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Published ahead of print on August 14, 2003, doi:10.1165/rcmb.2003-0208OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 220-227, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0208OC

Trafficking of Th1 Cells to Lung

A Role for Selectins and a P-Selectin Glycoprotein-1–Independent Ligand

Joan G. Clark, Janis B. Mandac-Dy, Anne E. Dixon, David K. Madtes, Kristin M. Burkhart, John M. Harlan and Daniel C. Bullard

Division of Pulmonary and Critical Care Medicine, and Division of Hematology, Department of Medicine, University of Washington, Seattle; Fred Hutchinson Cancer Research Center, Seattle, Washington; and Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama

Address correspondence to: Joan G. Clark, M.D., P.O. Box 19024 (D3–190), Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109. E-mail: jclark{at}fhcrc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Trafficking of lymphocytes to lung is a critical component of pulmonary immune defense and surveillance. Selectins, expressed on vascular endothelium, regulate T lymphocyte emigration into tissues, such as skin, but the role of the selectins in trafficking of T cells to lung has not been well characterized. Here, we used a model of lung inflammation induced by adoptive transfer of alloreactive Th1 cells to analyze the role of P- and E-selectin in Th1 cell trafficking to lung in vivo. We found that both P- and E-selectin play an important role in Th1 lymphocyte migration to lung. We confirmed that the Th1 cells express P-selectin glycoprotein ligand-1, which was functional in binding to P- and E-selectin in vitro. However, our studies reveal that a ligand distinct from P-selectin glycoprotein-1 also binds these selectins in vitro and appears to play a physiologic role in in vivo emigration of Th1 lymphocytes into the lung.

Abbreviations: 5-(and –6)-carboxyfluorescein diacetate, succimidyl ester, 5(6)-CFDA, SE • cutaneous lymphocyte-associated antigen, CLA • E-selectin ligand-1, ESL-1 • intercellular adhesion molecule-1, ICAM-1 • o-sialoglycoprotease, OSGP • P-selectin glycoprotein-1, PSGL-1


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The regulated trafficking of lymphocytes from the bloodstream to lung is a critical component of pulmonary immune surveillance and host defense. Localization of lymphocytes within the vasculature involves a stepwise interaction between molecules on the surface of lymphocytes and adhesion molecules on endothelium (1–5). Primary adhesion of lymphocytes may occur through members of the selectin family (i.e., P-, E-, and L-selectin) and their carbohydrate ligands on various glycoproteins such as P-selectin glycoprotein ligand-1 (PSGL-1), CD34, cutaneous lymphocyte-associated antigen (CLA), and E-selectin ligand-1 (ESL-1) (6–9). The selectins clearly are involved in lymphocyte homing to lymphoid tissue and skin (3, 10). The importance of this mechanism in lung lymphocyte interactions in the pulmonary microvasculature is not clear, but a role for selectins in margination of neutrophils in noncapillary microvessels of lung has been demonstrated (11). During lymphocyte recruitment to lung in response to particulate antigen in a murine model, P- and E-selectin expression was increased (12). Trafficking of cultured T lymphocytes to lung 2 d after intratracheal challenge with particulate antigen was reduced by deficiency of the T cell enzyme ({alpha} [1, 3]-fucosyltransferase) essential for selectin ligand biosynthesis (13), although the selectin ligand was not further characterized. A contribution of P- and/or E-selectin in lymphocyte recruitment to lung in this model also has been demonstrated using P- and E-selectin double-deficient mice (14).

Identification of selectin ligands has been the object of recent research. PSGL-1 has been most extensively characterized, and other ligands have been described and defined at least in part according to structural modifications such as sialation, glycosylation, and sulfation (9, 15–20). P- and E-selectin binding (through PSGL-1) has been described for Th1 cells, but not Th2 cells (21–23), and antigen-specific T cells in inflamed peritoneum (24). ESL-1 is a glycoprotein that binds specifically to E-selectin but not to P-selectin. It is expressed on mouse neutrophils, but its role, if any, in lymphocyte adherence in vivo is unknown (20, 25). An important question underlying these investigations is: what are the requirements for biologically relevant interactions of selectins and their ligands in vivo (17)?

