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*
British Heart Foundation Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom; and
Celltech Ltd., Slough, United Kingdom
4 Abbreviations used in this paper: EC, endothelial cells; Pt, platelets; CS, contact sensitivity; ID, injected dose; i.d., intradermal; OX, 4-ethoxymethyl-2-phenyl-2-oxazolin-5-one (oxazolone).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, or
IL-1ß. In contrast, during a contact sensitivity response to
oxazolone, expression of both selectins was prolonged, with distinct
peaks at 6 and 48 h. Experiments with P-selectin gene-targeted
mice showed that the P-selectin measured was exclusively expressed by
EC rather than platelets. The early and late phases of selectin
expression in contact sensitivity were differentiated in terms of their
requirement for prior sensitization, and the action of IL-1. Whereas
the early phase was a nonspecific ’irritant’ response to oxazolone,
the late phase was Ag specific and was partially IL-1 dependent.
Therefore, persistence of both E- and P-selectin expression in vivo can
occur as a result of sequential and distinct EC activation processes
that appear to be at least partially different from those previously
reported as stimulating ICAM-1 and VCAM-1 expression. The further
elucidation of mechanisms of EC activation in this model may help
determine the relative roles of selectins and ligands for leukocyte
integrins in the sequential recruitment of T cells and other leukocyte
subsets during ongoing immune-mediated inflammatory
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-granules. In contrast, TNF- in
vitro induces expression of both E- and P-selectin by EC, maximal at
2–4 h, that is dependent on gene transcription (2, 3).
The kinetics and mechanisms of expression of E- and P-selectin in
subacute and chronic inflammation are still unclear. The fraction of circulating leukocytes that roll and adhere on EC depends in large part on the density of expression of the various adhesion molecules involved (4). Therefore, precise quantification of EC adhesion molecule expression in vivo could lead to a greater understanding of their pathophysiological role in leukocyte migration. Accordingly, we and others have administered radiolabeled mAb against EC adhesion molecules i.v. and quantified their localization in different vascular beds by gamma counting. With this approach, it has been possible to measure the expression of endothelial selectins during the course of inflammatory responses in the pig (5) and the mouse (6).
The murine contact sensitivity (CS) response is a model of focal, T
cell immune-mediated inflammation, characterized by the migration of
leukocytes into the skin over 48 h (reviewed in Ref.
7). We recently reported that the EC adhesion molecules
ICAM-1 and VCAM-1 are up-regulated throughout the CS response elicited
by 4-ethoxymethyl-2-phenyl-2-oxazolin-5-one (oxazolone (OX)) in the
mouse. Even early in the response, this up-regulation was dependent on
prior sensitization to Ag, and it was inhibited by treatment before
challenge with a neutralizing anti-TNF- antiserum
(8). Recent studies have indicated that both E- and
P-selectins are involved in generating the elicitation phase of CS
(9, 10), whereas L-selectin may be necessary only for
induction of sensitization to the Ag (11). In this study,
we have compared the expression of E- and P-selectin over the time
course of elicited CS responses and have related the results to
previously demonstrated expression of ICAM-1 and VCAM-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MES1 is an IgG2a mAb against mouse E-selectin that was generated
from splenocytes of a rat immunized with mouse E-selectin-transfected
Chinese hamster ovary cells. RB40.3414 (RB40) is a rat IgG1 mAb against
mouse P-selectin and was supplied by Professor Dietmar Vestweber
(University of Münster, Münster, Germany). Rat hybridoma
cell lines producing Abs against DNP were obtained from Dr. David Gray
(Imperial College School of Medicine, London, U.K.) for the generation
of rat IgG1, IgG2a, and IgG2b mAb for use as irrelevant negative
control Abs. Hybridoma cell lines were cultured in RPMI 1640
supplemented with 10% heat-inactivated FCS, 2 mM
L-glutamine, 50 U/ml penicillin, and 50 µg/ml
streptomycin (all from Life Technologies, Paisley, U.K.). Hybridoma
supernatants were purified by gel filtration over Sepharose 4B followed
by affinity chromatography using protein G conjugated to Sepharose 4B
(Pharmacia, Uppsala, Sweden). Abs were eluted from the column with the
use of a glycine buffer (0.1 M, pH 3) and were dialyzed into HBSS (Life
Technologies). Polyclonal sheep anti-murine TNF- serum H92 and
recombinant murine TNF- were gifts from Dr. Tony Meager (National
Institute for Biological Standards and Control, Potters Bar, Hertford,
U.K.). Recombinant murine IL-1 and IL-1ß were purchased from
Peprotec (London, U.K.). The polyclonal sheep anti-murine IL-1
and IL-1ß antisera were supplied by Dr. Roberto Solari (Glaxo
Wellcome, Stevenage, U.K.).
