Adherence of adoptively transferred alloreactive Th1 cells in lung: partial dependence on LFA-1 and ICAM-1

Anne E. Dixon1,2, Janis B. Mandac1,2, Paul J. Martin2,3, Robert C. Hackman2,4, David K. Madtes1,2, and Joan G. Clark1,2

1 Division of Pulmonary and Critical Care Medicine, 3 Division of Oncology, Department of Medicine, and 4 Department of Pathology, University of Washington, Seattle 98195; and 2 Fred Hutchinson Cancer Research Center, Seattle, Washington 98109


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

T helper type 1 (Th1) cells are important effectors in a number of immune-mediated lung diseases. We recently described a murine model of lung injury induced by adoptive transfer of cloned alloreactive Th1 cells. To investigate mechanisms that result in injury to the lung, we studied the in vivo distribution of 51Cr-labeled Th1 cells. One hour after intravenous administration, >85% of injected radioactivity was left in the lung, and at 24 h, 40% of radioactivity was left in the lung. Adherence of Th1 cells in the lung was significantly inhibited by neutralizing antibody to lymphocyte function-associated antigen-1. Th1 cell adherence also was decreased in lungs of mice deficient in intercellular adhesion molecule-1 (ICAM-1). Th1 cell transfer further induced expression of ICAM-1 and vascular cell adhesion molecule-1 in the lung. Vascular cell adhesion molecule-1-immunoreactive protein was markedly induced in lung endothelium by alloreactive Th1 cells. These findings indicate that Th1 cells localize in normal lung by a mechanism involving lymphocyte function-associated antigen-1 and ICAM-1. Alloreactive cells further induce endothelial adhesion molecules that may facilitate recruitment of inflammatory cells to the lung and amplify Th1 cell-induced lung injury.

lymphocyte function-associated antigen-1; intercellular adhesion molecule-1; T helper type 1 and type 2 cells; homing; cell trafficking


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE DEFINITION of T helper (Th) subsets by their pattern of cytokine production has provided a framework to study mechanisms involved in immune-mediated lung injury. Th1 cells [producing interferon-gamma , interleukin-2 (IL-2), and members of the tumor necrosis factor family] have been implicated in a number of disease processes affecting the lung, including sarcoid, hypersensitivity pneumonitis, graft versus host disease, and autoimmune diseases such as Wegener's granulomatosis (2, 29, 33). However, the mechanisms by which Th1 cells cause lung inflammation are incompletely understood.

Chen et al. (12) and Clark et al. (15) recently described a murine model of Th1 cell-induced lung injury in which adoptive transfer of cloned alloreactive Th1 cells produced selective pulmonary inflammation. 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 into Ly5b mice produces lung inflammation characterized by vasculitis, alveolitis, and interstitial pneumonitis.

In previous experiments with bone marrow chimeric mice (Ly5a marrow into Ly5b mice and vice versa), Chen et al. (12) established that antigen expression on hematopoietic cells alone was sufficient for induction of lung injury. The mechanisms by which Th1 cell recognition of an antigen found throughout the hematopoietic system, but not on lung parenchymal cells, causes inflammation selectively in the lung are not known. However, the migration kinetics and adherence of cells in the lung may be an important factor in determining organ-specific inflammatory responses (10). Although it has been recognized that some cloned T cells localize in the lung after administration, the mechanisms underlying this observation have not been elucidated (16, 20, 28).

Adherence of lymphocytes within the vasculature involves a stepwise interaction between molecules on the surface of lymphocytes and adhesion molecules on the endothelium. The selectin family of adhesion molecules mediates transient rolling and tethering of lymphocytes on endothelial surfaces. Chemokines may then cause a rapid increase in affinity of adhesion molecules before stable adhesion of lymphocytes on endothelial cells, which requires the interaction of lymphocyte integrins [such as lymphocyte function-associated antigen-1 (LFA-1) and very late activating antigen-4 (VLA-4)] with endothelial adhesion molecules of the immunoglobulin superfamily [intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)] (5, 35). The selectins have been implicated in lymphocyte migration to the lymph nodes (36), skin (4), and gut (5), and recent evidence suggests that induction of selectins on the pulmonary vascular endothelium may be involved in lymphocyte influx in response to particulate antigen challenge in sensitized mice (37). The alpha 4-integrins are involved in lymphocyte recruitment to the brain (6), skin (14), and pancreas (7), and evidence exists for VLA-4 and VCAM-1 involvement in lymphocyte trafficking to the lung after antigenic challenge (1, 19, 37). LFA-1 and its endothelial counterreceptor have been implicated in lymphocyte recruitment to the skin (32), liver (25), and kidney (11) and also to the lung in models of allergic airway disease (13).

Several studies (3, 13, 38) have documented enhanced expression of adhesion molecules in the pulmonary vasculature during inflammation. The induction of adhesion molecules by cytokines elaborated in response to an initiating inflammatory challenge may amplify the inflammatory response by further facilitating leukocyte migration to the lung. However, the mechanisms by which leukocytes, including memory T cells, initially traffic to the lung are not well understood.

