatory drug–induced gastric mucosal injury. Finally, the concept that LTC4 is involved in pathogenesis is further supported by the fact that LTC4 infusion mimics the sequelae of inflammation. For example, in the gastrointestinal tract, LTC4 induces mild gastric mucosal lesions and greatly augments gastric ulcer formation and intestinal mucosal damage in response to irritants including ethanol16 and acidified acetylsalicylic acid.17
Many of the aforementioned pathologies are hallmarked by increased leukocyte infiltration. However, intravital microscopy revealed that topical application of LTC4 to either the mucosa of the hamster cheek pouch or hamster skin caused a profound dose-dependent increase in microvascular permeability but failed to stimulate leukocyte adhesion, suggesting that LTC4 is unlikely to recruit leukocytes to sites of inflammation.3 18 These observations are consistent with in vitro findings demonstrating that LTC4 does not induce neutrophil aggregation and adhesion to subendothelial matrices or other noncellular substrata.19 Although these data suggest that LTC4 does not directly affect leukocyte function, a substantial amount of literature would suggest that LTC4 may contribute significantly to leukocyte recruitment via activation of the endothelium. Treatment of endothelium with cysteinyl leukotrienes increased adhesivity of these cells for leukocytes.19 20 Further characterization of the increased endothelial adhesivity revealed that LTC4 caused endothelial cells to rapidly synthesize (within minutes) PAF, a phospholipid that is known to increase leukocyte adhesion by activating the ß2-integrin (CD18). However, while PAF receptor antagonists and monoclonal antibodies directed against CD18 prevented part of the adhesive interaction, there always remained significant PAF- and CD18-independent leukocyte adhesion. On the basis of preliminary anti–P-selectin antibody studies, Zimmerman et al19 proposed that this additional adhesive mechanism was mediated by P-selectin.
There is a growing body of evidence to suggest that P-selectin (also known as CD62P and GMP-140), a member of the selectin family of carbohydrate binding proteins, is responsible for a weak, transient, adhesive interaction known as leukocyte rolling, which is a necessary prerequisite for firm adhesion and subsequent emigration.21 22 23 24 P-selectin is stored in Weibel-Palade bodies of endothelial cells and, upon stimulation with such agents as histamine or thrombin, can be rapidly mobilized to the endothelial cell surface.25 Although the P-selectin–dependent leukocyte–endothelial cell interaction is manifested as adhesion in static assay systems, incorporation of P-selectin into a model membrane supports leukocyte rolling under shear conditions.21 22 More recent work in vivo has implicated a role for P-selectin as the adhesive moiety that supports leukocyte rolling under normal conditions as well as in various models of inflammation.26 27 28 Clearly, if LTC4-induces P-selectin–dependent leukocyte rolling, then this mediator may be critically involved in leukocyte recruitment during the inflammatory process.
We used intravital microscopy in a rat mesenteric preparation to visualize leukocyte behavior on-line in the presence and absence of LTC4. Our primary objective was to determine if LTC4 can indeed induce leukocyte rolling in single 20- to 40-µm postcapillary venules in vivo and to systematically assess the molecular mechanisms involved. We examined whether the LTC4-dependent leukocyte rolling was mediated by P-selectin and whether the potential P-selectin ligands, L-selectin, or the fucosylated oligosaccharide sLex29 was involved in the rolling interaction. Next, we determined whether leukocyte rolling was dependent on LTC4 or whether LTC4 had to be converted to LTD4 to mediate leukocyte rolling. Finally, we observed that LTC4 caused leukocyte rolling patterns quite distinct from other P-selectin inducers (histamine) and further characterized this unexpected observation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intravital Microscopy Experimentation
Male Sprague-Dawley rats (175 to 250 g) were maintained on a purified laboratory diet and fasted 18 to 24 hours before surgery. The animals were initially anesthetized with pentobarbital sodium (65 mg/kg body wt), and a right carotid artery and vein were cannulated to measure systemic arterial blood pressure (Statham P23A transducer and Grass physiological recorder) and drug administration, respectively. All animals were pretreated with sodium cromoglycate (5 mg/kg IV) to prevent baseline leukocyte rolling, as previously described.28 A midline abdominal incision was made, and a segment of the midjejunum was gently exteriorized and carefully placed over an optically clear viewing pedestal that permitted transillumination of a 2-cm2 segment of the mesentery. Single unbranched postcapillary venules (20 to 40 µm in diameter) were visualized, and the following parameters were measured: leukocyte rolling, leukocyte rolling velocity, leukocyte adhesion, centerline red blood cell velocity, and shear rates. This preparation has been used extensively by us28 30 31 and others.32 33 34
Experimental Protocol
Immediately after finding a venule of an appropriate size, the
image was recorded for 5 minutes, followed by three additional
5-minute recordings, during which the experimental
parameters were assessed. In the first series of
experiments, the mesentery was superfused with bicarbonate-buffered
saline for the first 5 minutes and then with various concentrations of
LTC4 (0, 2, or 20 nmol/L) for the remaining 55 minutes.
