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Department of Immunology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-selectin-deficient
(L-selectin-/-)4
mice have decreased trauma- and TNF-
-induced rolling of leukocytes
in venules (10, 11, 12). L-selectin-/-
mice also demonstrate decreased leukocyte recruitment into an inflamed
peritoneum at early and late time points, decreased delayed-type
hypersensitivity (DTH) responses, delayed rejection of allogeneic skin
transplants, and resistance to LPS-induced septic shock (10, 12, 13, 14, 15). Similarly, ICAM-1-/- mice have
significantly reduced numbers of infiltrating neutrophils during the
early stages of peritonitis, reduced susceptibility to LPS-induced
septic shock, and impaired DTH reactions, although allogeneic skin
graft rejection is normal (16, 17, 18). Moreover,
leukocyte-rolling velocities are significantly increased in
ICAM-1-/- mice during inflammation
(3). Recent studies in
P-selectin/ICAM-1-/- mice demonstrate a
requirement for ICAM-1 expression when L-selectin mediates leukocyte
rolling in the absence of P-selectin expression (4, 5).
P-selectin/ICAM-1-/- mice display a profound
decrease in trauma-induced leukocyte rolling that persists much longer
than in mice deficient in P-selectin alone (4), which
results in an almost complete lack of neutrophil emigration into an
inflamed peritoneum at early time points (5). By contrast,
recent studies in L-selectin/ICAM-1-/- mice
reveal complex interactions between P-selectin- and ICAM-1-mediated
adhesion during leukocyte rolling in the absence of L-selectin
(3). Elimination of ICAM-1 expression in
L-selectin-/- mice results in a further 50%
reduction in the flux of rolling leukocytes during TNF--induced
inflammation compared with what is observed with L-selectin deficiency
alone. Consistent with this, circulating neutrophil, monocyte, and
lymphocyte numbers are markedly increased in
L-selectin/ICAM-1-/- mice. Collectively, the
differences in leukocyte rolling behavior observed in these adhesion
molecule-deficient mice demonstrate that ICAM-1 expression is required
for optimal P- and L-selectin-mediated rolling in vivo.
The significant reduction in P-selectin-mediated leukocyte rolling observed in L-selectin/ICAM-1-/- mice has therefore prompted us to determine whether altered leukocyte rolling (i.e., increased velocity and/or decreased rolling flux fractions) would affect inflammatory responses in L-selectin/ICAM-1-/- mice. Moreover, would the reduction in P-selectin-mediated leukocyte rolling due to loss of ICAM-1 and L-selectin expression affect leukocyte entry into sites of inflammation beyond the effects resulting from the loss of ICAM-1 or L-selectin alone, or would this affect leukocyte entry under conditions where ICAM-1 expression does not appear to be required. To address these issues, the development of inflammation was assessed in L-selectin/ICAM-1-/- mice during thioglycollate-induced peritonitis, IL-8-induced neutrophil migration to the skin, contact hypersensitivity reactions, skin graft rejection, and LPS-induced septic shock. The results further confirm that the selectins and ICAM-1 have overlapping functions essential for optimal leukocyte rolling and entry into in vivo sites of inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-selectin-/- mice were produced as described (10). ICAM-1-/- mice (17) were from The Jackson Laboratory (Bar Harbor, ME). These ICAM-1-/- mice express residual amounts of ICAM-1 splice variants in the thymus and spleen but not in other organs such as brain, heart, liver, kidney, skin, and gut (19). Mice lacking both L-selectin and ICAM-1 were generated by crossing F1 offspring from crosses of homozygous L-selectin-/- mice with homozygous ICAM-1-/- mice. Lack of L-selectin surface expression was confirmed by direct immunofluorescence staining of blood leukocytes with FITC-conjugated LAM1–116 Ab (20). The presence of the mutated ICAM-1 gene was verified by PCR analysis of DNA from tail biopsies. The L-selectin/ICAM-1-/- mice were healthy, fertile, and did not display any evidence of infection or disease. All mice were backcrossed between 5 to 10 generations onto the C57BL/6 background (H-2b). BALB/c mice (H-2d) were purchased from The Jackson Laboratory. Mice were 7–12 wk old for all experiments, and age-matched wild-type littermates or C57BL/6 mice (The Jackson Laboratory) were used as controls. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Animal Care and Use Committee of Duke University Medical Center.