In this study, we analyzed the role of P- and E-selectin in the trafficking of adoptively transferred Th1 cells to lung in vivo. We found that both P- and E-selectin play an important role in Th1 lymphocyte migration to the lung. We confirmed that the Th1 lymphocytes express PSGL-1, which is functional in binding to P- and E-selectin in vitro. However, our studies reveal that a ligand independent of PSGL-1 also binds these selectins in vitro and appears to play a physiologic role in in vivo adherence of Th1 lymphocytes in lung.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
T cell clones specific for Ly5a (clone 8F5) and Ly5b (Clone 1A4) alleles were developed and maintained in culture as previously described (26). In brief, Ly5b mice (C57BL/6) and congenic Ly5a mice were immunized with 13-mer Ly5a and Ly5b peptides, respectively. The peptides were identical to a polymorphic region that differs by three amino acids. CD4+ helper T cells specific for the 13-mer peptides were elicited and cloned by limiting dilution. The clones used in these studies have a Th1 phenotype and produces interferon-{gamma} and tumor necrosis factor-{alpha}. They were noncytolytic in vitro. The T cell clones were expanded by periodic stimulation with peptide Ly5a or allogeneic irradiated splenocytes in the presence of congenic irradiated splenocytes and maintained in the presence of IL-2 (10 U/ml). Activated cells were harvested 1 d after stimulation; resting cells were harvested 14 d after stimulation. In some experiments, T cells were activated by incubation with mAb to CD3 as previously described (27).

Cytotoxic T lymphocyte clones specific for H2d alloantigens were generated as previously described (28). The cloned cells were CD8+ and had a type 1 cytokine profile (Tc1).

Control lymph node lymphocytes were obtained by mechanical disaggregation of peripheral lymph nodes from C57BL/6 mice. The cells were suspended in RPMI and used for in vivo migration studies on the same day.

Splenocytes for control adhesion assays were obtained by mechanical disaggregation of spleens from C57BL/6 mice. Splenocytes were cultured in Dulbecco's modified Eagle's medium–10% fetal calf serum and concanavalin A (4 µg/ml; Sigma, St. Louis, MO) for 48 h.

Mice
Normal C57BL/6 (Ly5b) mice and congenic Ly5a mice were obtained from the Jackson Laboratories (Bar Harbor, ME). The E-selectin, P-selectin, and E-/P-selectin double mutant mice used in these studies were backcrossed five or more generations onto either C57BL/6J or DBA/1J (29–31). Control mice were wild-type C57BL/6 or DBA/1, respectively. Both strains are Ly5b. Mice were used at 6–8 wk of age. The E-/P-selectin double mutant mice appeared normal at this age without cutaneous infections or histologic evidence of lung inflammation. Mice were housed in microisolator cages under specific pathogen–free conditions with free access to sterile water and chow.

In Vivo Migration Studies
In vivo migration of lymphocytes was analyzed as previously described (32, 33). Briefly, cloned Th1 cells were labeled with 20 µCi sodium chromate [51Cr] (Amersham Life Sciences, Arlington, IL) per milliliter for 1 h at 37°C. Excess chromium and dead cells were removed by density centrifugation though Nycodenz (Life Technologies, Grand Island, NY). Labeled cells (4 x 106 cells) were administered to mice by tail vein injection in a volume of 0.5 ml. For antibody blocking experiments, cells were incubated with neutralizing monoclonal antibody to PSGL-1 (clone 2PH1; Pharmingen, San Diego, CA) or isotype control antibody (clone R3–34; Pharmingen) at (10 µg/ml or 5 µg/mouse) for 15 min at room temperature before injection. In some experiments, Th1 cells were treated with enzyme before injection. Mice were killed at various time points. Organs were harvested and weighed, and distribution of radioactivity was measured for 1 min in a {gamma} scintillation counter (Packard, Downers Grove, IL).

