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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 580-589
Selective Requirements for Leukocyte Adhesion Molecules in Models of
Acute and Chronic Cutaneous Inflammation: Participation of E- and P-
But Not L-Selectin
By
Michelle D. Catalina,
Pila Estess, and
Mark H. Siegelman
From the Laboratory of Molecular Pathology, Department of Pathology,
The University of Texas Southwestern Medical Center at Dallas, Dallas,
TX.
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ABSTRACT |
Adhesion molecules borne by both endothelial cells and circulating
leukocytes are in large measure responsible for guiding the process of
extravasation. The selectin family has been primarily associated with
the early stages of adhesion involving initial contact and rolling. A
significant body of evidence has accumulated indicating a fundamental
role for the endothelial members of this family, E- and P-selectin, in
a variety of inflammatory states and models. Although originally
identified as the lymph node-specific lymphocyte homing receptor,
L-selectin has also been suggested to play an important role in
leukocyte recruitment to sites of inflammation. We have recently
demonstrated, using L-selectin-deficient mice, that defects in contact
hypersensitivity (CHS) responses are in essence due to the inability of
T cells to home to and be sensitized within peripheral lymph nodes,
whereas nonspecific effector cells are fully capable of entry into
sites of cutaneous inflammation (Catalina et al, J Exp Med
184:2341, 1996). In the present study, we perform an
analysis of adhesion molecule usage in two models of skin inflammation
and show in both L-selectin-deficient as well as wild-type mice that a
combination of P- and E-selectin is crucial for the development of both
acute (croton oil) and chronic (contact hypersensitivity) inflammation
at sites of the skin, whereas L-selectin does not appear to play a
significant role. Moreover,  4 integrins are shown to be
integral to a CHS but not an acute irritant response, whereas CD44 does
not significantly contribute to either. These results provide a
systematic examination in one study of major adhesion molecules that
are critical in acute and chronic skin inflammation. They reinforce the
essential role of the collaboration of E- and P-selectin in both
specific and nonspecific skin inflammatory responses and the importance of 4 in the specific response only. In addition, they substantiate only a limited role, if any, for L-selectin in these cutaneous effector
mechanisms and demonstrate the essential equivalence in this analysis
of L-selectin-deficient mice compared with normal mice treated with
blocking antibodies.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE RECRUITMENT OF leukocytes to sites of
inflammation is a critical component of the response to tissue injury;
thus, the elucidation of molecules mediating the extravasation of
leukocytes from the periphery into the inflamed tissue is central to
the understanding of the complex regulation of the host response to inflammation. Several families of adhesion receptors are thought to be
essential in the multistep model of extravasation for both nonspecific
effector cells as well as antigen-specific lymphocytes.1,2 The initial contact with and rolling on endothelium is mediated principally by the selectin family of molecules: L-selectin expressed on leukocytes and the endothelial P- and E-selectins.3 The endothelial selectins have clearly been shown in numerous model systems
using gene-targeting approaches to participate in an essential fashion
in the recruitment of leukocytes to sites of inflammation, including
sites of a cutaneous delayed-type hypersensitivity (DTH) response.4-9
Although L-selectin was originally described as the lymph node-specific
homing receptor,10-12 its role in the extravasation of
leukocytes at sites of inflammation has also been extensively investigated. L-selectin expression on circulating nonspecific effector
populations (ie, neutrophils and monocytes) has been suggested to be an
important mediator of the primary adhesion of these cells at sites of
inflammation.13-19 It has additionally been shown that,
although independent ligands clearly exist,3 carbohydrates
borne by L-selectin may function as ligands for neutrophils to bind to
E- or P-selectin on endothelial cells in the human,20,21
and it has been further suggested that L-selectin may exert its effect
at inflammatory sites by mediating leukocyte-leukocyte interactions
that facilitate the recruitment of neutrophils.22
We have previously generated L-selectin-deficient mice by targeted
disruption and demonstrated a strikingly impaired contact hypersensitivity (CHS) response to reactive haptens,23 in
agreement with others.24,25 However, our analysis indicated
that T cells, as well as neutrophil and monocyte effector populations,
are fully capable of entry into inflamed skin sites in the absence of
L-selectin and that the defect in CHS resides primarily in the
inability of antigen-specific T cells to home to and be activated
within lymph nodes, ie, in antigen sensitization rather than leukocyte recruitment. Indeed, L-selectin-deficient mice mounted completely normal CHS responses when alternate routes of immunization were used.