Thioglycollate-induced peritonitis
One milliliter of thioglycollate solution (3% w/v; Sigma, St. Louis, MO) was injected i.p. into mice as described (13). Mice were euthanized, and the leukocyte infiltrate was recovered by peritoneal lavage using 6 ml of warm (37°C) RPMI 1640 medium supplemented with 2% FCS, 5 mM EDTA, 10 mM HEPES, and 10 U/ml heparin. Viable cells were counted with a hemocytometer, and differential counts of peritoneal lavage smears were performed following staining to determine the relative percentages of neutrophils, macrophages, lymphocytes, and eosinophils.
In vivo lymphocyte migration assays
Single-cell suspensions of mouse splenocytes were prepared in RPMI 1640 medium (Sigma). Erythrocytes were lysed with a 0.83% ammonium chloride solution. Cells were then washed, counted, and labeled with either calcein-AM (Molecular Probes, Eugene, OR) or PKH26 (Sigma). Calcein labeling was conducted as previously described (21). Briefly, cells (5–10 x 107) were incubated on ice for 30 min in 2 ml RPMI 1640 medium containing 1 µM of calcein-AM. PKH26 labeling was conducted following the vendor’s recommendations. Cells (5 x 107) were resuspended in 1 ml of PKH26 diluent, immediately added to an equal volume of a 3 µM PKH26 dye solution, and allowed to incubate for 2 min at room temperature. Labeling was stopped by the addition of FCS. After labeling, the cells were washed three times with PBS, counted, and resuspended in PBS. Calcein-labeled lymphocytes (20 x 106, control or test populations) were mixed with an equal number of an internal standard population of PKH26-labeled control lymphocytes and injected in a volume of 400 µl into the lateral tail vein of recipient mice that had been injected i.p. with thioglycollate 24 h earlier. An aliquot of the injected cell mixture was also analyzed by flow cytometry to calculate the injected ratio of calcein-/PKH26-labeled cells (Ri). After 24 h, single-cell suspensions from spleen, peripheral lymph nodes (PLN, inguinal and axillary), mesenteric lymph nodes (MLN), blood, and peritoneal lavage were prepared and the percentage of calcein- and PKH26-labeled cells was determined by flow cytometry. Cells presenting light scattering properties of dead cells were excluded from the analysis. A minimum of 1000 viable fluorescence bright cells was counted for each sample, and the ratio of calcein-/PKH26-labeled cells was determined (Ro). Results were expressed as previously described (22) or as the total number of labeled cells in each sample.
Chemokine injections and immunohistochemistry
Recombinant endothelial cell-derived human IL-8 (Genzyme Diagnostics, Cambridge, MA) was reconstituted in endotoxin-free water (Sigma) and diluted to a final concentration of 100 µg/ml in PBS containing 0.1% low-endotoxin BSA (Sigma). One microgram of IL-8 (10 µl) was injected intradermally (i.d.) into a shaved flank region of anesthetized mice. An equal volume of 0.1% BSA in PBS was injected i.d. into a separate site at least 3 cm away from the IL-8 injection site to serve as a control. The injection sites were marked to facilitate subsequent accurate identification of the area. Four hours after injection, 4 mm full thickness skin biopsies were harvested and snap frozen in OCT compound (Miles, Elkhart, IN) and stored at -70°C until processed.
Serial 5-µm tissue sections of skin biopsies were acetone-fixed and then incubated with 10% normal goat serum in PBS (10 min, 37°C) to block nonspecific staining. Sections were then incubated with hybridoma culture supernatant fluid containing primary Ab used neat or diluted in PBS containing 1% BSA and 0.02% thiomerosal (30 min, 37°C). Abs used included rat mAbs specific for neutrophils (GR1, clone RB6-8C5, kindly provided by Dr. R. Coffman, DNAX, Palo Alto, CA) and monocytes (clone F4/80, American Type Culture Collection, Manassas, VA). Rat IgG (Southern Biotechnology Associates, Birmingham, AL) was used as a control for nonspecific staining. Sections were then incubated sequentially (20 min, 37°C) with a biotinylated goat anti-rat IgG secondary Ab (Southern Biotechnology Associates), then HRP-conjugated avidin-biotin complexes (Vectastain ABC method, Vector Laboratories, Burlingame, CA). Sections were washed three times with PBS between incubations, developed with 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and hydrogen peroxide, and then counterstained with hematoxylin. In all cases, multiple serial sections from each biopsy were compared with ensure that representative planes were assessed.