Enzymatic Treatments of Cells
Th1 cells were treated with enzymes as previously described (7). Briefly, to remove terminal cell-surface sialic acids, Th1 cells were incubated with 0.1 U/ml Vibrio Cholera neuraminidase (Calbiochem, San Diego, CA) for 30 min at 25°C in HBSS supplemented with 10 mM HEPES and 2 mM Ca2+ (H/H Ca2+ medium). To assess the role of O-sialo mucin-like ligands, Th1 cells were incubated for 40 min at 37°C in H/H Ca2+ medium supplemented with 2 mg/ml bovine serum albumin (Sigma) in the presence of 50 µg/ml of Pasturella haemolytica O-sialoglycoprotease (OSGP; Accurate Chemical and Scientific Corp., Westbury, NY). Control Th1 cells were incubated at 37°C in this medium in absence of enzymes. Reactions were terminated by washing cells twice with H/H medium + 5 mM EDTA.

Flow Cytometry
Th1 cells were incubated in buffer (1% fetal calf serum + 0.1% Azide, in phosphate-buffered saline) with 20 µg/ml murine P-selectin–human IgG fusion protein (Pharmingen), murine E-selectin–human IgG fusion protein (kindly provided by T. Hirata [23]), control human IgG Fc (Jackson ImmunoResearch, West Grove, PA), 10 µg/ml mAb to PSGL-1 (clone 2PH1), or isotype control mAb (R 3–34; Pharmingen). In some experiments, cells were treated with enzyme or incubated with anti–PSGL-1 mAb followed by incubation with E- or P-selectin-IgG chimera. In some experiments, cells were incubated with selectin fusion proteins in the presence of 10 mM EDTA. Cells were washed and labeled with secondary Abs (PE-conjugated goat anti-human IgG to detect E- or P-selectin–Ig, or FITC-conjugated mouse anti-rat IgG to detect PSGL-1 mAb; Jackson Immunochemicals) at a 1:100 dilution. Analysis was performed on a Becton Dickinson (San Jose, CA) FACSCalibur or FACScan and CellQuest analysis software.

Cell Adhesion Assays
Ninety-six-well plates (Nalge Nunc, Naperville, IL) were coated with 10 µg/ml selectin–IgG fusion protein for 2 h at 37°C and blocked with 1% bovine serum albumin in phosphate-buffered saline overnight at 4°C. The Th1 cells were labeled with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (5 (6)-CFDA, SE; Molecular Probes, Eugene, OR), added to each well (5 x 105 per well), and incubated for 20 min at 4°C under rotation (60 rpm). The plates were washed three times with HBSS with CaCl2 and MgCl2, and bound cells were quantitated using a Cytofluor II (Perseptive Biosystems, Inc., Framingham, MA). In some experiments, the cells were incubated with 10 µg/ml mAb to PSGL-1, isotype control mAb, or OSGP, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adoptively Transferred Th1 Cells Selectively Adhere in Lung
We have recently shown that our cloned Th1 cells are selectively localized in lung after adoptive transfer and that trafficking to lung is independent of alloreactivity (33). As a first step in analyzing whether the initial localization pattern was an adhesive molecular mechanism, we compared the in vivo migration pattern of 51Cr-labeled Th1 cells (Clone 8F5) and isolated lymph node cells 1 h after intravenous injection. The radioactivity detected in lung (50% of injected radioactivity) after Th1 cell administration was ~ 3-fold higher than radioactivity in lung after lymph node lymphocyte injection (Figure 1). Conversely, radioactivity in spleen was 30 times higher in mice that received lymph node lymphocytes compared with Th1 cells.



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  		Figure 1. Localization of 51Cr-labeled Th1 cells and lymph node lymphocytes after intravenous administration of cloned Th1 cells or freshly isolated peripheral lymph node (LN) lymphocytes. Aliquots of the labeled Th1 cells (Clone 8F5) (solid bars) or LN lymphocytes (open bars) containing equal amounts of radioactivity were injected intravenously into C57BL/6 mice. After 1 h, radioactivity was counted in organs as indicated. Values, expressed as cpm/mg tissue, are mean ± SEM (n = 4).