In support of this, an acute cutaneous inflammatory response to an
irritant was also completely normal.23 These results argued against a role for L-selectin in leukocyte migration into such inflamed
sites in these mutant mice, thus raising the issues of which adhesion
receptors were indeed responsible for such localization and whether
this is also true for normal mice. The degree to which L-selectin
participates in such responses at sites of inflammation has not been
fully resolved, and it has been suggested that L-selectin may be
responsible for the residual CHS responses seen in P/E-selectin deficient animals.7
In these studies, using both wild-type and L-selectin-deficient mice
we have investigated the use of adhesion molecules in models of both an
acute nonspecific inflammatory response as well as an antigen-specific
CHS response. We demonstrate that L-selectin does not significantly
contribute either in wild-type mice using antibody blockade or in
L-selectin-deficient mice to the recruitment of leukocytes at sites of
cutaneous inflammation. In contrast, antibodies to P- and E-selectin
together, but not singly, are necessary and sufficient to effectively
block the recruitment of neutrophils in an acute nonspecific irritant
response and to inhibit the ear swelling as well as the attendant
leukocyte recruitment to an antigen-specific allergic contact
dermatitis. The concordance of observations using these two approaches
suggests that leukocyte recruitment to sites of cutaneous inflammation
in L-selectin-deficient mice does not result from compensatory
mechanisms and that ligands distinct from L-selectin are clearly
operative in interactions with P- and E-selectin. Moreover, the results
indicate that blocking antibody to VLA-4 is strikingly inhibitory in a
CHS, but has no effect on the recruitment of neutrophils in acute
nonspecific inflammation. Together, these data suggest that the
mechanism for recruitment of nonspecific effector cells to the skin
relies heavily on expression of P- and E-selectins and their ligands, additionally on VLA-4 for antigen-specific responses, but not on
L-selectin for either acute or chronic inflammatory responses.
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MATERIALS AND METHODS |
Mice.
L-selectin-deficient mice ( /) were generated by targeted
disruption of the L-selectin locus, as described.23 Two- to
three-month-old 129Sv/C57Bl wild-type or L-selectin-deficient mice
were used and were housed in the animal resource center of the
University of Texas Southwest Medical Center (Dallas, TX).
Reagents and antibodies.
Croton oil, DNBS (2, 4-dinitrobenzene sulfonic acid), and DNFB (2, 4-dinitrofluorobenzene) were purchased from Sigma (St Louis, MO). The
following antimouse monoclonal antibodies (MoAbs) were used for
fluorescence-activated cell sorter (FACS) analysis
and/or immunohistochemistry: anti-GR-1-biotin (granulocytes and
neutrophils) and anti-Mac-1-biotin (CD11b; macrophages and neutrophils)
(Pharmingen, San Diego, CA); anti-Thy1.2-biotin (Becton Dickinson, San
Jose, CA); anti-L-selectin (MEL-14).10
Streptavidin-phycoerythrin (PE; Caltag, San Francisco, CA) was used as
a secondary reagent in FACS analyses. The following additional MoAbs
were used in in vivo blocking studies: anti-E-selectin
(9A9),26 blocking anti-P-selectin (anti-PB;
5H1),5 nonblocking anti-P-selectin (anti-PNB;
10A10),5 kindly provided by Dr B. Wolitzky,
Hoffman-LaRoche, Inc, Nutley, NJ; anti-L-selectin
(MEL-14),10 anti-VLA-4 (PS-227; kind gift of
Dr P. Kincade, Oklahoma Medical Research Foundation, Oklahoma City,
OK); anti-CD44 (KM81)28 and anti-CD45RB
(MB23G2)29 were obtained from the American Type Culture
Collection (Manassas, VA). Antibodies were affinity purified from
culture supernatants using protein-G-Sepharose according to the
manufacturer's instructions (Sigma).
CHS and acute nonspecific irritant responses.
CHS responses were induced in wild-type mice using DNFB as previously
described.30,31 Twenty-five microliters of 0.5% DNFB in
4:1 acetone:olive oil solution was painted on the shaved abdomens of
mice on days 0 and 1. Mice were challenged on day 5 by topical application of 20 µL of 0.2% DNFB (10 µL per side of the pinna). CHS responses in L-selectin-deficient mice were induced using DNBS
haptenated splenocytes, as described.23 Mice were immunized subcutaneously with 2 × 107 haptenated spleen cells
at two sites on the dorsum and were challenged on day 5 as described
above. For all CHS experiments, baseline ear thicknesses were
determined with a Fowler caliper (Lux Scientific Instrument Corp, New
York, NY) before sensitization. Ear swelling responses were measured at
24 hours after elicitation, and the change in ear thickness from
baseline measurement was computed. Each ear was measured five times and
the mean of these values was used. Statistical analysis was performed
using the Student's t-test for pairwise comparison between
groups and P values less than .05 were considered to be
statistically significant.