Leukocyte rolling and leukocyte rolling velocity were assessed over 60
minutes. Since 20 nmol/L LTC4 produced the maximum increase
in leukocyte rolling, this concentration was used for all subsequent
experiments. We directly compared our observations with
LTC4 to a second proinflammatory agonist, histamine (100
µmol/L), which is known to induce significant
P-selectin–dependent leukocyte rolling in vivo.28
In the next series of experiments, we studied the molecular mechanisms involved in the LTC4-induced leukocyte rolling. First, we examined a role for sLex, a sialylated fucosylated oligosaccharide that is known to bind to the lectin domain of selectins. Animals were pretreated with a soluble form of sLex (1 mg/100 g body wt, Alberta Research Council) before LTC4 exposure, and leukocyte rolling and rolling velocity were assessed. We also examined the effect of a control carbohydrate, NAcLac, which lacks the fucose sugar moiety and the 3'-sialyl group on the galactose residue normally found on sLex.
In another series of experiments, animals received an anti–P-selectin antibody, PB1.3 (P-selectin–blocking IgG1-clone 352; Dr James Paulson, Cytel Corp) at 2 mg/kg IV at 15 minutes of LTC4 exposure. We have previously demonstrated that this concentration of PB1.3 was most effective at preventing P-selectin–dependent leukocyte rolling in vivo.28 An isotype-matched control antibody had no effect on leukocyte kinetics. To study a role for L-selectin, animals were given an anti–L-selectin antibody, HRL3 (Upjohn Co), at 1 mg/kg IV as previously described.35 36
To further characterize the mechanisms underlying the
LTC4-induced leukocyte rolling, we examined the effect of
L-serine, which prevents the conversion of LTC4
to LTD4 by inhibiting the enzyme,
-glutamyltranspeptidase.37 In this series of
experiments, L-serine (100 µmol/L) or its inactive
enantiomer, D-serine, was superfused over the mesentery for
5 minutes before LTC4 superfusion, and leukocyte rolling
and leukocyte rolling velocity were assessed. In addition, we examined
the effect of MK 571 (Merck-Frosst, Canada Inc), a potent
LTD4 receptor antagonist that has been
demonstrated to antagonize a wide range of LTC4-mediated
effects in various species.38 In this series of
experiments, animals received MK 571 (30 mg/kg IV) just before
LTC4 superfusion.
The number of adherent leukocytes within a 100-µm segment of venule was also quantified before and after LTC4 superfusion. It has been demonstrated in vitro that LTC4 can induce the expression of endothelial cell-associated PAF20 and thereby support leukocyte adhesion in static assay systems. To study a role for PAF in our model of LTC4-induced leukocyte rolling and adhesion, we pretreated a group of animals with a PAF receptor antagonist, WEB 2086 (10 mg/kg IV, Boehringer-Ingelheim), and LTC4-induced leukocyte rolling flux, leukocyte rolling velocity, and leukocyte adhesion were observed over 60 minutes. To determine whether or not the LTC4-induced reduction in leukocyte rolling velocity increased the propensity of neutrophils to adhere when subsequently exposed to a chemotactic agent, we superfused PAF (1 nmol/L) at a concentration that does not normally cause adhesion of cells rolling at a control velocity. PAF was added to the LTC4 or the histamine preparation in these experiments, and rolling and adhesion were assessed.
Statistical Analysis
Data are presented as mean±SEM. A one-way ANOVA and
Student’s t test with Bonferroni correction were used for
multiple comparisons. Statistical significance was set at
P<.05.
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Results |
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The baseline hemodynamic parameters at 60 minutes in untreated and LTC4-treated (20 nmol/L) animals are summarized in Table 1
. In all animals,
venular diameter remained constant over the entire duration of the
experimental protocol. Red blood cell velocity and shear rates within
the venules of study did not change with time in either the untreated
or the experimental group, negating any effects of
hemodynamic factors on leukocyte behavior.
