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* Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,
Department of Pathology,
Comprehensive Cancer Center, and
Graduate Program in Immunology, University of Michigan Health Care System, Ann Arbor, MI 48109;
¶ Department of Genomics and Pathobiology, University of Alabama, Birmingham, AL 35294; and
|| Pulmonary and Critical Care Medicine Section, Medical Service, Department of Veterans Affairs Care System, Ann Arbor, MI 48105
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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60% early, but equivalent to wt
levels later. Surprisingly, many 
T cells were found in lungs and
blood of E-P- mice
but were undetectable in the lungs and blood of wt mice. Absolute
numbers of peripheral blood CD4, CD8, and B lymphocytes in
E-P- mice equaled or
exceeded the levels in wt mice, particularly after challenge.
Trafficking studies using 
T lymphoblasts confirmed that the
recruitment of circulating cells after challenge was markedly reduced
in E-P- mice.
Furthermore, Ag priming occurred normally in both the
selectin-deficient and wt mice, because primed lymphocytes from both
groups transferred Ag sensitivity into naive wt mice. Lung production
of mRNA for six CC and two CXC chemokines after challenge was
equivalent by RT-PCR analysis in wt and
E-P- mice. Therefore,
reduced lung accumulation of T cells and B cells in
E-P- mice did not
result from reduced delivery of circulating lymphocytes to the lungs,
unsuccessful Ag priming, or defective pulmonary chemokine production.
Selectin-dependent lymphocyte recruitment into the lungs following
i.t.-SRBC challenge is subset specific and time
dependent. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Such in vivo analysis is particularly important for the lungs, which possess a unique dual vascular supply that impacts directly on leukocyte recruitment. The pulmonary circulation carries the entire output of the right ventricle to the extensive capillary network investing the alveoli. The bronchial circulation branches off the aorta and supplies oxygenated blood directly to the peribronchial and submucosal tissues (2). Depending on the inciting agent and mode of entry, the pulmonary inflammatory response may be alveolar, bronchial and peribronchial, or a combination of both. Recruitment into alveoli generally occurs across capillaries where changes in leukocyte distensibility can arrest cells directly (3, 4, 5, 6), although the independence of this process from adhesion molecules has been contested recently (7, 8). In contrast, recruitment into the bronchial wall and peribronchial tissues most likely involves larger vessels, especially intact postcapillary venules (6, 9, 10, 11, 12), and hence may use the tethering receptors that mediate immune reactions at other sites with a similar microvascular organization.
To test this hypothesis, we chose an established model of CD4-dependent
alveolar and peribronchial lymphocyte recruitment in mice sensitized to
SRBC and then challenged with intratracheal (i.t.) SRBC
(13, 14, 15, 16, 17, 18). Initial studies from our group and others showed
that CD62E, CD62P, and VCAM are expressed on the lung microvasculature
in sensitized mice after challenge with i.t. SRBC (19, 20). All three receptors are expressed throughout the period of
initial lymphocyte influx (days 2–4). However, E-selectin expression
falls to baseline more rapidly than either P-selectin or VCAM. In
addition, i.t. SRBC challenge transiently increased the percentage of
circulating T cells expressing selectin ligands and resulted in the
accumulation of selectin ligand-positive T cells in the lung. Finally,
trafficking studies with cultured T lymphoblasts derived from
fucosyltransferase VII-deficient animals suggested that selectin
ligands and 4 integrins mediated independent
pathways of T cell recruitment into i.t. SRBC-challenged lungs
(16, 21). The current study used the SRBC model and mice
with gene-targeted deletions in both P- and E-selectins to directly
evaluate the contributions of the selectins to alveolar and
peribronchial lymphocyte accumulation. Substantial differences were
observed in the accumulation of CD4+ and
CD8+ T cells, B cells, and T cells in the
airways and interstitial tissue of the lung. The findings indicate that
selectins influence the accumulation of both T cells and B cells
but suggest that the contribution of selectins is subset and time
dependent.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following epitopes were assessed by three-color flow
cytometry analyses using directly conjugated mAbs (BD PharMingen, San
Diego, CA) (clone, isotype, and fluorochrome are indicated in
parentheses): CD4 (RM4-4; rat IgG2a; CyChrome), CD8 (53-6.72; rat
IgG2a; FITC), CD19 (1D3; rat IgG2a; PE), CD45 (30-F-11; rat IgG2b;
CyChrome), -TCR (GL3; hamster IgG2; CyChrome), Gr-1 (RB6-8C5; rat
IgG2b; PE), pan-NK cell (DX-5; rat IgM; FITC), CD14 (rmC5-3; rat IgG1;
FITC), and Mac-3 (M3/84; rat IgG1; FITC). The fluorochromes were chosen
based on the fluorescence intensities for each epitope and the level of
background observed with isotype-matched control reagents.