Croton oil was used to cause nonspecific inflammation, as
described.23,32 Ten microliters of 0.8% croton oil in
acetone was painted onto the pinna. The change in ear thickness from
baseline was measured at the indicated times as described for CHS
responses.
MoAbs were used to block nonspecific irritant and DNFB-induced CHS ear
swelling responses. Purified MoAb was administered intraperitoneally in a single dose of 200 µg/mouse in
500 µL of RPMI immediately after challenge with either croton oil or
DNFB. Ear swelling responses were determined as described above. In experiments using multiple antibodies, 200 µg of each MoAb was used.
Immunofluorescence analysis of peripheral blood granulocytes.
Twenty-five microliters of peripheral blood obtained by tail bleed at
indicated time points was collected into Alsever's solution (114 mmol/L dextrose, 27 mmol/L sodium citrate, 71 mmol/L NaCl, pH 6.1).
Cells were incubated with saturating amounts of biotinylated anti-GR-1
MoAb in 50 µL phosphate-buffered saline (PBS)/5% fetal calf serum
(FCS) for 20 minutes on ice and then washed with 3 mL of PBS/5% FCS.
Streptavidin-PE was added for 20 minutes, cells were again washed, and
red blood cells were lysed in NH4Cl lysis buffer (0.15 mmol/L NH4Cl, 1.0 mmol/L KHCO3, 0.1 mmol/L
EDTA, pH 7.2). Data were collected on a FACScan analytical instrument (Becton Dickinson, San Jose, CA) and analyzed using CELLQuest software.
Immunohistochemistry.
Ears were severed from euthanized mice, embedded in OCT compound, and
frozen. Serial cryostat sections were fixed in methanol and stained
with biotinylated anti-GR-1, anti-Mac-1, or anti-Thy1.2 as
described.23 Briefly, after incubation with antibody,
sections were rinsed with PBS and Streptavidin-horseradish peroxidase
was added. 1× DAB (0.5 µg/mL diaminobenzidene) and 0.03%
H2O2 in 50 mmol/L TrisHCl were added for 10 minutes and slides were washed in PBS/0.5% CuSO4 and
counterstained with methylene blue.
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RESULTS |
P- and E-selectin, but not L-selectin, are critical in a nonspecific
irritant response.
The role of key adhesion molecules in a croton oil-induced irritant
inflammatory response was investigated by examining the inhibition of
neutrophil migration in both L-selectin-deficient and wild-type mice.
In the background of L-selectin-deficient mice (/), no
defect in responsiveness was previously noted,23 and
neither a blocking anti-P-selectin nor a blocking anti-E-selectin MoAb alone was able to significantly block ear swelling
(Fig 1). In addition, a combination of a
nonblocking anti-P-selectin with a blocking E-selectin antibody also
had no significant effect. However, the combination of blocking
anti-P-selectin plus anti-E-selectin was able to inhibit ear swelling
from 80% to 90% in both wild-type and L-selectin-deficient mice,
indicating the very strong dependence of leukocyte recruitment on a
combination of these adhesion molecules. Additionally, MoAbs to VLA-4,
a molecule generally involved in secondary adhesion, or to CD44,
previously reported to be involved in an antigen-specific contact
hypersensitivity response,33 were also ineffective in
inhibiting ear swelling, as was also true of the additional isotype
control anti-CD45RB. In wild-type mice (+/+), it is
additionally notable that anti-L-selectin had no effect on croton oil
induced ear swelling, consistent with the normal ear swelling response
seen in the knockout mice. Together, the data strongly suggest that
L-selectin is not critical to a nonspecific irritant response and that
results in L-selectin-deficient mice do not result from the
supervention of compensatory adhesion mechanisms. Thus, among the
adhesion molecules tested, only a combination of P- and E-selectin
appears to be involved in this acute inflammatory ear swelling
response.
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| Fig 1.
Change in ear swelling induced in L-selectin-deficient
(A) and wild-type (B) mice by the nonspecific irritant croton oil.
Sixteen hours after croton oil application, the combination of blocking
anti-P-selectin (PB) plus anti-E-selectin was able to
significantly reduce ear swelling in both strains of animals (P < .001 compared with no antibody). Nonblocking anti-P-selectin
(PNB) in combination with anti-E-selectin had no effect.