 
In Vivo Localization of Th1 Cells Is Dependent on Both P- and E-Selectins
To directly test the involvement of P- and E-selectin in lymphocyte localization in lung, we adoptively transferred resting or in vitro activated 51Cr-labeled Th1 cells (clone 8F5) to mice deficient in P-, E-, or both P- and E-selectin–deficient mice (C57BL/6 background). The initial localization of resting cells, measured as CPM/mg tissue at 1 h after intravenous administration was reduced in the E-selectin–deficient mice (P < 0.03) and double P- and E-selectin–deficient mice (P < 0.02) (n = 3 mice/group) (Figure 2A). Likewise, the initial (1 h) as well as the prolonged (24 h) localization of activated Th1 cell was reduced by > 50% in the double P- and E-selectin–deficient mice (P < 0.05), but not in the single P- or E-selectin–deficient mice (Figure 2B). An additive effect of P- and E-selectin was suggested by analysis of both resting and activated cells, and confirmed in a repeat experiment, using selectin-deficient mice on an additional strain (DBA-1J) background.



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  		Figure 2. Localization of 51Cr-labeled Th1 cells (Clone 8F5) in lungs of P-, E-, and P/E-selectin–deficient (KO) mice. Resting Th1 cells (A) or in vitro activated Th1 cells (B) were labeled with 51Cr and administered intravenously to control (C57BL/6) or selectin KO mice. Organs were removed after 1 h (A, resting cells) or 1 and 24 h (B, activated cells), and radioactivity was measured. Localization of 51Cr-labeled Th1 cells was significantly reduced in lungs of P/E-selectin–deficient mice in all experiments (P < 0.05). Values, expressed as cpm/mg tissue, are mean ± SEM (n = 3 mice/group). Open bars, C57BL/6; dotted bars, P-KO; striped bars, E-KO; solid bars, P/E-KO.

 
Th1 Cells Bind E- and P-Selectin Fusion Proteins In Vitro
To further examine the role of the individual selectin molecules in Th1 cell adherence in lung, we analyzed P- and E-selectin binding of the Th1 cells by flow cytometry using soluble P- and E-selectin–human IgG fusion proteins. Both resting and in vitro CD3-activated Th1 cells bound P-selectin Ig (Figure 3A) (only resting cells are shown). Similar results were obtained with a different Th1 cell clone (1A4) and with cloned CD8+ cells with a type 1 cytokine phenotype (Clones 14C3, 7C11, 7C3, 14M4) (28) (not shown). In contrast, only a small population of control freshly isolated splenocytes bound P-selectin–Ig (not shown). A bimodal distribution of fluorescence intensity, indicating cell populations with high and intermediate levels of P-selectin binding, was apparent in most of the T cell clones. In similar flow cytometric analyses, both resting and CD3 mAb–activated Th1 cells also bound E-selectin–Ig (Figure 3B). In the presence of 10 mM EDTA, P- and E-selectin–Ig binding was nearly identical to the IgG control.



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  		Figure 3. Selectin-Ig fusion protein binding to Th1 cells. Resting Th1 cells (Clone 8F5) were incubated with murine P-selectin–Ig followed by PE-labeled antibody to human IgG (A). Controls were labeled with human IgG and the PE-labeled secondary antibody. Flow cytometric analysis showed bright staining of Th1 cells. In similar experiments, resting Th1 cells were incubated with murine E-selectin–Ig (B). Flow cytometric analysis showed positive staining compared with control IgG.

 
PSGL-1 Is Expressed on Th1 Cells
PSGL-1 is a well characterized high-affinity ligand for P-selectin (7). By flow cytometry using mAb to the glycoprotein core (Clone 2PH1; Pharmingen), we demonstrated expression of PSGL-1 on our cloned Th1 cells (Figure 4). PSGL-1 expression was also demonstrated in three additional clones that we tested: 1A4, 14M4, and 7C11. We also determined that antibody at 10 µg/ml (1 µg per 16 cells) is a saturating concentration in our flow cytometry analysis (i.e., no decrease in fluorescence intensity was seen with serial dilution of antibody down to 0.625 µg/ml).