Contact hypersensitivity responses
Mice were sensitized by application of 25 µl of oxazolone (100 mg/ml, 4-ethyoxymethylene-2-phenyloxazonone; Sigma) in acetone/sesame seed oil (4:1) for 2 consecutive days on a shaved hind flank as described (13). On day 5 after sensitization, mice were challenged with 10 µl of a 10 mg/ml solution of oxazolone in acetone/sesame seed oil (4:1) applied to the right ear (5 µl applied dorsally, 5 µl applied ventrally). An equal volume of acetone/sesame seed oil (4:1) was applied to the left ear in a similar manner. A calibrated digital thickness gauge (Mitsutoyo, Tokyo, Japan) was used to measure the thickness of the central portion of the ear lobes at 0, 12, 24, and 48 h after challenge. Each lobe was measured three times at each time point, and the mean of these values was used for analysis. The increase in ear thickness was determined by subtracting the ear thickness before challenge from the ear thickness after challenge. No significant increases in ear thickness were observed at any time point in the ear challenged with the acetone/sesame seed oil vehicle alone. Pinnae were harvested from some of the mice 24 h after challenge, cut longitudinally into equal halves, and fixed in 10% neutral-buffered formalin. Tissue sections were stained with hematoxylin and eosin.
Allogeneic skin grafts
Wild-type littermates generated from heterozygous breedings were used as controls to avoid any possible contributions by minor histocompatibility differences between test mice and controls as described (16). Briefly, dorsal skin was dissected from anesthetized donor mice after removal of hair with electric clippers and cleansing with 70% ethanol. Subcutaneous fascia was gently removed from the undersurface of donor skin with a no. 15 scalpel. Recipient mice were anesthetized with pentobarbital administered i.p. at a dose of 0.7 mg/g body weight. Following depilation, the dorsolateral skin of recipient mice was cleansed with 70% ethanol and painted with flexible collodian. Graft beds (8 mm diameter) were prepared by removing the epidermis and superficial dermis, leaving the fascia layer intact. Fitted skin grafts were then placed by trimming donor skin to fit graft beds. Grafts were dressed with petroleum-coated Telfa gauze (Johnson & Johnson Medical, Arlington, TX) held in place with circumferential bandages. Dressings were removed on day 3 after placement of grafts. Mice were monitored daily following surgery.
Duplicate BALB/c grafts were placed on each mouse. Duplicate syngeneic skin grafts placed on each mouse served as controls for nonspecific inflammation related to surgery. Grafts were followed daily for graft progression and time to rejection. Grafts were considered to be rejected at the time of sloughing or upon complete conversion to a hard avascular eschar. Time to rejection was monitored independently by two observers based upon the above criteria.
In vitro CTL assays
Specific in vitro CTL activity was measured by 51Cr release (23). Previous studies comparing direct CTL activity and CTL activity after in vitro preactivation in individual mice demonstrated good correlation between these methods. Because direct CTL activity was inconsistently detected and generally yielded low cpm with poor signal-to-background differentials, all assays were performed after preactivation in vitro (16). Spleens were harvested from three recipient mice from each genotype 14 days after placement of skin grafts. Splenocytes were isolated by density gradient centrifugation over Ficoll (Fico/Lite, Atlanta Biologicals, Norcross, GA) and then incubated with irradiated (2500 Rad) BALB/c splenocytes at an effector:stimulator ratio of 4:1 for 5 days at 37°C in 5% CO2. Primed effector cells were then harvested and incubated with 51Cr- (Amersham, Arlington Heights, IL) labeled BALB/c or syngeneic splenocyte targets at 100:1, 33:1, 10:1, 3:1 E:T ratios. Specific killing ability of effector cells was determined by measurement of 51Cr release over 4 h at 37°C. Spontaneous 51Cr release in culture medium and maximal 51Cr release after addition of 2% Triton X-100 were determined for each target population in all assays. Specific 51Cr release was determined by the following formula: specific 51Cr release = [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100.
All experiments were carried out in triplicate wells. Lytic units/106 effector cells were calculated from E:T lysis curves by standard methods whereby 1 lytic unit was defined as 50% of maximal lysis. This unit of measurement was selected as the majority of E:T lysis curves were linear at this value. Total lytic units/spleen were calculated based on lytic units/106 spleen cells.
Immunization of mice and T cell proliferation assays
Two-month-old mice were immunized in both hind limbs and at the base of the tail s.c. with DNP-conjugated keyhole limpet hemocyanin (DNP-KLH, 100 µg, Calbiochem-Novabiochem, La Jolla, CA) in CFA. Mice were bled from the retroorbital venous plexus before and 5 days after immunization and hapten-specific serum Ab levels were measured by ELISA as described (24). Relative levels of Ag-specific IgM and IgG were determined for each group of mice using individual and pooled serum samples.
