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4 Integrins Regulate Independent Pathways of T Lymphocyte Recruitment in the Pulmonary Immune Response1 ,2
,
*
Department of Pathology, University of Michigan Medical Center, Ann Arbor, MI 48109;
Department of Internal Medicine (Pulmonary and Critical Care Medicine Division), University of Michigan Medical Center, and the Pulmonary and Critical Care Medicine Section, Department of Veterans Affairs Medical Center, Ann Arbor, MI 48105;
Howard Hughes Medical Research Institute, Ann Arbor, MI 48109; and
§
Athena Neurosciences, South San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrins
to enter the airspace and interstitium during the response to SRBC.
Fluorescently labeled T lymphoblasts, derived via activation on CD3 and
growth in low dose IL-2, showed inflammation-specific recruitment into
lungs harvested 24 h after cell infusion. Their flux paralleled
the accumulation of host lymphocytes in the lung, with both peaking 2
to 4 days after SRBC challenge. Trafficking studies conducted over a
24-h period during peak lymphocyte accumulation in the lungs revealed
preferential recruitment of labeled T lymphoblasts expressing P- and
E-selectin ligands. In addition, mAb blockade of the 4
integrins and targeted deletion of an (1,3)fucosyltransferase
essential for selectin ligand synthesis each reduced labeled T
lymphoblast trafficking to a significant degree. Furthermore,
4 integrin blockade reduced the trafficking of the
selectin ligand-deficient cells into the airspace, confirming that its
contribution is in part independent from the vascular selectins. These
findings imply that both selectin ligands and 4
integrins participate in T lymphoblast recruitment during the pulmonary
immune response to IT SRBC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro, T lymphoblasts tether and roll on immobilized selectins (7, 8) and VCAM-1 (9, 10, 11, 12) under shear using carbohydrate-based ligands and
the 4 integrins, respectively. The 4
integrins also arrest the forward motion of lymphocytes once contact is
initiated (13). Animal studies confirm that the adhesion receptors
active in vitro mediate lymphocyte accumulation in a variety of
immunologic reactions in vivo. The selectins are implicated in T cell
recruitment into skin (14, 15, 16), glomeruli (17), joints (15), and
meninges (18). The 4 integrins contribute to T
lymphocyte accumulation during inflammation in the dermis (19), joint
(20), pancreas (21), and brain (22, 23). In the lung, blockade of the
4 integrins (24) and targeted deletion of
P-selectin (25) each partially reduced T lymphocyte accumulation in
OVA-induced airway inflammation. However, these studies examined
effects that developed over several days. Consequently, one cannot
determine whether the flux of cells from the circulation into the lung
(trafficking) or events after entry (e.g., proliferation, turnover, or
retention) accounted for the decrease in accumulation.
Recent studies from our laboratories implicate both the selectins and VCAM-1 in T cell recruitment during the pulmonary immune response to intratracheal (IT)4 challenge with SRBC (26). In C57BL/6 mice, IT SRBC challenge of primed mice induces marked peribronchovascular and airspace accumulation of activated CD4+ and CD8+ lymphocytes, transient pulmonary angiitis, alveolar macrophage activation, in situ maturation of specific Ab-secreting cells, and eosinophil recruitment (27, 28, 29, 30). Lymphocyte accumulation peaks at 3 to 4 days post-IT challenge and then resolves spontaneously within 3 wk (29, 31). In addition, Willis-Karp and associates report that IT SRBC challenge of primed A/J mice induces T lymphocyte (CD4+)-dependent airway hyper-responsiveness in conjunction with the chronic inflammatory infiltrate (32, 33). Thus, the murine response in this model shares several features in common with chronic asthma.