Mice
Mice containing null mutations for both E-selectin and P-selectin (E-P- mice) were generated by gene targeting in 129/Sv embryonic stem cells, as previously described (22). E-/-P- mice used in this study had been backcrossed to C57BL/6 mice for five generations. Pathogen-free female C57BL/6 mice for use as controls were purchased at 7–8 wk from Charles River Laboratory (Wilmington, MA). Although early reports of E-P- mice highlighted their frequent ulcerative skin lesion (22, 23), such lesions have been uncommon in our colony; no mice with visible skin lesions were used in this study. Mice were housed in the Animal Care Facility at the Ann Arbor VA Medical Center (Ann Arbor, MI), which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. This study complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHEW publication no. (NIH) 80-23) and followed a protocol approved by the Animal Care Committee of the local Institutional Review Board. Mice were fed standard animal chow (Rodent Lab Chow 5001; Purina, St. Louis, MO) and chlorinated tap water ad libitum. Mice were used at 8–14 wk of age.
Induction of pulmonary immune response
SRBC (sheep 4151; Colorado Serum, Denver, CO) were washed three times in 7 ml normal saline before use. Mice were Ag primed by i.p. injection with 1 x 108 SRBC in 0.5 ml normal saline. Beginning 2–4 wk later, mice were challenged i.t. with 5 x 108 SRBC in 50 µl normal saline as previously described (19).
Sample collection
At 4 or 7 days after i.t. Ag challenge, mice were deeply anesthetized with pentobarbital (80 mg/kg i.p.) and killed humanely by exsanguination and induction of bilateral pneumothoraces. Peripheral blood was collected by cardiac puncture. The peripheral blood specimens were centrifuged through Lympholyte-M (Accurate Chemical and Scientific, Westbury, NY), lysed in a UNOPETTE (BD Biosciences, San Jose, CA), washed, and resuspended in DMEM containing 0.1% azide and 0.1% BSA (DMEM+). Lungs were then processed in one of three ways. For cellular analysis, lungs were initially perfused in situ using normal saline to clear the pulmonary vasculature, and then the inflammatory cells in the airways and alveolar spaces were collected by bronchoalveolar lavage (BAL) (3). Next, the lavaged lungs were dissected free of extrapleural lymphatic tissue, and to release the interstitial inflammatory cells for further analysis, lungs were finely minced and enzymatically digested (collagenase plus DNase) (24). Aliquots of both BAL and lung mince preparations were washed and resuspended in DMEM+ before further analysis. Second, for histologic analysis, some lungs were inflated (1 ml) and immersed in 10% neutral buffered formalin for 18 to 24 h. Parasagittal slices of the fixed lungs were embedded in paraffin, sectioned at 5 µm thickness, and stained with H&E or Masson’s trichrome stain. Finally, the lungs for mRNA and protein analysis were perfused with normal saline; separated from the mainstem bronchi at the medial pleural surface, taking care to exclude extrapleural lymphatic tissue; snap frozen in liquid nitrogen; and stored at -70°C until use.
Flow cytometry analysis and determination of absolute lymphocyte counts
A minimum of three replicates was performed for each epitope. For each specimen, up to 1 x 105 events were collected without color compensation on a Beckman-Coulter (Fullerton, CA) EXCELL flow cytometer. Color compensation and analysis were conducted offline using the WinList analysis program (Verity Software House, Topsam, ME). Light scatter parameters were used to gate on the region containing the majority of lymphocytes in each specimen. The percentage of cells expressing each epitope was determined from one-parameter histograms. The threshold for positive events was based on isotype-matched control reagents. The percentage of positive events remaining in the control tube was subtracted from the percentage of positive events with each leukocyte-specific Ab to determine the prevalence (percentage) for the various subsets.
Preliminary studies indicated that 95–100% of the
CD45+ cells in the light scatter gate expressed
the CD4, CD8, CD19, TCR, or Gr-1 epitopes. Gr-1 (also known as
Ly-6G) is expressed by murine neutrophils and eosinophils;
consequently, the positive cells in the lymphocyte gate using this flow
cytometer are most likely degranulated granulocytes. These cells
accounted for a variable but significant percentage of the events in
the gated region (up to 25%), particularly in the lung specimens.