Selectin localization in vivo by mAb targeting
Animals were maintained and studied according to U.K. Home Office regulations. Female mice were used for all experiments and were age-matched. BALB/c and C57BL/6 mice were obtained from Harlan Olac (Oxon, U.K.) at 6–10 wk of age and housed in standard cages. P-selectin deficient C57BL/6J-selp (tm1 bay) (12) were obtained from The Jackson Laboratory (Bar Harbor, ME). Breeding pairs were housed in isolators, and progeny were housed in filter cages supplied with irradiated bedding, food, and acidified water. All mice were housed in proximity to sentinels, which were used for regular pathogen screening.
mAbs MES1 and RB40 and anti-DNP-negative control mAb, were differentially radiolabeled with 99mTc, 111In, and 125I to permit their detection in the skin after administration to mice, as previously described (8). They were then mixed together, and 2 µg of each were administered by tail vein injection. After 5 min, mice were placed under terminal anesthesia by i.p. injection of pentobarbitone sodium (Rhône Mérieux, Harlow, U.K.). The vasculature was then perfused with PBS containing heparin 10 U/ml (Leo Laboratories, Princes Risborough, U.K.). Tissues were harvested, weighed, placed in vials, and counted simultaneously in an automated gamma counter (Packard, Pangbourne, Berkshire, U.K.), with the use of separate channels for 99mTc, 111In, and 125I. Tissue cpm were corrected for background, spill between channels, and 99mTc decay over the counting period and for tissue sample weight in grams. They were then divided by the total cpm injected into that animal, which was calculated with the volume of injected material and the cpm in a standard dilution of the injection mixture. This value was either made into a percentage (referred to as the percent injected dose (ID) per gram; %ID/g) or, when experiments involved comparisons between knockout and wild-type mice, normalized for plasma volume by multiplying by the weight of the animal in grams (referred to as normalized %ID/g). Specific uptake of anti-selectin mAb was calculated by subtracting the %ID/g for the control mAb from that of the test mAb.
Alternatively, localization of i.v.-administered mAb was detected by immunohistochemistry. Mice received 50 µg of anti-selectin or control mAb; 5 min later they were placed under terminal anesthesia, and the vasculature was perfused as above. Harvested tissues were embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN) and snap frozen over liquid nitrogen. Then 5-µm sections were mounted on glass slides, air-dried, and fixed for 10 min in acetone. After blocking with 20% normal rabbit serum (Dakopatts, Glostrup, Denmark), rabbit anti-rat Ig Ab and alkaline phosphatase + anti-alkaline phosphatase complex (Dakopatts) were applied in succession. The sections then were developed with Fast Red (Sigma, Poole, U.K.), counterstained with Harris’ hematoxylin, and coverslipped with Aquamount (BDH, Poole, U.K.).
Models of skin inflammation
Acute dermal inflammation was induced by the intradermal (i.d.)
administration of TNF- or IL-1 as follows. Mice were anesthetized
i.m. with a 20-µl mixture of ketamine (Parke Davis, Gwent, U.K.),
xylazine (Bayer, Leverkusen, Germany), and PBS (4:1:5 v/v/v). The
flanks were then shaved, after which each flank received a single i.d.
injection of TNF- (5 ng), IL-1 (5 ng), or IL-1ß (5 ng) in HBSS
containing 0.01% Evans blue (Sigma). These concentrations of cytokines
were found to be optimal in preliminary experiments. Control mice
received HBSS, 0.01% Evans blue alone. After varying incubation
times, and 5 min after injection of radiolabeled mAb
mixture, mice were killed by cervical dislocation, and the flank skin
was removed. Any trace of blood was removed from the skin by blotting
with gentle pressure, and then injection sites were excised with a 5-mm
punch, weighed, and counted.