The present study was performed to examine the trafficking of adoptively transferred Th1 cells and its relationship to lung injury. We examined the contribution of L-selectin and VLA-4 and the interaction between LFA-1 and ICAM-1 in the adherence of Th1 cells in the lung. We studied the expression and induction of the vascular adhesion molecules VCAM-1 and ICAM-1 to understand their role in the initial adherence of lymphocytes in the lung and the changes that would facilitate subsequent recruitment of inflammatory cells to the lung.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice. C57BL/6 (Ly5b), Ly5a, and ICAM-1-deficient mice (40) were obtained from Jackson Laboratory (Bar Harbor, ME). The ICAM-1-deficient mice were on a C57BL/6 background. The mice were housed in microisolator cages under specific pathogen-free conditions with free access to sterile water and chow.

T cell clones and culture. T cell clones specific for the Ly5a allele were developed and propagated in culture as previously described (12). In brief, Ly5b mice were immunized with 13-mer Ly5a peptides. CD4+ Th cells specific for the 13-mer peptides were elicited and cloned by limiting dilution. The clone was of the Th1 phenotype and produced interferon-gamma and IL-2. T cell clones were maintained by periodic stimulation with Ly5a peptide in the presence of congenic irradiated splenocytes and were maintained in the presence of IL-2 (10 U/ml). Activated cells were harvested 1 day after stimulation, and resting cells were harvested 14 days after stimulation.

Flow cytometry. Th1 cells (106) were incubated in 50 µl of fluorescence-activated cell sorter (FACS) buffer (PBS with 1% FCS and 0.1% NaN3) with 1 µg of primary antibody for 30 min at 4°C. Primary antibodies included MEL-14 rat monoclonal antibody (MAb) to L-selectin (PharMingen, San Diego, CA), PS/2 rat MAb to the alpha 4-integrin chain of VLA-4 (kindly provided by Dr. W. Henderson, University of Washington, Seattle, WA), M17/4 rat MAb to the alpha L-integrin chain of LFA-1 (PharMingen), and rat IgG control (PharMingen). Cells were washed three times in FACS buffer and then stained with the secondary antibody, 1 µg of FITC-conjugated mouse anti-rat IgG (Jackson ImmunoResearch, West Grove, PA) in 50 µl of FACS buffer.

Flow cytometry was performed on a Becton Dickinson FACScan, and data were collected on 104 light-scatter gated events. The forward- and side-scatter properties of the cells were used to exclude dead cells from the analysis. Data were analyzed with CellQuest software (Becton Dickinson, Palo Alto, CA).

In vivo migration and adhesion of adoptively transferred Th1 cells. Cloned Th1 cells were resuspended at 107 cells/ml and labeled with 20 µCi/ml of sodium [51Cr]chromate (NEN, Boston, MA) for 1 h at 37°C. Viable cells were separated from dead cells and free chromium by density centrifugation through Nycodenz (GIBCO BRL, Life Technologies, Gaithersburg, MD) (22). 51Cr-labeled cells (5 × 106/mouse) were administered by tail vein injection. Animals were euthanized, and then the spleen, liver, kidney, thymus, lung, heart, and blood were harvested and weighed. The radioactivity in the organs and an aliquot of the injected cells were measured for 1 min in a gamma scintillation counter (Beckman, Fullerton, CA).

In some experiments, 51Cr-labeled cells (20 × 106) were incubated with either 10 µg of neutralizing MAb to LFA-1 (M17/4; PharMingen), isotype control (Thy-1.2, clone 53-2.1; PharMingen), or 10-40 µg of neutralizing MAb to VLA-4 (PS/2) for 30 min immediately before administration. We generated the F(ab')2 fragment of the LFA-1 and Thy-1.2 antibodies by papain digestion (Sigma, St. Louis, MO) and isolation on a Sephadex G-75 (Pharmacia Biotech, Uppsala, Sweden) column (24). Intact PS/2 antibody was used in this study because the intact antibody and F(ab')2 fragment have been shown to have similar effects in vivo (20). Intact Thy-1.2 antibody was then used as an isotype control.

RT-PCR of ICAM-1 and VCAM-1. To induce lung injury, 107 Ly5a-specific cells were administered to both Ly5a and Ly5b (control) mice. Mice were euthanized after 1, 24, and 48 h (3 mice/group). The lungs were harvested and snap-frozen in liquid nitrogen. RNA was extracted by the TRIzol method (Life Technologies, Grand Island, NY). RNA samples were treated with DNase to ensure complete removal of contaminating DNA. With a one-tube reaction method (Promega ACCESS system), 1 µg of RNA was reverse transcribed followed by immediate PCR. Primer sequences as previously described (27) were synthesized at the Biotechnology Center of the Fred Hutchinson Cancer Research Center (Seattle, WA). A 50-µl reaction contained 10 µl of the RNA template, 0.2 mM deoxynucleotide triphosphate, 1 mM MgSO4, 0.75 µM each sense and antisense primers, 5 U of avian myeloblastosis virus reverse transcriptase, and 5 U of tfl DNA polymerase in 1× avian myeloblastosis virus-tfl reaction buffer. RT was performed by a 45-min incubation at 48°C followed by an amplification cycle profile of denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min, and elongation at 72°C for 2 min for 30 cycles in a Perkin-Elmer thermal cycler (34). The amplified PCR product was visualized by ethidium bromide-agarose gel electrophoresis with an Eagle Eye II still video system (Stratagene, La Jolla, CA) and quantitated by ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Levels of ICAM-1 and VCAM-1 mRNA were normalized to beta -actin mRNA in the same sample.