L-selectin-deficient mice had ear swelling comparable to that of
wild-type animals and anti-L-selectin treatment had no effect on ear
swelling in +/+ mice. Each treatment group contained  5 animals.
(C) Neutrophil infiltrate into croton oil-treated ears is blocked by
anti-P-plus anti-E-selectin. Sixteen hours after the application of
croton oil to ears of wild-type mice, immunohistochemical staining of
ear sections with anti-GR-1 was performed. Cells stained brown are
positive for the marker (original magnification × 400). Combined
anti-endothelial selectin antibodies show a substantial diminution of
both GR-1 staining and ear swelling. L-selectin-deficient mice treated
in the same manner showed identical staining patterns (data not shown).
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Immunohistochemical staining of ear sections from mice treated with
croton oil show predominantly infiltrates of neutrophils.23 As with ear swelling, only the combination of blocking antibodies to P-
and E-selectin was able to significantly diminish the number of
infiltrating cells (Fig 1C and Table 1).
The dramatic inhibition of infiltrating cells with combined
anti-endothelial selectin antibodies also suggests that the slight
residual swelling after such treatment may not be due to inflammatory
cell emigration, but rather to edema more directly caused by the
irritant chemical. These data thus demonstrate that both P- and
E-selectin are essential for the recruitment of neutrophils in an
irritant response and that L-selectin does not significantly
contribute, when assessed either by antibody blockade in normal mice or
in knockout mice.
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Table 1.
The Effect of Antibody Treatment on the Number of
GR-1+ Cells in the Ears of Mice in Response to Topical
Application of Croton Oil
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Ear swelling response in L-selectin-deficient mice is not temporally
altered from wild-type and is blocked throughout by combined P- and
E-selectin MoAbs.
Some adhesion molecules, such as P-selectin, can be rapidly mobilizable
on endothelium upon stimulation and may serve as the initial ligand for
infiltrating cells during tissue damage and inflammation.3
In contrast, E-selectin is upregulated more gradually and also serves
as a ligand for emigrating leukocytes.3,34,35 Because the
upregulation of adhesion molecule expression on endothelium may be
sequential and could be additionally altered in L-selectin-deficient mice, it was possible that animals treated with MoAb would show differential ear swelling at different time points during the course of
the response. At all time points, the decrease in ear swelling with
combined anti-endothelial selectin MoAbs was dramatic in both
/ and +/+ mice (Fig 2).
Although treatment with anti-P-selectin alone resulted in slight
inhibition in mutant mice, only the 12-hour time point showed ear
swelling that was significantly less than that of untreated animals or
those treated with anti-E-selectin (P = .023; Fig 2A).
Anti-L-selectin treatment of wild-type mice had no effect in reducing
ear swelling at any time point (Fig 2B). Thus, although E-selectin
appears to have no independent role in ear swelling, P-selectin alone
may make a small contribution. However, E- and P-selectin clearly act
synergistically in ablating the formation of the ear swelling response
and together appear to be involved in neutrophil recruitment, even at
early time points.
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| Fig 2.
The effect of MoAb treatment on croton oil-induced ear
swelling throughout the response. (A) The change in ear swelling of
L-selectin-deficient mice. At each time point, mice treated with
combined anti-PB plus anti-E-selectin are significantly
different from each of the other treatment groups (all pairwise
comparisons P < .001). (B) The change in ear swelling of
wild-type mice. Anti-PB- plus anti-E-selectin inhibits ear
swelling at each time point (P < .005 compared with no
antibody control), whereas anti-L-selectin has no effect. Data are
from a representative experiment (n 3 mice/time point).
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Inflammatory response correlates with elevated granulocytes in
peripheral blood.