Single-cell suspensions of lymphocytes from the spleens, MLN, and pooled PLN pairs draining the sites of immunization (inguinal, popliteal, and para-aortic) were isolated from mice that had been immunized with KLH as described above. The lymphocytes were cultured in triplicate in 96-well plates in RPMI 1640 medium (2 x 105 cells/well in 200 µl) containing 10% FCS, 2 mM L-glutamine, 55 µM 2-ME, antibiotics, and varying concentrations of KLH. Cellular proliferation was quantified by the addition of 1 µCi of [3H]thymidine (Amersham) during the last 18 h of a 5-day culture, and incorporation of radioactivity was assayed by liquid scintillation counting.
Septic shock
Mice were injected i.p. with varying doses of LPS (Escherichia coli serotype 0111:B4; Sigma) and monitored for morbidity and mortality as described (18). Mice that appeared dehydrated were given water orally by dropper at least four times a day.
Statistical analysis
Data are shown as mean values ± SEM unless otherwise stated. ANOVA was used to analyze the data, and the Student’s t test was used to determine the level of significance of differences in population means.
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Results |
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The numbers of resident leukocytes within the peritoneum of
L-selectin/ICAM-1-/- mice are similar to those
of wild-type mice (3). However, 2 h after i.p.
injection of L-selectin/ICAM-1-/- mice with
thioglycollate, the concurrent loss of both adhesion molecules reduced
neutrophil transmigration far greater than observed with loss of either
molecule alone (3). To further assess the extent that
disrupted leukocyte rolling in
L-selectin/ICAM-1-/- mice affects leukocyte
entry, leukocyte influx into an experimental model of peritonitis was
further analyzed at early (4 h) and late (24 and 48 h) time
points. After 4 h of peritonitis, the loss of both L-selectin and
ICAM-1 resulted in a 69% decrease in infiltrating neutrophils relative
to wild-type mice (p < 0.01; Fig. 1
B).
L-selectin-/- and
ICAM-1-/- mice also had significant reductions
(55% and 75%, respectively) in the number of infiltrating neutrophils
(p < 0.01, Fig. 1B). By contrast,
normal numbers of neutrophils entered the peritoneum of
ICAM-1-/- mice by 24 h following the
injection of thioglycollate (Fig. 1B), but neutrophil
emigration was significantly inhibited in both
L-selectin-/- and
L-selectin/ICAM-1-/- mice (Fig. 1B,
p < 0.002). At 48 h, neutrophil emigration in
L-selectin/ICAM-1-/- mice was 66% lower
(p < 0.01) than in
L-selectin-/- mice. Eosinophil emigration was
significantly inhibited in both the
L-selectin-/- (by 52%) and
L-selectin/ICAM-1-/- (43%) mice compared with
ICAM-1-/- and wild-type mice at 48 h
(p < 0.002; Fig. 1E). These results
imply that the repertoire of functional adhesion molecules expressed at
different stages of inflammation varies and that the requirements for
overlapping L-selectin and ICAM-1 functions are dependent on the
relative expression densities of other adhesion molecules.
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C). Similarly,
emigration of monocytes into the peritoneum of
L-selectin/ICAM-1-/- mice was reduced to a
greater extent than in L-selectin-/- mice (48
h, p < 0.01; Fig. 1D). In fact, lymphocyte
and monocyte numbers at 24 and 48 h remained the same as at 4
h following thioglycollate administration. ICAM-1 loss alone did not
significantly impair leukocyte entry into the inflamed peritoneum at
these time points. These findings suggest that the emigration of
lymphocytes and monocytes during the later stages of
thioglycollate-induced peritonitis that occurs in the absence of
L-selectin expression is completely dependent on the expression of
ICAM-1.
Rather than reflecting adhesion molecule function, decreased lymphocyte
entry into the inflamed peritoneum of adhesion molecule-deficient mice
could result from decreased neutrophil entry and blunting of the
inflammatory response. This issue was addressed directly by quantifying
the migration of labeled lymphocytes injected into the circulation of
wild-type or ICAM-1-/- mice that had been
injected 24 h earlier with thioglycollate. Wild-type or
L-selectin-/- lymphocytes were calcein labeled
and mixed with equivalent numbers of PKH26-labeled wild-type
splenocytes. After 24 h of recirculation, the numbers and ratio of
calcein-/PKH26- labeled cells migrating into the peritoneum were
determined. L-selectin-/-
lymphocyte migration into the inflamed peritoneum of wild-type mice was
half that of wild-type splenocytes (see Table I; Fig. 2). L-selectin-/-
lymphocyte migration to PLNs and MLNs was mostly eliminated, whereas
these cells were found at higher levels in blood and spleen. Injection
of L-selectin-/- lymphocytes into
ICAM-1-/- mice with peritonitis generated
similar results (Table I, Fig. 2). Thus,
L-selectin was required for optimal lymphocyte migration into an
inflamed peritoneum, whereas ICAM-1 did not appear to contribute
substantially to migration during the later stages of peritonitis
(Figs. 1 and 2).
