The cell adhesion molecules (CAMs) required for T lymphocyte recruitment during pulmonary immune responses have not been defined. Our laboratories recently reported that intratracheal (IT) challenge of sensitized mice with SRBC induced prolonged expression of vascular P-selectin, E-selectin, and VCAM-1, particularly in areas of mononuclear leukocyte infiltration. A surge in the number of circulating T lymphocytes expressing selectin ligands preceded the peak accumulation of T cells in the lung. In addition, a significant percentage of the T cells recovered from the lung expressed selectin ligands as well. The current study demonstrates that cultured T lymphoblasts use both selectin ligands and α4 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 α4 integrins and targeted deletion of an α(1,3)fucosyltransferase essential for selectin ligand synthesis each reduced labeled T lymphoblast trafficking to a significant degree. Furthermore, α4 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 α4 integrins participate in T lymphoblast recruitment during the pulmonary immune response to IT SRBC.

Recruitment of lymphocytes from peripheral blood into lung supports both the clearance of viral pathogens and the development of immune-mediated lung diseases, including asthma, sarcoidosis, and pulmonary fibrosis. The first step in recruitment is the arrest of leukocytes within microvasculature in the inflamed tissue (1, 2). For neutrophils, both physical trapping in the pulmonary capillary bed (3) and adhesive interactions with activated endothelium (4, 5, 6) can initiate the process. The current study asks whether circulating T lymphoblasts rely on adhesive interactions for entry into an immunologically mediated inflammatory reaction in the lung.

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 α4 integrins, respectively. The α4 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 α4 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 α4 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 α4 integrins and VCAM-1 contribute as well.

The current study uses selectin ligand-deficient T lymphoblasts and mAb blockade of the α4 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 α(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 α4 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:α4 integrin adhesive interactions mediate in part independent pathways of recruitment into the lung.

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).

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 × 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.

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 × 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 × 106 cells/ml.

The cultured T lymphoblasts were washed twice in sterile saline and resuspended at 2 × 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 × 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 α4 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.

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.

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.

Table I.

Abs and concentrations used for flow cytometry

AgCloneSourceaIsotypeConcentrationb
CD4 RM4-5 PharMingen Rat IgG2a 5 μg/ml 
CD8 54-6.7 PharMingen Rat IgG2a 5 μg/ml 
CD90.2 53-2.1 PharMingen Rat IgG2a 5 μg/ml 
CD45 3.F11.1 PharMingen Rat IgG2a 5 μg/ml 
CD45 RB/220 RA3-6B2 PharMingen Rat IgG2a 5 μg/ml 
CD25 7D4 PharMingen Rat IgM 10 μg/ml 
CD69 H1.2F3 PharMingen Hamster IgG 10 μg/ml 
α4 integrins (CD49d) R 1/2 PharMingen Rat IgG2a 10 μg/ml 
L-selectin Mel-14 PharMingen Rat IgG2b 10 μg/ml 
CD11a M17 Ascites Rat IgG2a 1:500 
β2-integrins (CD18) M18 Ascites Rat IgG2a 1:1000 
CD44 KM81 Ascites Rat IgG2a 1:500 
E-selectin ligand Chimera (sup.) Ref 34 hIgM+mE-sel. 1:2 
P-selectin ligand Chimera (sup.) Ref 34 hIgM+mP-sel. 1:2 
AgCloneSourceaIsotypeConcentrationb
CD4 RM4-5 PharMingen Rat IgG2a 5 μg/ml 
CD8 54-6.7 PharMingen Rat IgG2a 5 μg/ml 
CD90.2 53-2.1 PharMingen Rat IgG2a 5 μg/ml 
CD45 3.F11.1 PharMingen Rat IgG2a 5 μg/ml 
CD45 RB/220 RA3-6B2 PharMingen Rat IgG2a 5 μg/ml 
CD25 7D4 PharMingen Rat IgM 10 μg/ml 
CD69 H1.2F3 PharMingen Hamster IgG 10 μg/ml 
α4 integrins (CD49d) R 1/2 PharMingen Rat IgG2a 10 μg/ml 
L-selectin Mel-14 PharMingen Rat IgG2b 10 μg/ml 
CD11a M17 Ascites Rat IgG2a 1:500 
β2-integrins (CD18) M18 Ascites Rat IgG2a 1:1000 
CD44 KM81 Ascites Rat IgG2a 1:500 
E-selectin ligand Chimera (sup.) Ref 34 hIgM+mE-sel. 1:2 
P-selectin ligand Chimera (sup.) Ref 34 hIgM+mP-sel. 1:2 
a

All commercial Abs were purchased labeled with FITC or biotin. Ascites were made in nude mice from hybridoma lines purchased from the American Type Culture Collection (Manassas, VA).

b

Final concentrations in 100 to 200 μl with 2.5 to 5 × 105 cells.