Elevated neutrophil counts have been shown to be characteristic of P-
and P/E-selectin-deficient mice.4,36 We therefore examined
whether the inhibition of neutrophil migration into an inflamed site
correlated with the accumulation of GR-1 positive cells in the
peripheral blood of mice treated with MoAb during their response to
croton oil. Figure 3 demonstrates the
percentages of GR-1+ cells in peripheral blood for each
MoAb-treated group of mice over a 20-hour time course. For
both / and +/+ mice, the percentage of granulocytes in
the peripheral blood increased in the initial 4 hours and then
gradually decreased during the remainder of the course of the response,
generally inversely related to what was observed in the amount of ear
swelling seen at each time point. Combined anti-endothelial selectin
antibodies increased the level of circulating GR-1+ cells
in L-selectin-deficient animals to an average of 69% by 12 hours
posttreatment, consistent with the inability of circulating leukocytes
to marginate and enter the site, compared with 15% in animals treated
with croton oil but not antibody (Fig 3A). Likewise, by the 12-hour
time point, wild-type animals treated with P- plus E-selectin
antibodies showed a fourfold increase in the percentage of circulating
GR-1+ cells, compared with animals not receiving antibody
(P < .001; Fig 3B). Combined anti-endothelial selectins also
had a greater effect on numbers of circulating granulocytes than either
anti-P-selectin or anti-E-selectin treatment alone in /
mice (Fig 3A). However, anti-P-selectin alone also has a moderate but
significant effect compared with croton oil only treated animals
(P < .03 at each time point), consistent with ear swelling
data, whereas anti-E-selectin only treatment did not. The striking
increase in the percentage of circulating granulocytes in anti-P- plus
anti-E-selectin treated mice further suggests that these antibodies
indeed act through blocking of extravasation is entirely consistent
with elevated circulating leukocytes in P/E-selectin-deficient
mice and further demonstrates the cooperative effect of these molecules
in the recruitment of cells into an inflamed site.
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| Fig 3.
The effect of MoAb treatment on the percentage of
granulocytes in peripheral blood after croton oil application to the
ears. Blood for FACS analysis was collected at indicated time points
after the application of croton oil and injection of antibody. (A)
L-selectin-deficient mice: Mice treated with combined endothelial
selectin antibodies have significantly increased neutrophils at each
posttreatment time point (P < .03 compared with control). By
20 hours, the percentage of circulating neutrophils is decreasing in
all animals. (B) Wild-type mice: combined anti-endothelial selectin
antibody increases the percentage of circulating granulocytes at each
time point (P < .005 compared with control). Anti-L-selectin
treatment does not significantly affect circulating granulocyte
percentages.
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Analysis of adhesion molecules required for a DTH response.
To examine leukocyte recruitment into a site of antigen-specific
dermatitis, DTH was induced using DNFB. Because L-selectin-deficient mice cannot be sensitized via topical immunization, they were sensitized subcutaneously with DNBS haptenized spleen cells, which results in DTH swelling and cellular infiltrates indistinguishable from
those of wild-type mice sensitized in the same way or via topical
sensitization with hapten.23 The amount of ear swelling in
response to cutaneous challenge with DNFB was determined with or
without MoAb treatment administered at the time of challenge on the
ear, ie, during the effector arm. As with the acute irritant response,
antibodies to the endothelial selectins in combination most
dramatically inhibited the ear swelling associated with contact dermatitis (Fig 4). Combined
anti-endothelial selectin treatment resulted in a 75% to 90% decrease
compared with untreated and antibody control animals in both wild-type
and knockout mice over elicitation alone. However, in contrast to the
results obtained with the croton oil response, antibody to VLA-4
resulted in a decrease in the CHS response comparable to blocking
endothelial selectins, indicating a role for 4 integrins in the
extravasation pathway in an antigen-specific response. When antibodies
to both endothelial selectins were combined with anti-VLA-4, no
significant additional inhibition of ear swelling was seen over
selectin antibodies alone in either type of animal. Although the
effects of antibody treatment in L-selectin-deficient and wild-type
mice were generally similar, the inhibitory effects by antibodies in
L-selectin-deficient animals appear slightly blunted compared with
wild-type, although this is not statistically significant. The results
nonetheless suggest that E- and P-selectin together play a vital role
in the mounting of a contact hypersensitivity response in normal mice and, in contrast to ear swelling induced by an irritant, VLA-4 appears
to have an equally important role in an antigen-specific response.
Antibody to L-selectin had no effect in this system; anti-CD44 also had
no effect in either knockout or wild-type animals, although it has been
reported by others to have an inhibitory effect in a CHS
response.33
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| Fig 4.
Antibody blocking of a CHS ear swelling response to DNFB.
Mice were treated as described and ears were measured 24 hours after
elicitation. (A) Wild-type mice: combined anti-endothelial selectin
antibody significantly reduces antigen-specific ear swelling compared
with both untreated and anti-CD45RB control treated
animals. Antibody to VLA-4 is equally effective, whereas neither
anti-L-selectin nor anti-CD44 treatment has any effect. (B)
L-selectin-deficient mice: treatment with combined anti-endothelial
selectin or anti-VLA-4 significantly reduces ear swelling. Anti-CD44
and control anti-CD45RB have no effect. n 4 for each
treatment group. *P < .001 compared with both untreated and
anti-CD45RB control treated animals.