Therefore, the lymphocyte subset percentages used in the following
calculations were corrected for the number of
Gr-1+ cells contaminating the analysis gate. This
type of adjustment was not performed in previous studies using this
model system (13, 14, 16, 18, 19, 24, 25), because the
light scatter characteristics of the flow cytometers used previously
did not result in such contamination of the lymphocyte gates
(24). The means of the replicate determinations for each
subset were then used to calculate the absolute number of cells as
detailed below. The absolute lymphocyte count in each specimen was
calculated from the white blood cell count and morphologic differential
count. The white blood cell counts were performed with a hemocytometer
as previously described (26). Morphologic differential
counts of the granulocytes, monocytes/macrophages, and lymphocytes in
each specimen were performed on stained filter preparations as
previously described (24). The number of lymphocytes in
each subset was then calculated from the absolute lymphocyte counts
(white blood cell count x percent of lymphocytes from the
morphologic differentials), and the lymphocyte subset percentages as
measured by flow cytometry. The results for each animal in a cohort
were then used for the statistical analyses reported in the figures and
tables.
Recruitment assay
Lymph node T cells from normal wild-type (wt) mice were activated using immobilized anti-CD3 mAb, and their numbers were expanded in IL-2 to produce T lymphoblasts, as previously described (16). After 5 days of expansion, T lymphoblasts were labeled with 5-chloromethylfluorescein diacetate (CMFDA) and transferred by tail vein injection into primed recipients, consisting of either wt or E-P- mice, which had been i.t. Ag challenged 4 or 7 days earlier.
Adoptive transfer assay
To confirm the capacity of T cells of E-P- mice to be Ag primed in vivo, in one experiment splenocytes from E-P- donor mice or wt donor mice that had been primed with 1 x 108 SRBC 6 days previously by the i.p. route were transferred to unprimed wt recipient (2 x 107 splenocytes per recipient mouse in 0.2 ml PBS by the i.v. route). Subsequent Ag-driven accumulation of lung lymphocytes was assayed in BAL 4 days after i.t. SRBC challenge.
Isolation of RNA
Lungs were homogenized in 2 ml of TRIzol reagent (Life Technologies, Gaithersburg, MD), and RNA was isolated as described in the TRIzol protocol. RNA was quantitated spectrophotometrically. To remove genomic DNA, all RNA samples were treated with DNase (DNA-free method; Ambion, Austin, TX). The integrity of individual RNA samples was confirmed by electrophoresing aliquots on a 2% agarose gel containing 0.5 µg/ml ethidium bromide and observing 28S and 18S rRNA bands. RNA samples were stored at -70°C.
RT-PCR detection of cytokine mRNA
Isolated RNA was reverse-transcribed to DNA and amplified by PCR
as previously described in detail (17). The primer
sequences used are defined in Table I
.
The amplification scheme was an initial 5 min at 95°C, repeated
cycles of 15 s at 95°C, 20 s at 58°C, 30 s at
72°C, and a final extension period of 6 min at 72°C. Positive and
negative controls were included in each assay, and the authenticity of
reaction products was verified by Southern analysis using a
chemiluminescent detection system (ECL; Amersham Life Science, Little
Chalfont, U.K.). The amplified DNA was analyzed by electrophoresis on
1.5% agarose containing 0.5 µg/ml ethidium bromide. To quantitate
cDNA bands, the ethidium bromide-stained agarose gels were photographed
using Polaroid 667 film (Polaroid, Cambridge, MA), scanned using a Scan
Jet IIcx (Hewlett Packard, Palo Alto, CA), and analyzed on a Macintosh
PowerPC G3 computer (Apple Computer, Cupertino, CA) using the public
domain National Institutes of Health Image software (version 1.6;
available at http://rsb.info.nih.gov/nih-image/). Results are expressed
as a ratio of OD signal for a given cytokine to that for cyclophilin in
the same sample.
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Data were expressed as mean ± SEM. Statistical calculations were performed using Statview and SuperANOVA programs (Abacus Concepts, Berkeley, CA) on a Macintosh PowerPC G3 computer. An unpaired Student t test (for two samples) or ANOVA (for multiple comparisons) was used to evaluate continuous ratio scale data with post hoc analysis by the Tukey-Kramer test (27). Percentage data were arcsine transformed before analysis to convert them from a binomial to a normal distribution using tables in the textbook of Zar (27). Results of RT-PCR experiments were analyzed by unpaired nonparametric Mann-Whitney test. Significant differences were defined as p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lung lymphocyte numbers did not differ significantly between wt
control mice and E-P-
mice before i.t. challenge, in either BAL (n = 6 mice
per group; p = 0.08, unpaired t test) or
minced lung preparations (p = 0.2) (Fig. 1). Within groups of either wt mice or
E-P- mice, there was no
statistical difference in total lymphocyte numbers between untreated
mice and mice receiving i.p. priming only, and these two groups were
pooled to derive day 0 numbers.