IT SRBC challenge of primed C57BL/6 mice increases the levels of
E-selectin, P-selectin, and VCAM-1 on the lung vasculature throughout
the period of peak lymphocyte recruitment (26). Furthermore, the number
of circulating T lymphocytes with binding sites for P-selectin
increases significantly within 1 to 2 days of IT challenge. Between
days 2 and 4, T lymphocytes with binding sites for both P- and
E-selectin accumulate in the airspace. This accumulation occurs in
concert with a fall in the number of circulating cells carrying
selectin ligands. These findings imply that de novo synthesis of
selectin ligands on T cells enhances the trafficking of circulating T
cells into the lung and raise the possibility that interactions between
4 integrins and VCAM-1 contribute as well.
The current study uses selectin ligand-deficient T lymphoblasts and mAb
blockade of the 4 integrins to test these hypotheses in
vivo. Trafficking assays are performed with fluorescently labeled T
lymphoblasts cultured from mice with and without targeted deletion of
the (1, 3)fucosyltransferase VII (FucT-VII) locus (34). The FucT-VII
blasts are devoid of selectin ligands but otherwise similar to T
lymphoblasts cultured from wild-type control mice. Trafficking studies
showed that both the selectin ligands and the 4
integrins are necessary for optimal T lymphoblast recruitment into
lungs. Furthermore, each receptor family alone initiated trafficking of
a significant number of exogenous T lymphoblasts. Consequently, the
selectin:selectin ligand and the VCAM-1:4 integrin
adhesive interactions mediate in part independent pathways of
recruitment into the lung.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All experiments were performed on specific pathogen-free mice, 10 to 16 wk of age. Female C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The development of the fucosyltransferase VII gene-deleted mice (FucT-VII KO) and its wild-type controls was described in detail previously (34). These genetically engineered mice have hybrid genetic backgrounds, with contributions from the DBA/2J, 129, and C57BL/6J strains. The FucT-VII KO and its wild-type control were derived from the same parental strain to minimize genetic differences away from the FucT-VII locus.
Mice were housed in specific-pathogen free animal rooms at University of Michigan or Ann Arbor Veterans Administration Animal Facilities (licensed by the American Association for Accreditation of Laboratory Animal Care). Mice were given routine animal chow (Rodent Lab Chow 5001, Purina, St. Louis, MO) and chlorinated tap water ad libitum. All procedures were performed according to a protocol approved by the animal care committees of the Veterans Administration and the University of Michigan Medical Centers. This study complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Department of Health, Education, and Welfare Publication 80-23, revised 1978, Office of Science and Health Reports, National Institutes of Health, Bethesda, MD).
Animal model
The secondary pulmonary immune response was induced in previously primed mice by intratracheal challenge with SRBC (Colorado Serum Co., Boulder, CO; sheep no. 4158), as described previously (31). Briefly, mice were primed by an i.p. injection of 108 saline-washed SRBC in a volume of 0.5 ml. Two to three weeks later, the animals were lightly sedated with pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL), and 5 x 108 SRBC in 0.05 ml of saline were deposited directly into the lungs via an intratracheal injection. This technique causes the majority of the SRBCs to be deposited and retained in the airspace and results in a highly reproducible, CD4-dependent pulmonary immune response (27). At various times after initiating the pulmonary response, mice were humanely killed by sodium pentathal overdose, and the bronchoalveolar lavage (BAL) and/or lung tissue were harvested for use in various assays.
Ex vivo lymphocyte expansion
Spleens and lymph nodes were harvested from mice at 10 to 16 wk
of age. Mononuclear cells were released by lacerating the capsule and
teasing the tissue from these organs. The cells were washed twice in
sterile saline and resuspended at 106 cells/ml in RPMI
(Life Technologies, Grand Island, NY) supplemented with 1 mM sodium
pyruvate (Sigma, St. Louis, MO), 1 mM L-glutamine (Life
Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin
(BioWhittaker, Walkersville, MD), and 55 µM 2-ME (Life Technologies;
RPMI+), containing 10% FCS (HyClone, Logan, UT). All
incubations and cell culture were conducted in a humidified chamber at
37°C with 5% CO2. Tissue culture flasks were incubated
with anti-murine CD3 (PharMingen, San Francisco, CA) in saline at 5
µg/ml for 
3 h to coat the plates. The mononuclear cells were
incubated in the coated flasks for 1 to 2 days, washed once, and
resuspended at 0.5 x 106 cells/ml in 1/2 AIM-V (Life
Technologies)/RPMI+ supplemented with 10 U/ml recombinant
murine IL-2 and 2 to 10% FCS. The cells were grown for an additional 2
to 3 days in medium containing either 10% FCS or decreasing amounts of
FCS (8% on day 3, 4% on day 4, 2% on day 5). In both cases, fresh
medium was added daily to maintain the cell concentration at 0.5
x 106 cells/ml.