Immunohistochemistry of ICAM-1 and VCAM-1. VCAM-1 immunohistochemistry was performed on frozen sections of mouse lung obtained from C57BL/6 (control) mice and C57BL/6 mice euthanized 3 days after receiving 107 anti-Ly5b cells. The excised lungs were inflated and embedded in Tissue-Tek optimum cutting temperature compound (Cryoform, IFC, Needham Heights, MA), snap-frozen in liquid nitrogen, and stored at -70°C. The cryosections were fixed in acetone and stained with an antibody to VCAM-1 (clone 429; PharMingen) or an isotype control. Biotinylated goat anti-rat IgG was used as a secondary antibody and was then visualized by streptavidin-peroxidase followed by diaminobenzidine with NiCl2 enhancement. Sections were counterstained with acridine orange-safranin O.

ICAM-1 immunohistochemistry was performed on sections obtained from untreated control C57BL/6 mice and C57BL/6 mice and ICAM-1-deficient mice 2 days after administration of 107 in vitro activated Th1 cells. Excised lungs were inflated, fixed in Formalin, and embedded in paraffin. Sections were incubated with a MAb to ICAM-1 (clone 3E2; PharMingen) and detected with biotinylated goat anti-hamster IgG (Jackson ImmunoResearch). The biotinylated antibody was then visualized by streptavidin-peroxidase followed by diaminobenzidine with NiCl2 enhancement. Sections were counterstained with acridine orange-safranin O.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adoptively transferred Th1 cells distributed preferentially to the lung. To determine the in vivo distribution of adoptively transferred Th1 cells, 51Cr-labeled Ly5a-specific Th1 cells were administered to Ly5a and Ly5b mice. Mice were euthanized at 1, 3, and 24 h, and radioactivity was measured in the blood, liver, spleen, kidney, lung, heart, and thymus. More than 95% of the injected radioactivity was recovered by this method. The distribution of radioactivity in the organs, expressed per milligram of tissue, showed that Th1 cells were preferentially localized in lung tissue (Fig. 1). One hour after instillation, >85% of the counts were in the lung. There was some redistribution over time, but at 24 h, >65% of the recovered radioactivity was still in the lung. The distribution was the same in both Ly5a and Ly5b mice, but only Ly5a mice developed lung injury. In vitro activated 51Cr-labeled cells had the same in vivo distribution after adoptive transfer (data not shown).


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Fig. 1.   Adoptively transferred T helper type 1 (Th1) cells localize in lung. 51Cr-labeled Ly5a-specific Th1 cells were administered to Ly5a and Ly5b mice. Recipients were euthanized after 1 (A and B), 3 (C and D), or 24 (E and F) h, and organs were harvested for measurement of radioactivity. cpm, Counts/min. Values are means ± SE, representative of 2 experiments; n = 4 mice/group. More than 95% of injected radioactivity was recovered. At 1 h, >85% of radioactivity was in the lung. There was some redistribution over time, but at 24 h, 65% of recovered radioactivity was still in the lung. The distribution was similar in both Ly5a and Ly5b animals.

Th1 cells expressed LFA-1 and VLA-4. To identify candidate molecules that might mediate adhesion of Th1 cells in the lung, we analyzed both resting and activated cells for expression of LFA-1, VLA-4, and L-selectin by flow cytometric analysis. Resting cells were positive for the integrins LFA-1 and VLA-4 (Fig. 2). There was no difference in the fluorescence intensity of either LFA-1 or VLA-4 in resting cells compared with that in activated cells. L-selectin expression was not detected in resting or activated cells (data not shown).


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Fig. 2.   Th1 cells express lymphocyte function-associated antigen (LFA)-1 (A) and very late activating antigen (VLA)-4 (B). Resting Th1 cells were incubated with rat monoclonal antibody (MAb) to the integrin alpha L chain (for LFA-1; A, solid line), alpha 4 chain (VLA-4; B, solid line), or isotype control (dashed lines). Cells were then stained with FITC-conjugated goat anti-rat IgG.