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). Total lung lymphocyte numbers differed
significantly between the two groups of mice at day 4 after i.t.
challenge both in the BAL (63% reduction relative to wt mice) and in
the minced lung preparation (44% reduction). Lung lymphocyte numbers
were also lower in the minced lung preparation at day 7 (34%
reduction), although this difference was not statistically significant
(p = 0.11). By contrast, total numbers of BAL
lymphocytes were essentially identical in the two groups of mice at day
7 after challenge (p = 0.8). Accumulation of
mononuclear phagocytes did not differ significantly between the two
groups of mice in either BAL or minced lung preparation at any time
point (data not shown).
Detailed analysis of lung lymphocyte subset distribution revealed the
full extent of the abnormal lymphocyte accumulation in the
selectin-deficient mice. Total leukocyte counts, leukocyte
differentials (200-cell count of stained filter preparation), and
lymphocyte subset differentials (flow cytometry) were performed on
leukocytes recovered from the BAL, interstitial mince, and peripheral
blood. The absolute counts for CD4, CD8, CD19, and T cell
subsets were then calculated as described in Materials and
Methods. CD4+ T cells,
CD8+ T cells, and B cells were reduced in both
the BAL and the lung interstitium of
E-P- mice throughout the
period of maximal lymphocyte recruitment (Fig. 2). The magnitude of the decrease was
subset dependent: accumulation of the CD4+ subset
was reduced by 2- to 3-fold, whereas the CD8+
subset and B cells fell by 8-fold to >10-fold at the 4-day time point.
The CD8+ subset and B cells remained low in both
compartments at the 7-day time point. In contrast, the
CD4+ subset remained low in the interstitial
compartment but returned to wt levels in the BAL.
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T cells from both lung compartments in the
E-P- mice after i.t.-SRBC
challenge (Fig. 2). Indeed, T cells accounted for 40% of all
lymphocytes recovered from the BAL of
E-P- mice at both day 4
and day 7. This subset constituted a much smaller percentage of the
lymphocytes recovered from the interstitium of the
E-P- mice, but the level
remained markedly higher than that obtained from wt animals. To further
investigate the significance of pulmonary T cell accumulation
after challenge, we compared numbers of lymphocytes in BAL and lung
mince in wt mice and E-P-
mice, both untreated and after i.p. priming only. There were no
differences between the two groups of mice from either treatment state
or in either lung compartment in numbers of CD4, CD8, or CD19 cells
(data not shown). In comparing the untreated with the primed state
within a given group of mice, slight (
2-fold) differences, which were
for the most part not statistically significant, were noted for each of
these three lymphocyte subsets in both lung compartment. By contrast,
T cells were significantly increased in numbers in both lung
compartments of untreated
E-P- mice relative to wt
mice (Table II). Similar disparity
between the two groups of mice was seen after i.p. priming in BAL, but
no significant difference was found between the two groups in numbers
of T cells recovered from lung minces after i.p. priming (Table II). Thus, E-P- mice had
baseline increases in numbers of lung T cells that further
increased markedly after i.t. challenge (in
E-P- mice, BAL T
cells, 1.55 ± 0.15 x 105 on day 4 and
2.63 ± 0.32 x 105 on day 7; in lung
mince T cells, 4.13 ± 0.04 x
105 on day 4 and 2.86 ± 0.05 x
105 on day 7).
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Before i.t. SRBC challenge, granulocyte numbers in
E-P- mice were elevated
3-fold (BAL) and >7-fold (in lung mince) relative to wt control mice
(Fig. 3). As with lymphocyte numbers,
within groups of either wt mice or
E-P- mice, there was no
statistical difference in granulocyte numbers between untreated mice
and mice receiving i.p. priming only, and these two groups were pooled
to derive day 0 numbers.
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A). However, the
number of granulocytes in the minced lung preparations of the
E-P- mice was markedly
greater than in wt mice at both the 4-day (p <
0.001) and 7-day time points (p < 0.001) (Fig. 3B). As in most previous studies in this model system, we
did not separate neutrophils and eosinophils in this enumeration, given
that distinguishing the two types of granulocytes in the mouse is
problematic. Caution should be exerted in interpreting these data.