5-Chloromethylfluorescein diacetate (CMFDA) labeling and infusion of lymphocytes
The cultured T lymphoblasts were washed twice in sterile saline
and resuspended at 2 x 107 cells/ml in either 500 nM
CMFDA or 5 µM 5-(and
6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR) for 30
min at room temperature (Molecular Probes, Eugene, OR). The cells were
then washed twice and resuspended in sterile saline for i.v.
administration. Trafficking studies were initiated by infusing 2
x 107 labeled cells in a volume of 0.2 ml into the tail
veins of C57BL/6 normal or SRBC-challenged mice. In some experiments,
the fluorescently labeled lymphocytes were preincubated before infusion
with either the 4 integrin-specific mAb PS/2 (35) or
polyclonal rat IgG (Accurate Chemical, Westbury, NY). The preincubation
was conducted for 30 min at 5°C using 1 mg of purified endotoxin-free
Ab/108 cells.
Leukocyte collection and purification
Lungs were perfused via the right ventricle with 5 ml of
saline until the tissue blanched completely. The trachea was then
cannulated with plastic tubing, and the lungs were lavaged with 10
successive 1-ml washes with PBS containing 0.5 mM EDTA (Sigma) as
described previously (26, 31). Lungs were excised at the medial pleural
surface, carefully excluding visible lymphoid tissue, and placed in PBS
on ice. BAL cells were washed once, resuspended in PBS, and kept on ice
until use. Lung tissue was minced finely and incubated with agitation
for 1 h at 37°C in PBS containing 175 U/ml collagenase IA
(Sigma) and 0.01% DNase type 1 (Sigma) in PBS (26, 31). After washing
twice in PBS, tissue particulates were allowed to settle out, and the
resulting single cell suspension was used for assay. Total cell counts
were performed with a hemocytometer. In some experiments, the
expression of adhesion receptors was measured on labeled cells
recovered from the lung using two-color flow cytometry. The samples
were washed, fixed with 2% paraformaldehyde, and stored at 4°C until
analysis by flow cytometry (FACScan, Becton Dickinson, Mountain View,
CA). Analysis was conducted within 48 h of cell collection. For
each sample, 100,000 events were collected and analyzed using the
WinList analysis program (Verity, Topsham, ME). The absolute number of
labeled cells per lung was calculated by multiplying the percentage of
CMFDA- or CMTMR-labeled cells by the total leukocyte count in a given
sample.
Staining for selectin ligand expression and cell surface markers
Leukocytes were washed and resuspended at 5 million cells/ml in
DMEM containing 0.1% BSA (Sigma) and 0.1% sodium azide (staining
buffer). The entire assay was conducted at 5°C. One
hundred-microliter aliquots of the cell suspensions were placed in a
96-well round-bottom plate (Corning, Corning, NY). After washing and
centrifuging once, the cells were vortexed and resuspended in 50 µl
of cell culture supernatant containing either murine selectin/human IgM
or murine CD45/human IgM chimeric protein (34). The chimera culture
supernatants contained 0.1 to 1.0 µg/ml of the chimeras. EDTA (10
mM) was added to some wells to prevent specific binding of the
selectins to their carbohydrate ligands. After 30-min incubation at
5°C, the cells were washed once with 150 µl of staining buffer and
resuspended in 50 µl of biotinylated goat anti-human IgM
secondary Ab (Zymed, South San Francisco, CA). The cells were incubated
for 30 min, washed once as described above, and resuspended in 50 µl
of a streptavidin/phycoerythrin conjugate (PharMingen, San Diego, CA).