Adherence of Th1 cells in lung was reduced by neutralization of LFA-1 or deficiency of ICAM-1. To examine the role of LFA-1 in mediating adherence of resting Th1 cells in lung, 51Cr-labeled Th1 cells preincubated with neutralizing monoclonal antibody to LFA-1 or Thy-1.2 (control) were administered to Ly5a mice. Mice were euthanized at three time points, and the organs were harvested for radioactivity. Neutralizing antibody to LFA-1 did not affect adherence of the cells at 1 or 3 h (Fig. 3A). Neutralizing antibody to LFA-1 significantly reduced adherence of cells in lung at 24 h compared with that in animals injected with untreated cells (P < 0.0005 by one-way ANOVA; Fig. 3A). The F(ab')2 fragment of the LFA-1 antibody similarly reduced the binding of resting cells in the lung at 24 h, excluding the possibility that nonspecific binding (of the intact antibody to Fc receptors) was affecting localization.


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Fig. 3.   Neutralizing MAb to LFA-1 inhibits adherence of Th1 cells in lung. 51Cr-labeled cells (control) or 51Cr-labeled cells incubated with F(ab')2 fragment of antibody to LFA-1 were administered to Ly5a mice. Mice were euthanized, and organs were harvested. A: resting cells. Values are means ± SE, representative of 2 experiments; n = 4 mice. * Adherence of resting Th1 cells in lung was reduced at 24 h by neutralizing antibody to LFA-1 compared with that by F(ab')2 control, P < 0.0005 by 1-way ANOVA. B: in vitro activated cells. Values are means ± SE, representative of 3 experiments; n = 4 mice. * Adherence of in vitro activated Th1 cells in lung was reduced at 1 h by neutralizing antibody to LFA-1 compared with that by isotype control, P < 0.005 by 1-way ANOVA.

To determine whether adherence of activated cells in the lung is dependent on LFA-1, in vitro activated 51Cr-labeled cells were incubated with the F(ab')2 fragment of either LFA-1 or Thy-1.2 (isotype control) and adoptively transferred. Adherence of activated cells in the lung was significantly reduced at 1 h (P < 0.005; Fig. 3B).

To further investigate the receptor-ligand interaction utilized by the Th1 cells, we examined Th1 cell adherence in mice deficient in ICAM-1, the major endothelial ligand for LFA-1. 51Cr-labeled in vitro activated Th1 cells were administered to ICAM-1-deficient and wild-type mice. Adherence of activated cells was significantly reduced in ICAM-1-deficient mice at 1 h (P < 0.005; Fig. 4). The histopathological response detected 1-3 days after Th1 cell transfer (i.e., mononuclear cell vasculitis and alveolitis) was only modestly reduced in the ICAM-1-deficient mice, suggesting that other adhesion pathways may compensate or dominate in the evolution of the inflammatory response.


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Fig. 4.   Adherence of activated Th1 cells in lung is reduced in intercellular adhesion molecule (ICAM)-1-deficient mice. In vitro activated 51Cr-labeled cells were administered to ICAM-1-deficient [knockout (KO)] and wild-type (control) mice. Values are means ± SE, representative of 2 experiments; n = 3 mice. * Adherence of Th1 cells in lung was significantly reduced at 1 h in the ICAM-1-deficient mice compared with that in control mice, P < 0.005 by 1-way ANOVA.

To examine the role of VLA-4 in the adherence of Th1 cells in lung, 51Cr-labeled cells were incubated with neutralizing antibody to VLA-4 or isotype control before adoptive transfer into mice. Neutralizing MAb to VLA-4 did not affect adherence of resting or activated cells in lung at 1 or 24 h (data not shown).

Administration of Th1 cell clone induced expression of vascular adhesion molecules. VCAM-1 and ICAM-1 are the endothelial ligands for VLA-4 and LFA-1, respectively. To assess the induction of VCAM-1 and ICAM-1 expression after Th1 cell transfer, we investigated the expression of endothelial adhesion molecule VCAM-1 and ICAM-1 mRNAs by RT-PCR in the lung of Ly5a and Ly5b mice at intervals after administration of 107 Ly5a-specific Th1 cells. VCAM-1 mRNA levels normalized to beta -actin mRNA were increased at 24 and 48 h in Ly5a mice compared with those in control Ly5b mice (P < 0.01 at 24 h; P < 0.0001 at 48 h; Fig. 5A). ICAM-1 mRNA was increased 1 h after transfer of cells into Ly5a animals compared with that in Ly5b animals and was persistently increased at 24 and 48 h (P < 0.05; Fig. 5B).


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Fig. 5.   Vascular cell adhesion molecule (VCAM)-1 (A) and ICAM-1 (B) mRNA levels were induced by administration of Ly5a-specific Th1 cells into Ly5a mice. VCAM-1 and ICAM-1 levels were analyzed by RT-PCR in both Ly5a and Ly5b animals after adoptive transfer of anti-Ly5a cells. The levels were normalized to beta -actin. Values are means ± SE; n = 3 mice. A: VCAM-1 message levels were increased in Ly5a mice compared with those in Ly5b mice. * P < 0.01 at 24 h. * P < 0.001 at 48 h (both by 1-way ANOVA). B: ICAM-1 levels were increased in Ly5a mice compared with those in Ly5b mice. * P < 0.05 at each time point by 1-way ANOVA.