Although the pulmonary vasculature of both groups of mice was perfused
in an identical manner,
E-P- mice have previously
been reported to have a marked peripheral blood leukocytosis
(28). Thus, we cannot exclude the possibility that
granulocyte numbers recovered by mincing lung tissue of
E-P- mice were spuriously
increased by incomplete removal of cells trapped in pulmonary
capillaries. Supporting this possibility, histologic analysis confirmed
identical overall anatomic distribution and magnitude of interstitial
inflammation in the two groups of mice (data not shown), with prominent
perivenous and peribronchoarterial accumulation of mononuclear cells
and granulocytes (9). Nevertheless, these data clearly
indicate that lung granulocyte accumulation after Ag challenge is not
impaired by absence of endothelial selectins. Peripheral blood analysis shows a leukocytosis in E-P- mice
To assess the delivery of circulating lymphocytes to the lung, PBL
counts in wt mice and
E-P- mice were compared.
In the absence of i.p. priming and of i.t. challenge, the numbers of
CD4+, CD8+, and
CD19+ cells were significantly higher in
E-P- mice than in wt mice
(Fig. 4A), and this difference
increased following SRBC sensitization and i.t. challenge. At the 7-day
time point, the absolute counts for these subsets were 2- to 4-fold
higher in E-P- mice than
in wt mice (Fig. 4B). T cells were below the level of
detection in both untreated wt mice and SRBC-challenged wt mice. In
contrast, T cells constituted 7% and 5% of the
circulating pool in untreated and i.t.-SRBC challenged
E-P- mice, respectively.
Thus, the decreased accumulation of CD4, CD8, and CD19 subsets in the
E-P- lungs did not result
from reduced numbers of circulating cells. In contrast, the increased
accumulation of T cells reflected, in part, a systemic expansion
of this subset in the
E-P- mice.
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Steady state lymphocyte counts in the lungs during a pulmonary
immune response reflect the dynamic balance between lymphocyte
recruitment and in situ proliferation on one hand vs lymphocyte
emigration and apoptosis on the other. To assess recruitment directly,
we next conducted short term trafficking assays using wt cultured T
lymphoblasts. T lymphoblasts were prepared from splenic mononuclear
cells under conditions that induced high levels of selectin-ligand
synthesis (16). The T lymphoblasts were labeled with the
fluorophore CMFDA and administered i.v. to syngeneic wt mice and
E-P- mice as previously
described (16) on either day 3 or day 6 after i.t. SRBC.
The overall level of trafficking into the BAL of wt mice was 5-fold
greater during the day 3–4 period than during the day 6–7 period, in
agreement with our previous findings (16). However, at
both time points, the number of fluorescent cells recovered from the
E-P- mice was at least
10-fold lower than the number recovered from wt mice (Fig. 5). Consequently, recruitment of wt T
lymphoblasts is significantly reduced in the absence of endothelial
selectins.
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Lung lymphocyte recruitment in the i.t.-SRBC model system is Ag
driven and requires that the same Ag be administered for both priming
and i.t. challenge (24). Therefore, reduced or absent
priming to SRBC Ags in the
E-P- mice could also
affect lymphocyte accumulation after i.t. challenge. To test this
possibility, lymphocytes harvested from the spleens of primed mice (wt
and E-P-) were adoptively
transferred into two groups of unprimed wt mice 3 days before i.t.
challenge with SRBCs. Subsequent lymphocyte recovery from the lungs of
these two groups of mice 4 days after i.t. challenge was virtually
identical, and both differed significantly from the modest levels
observed in control mice receiving i.t. SRBC challenge without previous
Ag priming (Fig. 6). Thus, wt and
E-P- mice showed equal
levels of priming after i.p. challenge with SRBC.