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During a CHS response, it is believed that antigen-specific T cells are
first recruited into the site in response to antigen and that their
recruitment causes the release of additional cytokines and activators
that mediate the subsequent recruitment of nonspecific effector cells
such as monocytes and granulocytes.37,38 To characterize
the cells recruited after topical challenge with DNFB, serial sections
of elicited ears from animals treated with antiadhesion receptor MoAbs
were examined to determine the composition and extent of the
infiltrates. Table 2 shows that, in
wild-type mice treated with either combined anti-endothelial selectins
or with anti-VLA-4, there is a severe reduction in the number of both
GR-1+ and Mac-1+ cells, as well as those
expressing Thy1.2. Moreover, mice treated with either anti-VLA-4 or
the anti-endothelial selectins in combination were essentially
indistinguishable from each other with respect to the extent and
composition of the infiltrate. Nonblocking anti-P-selectin in
combination with anti-E-selectin had no effect on the extent or type
of infiltrate; anti-L-selectin or anti-CD44 also had no effect. It is therefore apparent that the effect of
adhesion blockade is both on the swelling response as well as on both
early (T) and late (monocyte and granulocyte) inflammatory cell
recruitment.
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Table 2.
Effect of Antibody Treatment on the Influx of
Inflammatory Cells Into the Ears of Wild-Type Mice in a DNFB CHS
Response
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DISCUSSION |
The involvement of L-selectin in the trafficking of leukocytes to sites
of antigen-specific and nonspecific inflammation is complicated in part
by the contribution and overlapping functions of the endothelial
selectins. It has nonetheless been shown in vitro and in vivo within
venules, particularly after cytokine activation, that neutrophil and
monocyte rolling can be L-selectin-dependent.16,39-42 A
role for L-selectin in inflammation is further supported by a number of
studies that have reported that leukocyte recruitment into various
inflammatory sites, including lung,43 skin,
44and peritoneum,13,17,45 can be lessened by
treatment with anti-L-selectin antibodies. In addition,
L-selectin-deficient mice have been reported to exhibit defects in
recruitment to a site of peritoneal inflammation19 and to
sites of DTH, including a CHS response.23-25 Because of the
conflicting reports on the requirements for L-selectin during
inflammation and because our evidence in L-selectin-deficient mice
indicated that effector mechanisms were completely intact in CHS and
skin irritant responses, we sought to examine in detail which are the
relevant adhesion molecules in these models and also to explore whether
other mechanisms may compensate for L-selectin in our mutant animals
compared with normal mice. The evidence presented here indicates that
both wild-type mice treated with anti-L-selectin antibodies and
L-selectin-deficient mice show no substantial differences in effector
mechanisms and in the recruitment of leukocytes into the skin in either
an acute or chronic inflammatory model. This evidence is consistent
with an intravital study showing a lack of L-selectin involvement in recruitment to noninflamed dermal vessels.46 The
concordance of the neutralizing antibody and mouse mutant studies
indicate that L-selectin does not play a significant role in the
recruitment of nonspecific effector elements in the models of skin
inflammation studied.
In both of these inflammatory models, antibodies to P- and E-selectin
were required in combination to optimally block the ear swelling
response and recruitment of inflammatory cells, consistent with the
known cooperativity and overlapping functions of these molecules. Thus,
in an acute irritant response, these antibodies in combination
inhibited virtually all of the predominantly granulocytic infiltrate,
as well as the more heterogeneous infiltrate in a CHS response. These
neutralizing antibody studies are fully confirmatory of gene targeting
methods, which have indicated a requirement for both P- and E-selectin
in a CHS response.7 In mutant mice, sensitization and
effector phases are not separable; therefore, studies in them do not
distinguish the stage at which the molecules are required. In these
studies, neutralizing antibodies were administered only during the
effector phase of the response, giving a more direct demonstration of
the impact of the loss of P- and E-selectin function on effector cells
and mechanisms. These studies further reinforce the preeminent roles of
P- and E-selectin at this inflammatory site.