To examine the potential role of VCAM-1 and ICAM-1 in the initial adherence of Th1 cells as well as the ensuing inflammatory response, we examined VCAM-1 and ICAM-1 by immunohistochemistry. In the normal lung, VCAM-1 was detectable only in rare, isolated endothelial cells. In lung sections obtained 3 days after administration of alloreactive cells, VCAM-1 expression was dramatically increased and specifically localized in the endothelium associated with inflammatory foci (Fig. 6).


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Fig. 6.   Immunohistochemical detection of VCAM-1 in mouse lung. Frozen sections of mouse lung obtained from normal Ly5b mice or mice that were euthanized 3 days after receiving 107 anti-Ly5b cells were stained with MAb to VCAM-1 or MAb control. No staining was present with isotype control antibody (A; original magnification, ×40). Faint VCAM-1 staining of individual endothelial cells was present in normal lung (B; original magnification, ×25). Intense VCAM-1 staining was detected in the endothelium of T cell-treated mice, and this was associated with perivasculitis (C and D; original magnifications: ×25 and ×40, respectively).

ICAM-1 was expressed at high levels in the alveolar structures and vascular endothelium of normal untreated lungs. No ICAM-1 was detected in lung sections from ICAM-1-deficient mice stained in exactly the same manner. We did not detect major changes in the pattern of ICAM-1 expression by this method after Th1 cell administration (Fig. 7).


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Fig. 7.   Immunohistochemical detection of ICAM-1 in mouse lung. Sections were obtained from wild-type mice and embedded in paraffin. Sections were also obtained from both wild-type and transgenic mice deficient in ICAM-1 3 days after administration of 107 activated T cells. ICAM-1 staining was absent in ICAM-1-deficient mice (A and B; original magnifications: ×10 and ×20, respectively). Sections from wild-type untreated mice had widespread staining; ICAM-1 was detected throughout the alveolar epithelium, and endothelial staining was also noted (C and D; original magnifications: ×10 and ×20, respectively). In lung sections of wild-type mice injected with alloreactive Th1 cells, there was also widespread staining of the alveolar epithelium and vascular endothelium, but inflammatory cells in the perivascular infiltrate did not stain for ICAM-1 (E and F; original magnifications: ×10 and ×40, respectively).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study was designed to investigate the mechanisms for selective lung injury induced by adoptive transfer of alloreactive Th1 cells. The results demonstrate that Th1 cells distribute preferentially to the lung and that this adherence depends partly on the interactions between adhesion molecules expressed by Th1 cells and by the pulmonary vascular endothelium. The cloned Th1 cells express the integrin adhesion molecules LFA-1 and VLA-4 but not L-selectin. The initial adherence of in vitro activated cells in the lung was reduced by neutralizing antibody to LFA-1 and in ICAM-1-deficient mice. Adherence of Th1 cells in the lung was unaffected by neutralizing VLA-4. Our studies also demonstrated that the endothelial adhesion molecule VCAM-1 was expressed at low levels in the normal lung but was rapidly upregulated after Th1 cell administration. In contrast, ICAM-1 was constitutively expressed at high levels throughout the pulmonary epithelial and endothelial surfaces. ICAM-1 mRNA was further upregulated by administration of Th1 cells.

Other investigators (8, 31) have described similar trafficking of cloned T cells to the lung, but the mechanisms for this have not been previously delineated. Based on the high level of radiolabeled T cells in the lung immediately after intravenous administration, some (9, 28) have suggested that the cells are simply "trapped" in the pulmonary vasculature. However, the Th1 cells in our study were not merely retarded in transit through lung; their retention was stable over time. Twenty-four hours after administration of Th1 cells, 65% of recovered radioactivity was still localized in the lung. In addition, as other investigators (16) have also noted, the same distribution pattern was present after administration of resting or in vitro activated cells despite the much larger size of activated cells.

We found that ICAM-1, the major endothelial ligand for LFA-1, was expressed constitutively on normal pulmonary vascular endothelium and that adherence of activated Th1 cells in the lung is dependent on the interaction between LFA-1 and ICAM-1 as suggested in a previous report (21). The adherence of resting adoptively transferred cells was unaffected by neutralizing LFA-1 until 24 h after transfer. Taken together, the results suggest that LFA-1 on resting Th1 cells is in a low-affinity state and is not utilized for initial adherence but may be involved in later recruitment or retention of Th1 cells in the lung that become activated in vivo. In vitro activated cells utilize LFA-1 for initial adherence to ICAM-1, most likely because activation increases LFA-1 affinity (26). The initial adherence of Th1 cells was only partially dependent on LFA-1 and ICAM-1. Recent research has identified a number of vascular adhesion molecules (e.g., selectins) and T cell ligands (e.g., P-selectin glycoprotein ligand-1, CD44) that might contribute to lymphocyte adherence in tissue (4, 35, 37-39). Ongoing work in our laboratory suggests that both P- and E-selectins may participate in the initial adherence of adoptively transferred Th1 cells in our model.