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Lymphocyte recruitment is dependent on both endothelial CAMs and
chemokines. Therefore, CC and CXC chemokine production was analyzed
using semiquantitative RT-PCR on whole lung RNA extracts. The steady
state mRNA levels for the CC chemokines monocyte chemoattractant
protein (MCP)-1 (CC chemokine ligand (CCL2) in the recently proposed
nomenclature (29)), macrophage-inflammatory protein
(MIP)-1 (CCL3), MIP-1 (CCL4), RANTES (CCL5), MCP-3 (CCL7), and
eotaxin (CCL11) after i.t. SRBC challenge were equivalent in the two
groups of mice (Fig. 7). Additionally,
lung mRNA production of the CXC chemokines MIP-2 (CXC
chemokine ligand (CXCL2)) and IFN-inducible protein 10 (IP10) (CXCL10)
was equivalent except for a modest decrease in IP10 mRNA in the
E-P- mice at the 7-day
time point. Consequently, both groups developed virtually identical,
robust, and broad spectrum chemokine responses to i.p.-SRBC challenge.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells fromthe lungs of
E-P- lungs was markedly
(>>10-fold) increased relative to wt controls, reflecting, in part, a
previously unrecognized systemic expansion of this subset in the
selectin-deficient animals. The reduced T cell trafficking from the
circulation into i.t.-challenged
E-P- lungs was confirmed
using a polyclonal population of cultured T lymphoblasts. Direct
trafficking studies with T cells are problematic in the mouse;
however, our data indicate that both constitutive and
inflammation-induced trafficking of this subset into the lung is not
invariably selectin dependent in the mouse. The attenuated lymphocyte
accumulation in the E-P-
mice did not result from decreased numbers of circulating lymphocytes
of any of the subsets, inadequate Ag priming, or a global deficiency in
chemokine production. These findings extend previous work from our group showing that i.t.-SRBC challenge of sensitized mice induced P- and E-selectin expression on the pulmonary vasculature, increased the number of circulating T cells expressing selectin-ligands, and recruited selectin ligand-positive circulating T cells into the lung (16, 19, 21). The current results are in agreement with our previous demonstration that lung lymphocyte recruitment is partially dependent on binding to endothelial cell selectins (16). That conclusion was based on analysis of in vitro activated T lymphoblasts derived from gene-targeted mice lacking fucosyltransferase VII (FucT VII-/-), the rate-limiting enzyme in the biosynthetic pathway necessary to produce functional leukocyte selectin ligands (30). Absence of ligands for L-selectin in the FucT VII-/- mice leads to defects in lymphocyte entry into lymph nodes and in priming (30). Therefore, it is not feasible to compare the two genetic defects more directly by analyzing lung accumulation of individual lymphocyte subsets in response to i.t. SRBC in sensitized FucT VII-/- mice themselves. The current results are also consistent with those of Pan et al. (31), who found that ganglioside analogs of the endothelial selectin ligand sialyl-Lewis X inhibited lung inflammation in a murine model of hypersensitivity pneumonitis in response to Saccharopolyspora rectivirgula. Selectin-mediated Th1 lymphocyte recruitment has been documented in cutaneous and peritoneal immunologic lesions as well (32, 33, 34). However, selectins were not required for lymphocyte recruitment during immunologic reactions in the colon (35), liver (36), and brain (37). Indeed, Bartholdy et al. (38) found that meningeal accumulation of CD8+ T cells and CD8-mediated viral clearance from multiple organs was identical in E-P- and wt mice lethally infected with lymphocyte choriomeningitis virus.
These seemingly disparate results may reflect the partial redundancy of adhesion receptors involved in lymphocyte recruitment and changes in receptor expression/utilization that occur as immunologic reactions progress. On T cells, at least six receptors or receptor families mediate tethering interactions and at least two additional families can support the arrest/transmigration of tethered cells (39). Furthermore, several studies document changes in adhesion receptor expression and utilization during pulmonary immune responses. In SRBC-primed mice, i.t. SRBC challenge was followed by transient expression of E-selectin and prolonged expression of P-selectin and VCAM on the microvasculature of the lung (19). In OVA-sensitized mice, i.t. OVA-induced peribronchial inflammation was P-selectin dependent during the acute phase and CD49d dependent during the late phase response (40, 41, 42). In the current study, time-dependent changes were observed for the CD4 subset but not for the CD8 subset. In light of these complexities, it is not surprising that immune reactions use a wide variety of adhesion receptors and that receptor utilization is not uniform across organs, disease processes, or lymphocyte subsets.