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Fig 1 demonstrates the flux of rolling leukocytes in
response to varying concentrations of LTC4. In untreated
animals, the flux of rolling leukocytes remained below 15 cells per
minute throughout the entire experiment. A low concentration (2 nmol/L)
of LTC4 superfusion induced a small increase in the flux of
rolling leukocytes that reached significance only at the early time
point (15 minutes). LTC4 at 20 nmol/L, however, caused a
pronounced increase in leukocyte rolling that was maintained for the
entire duration of the experiment. Interestingly, in addition to the
increased number of rolling leukocytes, the average velocity of rolling
leukocytes was greatly reduced (Fig 2), so that the
cells appeared to be "creeping" along the length of the venule.
The surface area of contact between the rolling leukocytes and the
endothelium appeared to be greatly increased relative
to leukocytes rolling in the absence of LTC4. It should
also be noted that at 2 nmol/L LTC4, the few new
rolling leukocytes (at 15 minutes) and the few baseline rolling
leukocytes (throughout the 60-minute period) rolled at a slower
velocity. This was the first evidence that rolling velocity could be
dissociated from the actual recruitment of rolling leukocytes. The
LTC4-induced reduction in rolling velocity was independent
of any hemodynamic alterations, as red blood cell
velocity and shear rates remained unchanged during LTC4
superfusion (Table 1
).
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P<.05 relative to respective untreated
value.
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The increased flux of rolling leukocytes and decreased rolling velocity
were compared directly to the leukocyte rolling response elicited with
5000 times the concentration of histamine (Fig 3). This
concentration has previously been shown to be optimal for recruitment
of rolling leukocytes.28 Histamine administration at a
dose of 100 µmol/L induced a significant increase in leukocyte
rolling (Fig 3, top) but, unlike LTC4 (at either 2 or 20
nmol/L), had absolutely no effect on leukocyte rolling velocity (Fig 3,
bottom). Furthermore, even when histamine concentration was increased
to 1 mmol/L, leukocyte rolling velocity remained unchanged (data not
shown), suggesting that LTC4 and histamine exert distinct
leukocyte rolling profiles.
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Fig 4 summarizes the results for sLex on the
LTC4-induced increase in leukocyte rolling flux and
reduction in leukocyte rolling velocity. Pretreatment of animals with a
soluble form of sLex prevented the LTC4-induced
increase in leukocyte rolling flux (Fig 4, top). Moreover,
sLex pretreatment also prevented the
LTC4-induced reduction in leukocyte rolling velocity (Fig 4, bottom). In fact, the few remaining leukocytes that rolled after
sLex administration did so at a velocity that was above
baseline. A control carbohydrate, NAcLac, did not inhibit the flux of
rolling leukocytes; however, at 60 minutes, it did ablate the
LTC4-induced reduction in leukocyte rolling velocity
(44.4±8.8 µm/s with NAcLac pretreatment compared with 21.7±3.5
µm/s with LTC4 alone).
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We further examined the mechanisms of LTC4-induced
leukocyte rolling by testing a P- or L-selectin antibody. Fig 5, top, illustrates that the anti–P-selectin antibody
PB1.3 reversed the LTC4-induced increase in the flux of
rolling leukocytes, an effect that was maintained for the remainder of
the experiment, even in the presence of continued LTC4
superfusion. Fig 5, bottom, demonstrates that PB1.3 had no effect on
the LTC4-induced reduction in rolling velocity, suggesting
that P-selectin did not contribute to the reduced velocity with which
the remaining few leukocytes rolled. Fig 6 illustrates
the effect of anti–L-selectin antibody HRL3 on the
LTC4-induced leukocyte rolling and reduction in rolling
velocity. HRL3 administration, either as a pretreatment /(data not
shown) or as a posttreatment, had no effect on the
LTC4-induced increase in leukocyte rolling flux (Fig 6,
top) or the LTC4-induced reduction in rolling velocity (Fig 6, bottom). These data suggest that L-selectin is not involved in
LTC4-induced P-selectin–dependent alterations in
leukocyte behavior. HRL3, at the same concentration, did reduce
leukocyte rolling in other inflammatory models.35 36
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L-Serine pretreatment (which inhibits
-glutamyl-transpeptidase and thereby prevents the conversion
of LTC4 to LTD4) completely inhibited both the
LTC4-induced increase in the flux of rolling leukocytes
(Fig 7) and the LTC4-induced reduction in
rolling velocity (data not shown). These observations suggest that the
LTC4-induced leukocyte rolling is mediated via the
LTD4 receptor. D-Serine pretreatment had no
effect on either parameter (data not shown). The effects of
MK 571, an LTD4 receptor antagonist, was much
less impressive in reducing LTC4-induced leukocyte rolling.