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  		Figure 4. Flow cytometric analysis of Th1 cells for PSGL-1. Th1 cells (8F5) were stained with rat mAb to PSGL-1 or isotype control mAb and then developed with FITC-labeled goat anti-rat IgG. Both resting (A) and in vitro activated (B) Th1 showed similar positive staining for PSGL-1. PSGL-1 staining was completely eliminated by enzyme treatment of the Th1 cells with OSGP.

 
Enzyme Cleavage of Th1 Cell Ligands with OSGP or Neuraminidase Does Not Eliminate Th1 Cell Binding to P- and E-Selectin-Ig In Vitro
To determine the functional role of PSGL-1 on Th1 cell (Clone 8F5), the cells were treated with OSGP or neuraminidase. Desialylation with neuraminidase is expected to abolish binding by sialylated molecules, including PSGL-1 and related molecules (7). OSGP recognizes O-linked carbohydrate groups and cleaves the polypeptide chain nearby. PSGL-1, but not CLA or ESL-1, is susceptible (7, 25, 34–37).

We verified that enzyme treatment with OSGP resulted in complete loss of staining with mAb to PSGL-1 (Figure 4). Treatment with OSGP or neuraminidase resulted in reduction of high-intensity surface staining with P-selectin–Ig in resting cells. However, substantial P-selectin–Ig binding was retained in the entire cell population (Figure 5A). In activated cells, binding of P-selectin–Ig was reduced, but not eliminated (Figure 5B). After OSGP exposure, no loss of E-selectin–Ig binding was observed in either resting or activated Th1 cells (Figure 5C) (only resting cells are illustrated). Similar results were obtained with other T cell clones (1A4, 14M4, and 7C11; not shown). In repeated experiments in which Th1 cells were exposed to OSGP, we confirmed that staining with mAb to PSGL-1 was completely eliminated in both resting and activated cells, whereas binding to E- and P-selectin–Ig was retained. These results suggest that Th1 cell interactions with E- or P-selectin–Ig, in part, involve receptors distinct from PSGL-1.



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  		Figure 5. P-selectin–Ig fusion protein binding to Th1 cells. Th1 cells (Clone 8F5) were incubated with OSGP, neuraminidase, or media only. Cells were then incubated with murine P-selectin–Ig followed by PE-labeled antibody to human Ig. Controls were incubated with human IgG and the PE-labeled secondary antibody. (A) Resting Th1 cells (no enzyme treatment; heavy line) showed a bright staining population that was decreased in intensity by either OSGP (fine line) or neuraminidase (stippled line). However, a less intensively staining population was retained. (B) In vitro activated 8F5 cells also showed bright staining. Staining was decreased by treatment with neuraminidase or OSPG. However, a population of cells staining with P-selectin–Ig was still present. (C) Resting 8F5 cells were incubated with OSGP, followed by murine E-selectin–Ig and PE-labeled antibody to human Ig. Staining of the cells was not different in OSGP exposed cells.

 
Effect of Neutralizing mAb to PSGL-1 on Th1 Cell Binding to P-Selectin–Ig by Flow Cytometry
Because OSGP treatment of cells could be incomplete or have effects on ligands other than PSGL-1, we used a neutralizing mAb (clone 2PH1) to block PSGL-1. To confirm the neutralizing activity of the mAb to PSGL-1, we stimulated murine splenocytes with concanavalin A and determined that they express PSGL-1 by flow cytometry (not shown). We then used the splenocytes in a P-selectin–Ig adherence assay. The cells were fluorescence labeled and then incubated with PSGL-1 mAb or rat IgG isotype control. After incubation with control antibody, 21.5 ± 2.0% of the fluorescence-labeled cells adhered to P-selectin–Ig. Incubation with PSGL-1 mAb (10 µg/ml) reduced adherence to 7.0 ± 1.6%, which is similar to background adherence to plates coated with human Ig (5.4 ± 0.6%) (percentage values are mean ± SD, n = 8). This nearly complete neutralizing effect of PSGL-1 mAb was also seen at lower mAb concentrations of 1 and 5 µg/ml.