Mice were sensitized for CS responses by topical application of OX (Sigma) (50 µl) in acetone-olive oil vehicle (4:1 v/v) to the shaved right flank. Unless specified, the sensitizing and challenging concentrations of OX for BALB/c mice were 1%, whereas C57BL/6 were sensitized and challenged with 5% OX, because our preliminary studies had shown that the CS response to OX is more difficult to elicit in this strain (data not shown). Control mice received vehicle alone. From 5 to 7 days later, mice received a 10-µl challenge on one or both ears of varying concentrations of OX in acetone-olive oil or of vehicle alone. After various time periods, selectin expression in the ears was ascertained as above. The ears were transected at a line measuring 9 mm from the apex, and their thickness was measured with a micrometer (Cadar, Sheffield, U.K.), before weighing and gamma counting excised ears.
Our preliminary data suggested that small differences in the radiolabeled mAb preparations for each experiment resulted in some variation between experiments in the absolute level of the outcome measure, %ID/g. Therefore, no attempt was made to pool data from different experiments, and each experiment was designed to include appropriate internal controls. Individual comparisons between groups were made by Student’s t test; multiple comparisons were made using a one-tailed ANOVA followed by Bonferroni’s multiple comparison of means test.
Transfer of bone marrow cells
Mice selectively deficient in either platelet or endothelial P-selectin were created by the transfer of bone marrow cells between wild-type and P-selectin-deficient mice (13, 14) to determine the cell type responsible for uptake of i.v.-injected anti-P selectin mAb. Bone marrow cell recipients received a lethal dose of irradiation (9.5 Gy) from a cesium source (IBL 637; Cis Bio International, High Wycombe, Buckinghamshire, U.K.) 18 h before cell transfer. Bone marrow donor mice were killed by cervical dislocation, and then bone marrow cells were harvested from both femora and resuspended in sterile PBS. Each recipient was injected with 107 cells by i.v. tail vein injection and was left to generate bone marrow-derived cells for at least 8 wk before use. There was no mortality or morbidity among recipients. The blood cell phenotype of recipients was ascertained by purifying platelets from whole blood and determining their level of P-selectin expression with a solid phase ELISA and with RB40 as the primary Ab, as described (15).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first used immunohistochemistry to detect the luminal
expression of E- and P-selectin on vascular endothelium in mouse skin
of mice previously sensitized to 1% OX both under basal conditions and
48 h after local topical challenge with 1% OX (Fig. 1
). Control IgG, anti-E-selectin mAb
MES1, or anti-P-selectin mAb RB40 that had been administered i.v.
were localized in tissue sections using anti-rat Ig. Staining with
hematoxylin and eosin (Fig. 1, A and B) showed
typical changes of a CS response, with dermal edema, a mixed leukocyte
infiltrate, and epidermal microabscesses. E-selectin was not
detectable under resting conditions (Fig. 1E) but was
expressed on the lumina of small venules 48 h after OX challenge
(Fig. 1F). In contrast, P-selectin was detected on venular
EC in noninflamed (Fig. 1G) as well as inflamed skin (Fig. 1H). Control IgG did not localize to vascular endothelium
(Fig. 1, C and D).