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The overlapping roles of L-selectin and ICAM-1 in leukocyte
migration into other sites of inflammation was assessed by injecting
the four groups of mice i.d. with IL-8, a neutrophil chemoattractant.
This assay eliminates differences during the induction stage of
inflammatory responses in the different genotypes of mice. Therefore,
the effect of adhesion molecule loss on effector cell migration can be
assessed directly. Full-thickness skin biopsies were taken from
injection sites after 4 h and examined by immunohistochemistry.
Injection of IL-8 induced an intense influx of
GR1+ neutrophils into the dermis of wild-type
mice, with dense collections of neutrophils throughout the dermis (Fig. 3B). No influx of
F4/80+ monocytes was observed. In
L-selectin/ICAM-1-/- mice, the level of
neutrophil recruitment was reduced by >97% (p
< 0.001, Fig. 3) with minimal evidence of dermal thickening (Fig. 3E). Indeed, the IL-8 injection site in
L-selectin/ICAM-1-/- mice resembled BSA control
injection sites, which consistently contained few or no neutrophils. A
reduction in neutrophil recruitment into the IL-8 injection site was
also noted in L-selectin-/- (by 56%,
p < 0.001) and the ICAM-1-/-
mice (60%, p < 0.001), although considerable numbers
of neutrophils were still scattered throughout the dermis, which was
noticeably thickened as a result of inflammation (Fig. 3, C
and D). The >97% reduction observed in the
L-selectin/ICAM-1-/- mice suggests that these
two molecules function synergistically to mediate neutrophil migration
rather than reflecting the additive effects from blocking a
L-selectin-dependent first step and a subsequent independent,
nonoverlapping ICAM-1-dependent step.
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The ability of leukocytes to migrate into the skin following
contact hypersensitivity reactions was assessed by oxazolone challenge
on the ear 5 days following sensitization. Histologic examination of
ear biopsies taken 24 h after oxazolone challenge revealed that
the ears of wild-type mice were edematous and contained large numbers
of leukocytes in both the dermis and epidermis with frequent
microabscesses (Fig. 4B).
Edema was significantly reduced in ear sections from
L-selectin-/- or
ICAM-1-/- mice, although infiltrating
neutrophils and occasional microabscesses remained apparent (Fig. 4, C and D). By contrast, microabscesses were
completely absent in L-selectin/ICAM-1-/- mice,
and the ears of these mice were identical in appearance to ears
challenged with vehicle only (Fig. 4E). Reductions in ear
swelling were observed in L-selectin/ICAM-1-/-
mice at all time points examined compared with wild-type mice (by
76–98%, p < 0.001),
ICAM-1-/- mice (62–81%, p <
0.001) and L-selectin-/- mice (70–96%,
p < 0.02, Fig. 4). ICAM-1 deficiency significantly
reduced swelling by 31% at 12 h, but not at later time points, as
compared with wild-type mice (Fig. 4).
L-selectin-/- mice had significant reductions
(20–39%, p < 0.05) in ear swelling at all time
points following oxazolone challenge relative to wild-type littermates.
Again, the combined L-selectin/ICAM-1 loss resulted in a greater
reduction in leukocyte migration and edema than would be predicted if
L-selectin and ICAM-1 functioned in an independent manner.
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The roles of L-selectin and ICAM-1 in the migration of mononuclear
cells into allogeneic grafts were assessed by transplanting
MHC-disparate BALB/c (H-2d) skin onto adhesion
molecule-deficient mice. Rejection of BALB/c skin grafts by
L-selectin/ICAM-1-/- mice (mean ± SD,
16.25 ± 0.5 days) was significantly delayed when compared with
wild-type littermates (9.25 ± 0.5, p < 0.001)
and L-selectin-/- littermates
(p < 0.003, Fig. 5). Rejection of BALB/c skin grafts by
L-selectin-/- mice (mean ± SD, 14 ±
0 days) was significantly delayed when compared with wild-type
littermates (9.75 ± 0.5, p < 0.001, Fig. 5).
However, ICAM-1-/- mice (10.0 ± 0.8) and
their wild-type littermates (9.0 ± 2.2) rejected BALB/c skin
grafts similarly. Control syngeneic grafts remained healthy for up to 3
mo. Therefore, although skin graft rejection was normal in
ICAM-1-/- mice, skin graft rejection by
L-selectin/ICAM-1-/- mice was significantly
delayed beyond the delay associated with L-selectin deficiency
alone.