Samples were read within 36 h by one- or two-color flow cytometry and were analyzed using the WinList analysis program. The CD45/IgM chimera showed the same low level of binding to cells as the selectin/IgM chimeras in the presence of EDTA. This nonspecific reactivity was used to set the threshold for a positive reaction as detailed previously (26).

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.

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 α4 integrins, β2 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.

Table II.

Effect of serum on selectin ligand expression by murine T lymphoblasts

Culture Conditionsa% with P-Selectin Ligandsb% with E-Selectin Ligandsb
High FCS 8.7 ± 2.8 9.1 ± 3 
Low FCS 29.4 ± 5.3c 36 ± 5.6c 
Culture Conditionsa% with P-Selectin Ligandsb% with E-Selectin Ligandsb
High FCS 8.7 ± 2.8 9.1 ± 3 
Low FCS 29.4 ± 5.3c 36 ± 5.6c 
a

Mononuclear cells (pooled from the spleens and lymph nodes of normal C57BL/6 mice) were cultured in medium containing 10% FCS (high FCS) or in daily decreasing amounts of FCS (low FCS).

b

The expanded T lymphoblast populations were assayed for expression of P- and E-selectin ligands by flow cytometry using murine selectin/human IgM chimeric proteins. The % positive cells are shown as mean ± SEM of five to nine independent experiments.

c

p < 0.017 by Student’s t test compared with cells grown in high serum.

Trafficking studies commonly measure recruitment by infusing radiolabeled T lymphoblasts and then comparing the number of counts accumulated in intact normal and diseased target organs. However, short term trafficking studies by Austrup et al. (15) indicated that unfractionated samples of normal and diseased lungs contained equivalent numbers of radiolabeled lymphoblasts 1 h after i.v. infusion. This finding implied that analysis of unfractionated lung specimens at early time points may not be optimal for trafficking studies in the lung. Consequently, the trafficking of fluorescently labeled T lymphoblasts into normal and SRBC-challenged lungs was examined at two relatively late time points after i.v. infusion.

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. 1,A). In contrast, the prevalence of labeled cells in the minced and enzymatically digested lung samples was different at the 24 h point only (Fig. 1 B). 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 × 105 vs ∼4–10 × 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.

FIGURE 1.

Recovery of CMFDA-labeled T lymphoblasts from lungs at 4 and 24 h after infusion. Lymphocytes from normal C57BL/6J mouse spleens were cultured for 5 days and labeled with CMFDA as described in Materials and Methods. The CMFDA-labeled cells were infused (2 × 107/mouse) via the tail vein into normal C57BL/6J mice (▪) 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).

FIGURE 1.

Recovery of CMFDA-labeled T lymphoblasts from lungs at 4 and 24 h after infusion. Lymphocytes from normal C57BL/6J mouse spleens were cultured for 5 days and labeled with CMFDA as described in Materials and Methods. The CMFDA-labeled cells were infused (2 × 107/mouse) via the tail vein into normal C57BL/6J mice (▪) 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).

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As noted above, the culture conditions promoted CD8 proliferation primarily. The CD8 subset of cultured T lymphoblasts also showed the most active trafficking activity. For example, the CD4:CD8 ratio of infused wild-type T lymphoblasts recovered from BAL 24 h after infusion was <0.25 during peak recruitment (days 3–4 post-SRBC challenge; data not shown). The low CD4:CD8 ratio is an artifact of the culture conditions, since the CD4:CD8 ratio of host lymphocytes in BAL at this time ranged from 1 to 2. Consequently, trafficking assays performed with T lymphoblasts cultured as described herein are biased toward the CD8 subset.

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.

FIGURE 2.

The recruitment of labeled T lymphoblasts parallels the accumulation of host T cells in inflamed lung. CMFDA-labeled syngeneic T lymphoblasts (see Fig. 1) were infused into SRBC-primed mice either before (day 0) or after intratracheal challenge with SRBCs. The mice were sacrificed 24 h later and processed as described in Materials and Methods. The administration of IT SRBCs was staggered so that all animals received labeled T lymphoblasts from the same population. The time points on the x-axis give the day post-IT that the lungs were processed. At each time point, the numbers of CMFDA-labeled (infused) and nonlabeled (host) lymphocytes in the BAL (A) and lung mince (B) were determined as described in Materials and Methods. The data are expressed as the mean number of labeled (solid symbols) and unlabeled (open symbols) lymphocytes per mouse lung ± SEM based on four to six mice per time point.