In fact, MK 571 pretreatment of animals only attenuated the
LTC4-induced increase in leukocyte rolling flux for the
first 15 minutes (data not shown). A 10-fold greater increase in MK 571
did not further affect the LTC4-induced leukocyte
responses.
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Table 2 summarizes changes in leukocyte rolling flux,
leukocyte rolling velocity, and leukocyte adhesion under control
conditions (0 minutes) and after 60 minutes of exposure to
LTC4 in the absence and presence of WEB 2086. WEB 2086 had
no effect on leukocyte rolling flux in untreated animals or in animals
exposed to LTC4. The LTC4-induced reduction in
leukocyte rolling velocity, however, was inhibited by WEB 2086
pretreatment. LTC4 induced a slight, yet significant,
increase in leukocyte adhesion, which was significantly attenuated with
WEB 2086 pretreatment. These observations suggest that PAF may be
involved in the reduction in leukocyte rolling velocity and the small
increase in adhesion observed with LTC4.
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To determine whether the reduction in rolling velocity facilitates leukocyte adhesion, animals were treated with either histamine or LTC4 and then exposed to the chemotactic agent PAF (1 nmol/L). PAF superfusion induced a significantly greater increase in leukocyte adhesion with LTC4 when compared with animals treated with histamine. Histamine, more so than LTC4, induces the endogenous production of PAF, which may contribute to the subsequent desensitization of PAF receptors on leukocytes.19 Therefore, to ensure that the lack of response to exogenous PAF with histamine was not simply specific for PAF, we used a second exogenous chemoattractant, fMLP (10 nmol/L). The data revealed that fMLP induced many more rolling leukocytes to adhere with LTC4 exposure than with histamine exposure (data not shown). These data suggest that the LTC4-induced slow rolling cells are more likely to adhere than the faster rolling cells associated with histamine. In both groups, the flux of rolling leukocytes was the same (data not shown).
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Discussion |
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It is generally accepted that under normal conditions there is little leukocyte rolling through postcapillary venules. However, at the time of surgical preparation that is necessary to visualize leukocyte behavior in vivo, there is a dramatic increase in leukocyte rolling.39 It is well documented that baseline rolling is induced in invasive preparations associated with the mesentery, cheek pouch, and cremaster microcirculation. P-selectin appears to be an important component of baseline rolling, inasmuch as inhibition of P-selectin with selective antibodies26 or various P-selectin binding moieties24 or by knocking out the gene for P-selectin in mice27 decreases baseline rolling. These observations implicate P-selectin in baseline rolling, but at the same time, the artificially elevated level of P-selectin–dependent leukocyte rolling after surgery does not allow for further induction of leukocyte rolling in response to proinflammatory agonists. Therefore, it is not surprising that despite the fact that various potential P-selectin inducers, including histamine and LTC4, have been previously studied by use of intravital microscopy, changes in leukocyte behavior were not noted despite profound changes in other microvascular parameters.40 We recently developed a model that circumvented the problem of high baseline rolling by identifying mast cell degranulation as a major contributor to the high background rolling.28 Stabilization of the mast cells before any surgical manipulation significantly reduced baseline rolling, and this has allowed us to investigate potential mediators of P-selectin in vivo. Alternative approaches include either the use of animals with very low baseline rolling41 or the examination of leukocyte rolling immediately upon exteriorization of the mesentery.42
Our results demonstrate that LTC4 rapidly increases leukocyte rolling via a P-selectin–dependent event. This contention is based on the observation that increased leukocyte rolling was completely reversed by the administration of an anti–P-selectin antibody despite continuous LTC4 superfusion. These results are entirely consistent with the view that P-selectin can be rapidly (within minutes) mobilized to the endothelial cell surface in response to various proinflammatory mediators, including histamine, thrombin, and oxidants.25 Although LTC4 has also been postulated to induce P-selectin expression,19 data to support this view have not been reported to date. Our data for the first time demonstrate a functional role for P-selectin in vivo after exposure of the microvasculature to LTC4. This work also suggests that LTC4 may be far more effective at inducing P-selectin than are mediators such as histamine. In the present study, we demonstrate that LTC4, at <5000 times the concentration of histamine, induced an equivalent increase in leukocyte rolling. A possible explanation for the greater apparent sensitivity of the endothelium to LTC4 versus histamine may be the high plasma levels of endogenous histaminase, which might greatly reduce the amount of intact histamine that contacts the endothelium.