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). These findings suggest
that delayed rejection of allogeneic grafts in adhesion
molecule-deficient mice resulted from decreased migration of CTL
effector cells into the skin rather than from reduced or impaired
priming of lymphocytes during the sensitization phase of allograft
rejection.
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Whether adhesion molecule deficiencies reduced or impaired priming
of lymphocytes during the sensitization phase of contact
hypersensitivity or allograft rejection responses was assessed further
by examining T cell responses to KLH immunization. Five days after mice
were immunized with KLH, their MLN, spleen, and draining PLN T cells
were isolated and cultured in vitro with KLH for 5 days. Significant T
cell proliferation was observed for T cells from all tissues, although
the highest levels of proliferation were observed for PLN T cells and
the least for MLN T cells (Fig. 6). In
three experiments, similar levels of proliferation were observed for T
cells from wild-type, L-selectin-/-,
ICAM-1-/-, and
L-selectin/ICAM-1-/- mice. The only exception
was that T cell proliferation by splenocytes from
L-selectin/ICAM-1-/- mice was consistently
higher than for T cells from mice of other genotypes.
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Despite the inability of L-selectin-/-
lymphocytes to enter PLNs across high endothelial venules (HEV) during
Ag challenge (24), the high frequency of KLH-reactive T
cells within the draining PLNs of adhesion molecule-deficient mice is
explained by their entry through the afferent lymphatics. Although PLN
cellularity was greatly reduced in adhesion molecule-deficient mice,
node cellularity increased 
400–1300% following Ag challenge (Table III). By contrast, MLN and spleen
cellularity did not change significantly following Ag challenge.
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To further assess immune responses in adhesion molecule-deficient
mice, serum Ab responses were assessed 5 days following KLH
immunization. All mice generated significant Ab responses (Fig. 7). IgM and IgG Ab responses were reduced
slightly in both L-selectin-/- and
L-selectin/ICAM-1-/- mice as previously
reported for L-selectin-/- mice
(24), but ICAM-1 deficiency had no apparent effect on
humoral responses.
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Susceptibility to lethal endotoxin shock was assessed in adhesion
molecule-deficient mice by the i.p. injection of LPS. Wild-type mice
and adhesion molecule-deficient mice demonstrated similar symptoms of
shock including ruffled fur, shivering, lethargy, diarrhea, and watery
eyes within 12 h of LPS injection as described previously for
L-selectin-/- and
ICAM-1-/- mice (13, 18). Most mice
succumbed to shock within 3 days. For wild-type mice, the
LD50 for LPS treatment was 2 x
105 endotoxin units (EU)/g of body weight (Fig. 8). By contrast, the
LD50 for ICAM-1-/- and
L-selectin-/- mice was about 10-fold higher,
1.5–2.0 x 106 EU/g of body weight. The
double adhesion molecule deficiency was slightly more protective with
an LD50 of 2.7 x
106 EU/g of body weight. Thus, the protective
effects of L-selectin and ICAM-1 deficiencies appear to operate through
overlapping pathways or mechanisms in this in vivo model of systemic
inflammation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). Although ICAM-1 deficiency alone did not significantly
affect neutrophil migration into an inflamed peritoneum at 24 and
48 h, the majority of neutrophil entry into the peritoneum was
eliminated in L-selectin/ICAM-1-/- mice (Fig. 1B). Similarly, lymphocyte and monocyte migration into the
peritoneum during peritonitis was essentially eliminated in
L-selectin/ICAM-1-/- mice, whereas ICAM-1
deficiency alone had no significant effect (Fig. 1, C
and D). These results demonstrate a high degree of
cooperation between the adhesion molecules that mediate leukocyte
rolling and those that were previously presumed to only mediate firm
adhesion. This also suggests a significant role for ICAM-1 in
regulating leukocyte rolling mediated by P-selectin during inflammation
in vivo.
The effects from loss of both L-selectin and ICAM-1 were more than
would be expected if these molecules functioned independently.
L-selectin/ICAM-1 loss resulted in dramatically reduced inflammatory
responses in the skin following IL-8 treatment (Fig. 3), contact
hypersensitivity responses (Fig. 4), and allogeneic skin
transplantation (Fig. 5). The ICAM-1-/- mice
used in this study do not express alternate isoforms of ICAM-1 in the
resting skin, although systemic LPS treatment induces significant
expression of hypomorphic ICAM-1 molecules (19). However,
the in vivo physiological significance of hypomorphic ICAM-1 expression
is unknown because IgG fusion proteins generated with hypomorphic
ICAM-1 isoforms have only been shown to support LFA-1-dependent binding
in vitro. In fact, the finding that the
ICAM-1-/- mice are extremely resistant to
LPS-induced septic shock (Fig. 8 and Ref. 18) argues
against these ICAM-1 isoforms playing a dominant role in vivo.