We did not demonstrate a role for the VLA-4 integrin in the initial adherence of the Th1 cell clone in the lung. This finding likely is due in part to absent or low-level expression of the endothelial receptor VCAM-1 on normal resting lung endothelium. However, VLA-4 and its endothelial counterreceptor VCAM-1 have been implicated in lymphocyte recruitment to the lung in models of pulmonary inflammation such as intratracheal challenge with sheep red blood cells (37). Intratracheal instillation of the blood cells causes a rapid induction of endothelial VCAM-1 (38) and may then be important in facilitating leukocyte recruitment to the lung. We demonstrated a similar rapid induction of VCAM-1 expression after administration of Th1 cells. Although we were unable to demonstrate a role for VLA-4 and VCAM-1 interactions in the initial adherence of the Th1 cell clone, VCAM-1 may be important in the amplification of Th1 cell-induced inflammation that involves recruitment of recipient mononuclear cells to the lung.

The adherence of Th1 cells in the lung may be necessary for lung injury in our model, but it is not sufficient. Ly5a-specific cells but not Ly5b-specific cells induce lung injury in Ly5a mice. Other models of lung injury induced by adoptive transfer of antigen-specific T cells have been described (18, 23, 33). In these models, the antigen is predominantly or exclusively present in the lung, and homing of transferred cells to the lung could be caused by the presence of antigen. Our results suggest an alternative interpretation. Antigen-specific "memory" T cells may preferentially circulate to the lung by intrinsic mechanisms involving specific adhesion molecules. If antigen is encountered, effector mechanisms come into play. If not, the cells circulate to regional lymph nodes or other sites. Although this pathway may be apparent after adoptive transfer of cells through an intravenous route, it could also be important for recirculation of memory lymphocytes and immune surveillance (30, 31).

Cytotoxic CD8 cells may be similarly classified as type 1 (Tc1) or type 2 (Tc2) according to their cytokine profile. An adoptive transfer study (17) indicated that the preferential adherence of Tc1 cells in the lung may enhance clearance of viral infections and pulmonary metastases (17). This suggests that intrinsic trafficking patterns of lymphocyte subsets may have important functional implications for novel therapeutic approaches using adoptive immunotherapy for treatment of viral infections and malignancies.

T cells play a central role in a number of pulmonary diseases as well as mediating normal host defense. Elucidating the mechanisms by which T cells and T cell subsets incite pulmonary immune responses is essential to understanding these diseases. Th2 cells in the lung have been the subject of much recent interest because these cells may augment airway hyperactivity and inflammation characteristic of asthma. Efforts to ameliorate Th2-induced responses by adoptive transfer of Th1 cells have been thwarted by the finding of exacerbated parenchymal injury induced by Th1 cells (23). The mechanisms underlying this injury are incompletely understood, but trafficking of adoptively transferred Th1 lymphocytes to the lung with subsequent activation and inflammation may be important. A recent study (39) suggested that trafficking patterns of Th1 cells are different from those of Th2 cells, a characteristic that may depend on adhesion molecule and chemokine receptor expression.

In conclusion, we have shown that cloned alloreactive Th1 cells localize in the lung by a mechanism involving LFA-1 and ICAM-1 and then induce expression of vascular adhesion molecules, leading to a progressive mononuclear vasculitis, alveolitis, and interstitial pneumonitis. The further study of adherence of T cell subsets in lung and the mechanisms by which they provoke an immune reaction may provide important insights into the pathogenesis of immune-mediated pulmonary diseases.


    ACKNOWLEDGEMENTS

We thank Mary Beauchamp, Andrew Elston, and Caroline Sawe for technical advice and assistance and Heather Peake for assistance with preparation of the manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-55200 and K32-HL-07237

Address for reprint requests and other correspondence: J. G. Clark, Pulmonary and Critical Care Medicine, Mailstop D3-190, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Seattle, WA 98109 (E-mail: jclark{at}fhcrc.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 14 February 2000; accepted in final form 31 March 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abraham, WM, Sielczak MW, Ahmed A, Cortes A, Lauredo IT, Kim J, Pepinsky B, Benjamin CD, Leone DR, Lobb RR, and Weller PF. alpha 4-Integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep. J Clin Invest 93: 776-787, 1994[ISI][Medline].

2.   Agostini, C, Cassatella M, Zambello R, Trentin L, Gasperini S, Perin A, Piazza F, Siviero M, Facco M, Dziejman M, Chilosi M, Quin S, Luster AD, and Semenzato G. Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. J Immunol 161: 6413-6420, 1998[Abstract/Free Full Text].

3.   Anderson, JA, Lentsch AB, Hadjiminas DJ, Miller FN, Martin AW, Nakagawa K, and Edwards MJ. The role of cytokines, adhesion molecules, and chemokines in IL-2-induced lymphocytic infiltration in C57BL/6 mice. J Clin Invest 97: 1952-1959, 1996[Abstract/Free Full Text].