This experimental model system examines the pulmonary response to the classic particulate Ag, SRBC (21). This system is relevant to human lung disease because many naturally occurring inhaled or aspirated substances are complex particulates, the response to which may be poorly simulated by results from experimental systems using soluble Ags of lower complexity. SRBC do not proliferate or directly cause tissue damage but do present a variety of glycopeptide and glycolipid moieties (notably the Forsmann Ag) in a three-dimensional context that may stimulate recognition by the innate immune system (43). The response to i.t. challenge is dose dependent and Ag specific (24, 44). An i.t. challenge induces intense inflammatory cell accumulation, predominantly in the bronchovascular bundles and around veins (9), that most closely resembles a type IV Gell and Coombs response. Although priming does not involve adjuvants, initial i.t. challenge induces a predominately type 2 cytokine response with prominent lung eosinophilia (17, 26). Repeated i.t. challenge evolves to a distinctly type 1 and waning pulmonary response (17), thus appearing to duplicate the spontaneous tolerance seen in most animal models of repeated i.t. Ag challenge (45, 46, 47, 48). The SRBC model system has been used by a number of laboratories to analyze the anatomic and molecular mechanisms of lymphocyte recruitment to lung parenchyma (19, 20), the cytokine requirements for airways hyperresponsiveness (49), and the role of neuropeptides in development of lung inflammation (15).
In the SRBC model, lung lymphocyte accumulation is dependent on continuous recruitment from the periphery. This conclusion is based on the high rate of lymphocyte elimination by in situ apoptosis (50), the very low rate of in situ proliferation (26), and direct evidence that circulating T lymphoblasts are trafficking into the lung throughout the immune response (16). Furthermore, previous experiments in this model indicated that systemic depletion of CD4+ T cells before i.t. challenge with SRBC reduced the peribronchial accumulation of all leukocytes except CD8+ T cells (13). Conversely, CD8 depletion before i.t. SRBC challenge did not alter the accumulation of either the CD4 subset or B cells (25). Thus, the early recruitment of the CD4 and CD8 subsets in the SRBC model are mutually independent. These findings, coupled with the persistence of multiple T cell-directed chemokines in the lungs of E-P- animals, support the hypothesis that recruitment of CD8 and, to a lesser extent, the CD4 subset of T cells is initially selectin dependent following i.t. SRBC challenge in primed mice.
B cell accumulation in the lungs, in contrast, is dependent on
CD4+ T cells in the SRBC model (13).
This dependence is distinct from the T cell help required for Ag
priming. Therefore, the profound reduction in B cell accumulation in
selectin-deficient mice may reflect either direct involvement of
selectins in B cell recruitment or indirect changes in the lung
microenvironment resulting from the altered trafficking of
CD4+ T cells. Ligands for both E- and P-selectin
are synthesized by human B cells and mediate tethering interactions in
vitro (51, 52, 53); therefore, selectin-mediated B cell
recruitment in vivo is a theoretical possibility. Alternatively,
diminished accumulation of a critical T cell subset might delay
expression of endothelial adhesion receptors or chemokines essential
for subsequent B cell recruitment. For example, Gonzalo et al.
(40) proposed that CD4+ T cells
recruited early following i.t. OVA administration in sensitized mice
induced subsequent VCAM-mediated eosinophil recruitment. B cells
express 4 integrins and interact with VCAM in
vitro (54, 55). The 4
integrins clearly mediate memory B cell trafficking in vivo, as shown
for 47:mucosal
addressin CAM-dependent localization of IgA-specific B cells in the
gastrointestinal tract (56, 57). Thus, the current results
are compatible with either a direct or an indirect role for selectins
in B cell recruitment.
The existence of large numbers of T cells in the peripheral
blood and alveoli of E-P-
mice has not been reported previously. The generalized
increased in myelopoiesis observed in selectin-deficient animals has
been attributed, in part, to increased production of key growth factors
including IL-3 and GM-CSF (23). This dysregulation may
account for the expansion of T cells as well; however, a
compensatory increase due to deficiencies in either innate or acquired
immunity cannot be ruled out. Although uncommon in rodents and humans,
T cells comprise the major circulating lymphocyte in newborn
ruminants (58). Interestingly, Jutila et al.
(59) have shown that P- and E-selectin support
shear-dependent rolling of bovine T cells in vitro and mediate
recruitment into some immune reactions (58, 60).
Nevertheless, the current findings indicate that T cells can
enter the lungs without the endothelial selectins in the mouse, both in
the unchallenged state and during lung inflammation.
The finding of abundant T cells in
E-P- mice is noteworthy
because this cell type may contribute to host responses as both an
effector and regulatory cell (61). T cells react
directly with unprocessed Ag (62) and mediate effector
functions including cytokine production, cytotoxicity, and presentation
of Ag to T cells (63, 64, 65). Pulmonary T cells
comprise a heterogeneous population that are found both in normal mice
and in a variety of infectious models (66, 67, 68). Unlike the
case in the skin, where T cells lack TCR diversity, pulmonary
T cells show considerable clonal diversity (66, 69). T cells may also play important immunoregulatory
roles in autoimmunity and allergic inflammation (70). In
both murine and rat model systems, small numbers of cells from
Ag-exposed donors transferred Ag-specific tolerance to naive recipients
via immune deviation (71, 72). In addition,
T cells augmented IL-4 dependent, type 2-mediated
airway inflammation to peptide Ags in some murine models
(73). Therefore, the elevated numbers of T cells in
E-P- mice and their
capacity for recruitment to the lungs in the absence of endothelial
selectins must be considered when using these mice for in vivo
experimental model systems.