FIGURE 2.

The recruitment of labeled T lymphoblasts parallels the accumulation of host T cells in inflamed lung. CMFDA-labeled syngeneic T lymphoblasts (see Fig. 1) were infused into SRBC-primed mice either before (day 0) or after intratracheal challenge with SRBCs. The mice were sacrificed 24 h later and processed as described in Materials and Methods. The administration of IT SRBCs was staggered so that all animals received labeled T lymphoblasts from the same population. The time points on the x-axis give the day post-IT that the lungs were processed. At each time point, the numbers of CMFDA-labeled (infused) and nonlabeled (host) lymphocytes in the BAL (A) and lung mince (B) were determined as described in Materials and Methods. The data are expressed as the mean number of labeled (solid symbols) and unlabeled (open symbols) lymphocytes per mouse lung ± SEM based on four to six mice per time point.

Close modal

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 α4 integrins resulted in an ∼50% reduction (p < 0.05) in the ability of labeled cells to traffick into both the pulmonary airspace (Fig. 3,A) and interstitial tissues (Fig. 3 B) 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).

FIGURE 3.

Blocking α4 integrins reduces T lymphoblast recruitment into lung. CMFDA-labeled T lymphoblasts were preincubated with the rat IgG (▪) or anti-α4 mAb PS/2 (□) as described in Materials and Methods. The cells and Ab were then injected into the tail veins of SRBC-primed mice 3 days after IT challenge with SRBC (the time of peak host lymphocyte recruitment; see Fig. 2). The absolute number of labeled lymphoblasts in the BAL (A) and minced lung tissue (B) was determined 24 h later (note difference in scales of y-axes). The data are expressed as the mean number of labeled cells ± SEM per mouse lung. ∗, p < 0.03; ∗∗, p = 0.041 (by Student’s unpaired t test based on 10–16 mice from three independent experiments).

FIGURE 3.

Blocking α4 integrins reduces T lymphoblast recruitment into lung. CMFDA-labeled T lymphoblasts were preincubated with the rat IgG (▪) or anti-α4 mAb PS/2 (□) as described in Materials and Methods. The cells and Ab were then injected into the tail veins of SRBC-primed mice 3 days after IT challenge with SRBC (the time of peak host lymphocyte recruitment; see Fig. 2). The absolute number of labeled lymphoblasts in the BAL (A) and minced lung tissue (B) was determined 24 h later (note difference in scales of y-axes). The data are expressed as the mean number of labeled cells ± SEM per mouse lung. ∗, p < 0.03; ∗∗, p = 0.041 (by Student’s unpaired t test based on 10–16 mice from three independent experiments).

Close modal

The effect of PS/2 on lung recruitment was specific, since splenic lymphocytes preincubated with the Ab showed normal levels of L-selectin-dependent trafficking into peripheral lymph nodes (Table III). Hamann et al. also found that PS/2 inhibited α4 integrin-dependent trafficking exclusively. These investigators reported that the Ab blocked the α47-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 α4 integrins participate in the SRBC-induced recruitment of T lymphoblasts into the lung but cannot account for all trafficking activity.

Table III.

Specificity of the intact PS/2 mAb for in vivo trafficking studiesa

Target TissueIsotype ControlbPS/2b
Peripheral lymph node 540 ± 60 600 ± 80 
Peyer’s patches 200 ± 40 10 ± 5c 
Target TissueIsotype ControlbPS/2b
Peripheral lymph node 540 ± 60 600 ± 80 
Peyer’s patches 200 ± 40 10 ± 5c 
a

Pooled splenic and lymph node lymphocytes from normal C57BL/6J mice were labeled with CMFDA and infused (2 × 107/mouse) via the tail vein into syngeneic animals as described in Materials and Methods. At 24 h after infusion, cell suspensions from peripheral lymph nodes and Peyer’s patches were analyzed by flow cytometry.

b

The data are expressed as the mean number of labeled cells per 104 events ± SEM based on three to five mice per group.

c

p<0.05 by unpaired Student’s t test.

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. 4,A). 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. 4 B). 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.

FIGURE 4.

Expression of ligands for P- and E-selectin correlates with enhanced recruitment into inflamed lung. T lymphoblasts were expanded in vitro as described in Materials and Methods. These cells were assayed for expression of P- (A) and E-selectin (B) ligands by flow cytometry using mouse selectin/human IgM chimeric proteins (initial population, ▪). After labeling with CMFDA, the blasts were injected i.v. into SRBC-primed mice 3 days after IT challenge with SRBC. The labeled lymphocytes recovered 24 h later from the airspace (BAL, □) and minced lung tissue (lung mince, ▨) 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).