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To achieve more quantitative data on E- and P-selectin expression,
we administered radiolabeled mAb and determined their uptake in skin
tissue by gamma counting. Initially, we performed experiments to
validate radiolabeled mAb targeting as a means of quantifying
E-selectin expression in vivo. We chose to study the effect over time
of i.d. TNF-, in that we previously had shown a marked, transient
up-regulation in E-selectin expression after this stimulus in pigs
(16). Fig. 2 shows that
after i.d. injection of 5 ng TNF-, the specific uptake of
anti-E-selectin mAb (MES1) increased to a peak at 2–4 h, after
which it declined to a low but significant level that was maintained
until at least 24 h. A further experiment (data not shown) showed
that radiolabeled mAb MES1 localization was also increased 24 h
into the elicitation phase of a CS response to OX and that this
increase could be completely inhibited by coadministration of excess
unlabeled MES1.
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or
IL-1
We then sought to validate similarly the technique for P-selectin
by quantifying skin expression of P-selectin at various times after
i.d. TNF- administration using 125I-labeled
anti-P-selectin (mAb RB40). Fig. 3A shows that TNF- led to
transient up-regulation of P-selectin expression, with kinetics broadly
similar to those of E-selectin, peaking at 4 h, and returning to
baseline at 24 h. We then determined P-selectin expression 4
h after i.d. IL-1 and IL-1ß, using TNF- as a positive control.
Fig. 3B shows that P-selectin was up-regulated in response
to IL-1- and IL-1-ß to a comparable degree to that seen after i.d.
TNF-.
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compared
with vehicle alone (Fig. 3
C). In contrast, there was no
detectable specific anti-P-selectin mAb uptake in the skin of
P-selectin -/- or Pt+,
EC- mice (Fig. 3C). However, in
Pt-, EC+ mice, the
anti-P-selectin uptake was not significantly different from that
seen in +/+ mice. This result indicated that mAb RB40 was specifically
targeting P-selectin expressed by EC rather than platelets. The finding
that +/+ skin injected with HBSS vehicle showed significantly more
P-selectin expression than the TNF--treated skin of P-selectin -/-
mice (Fig. 3C) suggested that P-selectin is normally basally
expressed in the skin (cf. Fig. 1). However, it was possible that i.d.
application of vehicle alone was sufficient to induce P-selectin
expression. We therefore proceeded to evaluate P-selectin expression in
entirely untreated ear skin, of +/+ and -/- mice. Fig. 3D
clearly shows a significant low level constitutive luminal expression
of P-selectin in the skin of untreated C57BL/6 mouse ears. E-selectin expression in CS responses of P-selectin-deficient mice
Previous work with E and P-selectin gene-targeted mice has raised
the possibility that disruption of the gene encoding one selectin
molecule might cause compensatory changes during ontogeny in the
control of expression of other selectin molecules
(17, 18, 19). We were able to directly address this question
by quantifying E-selectin expression in wild-type and P-selectin -/-
mice in both the TNF- skin spot model (Fig. 4A) and in CS responses to OX
(Fig. 4B). We found no differences in TNF-- or OX-induced
E-selectin expression between wild-type and P-selectin -/- mice,
suggesting that a compensatory up-regulation of E-selectin in response
to P-selectin gene-targeting had not occurred.
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We delineated the precise kinetics of E- and P-selectin expression
during the CS response to OX in BALB/c mice. Fig. 5A shows that E-selectin
expression had two overlapping phases of expression, with peaks around
6 and 48 h, after which expression returned toward baseline. The
kinetics of P-selectin expression closely paralleled that of E-selectin
(Fig. 5B). In contrast to the biphasic kinetics of selectin
expression, ear thickness increase measured during this period (i.e.,
6–72 h) was monophasic (Fig. 5C). We then determined the
earliest time point at which increased P-selectin expression was
detectable during the onset of the CS response. As shown in Fig. 5D, significant P-selectin up-regulation was first noted at
2 h after application of OX. No difference was detected between
the time of onset of P-selectin and that of E-selectin expression (data
not shown).
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was
designed to establish the contribution of Ag-specific immunity to the
two phases of expression of E- and P-selectin. OX-sensitized mice were
compared with those sham-sensitized with vehicle alone. Responses were
then elicited with 1% OX and with a lower OX challenge dose of 0.25%.
The late phase of expression of both E-selectin (Fig. 6, A
and B) and P-selectin (Fig. 6, C and
D) observed in sensitized mice was not detected in
sham-sensitized mice. In contrast, at 4 h, selectin expression did
not differ significantly with respect to previous exposure to OX.