Early work proposed that L-selectin may be the endogenous ligand for P-selectin. This was based on the finding that L-selectin antibodies prevented neutrophil binding to P-selectin–transfected COS cells.43 Our observations do not support this hypothesis; a rat monoclonal antibody to L-selectin did not prevent the P-selectin–dependent (LTC4-induced) leukocyte rolling in the rat mesenteric microcirculation. This monoclonal antibody does reduce leukocyte rolling in more chronic models of inflammation36 but clearly is not important in P-selectin–dependent leukocyte rolling. This is in agreement with more recent data suggesting that L-selectin is unlikely to be the ligand for P-selectin in vivo. For example, Nolte et al44 recently demonstrated that P-selectin is essential for leukocyte rolling in the mouse cremaster and skin, whereas L-selectin appeared not to play a significant role. Ley et al45 observed that L-selectin–deficient mice had significant baseline rolling, whereas there was a lack of rolling in P-selectin–deficient mice in the early phase of the experimental protocol. These data support a role for P-selectin, but not L-selectin, in the early leukocyte rolling event in postcapillary venules.
A potential ligand for P-selectin may be a fucosylated
oligosaccharide, sLex, or a closely
related sugar,29 displayed by more complex
glycoprotein structures, including P-selectin
glycoprotein ligand-1.46 Zhou et
al29 demonstrated that HL60 cells and Chinese hamster
ovary cell lines transfected with 
-1.3/4-fucosyltransferase to
express sLex bound avidly to P-selectin. Our data strongly
support this hypothesis; soluble sLex entirely prevented
the LTC4-induced P-selectin–dependent increase in
leukocyte rolling. These data are consistent with the work of
Asako et al,41 who reported that the rise in
histamine-induced leukocyte rolling (P-selectin dependent) could be
inhibited with soluble sLex. Moreover, Mulligan et
al47 reported that soluble sLex reduced
P-selectin–dependent neutrophil recruitment and lung injury
associated with cobra venom factor, further supporting a role for
P-selectin–sLex interactions in postcapillary
venules.
A very obvious and consistent finding in the present study was that LTC4 induced a very significant reduction in leukocyte rolling velocity. Qualitatively, rolling leukocytes exposed to LTC4-treated endothelium had greater surface area attachment to endothelium than did leukocytes rolling under control conditions or those exposed to histamine. These cells appeared to be "crawling" or "creeping" rather than rolling. The reduction in leukocyte rolling velocity occurred without a change in the hydrodynamic dispersal forces (shear forces) that tend to push leukocytes along the length of postcapillary venules, suggesting the involvement of an adhesive mechanism rather than a simple reduction in shear. The data in the present study would suggest that LTC4 induced a unique adhesive interaction, independent of P-selectin but entirely dependent on sLex. Clearly, sLex may serve as a ligand for P-selectin to induce rolling (flux) but also may serve as a ligand for an unidentified adhesion molecule to reduce the rolling velocity. It is noteworthy that a very similar slow leukocyte rolling profile has been observed in vitro on E-selectin but not P-selectin.48 It is also well known that E-selectin binds avidly to sLex, perhaps supporting the notion of E-selectin as a potential ligand. However, all of the data to date would suggest that E-selectin is not induced rapidly on endothelium and therefore is an unlikely candidate in the present study.
Although almost no attention has been given to the role of adhesion molecules responsible for leukocyte rolling velocity, this event may be functionally just as important as leukocyte rolling flux. For example, reduced leukocyte rolling velocity may give rolling leukocytes a higher propensity to adhere in the presence of an appropriate stimulus. In fact, the present data support this hypothesis, inasmuch as 1 nmol/L PAF superfusion induced a significantly greater increase in leukocyte adhesion in the presence of LTC4 (when the rolling leukocytes were rolling very slowly) but not histamine, which increased the number of rolling cells but had no effect on leukocyte rolling velocity. Therefore, simply targeting the molecules responsible for leukocyte rolling velocity may be as effective at inhibiting subsequent adhesion and vascular dysfunction as preventing leukocyte rolling per se.