In some assays, this final step was conducted in the presence of 5
µg/ml FITC-labeled anti-mouse CD4 or CD8 (PharMingen). After a
final wash to remove unbound reagents, the cells were resuspended in
fixative (2% paraformaldehyde in PBS containing 1 g/l
CaCl2 and MgCl2) and stored at 5°C. Staining
for other cell surface markers was conducted in essentially the same
manner using the commercial Abs listed in Table I
.
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Statistical analyses
Experiments with multiple treatment groups were analyzed by one-way analysis of variance followed by Student-Newman-Keuls post-hoc t test. Assays with two treatment groups were analyzed by unpaired or paired Student’s t test, as applicable. In all data analyses, p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine splenic lymphocytes were expanded for 5 days ex vivo using
plate-immobilized anti-CD3 followed by exogenous IL-2 stimulation.
Previous studies (7) showed that serum-free medium increased the
synthesis of selectin ligands during the ex vivo expansion of human T
cells. Murine T cells did not grow well under these conditions;
however, gradually reducing the amount of FCS in the murine cultures
enhanced selectin ligand synthesis while maintaining viability (Table II). The culture conditions resulted in
expansion of the CD8 subset primarily with the CD4:8 ratios varying
from 0.2 to 1 at the time of infusion (data not shown). The cultured
cells showed unimodal distributions of the 4 integrins,
ß2 integrins, CD11a, CD25 (IL-2R), and CD44 (Pgp-1).
CD62L was down-regulated on most cells, whereas selectin ligands were
up-regulated on 20 to 50% of the population. Both CD8 and CD4 subsets
expressed selectin ligands after culture; however, the prevalence and
density of ligands were generally highest on the CD8 subset (data not
shown). Therefore, the culture conditions induced expression of
adhesion receptors capable of interacting with the vascular adhesion
molecules detected on the pulmonary vasculature following SRBC
challenge (selectins and VCAM-1) (26). Consequently, the population was
suitable for determining whether these vascular adhesion receptors
participated in the recruitment of circulating T lymphoblasts.
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The prevalence of labeled cells in the BAL of SRBC-challenged lungs was
greater than that in unchallenged lungs both 4 and 24 h after
infusion; however, the increase was most significant at the 24 h
point (Fig. 1A). In contrast, the
prevalence of labeled cells in the minced and enzymatically digested
lung samples was different at the 24 h point only (Fig. 1B). Furthermore, the prevalence of labeled cells in the
enzymatically digested normal lungs fell 90% between the 4 and 24
h points, suggesting transient retention of the infused cells. It
should be noted that 7 to 10 times more leukocytes (7 x
105 vs 4–10 x 106) were recovered
from the BAL and enzymatically digested tissues of the SRBC-challenged
lungs than from these samples in unchallenged lungs (data not shown).
Consequently, the absolute number of fluorescently labeled lymphoblasts
in the inflamed lungs greatly exceeded the number in normal lungs
whether the trafficking study was stopped after 4 or 24 h.
However, the accumulation of T lymphoblasts was most specific and
significant after 24 h in situ; therefore, subsequent trafficking
studies used this time point.
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The flux of labeled T lymphoblasts parallels the accumulation of host T lymphocytes
The flux of labeled T lymphoblasts was compared with the
accumulation of host T lymphocytes in inflamed lung over a 7-day period
(Fig. 2). Groups of primed mice were IT
challenged on various days, and then all mice were infused on the same
day with labeled cells from a single blast population. The numbers of
labeled cells in the lungs were assessed 24 h later. The flux of
labeled T lymphoblasts during each 24-h period mirrored the
accumulation of host T lymphocytes during the same interval. Labeled T
lymphoblasts were detected in very low numbers in both the BAL and the
lung mince of unchallenged lungs 24 h after infusion (day 0
post-IT). Trafficking into both compartments increased steadily
following IT SRBC challenge, peaking at 20-fold above that in the
unchallenged lung on days 3 to 4 post-IT (p <
0.01 vs day 0). Specifically, both the trafficking of labeled cells and
the accumulation of endogenous T cells were lowest on day 1 post-IT.