Nonetheless, that the combined loss of L-selectin and ICAM-1
dramatically reduced inflammation in four different experimental models
demonstrates that L-selectin and ICAM-1 mediate optimal leukocyte
accumulation during inflammation through overlapping as well as
synergistic functions, despite these receptors having distinct
mechanical capacities. A deficiency in ICAM-1 alone did not
significantly affect neutrophil or lymphocyte migration into an
inflamed peritoneum at 24 and 48 h (Figs. 1 and 2, Table I) or
affect allogeneic skin graft rejection (Fig. 5). Therefore, in the
absence of ICAM-1 expression, P-selectin, E-selectin, LFA-1/ICAM-2, or
4ß1 integrin
interactions may be sufficient to facilitate optimal rolling when
L-selectin function is intact. The differential requirements for
simultaneous L-selectin and ICAM-1 expression at different time points
also emphasizes that the collective array of adhesion molecules
expressed at each time point dictates the requirement for function of
individual adhesion receptors during these dynamic processes.
That excessive leukocyte accumulation can lead to a variety of pathologic inflammatory disorders is well demonstrated in septic shock, a systemic response to infection with a very high mortality rate. Previous studies have demonstrated that L-selectin-/- and ICAM-1-/- mice are dramatically resistant to the lethal effects of high-dose endotoxin shock (13, 18). In both cases, the protective mechanism appears to be distal from endotoxin-initiated cytokine production, but is related to the inhibition of leukocyte accumulation in tissues, an event that precedes the lethal pathophysiological response. Surprisingly, the combined loss of L-selectin and ICAM-1 did not result in remarkably extended survival. This finding suggests that blockade of adhesion receptor function may be protective to a major extent, but that high-dose LPS-induced shock may be refractory to this treatment due to the complexity of this multiorgan inflammatory disorder. Nonetheless, efficacious inhibitors of selectin and integrin function may have a considerable impact on multiple acute and chronic inflammatory conditions when used in combination during therapy.
A central role for ICAM-1 in the initiation and generation of immune
responses raises the issue of whether the current findings with
ICAM-1-/- mice result from a lack of
sensitization or the appropriate generation of effector cells during
inflammatory responses (17). Similarly, the requirement
for lymphocytes to express L-selectin to enter PLNs across HEVs
(10, 24) has lead to the suggestion that
L-selectin-/- mice are immunocompromised
(14). However, there is ample experimental evidence in
both cases to suggest that the decreases in inflammation observed in
the current study results from a lack of leukocyte entry into sites of
inflammation rather than simply a deficiency in effector cells. In the
current study, ICAM-1 deficiency did not inhibit the in vivo generation
of T cells able to mount in vitro proliferative responses to KLH (Fig. 6) or inhibit humoral immune responses following KLH immunization (Fig. 7). Similarly, wild-type numbers of lymphocytes were retained within
draining PLNs following KLH immunization (Table III). CTL responses
were also generated at wild-type levels in
ICAM-1-/- skin allograft recipients (Table II).
Furthermore, the absence of ICAM-1 expression on donor tissue or in the
recipients of allografts had no effect on skin transplant rejection
(Fig. 5 and Ref. 16), and does not significantly prolong
cardiac allograft survival (26). Ab (IgG) and cellular
(DTH) immune responses to type II collagen are also similar between
ICAM-1-/- and wild-type mice (27).
Although APC from ICAM-1-/- mice are poor
stimulator cells in vitro, ICAM-1-/- T cells
respond well in mixed lymphocyte reactions (17). These
findings suggest that the reductions in inflammation observed in these
ICAM-1-/- mice are due to leukocyte migration
defects rather than immunodeficiency.
Although defective neutrophil migration during inflammation in
L-selectin-/- mice is well accepted, concern
has been raised that decreased lymphocyte migration into sites of
inflammation is a secondary consequence due to disrupted
neutrophil entry or immunodeficiency rather than a requirement for
lymphocyte expression of L-selectin. To address this directly, the
ability of labeled adhesion molecule-deficient lymphocytes to enter the
inflamed peritoneum of wild-type mice was assessed. L-selectin
deficiency significantly inhibited the ability of lymphocytes to enter
the peritoneum between 24 and 48 h after the initiation of
inflammation (Fig. 2, Table I). Therefore, there are
L-selectin-dependent pathways through which lymphocytes enter sites of
inflammation. However, some L-selectin-/-
lymphocytes do traffic to sites of peritonitis (Fig. 1C, and
Ref. 13), DTH reactions (Fig. 4C), allogeneic
skin transplantation (16), and do enter PLNs through the
afferent lymphatics that drain sites of Ag challenge (Table III, and
Ref. 24). Therefore, although there are
L-selectin–independent pathways through which lymphocytes enter sites
of inflammation, a substantial portion of L-selectin-bearing
lymphocytes require L-selectin function for effective migration.