Th1 cells were incubated with the neutralizing mAb to PSGL-1, and then their ability to bind P-selectin–Ig was tested by flow cytometric analysis(21,38). No significant decrease in P-selectin-Ig binding was detected. Experiments with other T cell clones (1A4, 14M4, and 7C11) revealed similar results (not shown). Although these results were reproducible, we would have expected some mAb-mediated decrease in T cell binding to P-selectin–Ig based on our analysis of OSGP treatment and analysis of the neutralizing capacity of the mAb described above. It remains possible that in this static assay, the binding kinetics of P-selectin–Ig and mAb are such that functional PSGL-1 binding is underestimated.

Th1 Cell Adherence to P- and E-Selectin–Ig under Rotating Nonstatic Conditions
In cell adhesion assays, Th1 cells bound to immobilized P-selectin–Ig (Figure 6). Treatment with neutralizing mAb to PSGL-1 (10 µg/ml) partially decreased adherence of resting cells to P-selectin–Ig (compared with cells treated with isotype control mAb; P < 0.001), but did not decrease adherence of activated cells (three experiments). Treatment of the cells with OSGP decreased adherence of both resting and activated cells to P-selectin–Ig (compared with untreated cells; P < 0.00001). The percentage of cells adhering was low, but OSGP treatment did not totally abolish cell adherence to P-selectin–Ig compared with background levels (OSGP-treated cells binding to IgG) in three separate experiments (P < 0.002). Even allowing for multiple comparisons, by the Bonferroni correction, the difference is significant (P < 0.01).



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  		Figure 6. In vitro adherence of Th1 cells to P-selectin–Ig. Th1 cells were labeled with 5(6)-CFDA, SE. The cells were then incubated with mAb to PSGL-1, isotype control mAb, media only, or OSGP. Resting (A) or activated (B) Th1 cells were added to 96-well plates coated with human IgG or P-selectin–Ig. The plates were incubated for 20 min at 4°C under rotating conditions, unbound cells were removed, and fluorescence per well was determined. Values are mean ± SEM (n = 8 wells). Open bars, untreated; striped bars, OSGP; dotted bars, rat IgG; solid bars, PSGL-1 mAb.

 
The Th1 cells also adhered to E-selectin–Ig (Figure 7). Neutralizing antibody to PSGL-1 did not decrease adherence of resting cells to E-selectin–Ig, but partially decreased adherence of activated cells to E-selectin–Ig (P < 0.001). Treatment with OSGP partially decreased adherence of both resting and activated cells to E-selectin (P < 0.001 under both conditions), but with substantial adherence above background (binding to IgG) remaining. The results are representative of three separate experiments.



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  		Figure 7. In vitro adherence of Th1 cells to E-selectin-Ig. Resting (A) or activated (B) Th1 cells were analyzed as described in Figure 6 except plates were coated with E-selectin–Ig. Open bars, untreated; striped bars, OSGP; dotted bars, rat IgG; solid bars, PSGL-1 mAb.

 
Neither Enzyme Cleavage of Th1 Cell Ligands with OSGP nor Neutralizing mAb to PSGL-1 Inhibits Localization of 51Cr-Labeled Th1 Cells in Lung In Vivo
To determine if our in vitro observations translated into similar in vivo findings, we treated 51Cr-labeled Th1 cells with (i) OSGP or medium only (control) or (ii) mAb to PSGL-1 or isotype control mAb. Neither treatment significantly decreased Th1 cell localization in lung 1 h after injection (Figures 8 and 9). Th1 cell adherence in lung was not reduced at later times (3, 24 h) after injection (not shown). These results are representative of two experiments with both OSGP and mAb to PSGL-1.



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  		Figure 8. In vivo localization of 51Cr-labeled Th1 cells after OSGP digestion. Resting Th1 cells (A) or in vitro activated Th1 cells (B) were labeled with 51Cr, incubated with OSGP or control medium, and administered to C57BL/6 mice (n = 4 mice/group). Organs were removed after 15 min or 1 h, and radioactivity was measured. No significant differences were observed after OSGP treatment of cells. Values, expressed as cpm/mg tissue, are mean ± SEM (n = 4). Open bars, control; solid bars, OSGP-treated T cells.