These values rose in unison to a peak on days 3 to 4 post-IT and showed
a downward trend by the 7 day point.
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4 integrins participate in T lymphoblast recruitment
Labeled T lymphoblasts were preincubated with either the PS/2 mAb
(36) or pooled rat IgG before infusion into SRBC-challenged mice (Fig. 3). This and all subsequent trafficking
studies in the IT SRBC challenged mice were conducted during the period
of peak lymphocyte recruitment into the lungs (see Fig. 2
).
Specifically, a bolus of labeled cells was administered i.v. 72 h
post-IT (day 3), and the lungs were harvested 24 h later (day 4).
Blockade of the 4 integrins resulted in an 50%
reduction (p < 0.05) in the ability of labeled
cells to traffick into both the pulmonary airspace (Fig. 3A)
and interstitial tissues (Fig. 3B) compared with that of
cells treated with the control Ab. Analysis of the labeled T
lymphoblasts recovered from BAL showed that recruitments of the CD4 and
CD8 subsets were equally affected (p < 0.03 vs
IgG control; data not shown).
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). Hamann et al. also found that PS/2
inhibited 4 integrin-dependent trafficking exclusively.
These investigators reported that the Ab blocked the
4/ß7-mediated trafficking of lymphocytes
into Peyer’s patches and mesenteric nodes, but did not inhibit
lymphocyte entry into the peripheral lymph nodes or visceral organs of
normal mice (35). Consequently, the current findings indicate that
4 integrins participate in the SRBC-induced recruitment
of T lymphoblasts into the lung but cannot account for all trafficking
activity.
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The relationship between selectin ligand expression on the infused
T lymphoblasts and trafficking into SRBC-challenged lungs is shown in
Figure 4. The labeled T lymphoblasts
recovered from both the BAL and, to a lesser extent, the lung mince
expressed selectin ligands in a higher percentage of cells than the
infused population. This was most apparent when T lymphoblasts were
expanded in medium containing 10% FCS. Under these conditions, T
lymphoblasts with P-selectin ligand accounted for <3% of the infused
cells but >35% of the labeled cells recruited into the BAL on day 4
post-IT (Fig. 4A). Similarly, T lymphoblasts expressing
E-selectin ligands made up 2% of the input population, but 7 to
10% of the cells recovered from BAL (Fig. 4B). The
percentage of labeled cells with P-selectin ligands was consistently
higher in BAL than in the enzymatically digested lung mince
preparations. However, labeled cells recovered by maceration of lung
tissue, rather than by enzymatic digestion, expressed selectin ligands
at levels similar to those in the labeled cells in the BAL (data not
shown). Unfortunately, this method of tissue processing resulted in low
cell yields and viability; therefore, it was not suitable for
quantitative studies. Nonetheless, it appears that the difference in
the number of P-selectin ligands on lymphocytes recovered from the BAL
and the enzymatically treated lung mince results in part from
degradation of the ligand during processing.
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These findings suggested that selectin ligands on the infused T
lymphoblasts enhanced trafficking into the lung. Alternatively,
selectin ligand expression might increase on the labeled lymphoblasts
following entry into the lung. These alternative hypotheses were tested
using T lymphoblasts derived from FucT-VII KO mice. The FucT-VII locus
is required for synthesis of ligands for P- and E-selectin (34). Flow
cytometry assays confirmed that cultured splenic T lymphoblasts from
the FucT-VII KO mice had virtually no binding sites for E- or
P-selectin on their surfaces (Table IV).
In contrast, 30% of the cultured T lymphoblasts from the
genetically matched, wild-type control animals expressed selectin
ligands. The blast cells from the FucT-VII KO and the control mice
showed equivalent levels of other relevant adhesion receptors,
including L-selectin, 4 integrins, and ß2
integrins. Furthermore, the two blast populations did not differ
measurably in their chemotactic responses to zymosan-activated serum or
in their growth rates in vitro (data not shown).