Furthermore, this early expression was highly dose dependent, given
that it was not detected with the 0.25% OX challenge (Fig. 6, B and D). Thus, selectin expression at 4 h
after the elicitation of CS was Ag nonspecific, whereas by 24 h
expression of the two selectins was largely dependent on prior
immunization.
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, both IL-1 and
IL-1ß, or all three cytokines on selectin expression during CS, using
specific sheep polyclonal neutralizing antisera. These had been shown
to inhibit the expression of ICAM-1 and VCAM-1 (8) and E-
and P-selectin (data not shown) in response to i.d. injection of the
appropriate recombinant cytokines. Cytokine blockade during the CS
response was achieved by i.p. administration of antisera 24 h
before OX challenge and thereafter every 24 h until termination of
the experiment. As expected, the antisera significantly reduced the
increase in ear thickness seen in control mice at 48 h after
challenge (from 0.11 ± 0.012 mm to 0.060 ± 0.019 mm for
TNF- and to 0.074 ± 0.028 mm for IL-1; p <
0.005 and p < 0.05 by Student’s unpaired
t test, respectively). As shown in Fig. 7, A and C,
expression of both E- and P-selectins at 4 h after challenge
was not detectably affected by inhibition with these
anti-cytokine antisera. In contrast, at 48 h there was a
modest but significant inhibition of P-selectin expression with
anti-IL-1 and -ß and with a combination of anti-IL-1 and
anti-TNF-. In contrast, we observed no statistically significant
effect of IL-1 and TNF- blockade on E-selectin expression (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Besides providing data on the expression of P-selectin, experiments with the P-selectin -/- mice also allowed us to clarify whether the relatively small effect of targeted disruption of the P-selectin gene on leukocyte trafficking into inflamed skin is caused by a developmental compensatory increase in E-selectin expression. We found no difference in E-selectin expression between wild-type and P-selectin -/- mice, supporting the view that in the mouse the two EC selectins are to some extent redundant (9, 10, 18).
In CS, we found prolonged up-regulation of both selectins throughout
the time course of the elicited response. Up-regulation was detected in
two phases: an early phase peaking at 4–6 h; and a later phase at
24–48 h. Our data support previous observations on E-selectin
expression made using the radiolabeled mAb technique in pig skin
(5) and by immunohistochemistry in the mouse
(26). It now appears that the kinetics of P-selectin
expression is similar to that of E-selectin. We have distinguished
mechanisms underlying the early and the late phase. The early phase was
independent of immunity to Ag and highly OX dose dependent, consistent
with an "irritant" effect of the hapten (27), whereas
the late phase was immune dependent. Unlike the early phase, the late
phase was inhibited by mAb to 4 and
ß2 integrins (data not shown), suggesting a
dependence on the migration of leukocytes into the lesion for its
expression. Local elaboration of both TNF- and IL-1 have been shown
to be important in mediating inflammation in CS (28, 29).
Our studies attempting to establish roles for these cytokines in the
stimulation of E- and P-selectin showed evidence for IL-1 mediating
P-selectin expression in the late phase, but otherwise were negative.
It is possible that the inhibitory effects of anti-cytokine
antisera lay below the level of detection in this experimental system.
However, the failure of anti-TNF- to inhibit E-selectin
expression is consistent with our experience in the porcine
delayed-type hypersensitivity response to tuberculin purified protein
derivative (30). The exact mediators responsible for
up-regulation of selectins in the two phases therefore remain to be
determined but, in the case of the early phase, could include a direct
effect on EC of hapten itself (31).
Comparison with the results of our earlier study (8) shows
that expression of the EC selectins differs from that of ICAM-1 and
VCAM-1 in at least three ways: 1) expression of the selectins is more
obviously biphasic; 2) expression of the integrin ligands was immune
dependent in both the early and later phases of the CS response,
whereas the early expression of E- and P-selectins was not Ag-specific;
3) expression of ICAM-1 and VCAM-1 at 4 h after challenge was
inhibited by anti-TNF-, which had no detectable inhibitory
effect on expression of the selectins at this or later time points.