One interpretive concern in P/E-selectin double mutants was that the
L-selectin surface density on all leukocytes was markedly diminished,
which is thought to be due to cell activation, and that this could have
significantly contributed to defects in inflammatory cell
recruitment.8 Moreover, residual ear swelling found in these mutants,7 as well as in E-selectin-deficient
mice,5 had been suggested to derive from the participation
of L-selectin. Although L-selectin has been demonstrated to serve as a
major ligand on neutrophils for E-selectin in the
human,20,21 this has recently been demonstrated not to be
the case in mice,21 consistent with our results showing no
impact of the loss of L-selectin in these murine models. However,
because LADII patients have been reported to mount antigen-specific
T-cell recruitment to the skin comparable to normal
individuals,47 it is clear that the situation is more
complex in the human and that other adhesion pathways not involving
selectins or their traditional ligands may be used. L-selectin has
further been shown to potentially contribute to and augment
inflammatory cell recruitment via neutrophil/neutrophil interactions,22 but again, such augmentation does not
appear necessary under the conditions used here. Thus, our evidence
alleviates the concerns about L-selectin in the previous studies of
selectin knockout mice and suggests that L-selectin does not represent a significant contributor to the observations in CHS responses using
such mice.
To assess whether selectins contributed differentially over time during
the course of a response, we examined the effect of antibody treatment
on an acute irritant response at intervals over its course. E-selectin
expression on human umbilical vein endothelial cells
(HUVEC) has been shown to peak between 3 to 6 hours after
stimulation with tumor necrosis factor .35 P-selectin is
also induced by proinflammatory stimuli and is thought to be important
at later as well as very early time points of cell
recruitment.48,49 Therefore, defects in trafficking might
be more apparent at different times when inhibiting these various
adhesion molecules. However, anti-E-selectin had no significant effect
at any interval in an acute irritant response, and the modest effect of
anti-P-selectin did not appear accentuated at any point.
P-selectin-deficient mice have previously been shown to demonstrate
only slight effects on ear swelling (diminished, but not significantly
so) with a significant inhibition of inflammatory cell
recruitment.6 Our results similarly showed a modest
decrease in ear swelling with anti-P-selectin, and at the 12-hour
point the differences were statistically significant (Fig 2). In any
case, even at the earliest time point of 4 hours, the synergistic
effect of both anti-E- and anti-P-selectin is evidenced by the marked
decrease in ear swelling (Fig 2) as well as the increase in the
percentage of circulating granulocytes when compared with
anti-P-selectin treated or control mice (Fig 3). Thus, it appears that
the coordinated function of P- and E-selectin in an acute irritant
response has its onset early and continues throughout the response.
Integrins containing the 4 chain have been shown to be quite
versatile with respect to the adhesion cascade and are able to mediate
tethering and rolling50-52 as well as firm
arrest.53 VLA-4 is expressed on T cells, monocytes,
eosinophils, and, in some studies, neutrophils and has been shown to
have a role in many inflammatory models involving all of these cell
types.54-60 In these studies, a blocking anti-4 antibody
was able to inhibit the preponderance of an antigen-specific response
(Fig 4A and B), but had no effect on an acute response at the same site
(Fig 1). Despite potential expression on numerous inflammatory cell types, our evidence for a role for this molecule at a single anatomic site, cutaneously on the ear, in an antigen-specific response only is
highly suggestive that the inhibition in these studies operates at the
level of effector T cells. Furthermore, analysis of the infiltrate
present after a CHS response in the ears of antibody-treated mice
demonstrated that T cells as well as the nonspecific effector elements
are dramatically reduced with anti-VLA-4 treatment (Table 2), further
supporting this interpretation. However, we cannot formally rule out
that the activation state of 4 on monocytes and/or
neutrophils differs between these cutaneous models or that different
subpopulations of these cells could be involved under the two
circumstances. Our analysis agrees with previous use of VLA-4
antibodies in CHS studies using oxazolone or DNFB in Balb/c
mice,61,62 and the major cell surface VLA-4 ligand VCAM-1
has been implicated in DTH in a primate model.63 In a rat
model of skin DTH, although significant inhibition was seen with
anti-VLA-4 MoAbs, both anti-VLA-4 and LFA-1 were required to
maximally block in vivo.64 It should be cautioned that in addition to direct ligand blocking, anti-VLA-4 can also have
intracellular signaling effects,65-68 and such secondary
effects in these studies cannot be ruled out. The extent of the effect
both on ear swelling and cellular infiltration with anti-4 that we
saw was similar to that using both endothelial selectin antibodies, and
the use of both the endothelial selectin antibodies plus anti-4 did
not result in a significantly greater decrease in ear swelling or infiltrates, suggesting again that these antibodies do not act in
independent pathways. Thus, whereas anti-VLA-4 treatment appears to
have no effect on the recruitment of neutrophils into an
antigen-independent site of acute inflammation, it does inhibit the
majority of an antigen-specific DTH response, supporting the contention
that anti-VLA-4 blocks the entry of T cells, which are in turn
necessary for the recruitment of nonspecific effector cells.