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compares the trafficking of T
lymphoblasts from the FucT-VII KO and its wild-type control mice in
SRBC-challenged C57BL/6J mice. The FucT-VII-deficient blast cells were
devoid of selectin ligands (Fig. 5A) and showed a 64%
reduction in trafficking into BAL relative to control T lymphoblasts
(Fig. 5B). An additional assay was performed in which the
control and FucT-VII KO blast populations were labeled red and green,
respectively, using the fluorochromes CMTMR and CMFDA. The labeled cell
populations were mixed in equal parts and infused into IT-challenged
C57BL/6J hosts. Two-color flow cytometry was used to measure the number
of control and FucT-VII-deficient blast cells in the lung 24 h
after infusion. This paired experimental design revealed highly
significant reductions in the trafficking of FucT-VII-deficient blasts
into both the lung airspace (Fig. 6A; p = 0.002) and
interstitial tissues (Fig. 6B; p < 0.001).
The findings imply that synthesis of selectin ligands enhances T
lymphoblast trafficking into SRBC-challenged lungs.
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4 integrins make independent
contributions to T lymphoblast recruitment
The final studies examined the impact of 4 integrin
blockade on the trafficking of T lymphoblasts derived from FucT-VII KO
mice and their wild-type controls (Fig. 7).
Blocking the 4 integrins on the control blasts decreased
recruitment into BAL by 50% compared with that of cells treated
with polyclonal IgG. Furthermore, the recruitment of IgG-treated
FucT-VII KO blasts was reduced by >60% compared with that of
IgG-treated wild-type blasts (p < 0.05).
Finally, blockade of the 4 integrins on the FucT-VII KO
blasts reduced recruitment by another 60% relative to that of the
IgG-treated FucT-VII KO blasts. Consequently, the combined suppression
of selectin ligand synthesis and 4 integrin function
decreases SRBC-induced T lymphoblast recruitment into BAL by >85% in
short term trafficking studies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrins mediate inflammation-dependent T lymphoblast
trafficking into the lung. Short term trafficking assays demonstrated
that optimal recruitment of cultured T lymphoblasts into
SRBC-challenged lungs required both selectin ligands and
4 integrins. Blocking either 4 integrin
function or selectin ligand synthesis alone resulted in an 45 to
65% reduction in the trafficking of cultured T lymphoblasts into the
lung. However, concurrent suppression of selectin ligand synthesis and
4 integrin function on the blasts reduced trafficking
more than interdiction of either receptor family alone (>85%).
Consequently, the 4 integrins both augment
selectin-dependent trafficking and initiate recruitment on their own. Short term trafficking assays provide the best measure of cell entry from the circulation into tissue. Previous attempts to measure the flux into inflamed lungs are difficult to interpret due to the high, nonspecific retention of infused cells in normal lungs (15). This study demonstrates that polyclonal T lymphoblasts, activated on plate-immobilized CD3 and expanded in low dose IL-2, show inflammation-specific trafficking into the lung. Nonspecific retention of these cells occurred in normal lungs up to 4 h after infusion. However, this retention was transient, since the number of labeled cells recovered from normal lungs fell by 90% between 4 and 24 h. Consequently, 24 h after infusion, significant accumulation of labeled cells occurred in SRBC-primed and IT-challenged animals only. The earliest and most significant accumulations occurred in the BAL. This may reflect the fact that circulating labeled cells must cross at least one vascular bed to enter the airspace. In contrast, cells recovered from enzymatically digested lung, even after exhaustive lavage of the vasculature, are likely to contain cells trapped in the extensive pulmonary capillary bed. These cells may not express the adhesion receptor profile needed for entry into tissues. Therefore, accumulation of labeled cells 24 h after infusion, particularly in the BAL, provides the best quantitative measure of inflammation-dependent trafficking into the lung.