These differences were also observed in our study comparing E-selectin
and VCAM-1 expression in the porcine delayed-type hypersensitivity
response to purified protein derivative (30), suggesting
that they are not unique to the murine model.
Taken together, our studies suggest that the two EC selectins perform a different function to the integrin ligands in the control of leukocyte recruitment in CS responses. The ability of EC to express selectins transiently in response to irritant stimuli may allow leukocytes to be exposed to factors in the local vascular microenvironment without committing them to firm adhesion. In sensitized mice, however, the additional up-regulated expression of ICAM-1 and VCAM-1 may facilitate the firm adhesion and transendothelial migration of activated CS-effector T cells critical to the development of the response.
In conclusion, this study has demonstrated that the persistence of both E- and P-selectin expression in vivo can occur in cutaneous immune responses as a result of sequential EC activation, a process likely to be relevant in chronic, immune-mediated inflammatory diseases. We postulate that selectin expression in response to different stimuli enables leukocyte rolling in a number of pathophysiological states, whereas the more tightly controlled expression of ICAM-1 and VCAM-1 may be a rate-limiting step for in the leukocyte adhesion cascade leading ultimately to recruitment across the endothelium and into the tissues.
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Footnotes |
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2 Current address: Section of Allergy and Clinical Immunology, Department of Medicine, Yale University School of Medicine, New Haven, CT 06510.
3 Address correspondence and reprint requests to Dr. Dorian O. Haskard, British Heart Foundation Cardiovascular Medicine Unit, NHLI, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, U.K. E-mail address:
4 C. J. Stocker, K. L. Sugars, O. A. Harari, R. C. Landis, B. J. Morley, and D. O. Haskard. TNF-, IL-4 and IFN-
regulate differential expression of P- and E-selectin expression by porcine aortic endothelial cells. Submitted for publication.
Received for publication July 8, 1999. Accepted for publication October 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Immunol. 162:1648.- or lipopolysaccharide-induced expression of the murine P-selectin gene in endothelial cells involves novel 
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 C. Tremblay, M. Paradis, and M. Dore Expression of E- and P-selectin in Tumor Necrosis Factor-induced Dermatitis in Dogs Vet. Pathol., May 1, 2001; 38(3): 261 - 268. [Abstract] [Full Text] [PDF]
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 P. Bauer, T. Welbourne, T. Shigematsu, J. Russell, and D. N. Granger Endothelial expression of selectins during endotoxin preconditioning Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2000; 279(6): R2015 - R2021. [Abstract] [Full Text] [PDF]
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C. J. Stocker, K. L. Sugars, O. A. Harari, R. C. Landis, B. J. Morley, and D. O. Haskard TNF-{alpha}, IL-4, and IFN-{gamma} Regulate Differential Expression of P- and E-Selectin Expression by Porcine Aortic Endothelial Cells J. Immunol., March 15, 2000; 164(6): 3309 - 3315. [Abstract] [Full Text] [PDF]
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) or
vehicle (
) (n = 5 per group). The normalized
%ID/g represents the %ID/g corrected for mouse weight (see
Materials and Methods). Bars represent mean + SD.
P-selectin expression was not detectable in TNF-
).
Columns represent mean + SD. From 24 h after 0.25% OX and 48
h after 1% OX challenge, selectin up-regulation was detected only in
sensitized mice. At 4 h after 1% OX challenge, both E-
(A) and P-selectin (C) up-regulation was
detected at similar levels in sensitized and sham-sensitized mice;
0.25% OX was not sufficient to induce a detectable early selectin
up-regulation. Groups were compared by one-tailed ANOVA with Bonferroni
correction. *, p < 0.05, **,
p < 0.001 in comparison with the 0-h group; ##,
p < 0.001 comparing OX- vs sham-sensitized groups
at 48 h.
) in
%ID/g due to the CS response. Mice were pretreated by i.p. injection
of neutralizing sheep antisera to TNF-