 


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  		Figure 9. In vivo localization of 51Cr-labeled Th1 cells after incubation with neutralizing mAb to PSGL-1. Resting Th1 cells (A) or in vitro activated Th1 cells (B) were labeled with 51Cr, incubated with neutralizing mAb to PSGL-1 or isotype control mAb, and administered to C57BL/6 mice (n = 4 mice/group). Organs were removed after 1 h, and radioactivity was measured. No significant differences were observed after mAb treatment of cells. Values, expressed as cpm/mg tissue, are mean ± SEM (n = 4). Open bars, isotype control; solid bars, PSGL-1 mAb.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We recently described a murine model of Th1 cell induced lung injury in which adoptive transfer of cloned alloreactive Th1 cells produces selective pulmonary inflammation (26, 27). The alloreactive Th1 cells recognize Ly5, an antigen expressed exclusively on hematopoietic cells. Two forms of Ly5 exist in mice, Ly5a and Ly5b. Adoptive transfer of Ly5a-specific Th1 cells into Ly5a mice, but not Ly5b mice, produces lung inflammation characterized by vasculitis, alveolitis, and interstitial pneumonitis.

In previous experiments using bone marrow chimeric mice (Ly5a marrow into Ly5b mice and vice versa), we established that antigen expression on hematopoietic cells alone was sufficient for induction of lung injury (26). We also determined that the adoptively transferred Th1 cells are preferentially localized in lung for at least 24 h after and might thereby elicit an inflammatory response selectively in lung. Other investigators have suggested that the migration kinetics and adherence of cloned cells in lung may be an important factor in determining organ-specific inflammatory responses (39). Previous studies also showed that some cloned T cells localize in lung after administration (40–42). Studies of the migratory behavior of T cells freshly isolated from lymphoid tissue also indicate that memory T cells (expressing low levels of CD45RB, low levels of L-selectin or high levels of CD44) traffic preferentially to lung, whereas naive T cells accumulate in lymph nodes (43). Because the size (by light scatter profile) of the T cells was similar in the two populations, an increased affinity of memory cells for the lung capillary bed was suggested. The mechanisms underlying these observations have not been elucidated.

In this study, we have established a role for both P- and E-selectin in Th1 lymphocyte adherence in lung. In vitro the Th1 cells bound to both P- and E-selectin molecules as we detected by flow cytometry and in nonstatic adherence assays. In vivo localization of the adoptively transferred Th1 cells in lung was markedly reduced in mice with P- and E-selectin deficiency. The reduction in Th1 cell localization was most notable in double-deficient mice, suggesting a cooperative functioning of P- and E-selectin as has been shown with lymphocyte migration in skin models of contact hypersensitivity (10, 44). However, these models involve inflammatory stimulation of the skin tissue and localized increase in vascular P- and E-selectin. In our model, Th1 cells are administered to mice under unstimulated conditions. It is likely that constitutive expression of P-selectin in lung, as reported by others (45, 12) facilitates selective adherence in lung. Constitutive E-selectin expression has not been demonstrated, but could be present at low but functional levels.

Despite the substantial reduction of Th1 lymphocyte adherence in the lungs of double P- and E-selectin–deficient mice, significant Th1 cell adherence was still detected. This result leads to the speculation that other adhesion molecules previously thought to be involved in only firm adhesion, such as intercellular adhesion molecule-1 (ICAM-1), can substitute for or cooperate with the selectins in the deficient mice (46, 47). In previous studies, we have detected constitutive expression of ICAM-1 in mouse lung (33). We also showed in our model that adherence and retention of adoptively transferred (activated) Th1 lymphocytes was impaired when leukocyte function antigen-1 (LFA-1) was neutralized or when ICAM-1 was deficient (33). Other Th1 cell ligands, such as VLA-4 and CD44, might also be involved in lymphocyte adherence in lung. Although neutralizing antibodies to these individual ligands do not impair Th1 cell migration to lung in our model (33) (Clark and colleagues, unpublished data), a possible cooperative role with the selectins has not been excluded.