The trafficking of the selectin ligand-deficient blasts into both airspace and interstitium was reduced 50 to 65% relative to that of selectin ligand-positive, wild-type (WT) blasts, implying that 50% or more of the WT cells used selectin ligands for entry. However, less than half of the labeled WT blasts recovered from lung, particularly from enzymatically digested tissue, expressed selectin ligands. Several factors contribute to this apparent discrepancy. The low affinity and rapid off time for selectin:selectin ligand interactions results in the loss of Ig chimera from specific binding sites during the staining procedure. This conclusion is based on the observation that selectin chimeras detach from the cell surface more rapidly than mAbs after washing (R. N. Knibbs and L. M. Stoolman, unpublished observations). Furthermore, the enzymatic treatment used to harvest lymphocytes from the lung interstitium partially destroys Ig chimera binding activity. Lymphocytes released by maceration of lung tissue showed selectin chimera reactivity similar to that of lymphocytes in BAL (data not shown). Maceration was not used routinely due to inconsistent lymphocyte recoveries. However, the experiment provides additional evidence that immunofluorescent detection of selectin chimera attachment probably underestimates the percentage of selectin ligand-positive lymphocytes in inflamed lung tissue. Consequently, the trafficking studies with the selectin ligand-deficient T lymphoblasts provide the best estimate of selectin activity in this model.
A previous study in this model revealed a surge in selectin ligand-positive T cells in the circulation shortly before the period of maximal recruitment into the lung. Furthermore, 20 to 50% of the host cells recovered from the lung (both CD4 and CD8 subsets) expressed selectin ligands, implying that these receptors contributed to recruitment. The accumulation of cultured, selectin ligand-positive T lymphoblasts in SRBC-challenged lung and the marked reduction in recruitment of cultured, selectin ligand-deficient T lymphoblasts support this hypothesis. As noted previously, the culture conditions promoted the growth of CD8 lymphoblasts primarily. Since the CD4 and CD8 subsets responded differently to the culture conditions, no attempt was made to compare selectin usage by the two subsets. Furthermore, the current study did not directly evaluate the impact of adhesion receptor blockade on host cell accumulation. These important questions are currently under investigation in selectin-deficient animals responding to SRBC challenge. None the less, the short term trafficking assays provide clear and direct evidence that selectins expressed by the pulmonary vasculature augment recruitment of selectin ligand-positive T lymphoblasts in the circulation.
The current study does not separate the contributions of P- and E-selectin to lymphocyte recruitment into the lung. The literature indicates that these receptors work both independently and together to initiate lymphocyte recruitment in vivo. DeSantis and colleagues report that deletion of P-selectin alone reduces T cell accumulation in the BAL following OVA challenge (25). In contrast, E- and P-selectin appear redundant in delayed-type hypersensitivity lesions in the skin, since both must be blocked before either host leukocyte accumulation or cultured Th1-lymphoblast trafficking is significantly reduced (14, 15).
In the SRBC model, the contributions of the selectins and VCAM-1 to recruitment are likely to change as the lung lesions evolve. Previous studies found that E-selectin message in lung extracts dropped to basal levels before P-selectin message (day 4 vs day 7 post-IT) (26). In addition, the intensity of vascular E-selectin staining fell more rapidly than staining for either P-selectin or VCAM-1 (26). Consequently, vascular P-selectin and VCAM-1 are more widely distributed and expressed at higher levels in the lung than E-selectin during peak lymphoblast recruitment (2–4 days post-IT). The trafficking studies reported here also suggest a greater role for P- than E-selectin during this period, since more labeled T lymphoblasts recovered from the lung expressed P-selectin ligands than E-selectin ligands. On-going studies in selectin-deficient animals will address this issue in more detail.