FIGURE 4.

Expression of ligands for P- and E-selectin correlates with enhanced recruitment into inflamed lung. T lymphoblasts were expanded in vitro as described in Materials and Methods. These cells were assayed for expression of P- (A) and E-selectin (B) ligands by flow cytometry using mouse selectin/human IgM chimeric proteins (initial population, ▪). After labeling with CMFDA, the blasts were injected i.v. into SRBC-primed mice 3 days after IT challenge with SRBC. The labeled lymphocytes recovered 24 h later from the airspace (BAL, □) and minced lung tissue (lung mince, ▨) 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).

Close modal

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, α4 integrins, and β2 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).

Table IV.

Expression of cell surface markers on T lymphoblasts grown from the FucT-VII KO and its wild-type control mice

AgbWild-Type Control BlastsaFucT-VII KO Blastsa
% positiveMCF% positiveMCF
E-selectin ligand 22 ± 6 207 ± 30 2 ± 1c NA 
P-selectin ligand 29 ± 9 390 ± 9 1 ± 1c NA 
L-selectin 21 ± 11 43 ± 7 29 ± 14 42 ± 8 
α4 integrins 69 ± 4 41 ± 9 53 ± 9 38 ± 9 
β2 integrins 97 ± 2 310 ± 110 92 ± 1 360 ± 20 
CD25 83 ± 8 377 ± 100 72 ± 2 522 ± 122 
AgbWild-Type Control BlastsaFucT-VII KO Blastsa
% positiveMCF% positiveMCF
E-selectin ligand 22 ± 6 207 ± 30 2 ± 1c NA 
P-selectin ligand 29 ± 9 390 ± 9 1 ± 1c NA 
L-selectin 21 ± 11 43 ± 7 29 ± 14 42 ± 8 
α4 integrins 69 ± 4 41 ± 9 53 ± 9 38 ± 9 
β2 integrins 97 ± 2 310 ± 110 92 ± 1 360 ± 20 
CD25 83 ± 8 377 ± 100 72 ± 2 522 ± 122 
a

Pooled mononuclear cells from FucT-VII KO and wild-type control mice were expanded in daily decreasing amounts of FCS (see Table II) to maximize selectin ligand expression.

b

The expanded T lymphoblast populations were assayed for expression of P- and E-selectin ligands and other Ags as described in Materials and Methods. The % positive cells and MCF (mean channel of fluorescence) are expressed as the mean ± SEM of four replicates.

c

p < 0.05 by Student’s t test compared with cells from the wild-type controls.

Figure 5 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. 5,A) and showed a 64% reduction in trafficking into BAL relative to control T lymphoblasts (Fig. 5,B). 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. 6,A; p = 0.002) and interstitial tissues (Fig. 6 B; p < 0.001). The findings imply that synthesis of selectin ligands enhances T lymphoblast trafficking into SRBC-challenged lungs.

FIGURE 5.

Selectin ligand expression and trafficking of T lymphoblasts cultured from FucT-VII-deficient mice. T lymphoblasts were cultured from FucT-VII KO mice or wild-type controls with normal alleles at the FucT-VII locus (WT control). The cultured lymphoblasts were assessed for P- and E-selectin ligands as described in Materials and Methods (A). Selectin ligands were expressed by ∼30% of the WT control blasts and virtually none of the FucT-VII KO blasts in this experiment (see Table IV for statistical comparison). These T lymphoblasts were labeled with CMFDA and infused into different groups of SRBC-primed C57BL/6J mice 3 days after IT challenge with SRBC. The numbers of labeled cells in the BAL of the two treatment groups were determined 24 h later (B). The data are presented as the mean number of labeled cells ± SEM per mouse lung in animals receiving WT control (▪) and FucT-VII KO (□) blasts. ∗, p < 0.001 (by Student’s unpaired t test based on eight mice per group pooled from two independent experiments).

FIGURE 5.

Selectin ligand expression and trafficking of T lymphoblasts cultured from FucT-VII-deficient mice. T lymphoblasts were cultured from FucT-VII KO mice or wild-type controls with normal alleles at the FucT-VII locus (WT control). The cultured lymphoblasts were assessed for P- and E-selectin ligands as described in Materials and Methods (A). Selectin ligands were expressed by ∼30% of the WT control blasts and virtually none of the FucT-VII KO blasts in this experiment (see Table IV for statistical comparison). These T lymphoblasts were labeled with CMFDA and infused into different groups of SRBC-primed C57BL/6J mice 3 days after IT challenge with SRBC. The numbers of labeled cells in the BAL of the two treatment groups were determined 24 h later (B). The data are presented as the mean number of labeled cells ± SEM per mouse lung in animals receiving WT control (▪) and FucT-VII KO (□) blasts. ∗, p < 0.001 (by Student’s unpaired t test based on eight mice per group pooled from two independent experiments).