We have recently demonstrated that CD44 can mediate the primary
adhesion of T lymphocytes to endothelium expressing its principal ligand hyaluronate (HA),69 that the activation of CD44 to
bind HA is induced via TCR signaling,70 and that
extravasation of such activated T cells in an in vivo model of
peritoneal inflammation is critically dependent on T-cell CD44
interactions with HA.71 It is therefore of particular
interest that use of the HA-blocking anti-CD44 MoAb, KM81, results in
no decrease or delay in either the acute irritant or the CHS response
when used either alone (Figs 1 and 4) or in conjunction with other
antibodies (data not shown). This is in contrast to previous reports in
which an inhibitory effect on CHS responses to DNFB was
found.33 It is possible that the antibodies used in those
studies may have had additional significant cell surface modulating
and/or signaling effects on those cells. Such modulation in our
experience is considerably reduced with KM81. Furthermore, it has
recently been reported that mice in which CD44 has been disrupted by
gene targeting show no defect in CHS responses,72
consistent with our results. Thus, although it is clear that the
CD44/HA interaction can be important for aspects of effector T-cell
extravasation, this may not be the case at the particular site of the
skin in mice. Because Th1 helper cells entering the skin are in fact
dependent on P- and E-selectin,73 additional primary
adhesion mechanisms may not be required at this site.
It has become clear that caution must be exercised in interpreting
phenotypes of mice with disrupted adhesion molecule genes. Whereas
E-selectin knockouts displayed a relatively mild
phenotype,5 the importance of this molecule and its close
cooperativity with P-selectin was made apparent after using P-selectin
blocking MoAbs in these mice, and particularly after the development of
P/E-selectin double knockout mice.4,8 It is further
possible that compensatory mechanisms may supersede under circumstances
of genetically deficient animals, and discrepancies have been noted
between antibody blocking studies and observations in knockout mice.
For instance, in models of acute lung injury, analysis of mice
deficient in ICAM-1, P-selectin, or both led to significantly different
conclusions than using blocking antibodies to these molecules in both a
cobra venom factor-induced lung injury model and Pseudomonas
infection.74,75 Thus, the use of both approaches can be
quite informative. In using a combination of L-selectin-deficient mice
and blocking MoAb in wild-type mice, we found complete concordance of
results between our own or previous knockout mouse studies and the use
of blocking MoAbs in two standard models of skin inflammation, further
underscoring the necessity for examined such issues on a case by case
basis.
In summary, these studies examine within a single system the major
adhesion molecules contributing to an acute irritant as well as a CHS
response using a combination of mice genetically deficient in
L-selectin and antibody blockade. The evidence emphasizes (1) the
crucial importance of the P- and E-selectin collaboration to these
responses, (2) that L-selectin does not contribute to the effector
phase of either of these responses in a significant way by either
antibody blockade or knockout approaches, (3) that VLA-4 appears
central to an antigen-specific but not acute irritant response at skin
sites, and (4) that, although caution must be used in using knockout
models, antibody blockade and knockout approaches gave quite similar
results in these studies.
| |
ACKNOWLEDGMENT |
The authors thank Dr Barry Wolitzky (Hoffman-LaRoche, Inc, Nutley, NJ)
for kindly providing anti-P- and anti-E-selectin reagents and Helen
Arizpe for technical assistance.
| |
FOOTNOTES |
Submitted June 18, 1998;
accepted September 10, 1998.
Supported by grants from the National Institutes of Health (Grants No.
CA57571 and HL56746), the Welch Foundation (I-227), the Arthritis
Foundation, and the Texas Higher Education Coordinating Board
(003660-076). M.H.S. is an Established Investigator of the American
Heart Association. M.D.C. was a student in the Graduate Program of
Immunology in the Graduate School of Biomedical Sciences, UTSWMCD.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mark Siegelman, MD, PhD, Department of
Pathology, University of Texas Southwestern Medical Center at Dallas,
5323 Harry Hines Blvd, Dallas, TX 75235-9072; e-mail:
siegelman{at}utsw.swmed.edu.
| |
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S. Kumar and S. Ponnazhagan
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A. Schwarz, A. Maeda, M. K. Wild, K. Kernebeck, N. Gross, Y. Aragane, S. Beissert, D. Vestweber, and T. Schwarz
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January 15, 2004;
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December 15, 2003;
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C. Zaph and P. Scott
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Top
Abstract
Introduction