The trafficking of labeled, infused T lymphoblasts paralleled the accumulation of endogenous T lymphocytes during the pulmonary response to SRBC. The maximal flux of infused cells and the maximal accumulation of endogenous T cells occurred on days 2 to 5 post-IT. This observation is consistent with the view that recruitment from the circulation, rather than local proliferation, accounts for most of the cells recovered from the lung during the immune response. The finding that <5% of the T lymphocytes recovered from the lung after IT SRBC challenge incorporated bromodeoxyuridine into nuclear DNA provides further support for this hypothesis (37).
Previous studies in this model showed that the endothelial selectins
and VCAM-1 peaked on the pulmonary vasculature on day 1 post-IT (26).
The endothelial CAMs remained elevated for 4 to 7 days, but never
exceeded the level observed on day 1. However, the current study found
that the maximal flux of infused T lymphoblasts occurred during days 2
to 4 post-IT. Thus, the endothelial selectins and VCAM-1 are required
for T cell entry at the time of maximal recruitment, but their levels
peak in the endothelium 2 days earlier. Since the same batch of
labeled T lymphoblasts was used for measurements at each time point,
the gradual rise in T lymphoblast trafficking into the lung and its
subsequent fall must reflect local changes during the immune response.
Up-regulation of chemokines that increase adhesion, arrest, and
subset-specific transmigration in vitro (38, 39, 40, 41, 42) are potential
contributing factors. These finding confirm that lymphocyte recruitment
in vivo involves sequential, independently regulated steps.
In conclusion, the current study provides direct evidence that selectin
ligands enhance T lymphoblast recruitment into the lung following
airway challenge with particulate Ag and reveals a selectin-independent
recruitment pathway involving the 4 integrins. Together
these adhesion receptors control >85% of the exogenous T lymphoblast
traffick into the BAL during the period of peak recruitment. Whether
the receptors mediate recruitment of discrete subsets of committed T
cells, as suggested for Th1 and Th2 entry into cutaneous delayed-type
hypersensitivity lesions (15, 16) or represent redundant mechanisms for
the initial recruitment of uncommitted precursors remains to be
determined. In either event, blockade of multiple receptors may be
needed to optimally inhibit pathologic immune responses in the lung.
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Footnotes |
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2 The substance of this report was presented orally and in abstract form at Keystone Conference on Molecular Mechanisms of Leukocyte Trafficking, March 22–28, 1998, and Experimental Biology 1998 Immunology Block Symposium: Selectins and Endothelial Binding, April 18–22.
3 Address correspondence and reprint requests to Dr. Lloyd M. Stoolman, Department of Pathology, University of Michigan, Room 4224, Medical Sciences Building 1, 1301 Catherine Rd., Ann Arbor, MI 48109-0602. E-mail address:
4 Abbreviations used in this paper: IT, intratracheal; FucT-VII, (1,3)fucosyltransferase VII; FucT-VII KO, fucosyltransferase VII gene-deleted mice; BAL, bronchoalveolar lavage; CMFDA, 5-chloromethylfluorescein diacetate; CMTMR, 5-(and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine.
Received for publication January 29, 1998. Accepted for publication June 22, 1998.
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inducible protein-10, stimulate transendothelial chemotaxis of T lymphocytes. Eur. J. Immunol. 25:3482.[Medline]
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) or SRBC-primed mice 2 days after IT challenge with
SRBC (
). Cells were collected 4 and 24 h after infusion by BAL
(A) or enzymatic digestion of minced lung tissues
(B). The percentage of labeled T lymphoblasts in each
sample was identified by flow cytometry. The data are expressed as the
mean percentage of labeled cells per mouse lung ± SEM based on
three to five mice per group. At the 24 h point, the percentage of
labeled cells in SRBC-challenged lungs was significantly different from
that in normal lungs (p < 0.05, by analysis of
variance/Student-Newman-Keuls test).
) were assayed for expression of selectin ligands using a
phycoerythrin-conjugated secondary to detect the chimeras. The data are
expressed as the mean percentage of positive cells ± SEM. *,
p < 0.01 (compared with the initial population by
analysis of variance/Studen-Newman-Keuls test based on four
replicates).
). p < 0.05 (by analysis of
variance/Student-Newman-Keuls test based on four mice per group).