Although it is tempting to conclude that the reduced rolling velocity
with LTC4 (not histamine) is the reason for the greater
adhesive response to PAF, other explanations exist. It is known from in
vitro studies that both histamine and LTC4 induce PAF
production from endothelial cells, with greater
amounts of PAF produced with histamine than with
LTC4.49 This PAF remains cell-associated
and may contribute to PAF receptor desensitization on
leukocytes.19 Therefore, another explanation for the
differences between histamine and LTC4 depicted in Fig 8 may be that histamine is more effective at causing
desensitization of the PAF receptor, thereby blunting the response to
exogenously administered PAF. However, observations from our laboratory
with another exogenous chemotactic agent, fMLP, would not support this
hypothesis. A low dose of fMLP (10 nmol/L) also induces greater
leukocyte adhesion in animals exposed to LTC4 compared with
those treated with histamine, suggesting that the observation is not
particular to PAF. Finally, another explanation for the results in Fig 8 could be that histamine may be more potent than LTC4 at
inducing the generation of an anti-inflammatory agonist (eg, nitric
oxide or prostacyclin), which would then blunt the response to
subsequent exposure to chemotactic stimuli. This possibility warrants
further attention.
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Previous in vitro work has demonstrated that LTC4-induced PAF synthesis on the surface of endothelium promotes CD18-dependent leukocyte adhesion.19 Although one might predict from these in vitro experiments a significant increase in leukocyte adhesion in postcapillary venules treated with LTC4, the magnitude of the LTC4-induced adhesion in the present study was subtle compared with that observed with exogenous PAF or various other chemotactic agents.50 When animals were pretreated with WEB 2086, not only was the LTC4-induced adhesion decreased, but the rolling velocity also returned to baseline levels. These data suggest that PAF may also be involved in the reduced rolling velocity associated with LTC4. This is consistent with observations that WEB 2086 inhibits the reduction in leukocyte rolling velocity and adhesion in various inflammatory conditions, including ischemia/reperfusion and mast cell–dependent leukocyte recruitment.51 52
Once released from a cell, LTC4 is rapidly and
predominantly metabolized to LTD4 via
-glutamyltranspeptidase and then to LTE4 via
aminopeptidase.37 53 54 Depending on the
species and route of elimination, very small amounts of
LTC4 are actually detectable
systemically.1 55 56 In humans, urinary levels of
LTD4 and LTE4 are often used as estimates of
systemic LTC4 generation in
pathophysiological states, including
inflammatory bowel disease, glomerulonephritis, and rheumatoid
arthritis.12 57 58 In animal models, investigators have
primarily studied the effect of LTC4 on bronchial smooth
muscle contraction and microvascular permeability in lung tissue and
have demonstrated that although selective receptors for
LTC4 may exist,38 53 59 the majority of the
actions of LTC4 are in fact mediated via the
LTD4 receptor.38 54 In the present study,
we demonstrate that inhibition of the LTC4 bioconversion to
LTD4 and the subsequent bioconversion to LTE4
prevent the LTC4-induced increase in leukocyte rolling and
reduction in rolling velocity, suggesting that these responses are
indeed mediated via the LTD4 receptor. However, the
possibility that an LTE4 receptor exists and mediates this
response cannot be excluded. The reduction in LTC4-induced
leukocyte rolling with L-serine was as effective as that
with sLex and raises the possibility that targeting the
enzyme responsible for LTC4 conversion to LTD4
(-glutamyltranspeptidase) may be a rational approach to
anti-inflammatory therapy. A specific LTD4 receptor
antagonist did not entirely prevent the
LTC4-induced leukocyte–endothelial
cell interactions; however, this may be explained by the fact that
LTD4 receptors display significant species
specificity.60 61 62 63
In conclusion, our data provide the first evidence that LTC4 can indeed induce leukocyte rolling in postcapillary venules via a P-selectin–dependent and sLex-dependent mechanism. These events are likely to be mediated by LTC4 bioconversion to LTD4. Moreover, LTC4 induced a rapid and significant sLex-dependent reduction in leukocyte rolling velocity, which further increased the likelihood that a rolling leukocyte would adhere.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the Alberta Heritage Foundation for Medical Research (AHFMR) and the Canadian Medical Research Council (MRC). Dr Kubes is an AHFMR and MRC scholar.
Received May 24, 1995; accepted August 17, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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