The current study found that granulocyte recovery from the lungs of E-P- mice was either unaltered (BAL) or significantly increased (minced tissues) compared with wt animals. This finding is consistent with previous reports documenting selectin-independent pathways for neutrophil recruitment into the lungs (3, 4, 5, 6, 74). However, one cannot completely rule out a role for selectins in neutrophil recruitment. As previously reported (28), neutrophil counts were constitutively elevated in the E-P- mice. After i.t. SRBC challenge, the absolute number of circulating neutrophils reached levels as high as 10- to 50-fold above the measurements in wt control animals (data not shown). Because neutrophil delivery to the lungs is markedly increased in E-P- mice, the relatively low numbers of neutrophils recovered from the BAL in particular may indicate that the extraction efficiency is actually decreased in the absence of endothelial selectins.
In summary, the absence of endothelial selectins significantly
reduces lymphocyte accumulation in the lungs following i.t. Ag
challenge of sensitized mice. The impact is greatest on
CD8+ cells and B cells, less marked on
CD4+ cells, and without apparent effect on
T cells. Thus, agents designed to block the endothelial selectins may
both diminish and skew pulmonary immune responses.
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Acknowledgments |
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Footnotes |
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2 Portions of these data were presented at the Midwest Autumn Immunology Meeting, Chicago, IL, November 20, 1999, and at the annual meeting of the American Association of Immunologists, Seattle, WA, May 15, 2000 (75 ).
3 The Curtis and Stoolman laboratories contributed equally to this study.
4 Address correspondence and reprint requests to Dr. Jeffrey L. Curtis, Pulmonary and Critical Care Medicine Section (111G), Department of Veterans Affairs Medical Center, 2215 Fuller Road, Ann Arbor, MI 48105-2303. E-mail address: jlcurtis{at}umich.edu
5 Abbreviations used in this paper: CAM, cell adhesion molecule; BAL, bronchoalveolar lavage; E-P- mice, gene-targeted mice lacking both E-selectin and P-selectin; FucT VII-/- mice, gene-targeted mice lacking fucosyltransferase VII; i.t., intratracheal; CMFDA, 5-chloromethylfluorescein diacetate; MCP, monocyte chemoattractant protein; MIP, macrophage-inflammatory protein; CCL, CC chemokine ligand; CXCL, CXC chemokine ligand; IP10, IFN-inducible protein 10; wt, wild type.
Received for publication December 27, 2001. Accepted for publication June 14, 2002.
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  J. L. Curtis, C. M. Freeman, and J. C. Hogg The Immunopathogenesis of Chronic Obstructive Pulmonary Disease: Insights from Recent Research Proceedings of the ATS, October 1, 2007; 4(7): 512 - 521. [Abstract] [Full Text] [PDF]
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 S. Ghosh, A. A. Chackerian, C. M. Parker, C. M. Ballantyne, and S. M. Behar The LFA-1 adhesion molecule is required for protective immunity during pulmonary Mycobacterium tuberculosis infection. J. Immunol., April 15, 2006; 176(8): 4914 - 4922. [Abstract] [Full Text] [PDF]
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T. Schreiber, S. Ehlers, S. Aly, A. Holscher, S. Hartmann, M. Lipp, J. B. Lowe, and C. Holscher Selectin Ligand-Independent Priming and Maintenance of T Cell Immunity during Airborne Tuberculosis J. Immunol., January 15, 2006; 176(2): 1131 - 1140. [Abstract] [Full Text] [PDF]
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, wt C57BL/6 mice; 
,
E-P- mice. Absolute lymphocyte numbers were
determined as the product of total BAL cell count (by hemocytometer)
and lymphocyte percentage on differential cell count of H&E-stained
preparations. Note differences in scales between A and
B. Data represent mean ± SEM of 6 mice per group
(days 4 and 7) or 11–12 mice per group (day 0) assayed individually in
at least 2 experiments. **, p < 0.01, unpaired
Student t test.
, CD8 cells; 
, 