4.   Austrup, F, Vestweber D, Borges E, Lohning M, Brauer R, Herz U, Renz H, Scheffold A, Radbruch A, and Hamann A. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues. Nature 385: 81-83, 1997[ISI][Medline].

5.   Bargatze, RF, Jutila MA, and Butcher EC. Distinct roles of l-selectin and integrins alpha 4beta 7 and LFA-1 in lymphocyte homing to Peyer's patch-HEV in situ: the multistep model confirmed and refined. Immunity 3: 99-108, 1995[ISI][Medline].

6.   Baron, JL, Madri JA, Ruddle NH, Hashim G, and Janeway CR. Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma. J Exp Med 177: 57-68, 1993[Abstract].

7.   Baron, JL, Reich EP, Visintin I, and Janeway CA. The pathogenesis of adoptive murine autoimmune diabetes requires an interaction between alpha 4-integrins and vascular cell adhesion molecule-1. J Clin Invest 93: 1700-1708, 1994[ISI][Medline].

8.   Berman, JS, Beer DJ, Theodore AC, Kornfeld H, Bernardo J, and Center DM. Lymphocyte recruitment to the lung. Am Rev Respir Dis 142: 238-257, 1990[ISI][Medline].

9.   Carroll, AM, Palladino MA, Oettgen H, and De Sousa M. In vivo localization of cloned IL-2-dependent T cells. Cell Immunol 76: 69-80, 1983[ISI][Medline].

10.   Cerwenka, A, Morgan TM, Harmsen AG, and Dutton RW. Migration kinetics and final destination of type 1 and type 2 CD8 effector cells predict protection against pulmonary virus infection. J Exp Med 189: 423-434, 1999[Abstract/Free Full Text].

11.   Chakravorty, SJ, Howie AJ, Cockwell P, Adu D, and Savage CO. T lymphocyte adhesion mechanisms within inflamed human kidney: studies with a Stamper-Woodruff assay. Am J Pathol 154: 503-514, 1999[Abstract/Free Full Text].

12.   Chen, W, Chatta GS, Rubin WD, Clark JG, Hackman RC, Madtes DK, Liggitt DH, Kusunoki Y, Martin PJ, and Cheever MA. T cells specific for a polymorphic segment of CD45 induce graft-versus-host disease with predominant pulmonary vasculitis. J Immunol 161: 909-918, 1998[Abstract/Free Full Text].

13.   Chin, JE, Winterrowd GE, Hatfield CA, Brashler JR, Griffin RL, Vonderfecht SL, Kolbasa KP, Fidler SF, Shull KL, Krzesicki RF, Ready KA, Dunn CJ, Sly LM, Staite ND, and Richards IM. Involvement of intercellular adhesion molecule-1 in the antigen-induced infiltration of eosinophils and lymphocytes into the airways in a murine model of pulmonary inflammation. Am J Respir Cell Mol Biol 18: 158-167, 1998[Abstract/Free Full Text].

14.   Chisholm, PL, Williams CA, and Lobb RR. Monoclonal antibodies to the integrin alpha-4 subunit inhibit the murine contact hypersensitivity response. Eur J Immunol 23: 682-688, 1993[ISI][Medline].

15.   Clark, JG, Madtes DK, Hackman RC, Chen W, Cheever MA, and Martin PJ. Lung injury induced by alloreactive Th1 cells is characterized by host-derived mononuclear cell inflammation and activation of alveolar macrophages. J Immunol 161: 1913-1920, 1998[Abstract/Free Full Text].

16.   Dailey, MO, Fathman CG, Butcher EC, Pillemer E, and Weissman I. Abnormal migration of T lymphocyte clones. J Immunol 128: 2134-2136, 1982[Abstract/Free Full Text].

17.   Dobrzanski, MJ, Reome JB, and Dutton RW. Therapeutic effects of tumor-reactive type 1 and type 2 CD8+ T cell subpopulations in established pulmonary metastases. J Immunol 162: 6671-6680, 1999[Abstract/Free Full Text].

18.   Enlow, RI, Mohammed AZ, Stoler MH, Liu AN, Young JS, Lou Y, and Braciale TJ. Structural and functional consequences of alveolar cell recognition by CD8+ T lymphocytes in experimental lung disease. J Clin Invest 102: 1653-1661, 1998[Abstract/Free Full Text].

19.   Gonzalo, JA, Lloyd CM, Kremer L, Finger E, Martinez-A C, Siegelman MH, Cybulsky M, and Gutierrez-Ramos JC. Eosinophil recruitment to the lung in a murine model of allergic inflammation. The role of T cells, chemokines, and adhesion receptors. J Clin Invest 98: 2332-2345, 1996[Abstract/Free Full Text].

20.   Hamann, A, Andrew DP, Jablonski-Westrich D, Holzmann B, and Butcher EC. Role of alpha 4-integrins in lymphocyte homing to mucosal tissues in vivo. J Immunol 152: 3282-3293, 1994[Abstract/Free Full Text].