PSGL-1 has been extensively investigated as a major ligand that mediates neutrophil rolling on the vascular endothelium (25). More recently, PSGL-1 expression on lymphocytes (Th1 phenotype) has been reported and shown to be involved in binding to both P- and E-selectin in a model of skin contact hypersensitivity, but PSGL-1–independent ligands were also suggested (23). However, both the in vivo function of PSGL-1 and the existence of PSGL-1–independent ligands on lymphocytes are the object of ongoing research. Part of the debate relates to the realization that expression of selectin ligands on cells does not predict that they will be used, and in vitro assays do not necessarily predict function in vivo (17). In addition, requirements for selectin ligands in cell trafficking may differ in different vascular beds. For example, core 2 oligosaccarides mediate neutrophil recruitment to the peritoneum, but not lung, in response to lipopolysaccaride (48).

We detected PSGL-1 expression on our Th1 cells as well as binding of both P- and E-selectin–Ig chimeric proteins by the Th1 cells by flow cytometry. However, neither enzyme digestion nor neutralizing mAb to PSGL-1 eliminated binding to the selectin chimeras. Although neutralizing mAb arguably may not be completely effective, we demonstrated complete loss of PSGL-1 staining after enzyme digestion with OSGP. Because the detection antibody is to the glycoprotein core essential for PSGL-1 function, the results suggest a PSGL-1–independent ligand(s) for both P- and E-selectin. These results were partially confirmed in a nonstatic adherence assay in which we observed binding of Th1 lymphocytes to both P- and E-selectin. The Th1 cell binding to E-selectin was modestly reduced by enzymatic pretreatment of the cells with OSGP or neutralizing mAb to PSGL-1, consistent with previous reports that PSGL-1 is a ligand for E-selectin, but that PSGL-1–independent ligands for E-selectin are present (23). Neutralizing Ab to PSGL-1 also decreased Th1 cell binding to P-selectin, but to a lesser extent than expected. Enzyme pretreatment of the Th1 cells substantially reduced cell binding to P-selectin, but statistically significant binding above background control binding was still present in three separate experiments. The results are consistent with significant functional binding to P-selectin that is OSGP-sensitive. In addition, the significant binding in the presence of mAb to PSGL-1 or after OSGP suggests that PSGL-1–independent ligands are expressed, consistent with our flow cytometry analyses.

Our in vivo experiments also indicate that PSGL-1–independent ligands play an important functional role in adherence of Th1 lymphocytes to lung vasculature. Neither neutralizing mAb to PSGL-1 nor OSPG pretreatment of the Th1 cells before adoptive transfer diminished the adherence of resting or activated Th1 cells in lung. It is unlikely that LFA-1 and ICAM-1 are substituting for the selectin ligand interactions in these experiments because LFA-1 neutralization decreased adherence of only activated Th1 cells in our previous studies. Taken together with our in vitro studies, we think that PSGL-1–independent ligands, interacting with P- and/or E-selectin, account for the preserved ability of the Th1 cells to adhere to lung. The preponderance of our results suggest that although PSGL-1 is an important ligand for P-selectin, a PSGL-1–independent ligand which may interact primarily with E-selectin, provides overlapping functional binding of Th1 cells to the lung vasculature. The role of PSGL-1 independent ligand interactions with both E- and P-selectin, as well as the contribution of LFA-1 and ICAM-1 to lymphocyte trafficking to the lung vasculature, warrant further study. In addition, the molecular nature of selectin ligands other than PSGL-1 needs to be determined.


   Acknowledgments
 
The authors thank Takako Hirata (Boston, MA) for providing selectin-IgG chimeric proteins, Paul J. Martin (Seattle, WA) for providing additional T cell clones, and Heather Peake for assistance with preparation of the manuscript. This work was supported by National Institutes of Health Grants R01 HL55200, R01 HL69422, K32 HL07237, R01 AR46404 and an American Heart Association Grant In Aid.

Received in original form May 30, 2003

Received in final form August 8, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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