Defective effector lymphocyte generation does not explain decreased
lymphocyte migration into sites of inflammation in
L-selectin-/- mice. L-selectin deficiency did
not inhibit the in vivo generation of T cells able to mount in vitro
proliferative responses to KLH (Fig. 6) or the generation of CTL
responses in skin allograft recipients (Table II, and ref.
16). L-selectin deficiency did not significantly inhibit
specific IgM immune responses following immunization with KLH (Fig. 7),
and has previously been shown to either augment or delay humoral
responses to haptenated Ags depending on the immunization regime
(24). In addition, germinal centers are generated in PLNs
draining sites of Ag challenge (24). Similarly,
L-selectin-/- mice are sensitized normally to
OVA during the induction of hypersensitivity responses, although
L-selectin deficiency does inhibit inflammation-induced recruitment of
leukocytes into tissues during subsequent Ag challenge
(28). Current dogma dictates that only memory or activated
lymphocytes enter inflamed tissues and thereby enter the draining lymph
nodes via the afferent lymphatics while restricting naive lymphocytes
to HEV-dependent entry. However, naive T cells migrate into both lymph
nodes and extra-lymphoid tissues such as skin to the same extent
(29, 30). In addition, at least 10% of the T cells
draining chronically inflamed tissue sites have a naive phenotype
(31). Therefore, memory and naive lymphocytes each use
multiple migration pathways (reviewed in Refs. 32 and
33). Thus, the significant decreases in lymphocyte entry
into sites of inflammation in L-selectin-/-
mice and in combination with ICAM-1 deficiency are most likely due to
interrupted lymphocyte-endothelial interactions rather than a lack of
immune response.
Although previous studies have suggested that L-selectin and ICAM-1 are engaged in series by leukocytes, rather than in parallel, the current results are consistent with previous intravital microscopy studies demonstrating that L-selectin and ICAM-1 function synergistically to facilitate leukocyte-endothelial interactions in vivo (3). In the absence of optimal adhesion molecule expression, increases in leukocyte rolling velocities decrease the frequency and efficiency of leukocyte/endothelial interactions and impair leukocyte emigration at sites of inflammation in vivo (8, 11, 34, 35). Thus, when multiple adhesion pathways are blocked, as in the case of L-selectin/ICAM-1 (3) or P-selectin/ICAM-1 deficiencies (4, 5), leukocyte interactions with endothelial cells are sufficiently destabilized so that other adhesion molecule pairs are unable to retard leukocyte rolling velocities enough to facilitate leukocyte entry into sites of inflammation (6). Therefore, leukocyte rolling, firm adhesion, and emigration during inflammation are interrelated events rather than separate processes mediated by interactions between multiple families of adhesion molecules.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Douglas A. Steeber, Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address:
3 D.A.S. and M.L.K.T. contributed equally to this study and share first authorship.
4 Abbreviations used in this paper: L-selectin-/-, L-selectin-deficient; DTH, delayed-type hypersensitivity response; HEV, high endothelial venule; ICAM-1-/-, ICAM-1-deficient; KLH, keyhole limpet hemocyanin; L-selectin/ICAM-1-/-, L-selectin/ICAM-1-deficient; MLN, mesenteric lymph node; PLN, peripheral lymph node; P-selectin/ICAM-1-/-, P-selectin/ICAM-1-deficient; Ri, ratio of calcein-labeled test cells to PKH26-labeled internal control cells injected into mice for migration assays; Ro, ratio of calcein-labeled test cells to PKH26-labeled internal control cells within each tissue after migration assays; i.d., intradermally; EU, endotoxin units.
Received for publication March 5, 1999. Accepted for publication June 9, 1999.
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; L-selectin-/-, 
;
ICAM-1-/-, 
; L-selectin/ICAM-1-/-, 
).
Data points were collected from three independent experiments using a
total of 13 wild-type, 12 L-selectin-/-, 11
ICAM-1-/-, and 10 L-selectin/ICAM-1-/-
mice. *, p < 0.05, significantly different from
wild-type mice. #, p < 0.05, significantly
different from all other genotypes. Additional statistical analysis
results are presented in the Results section.