21.   Hamann, A, Jablonski-Westrich D, Duijvestifn A, Butcher EC, Baisch H, Harder R, and Thiele HG. Evidence for an accessory role of LFA-1 in lymphocyte-high endothelium interaction during homing. J Immunol 140: 693-699, 1988[Abstract/Free Full Text].

22.   Hamann, A, and Jonas P. Lymphocyte migration in vivo: the mouse model. In: Immunology Methods Manual, edited by Lefkovits I.. San Diego, CA: Academic, 1997, p. 1334-1341.

23.   Hansen, G, Berry G, DeKruyff RH, and Umetsu DT. Allergen-specific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation. J Clin Invest 103: 175-183, 1999[Abstract/Free Full Text].

24.   Harlow, E, and Lane D. Digesting antibodies with papain to isolate Fab fragments. In: Antibodies: A Laboratory Manual, edited by Harlow E, and Lane D.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1988, p. 628-629.

25.   Howell, CD, Li J, and Chen W. Role of intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 during nonsuppurative destructive cholangitis in a mouse graft-versus-host disease model. Hepatology 29: 766-776, 1999[ISI][Medline].

26.   Lasky, LA. How integrins are activated. Nature 390: 81-85, 1997[ISI][Medline].

27.   Lindsey, JW, and Steinman L. Competitive PCR quantification of CD4, CD8, ICAM-1, VCAM-1, and MHC class II mRNA in the central nervous system during development and resolution of experimental allergic encephalomyelitis. J Neuroimmunol 48: 227-234, 1993[ISI][Medline].

28.   Lotze, MT, Line BR, Mathisen DJ, and Rosenberg SA. The in vivo distribution of autologous human and murine lymphoid cells grown in T cell growth factor (TCGF): implications for the adoptive immunotherapy of tumors. J Immunol 125: 1487-1493, 1980[Free Full Text].

29.   Ludviksson, BR, Sneller MC, Chua KS, Talar-Williams C, Langford CA, Ehrhardt RO, Fauci AS, and Strober W. Active Wegener's granulomatosis is associated with HLA-DR+CD4+ T cells exhibiting an unbalanced Th1-type T cell cytokine pattern: reversal with IL-10. J Immunol 160: 3602-3609, 1998[Abstract/Free Full Text].

30.   Mackay, CR, Marston WL, and Dudler L. Naive and memory T cells show distinct pathways of lymphocyte recirculation. J Exp Med 171: 801-817, 1990[Abstract].

31.   McDermott, MR, Lukacher AE, Braciale VL, Braciale TJ, and Bienenstock J. Characterization and in vivo distribution of influenza-virus-specific T-lymphocytes in the murine respiratory tract. Am Rev Respir Dis 135: 245-249, 1987[ISI][Medline].

32.   Schiltz, PM, Giorno RC, and Claman HN. Increased ICAM-1 expression in the early stages of murine chronic graft-versus-host disease. Clin Immunol Immunopathol 71: 136-141, 1994[ISI][Medline].

33.   Schuyler, M, Gott K, Cherne A, and Edwards B. Th1 CD4+ cells adoptively transfer experimental hypersensitivity pneumonitis. Cell Immunol 177: 169-175, 1997[ISI][Medline].

34.   Shankar, G, Bryson JS, Jennings CD, Kaplan AM, and Cohen DA. Idiopathic pneumonia syndrome in mice after allogeneic bone marrow transplantation in mice. Role of pretransplant radiation conditioning. Am J Respir Cell Mol Biol 20: 1116-1124, 1999[Abstract/Free Full Text].

35.   Springer, TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76: 301-314, 1994[ISI][Medline].

36.   Warnock, RA, Askari S, Butcher EC, and Von Andrian UH. Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J Exp Med 187: 205-216, 1998[Abstract/Free Full Text].

37.   Wolber, FM, Curtis JL, Maly P, Kelly RJ, Smith P, Yednock TA, Lowe JB, and Stoolman LM. Endothelial selectins and alpha 4 integrins regulate independent pathways of T lymphocyte recruitment in the pulmonary immune response. J Immunol 161: 4396-4403, 1998[Abstract/Free Full Text].

38.   Wolber, FM, Curtis JL, Milik AM, Fields T, Seitzman GD, Kim K, Kim S, Sonstein J, and Stoolman LM. Lymphocyte recruitment and the kinetics of adhesion receptor expression during the pulmonary immune response to participate antigen. Am J Pathol 151: 1715-1727, 1997[Abstract].

39.   Xie, H, Lim YC, Luscinskas FW, and Lichtman AH. Acquisition of selectin binding and peripheral homing properties by CD4+ and CD8+ T cells. J Exp Med 189: 1765-1776, 1999[Abstract/Free Full Text].

40.   Xu, H, Gonzalo JA, St. Pierre Y, Williams IR, Kupper TS, Contran RS, Springer TA, and Gutierrez-Ramos JC. Leukocytosis and resistance to septic shock in intercellular adhesion molecule 1-deficient mice. J Exp Med 180: 95-109, 1994[Abstract].


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