Close modal
FIGURE 6.

Simultaneous tracking of T lymphoblasts from FucT-VII-deficient mice and wild-type controls. T lymphoblasts cultured from the FucT-VII KO mice and the wild-type controls (WT Control) were fluorescently labeled with green CMFDA and red CMTMR, respectively. The labeled cells were mixed in equal parts and injected into the tail veins of SRBC-primed C57BL/6J mice 3 days after IT challenge with SRBC. The numbers of labeled cells in the BAL (A) and lung mince (B) were measured 24 h later. The data are presented as the mean number of labeled WT Control (▪) and FucT-VII KO (□) cells per mouse lung ± SEM. ∗, p < 0.002 (by Student’s paired t test based on nine mice per group).

FIGURE 6.

Simultaneous tracking of T lymphoblasts from FucT-VII-deficient mice and wild-type controls. T lymphoblasts cultured from the FucT-VII KO mice and the wild-type controls (WT Control) were fluorescently labeled with green CMFDA and red CMTMR, respectively. The labeled cells were mixed in equal parts and injected into the tail veins of SRBC-primed C57BL/6J mice 3 days after IT challenge with SRBC. The numbers of labeled cells in the BAL (A) and lung mince (B) were measured 24 h later. The data are presented as the mean number of labeled WT Control (▪) and FucT-VII KO (□) cells per mouse lung ± SEM. ∗, p < 0.002 (by Student’s paired t test based on nine mice per group).

Close modal

The final studies examined the impact of α4 integrin blockade on the trafficking of T lymphoblasts derived from FucT-VII KO mice and their wild-type controls (Fig. 7). Blocking the α4 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 α4 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 α4 integrin function decreases SRBC-induced T lymphoblast recruitment into BAL by >85% in short term trafficking studies.

FIGURE 7.

Endothelial selectins and α4 integrins participate in independent recruitment pathways. T lymphoblasts cultured from the FucT-VII KO mice and the wild-type controls were fluorescently labeled with CMFDA. These cells were incubated with pooled rat IgG or the anti-α4 mAb PS/2 and injected into four groups of SRBC-primed and IT-challenged C57BL/6 mice as described in Figure 3. The number of labeled cells in the BAL was measured 24 h later in the four treatment groups. The data are presented as the mean number of labeled cells ± SEM in mice receiving WT control blasts and IgG (▪), WT control blasts and anti-α4 (□), FucT-VIIKO blasts and IgG (▨), and FucT-VIIKO blasts and anti-α4 (▧). p < 0.05 (by analysis of variance/Student-Newman-Keuls test based on four mice per group).

FIGURE 7.

Endothelial selectins and α4 integrins participate in independent recruitment pathways. T lymphoblasts cultured from the FucT-VII KO mice and the wild-type controls were fluorescently labeled with CMFDA. These cells were incubated with pooled rat IgG or the anti-α4 mAb PS/2 and injected into four groups of SRBC-primed and IT-challenged C57BL/6 mice as described in Figure 3. The number of labeled cells in the BAL was measured 24 h later in the four treatment groups. The data are presented as the mean number of labeled cells ± SEM in mice receiving WT control blasts and IgG (▪), WT control blasts and anti-α4 (□), FucT-VIIKO blasts and IgG (▨), and FucT-VIIKO blasts and anti-α4 (▧). p < 0.05 (by analysis of variance/Student-Newman-Keuls test based on four mice per group).

Close modal

This study provides direct evidence that both selectin ligands and α4 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 α4 integrins. Blocking either α4 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 α4 integrin function on the blasts reduced trafficking more than interdiction of either receptor family alone (>85%). Consequently, the α4 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 α4 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.

1

This work was supported in part by the following U.S. Public Health Service grants: Specialized Center for Research in Occupational and Immunologic Lung Diseases P50HL46487 (to J.L.C. and L.M.S.), HL56309 (to L.M.S.), and AI33189 (to L.M.S. and J.B.L.). Additional support was provided by research funds from the Department of Veterans Affairs. J.L.C. is a Career Investigator with the American Lung Association of Michigan. J.B.L. is an Investigator with the Howard Hughes Medical Institute.

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.

4

Abbreviations used in this paper: IT, intratracheal; FucT-VII, α(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.

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