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The Journal of Immunology, 2001, 166: 293-303.
Copyright © 2001 by The American Association of Immunologists

Bi-Directional Activation Between Human Airway Smooth Muscle Cells and T Lymphocytes: Role in Induction of Altered Airway Responsiveness1

Hakon Hakonarson2,*, Cecilia Kim*, Russel Whelan*, Donald Campbell{dagger} and Michael M. Grunstein*

Divisions of * Pulmonary Medicine and {dagger} Allergy, Immmunologic, and Infectious Diseases, The Joseph Stokes Jr. Research Institute, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because both T lymphocyte and airway smooth muscle (ASM) cell activation are events fundamentally implicated in the pathobiology of asthma, this study tested the hypothesis that cooperative intercellular signaling between activated T cells and ASM cells mediates proasthmatic changes in ASM responsiveness. Contrasting the lack of effect of resting human T cells, anti-CD3-activated T cells were found to adhere to the surface of naive human ASM cells, increase ASM CD25 cell surface expression, and induce increased constrictor responsiveness to acetylcholine and impaired relaxation responsiveness to isoproterenol in isolated rabbit ASM tissues. Comparably, exposure of resting T cells to ASM cells prestimulated with IgE immune complexes reciprocally elicited T cell adhesion to ASM cells and up-regulated T cell expression of CD25. Extended studies demonstrated that: 1) ASM cells express mRNAs and proteins for the cell adhesion molecules (CAMs)/costimulatory molecules, CD40, CD40L, CD80, CD86, ICAM-1 (CD54), and LFA-1 (CD11a/CD18); 2) apart from LFA-1, ASM cell surface expression of the latter molecules is up-regulated in the presence of activated T cells; and 3) pretreatment of ASM cells and tissues with mAbs directed either against CD11a or the combination of CD40 and CD86 completely abrogated both the activated T cell-induced changes in expression of the above CAMs/costimulatory molecules in ASM cells and altered ASM tissue responsiveness. Collectively, these observations identify the presence of bi-directional cross-talk between activated T cells and ASM cells that involves coligation of specific CAMs/costimulatory molecules, and this cooperative intercellular signaling mediates the induction of proasthmatic-like changes in ASM responsiveness.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A wealth of information gathered over the past decade has established a crucial role for CD4+ Th lymphocytes, most notably of the Th2 phenotype, in the pathogenesis of allergic asthma (1, 2, 3, 4). Specifically, Th2 cell-derived cytokines, such as IL-4, IL-13, and IL-5, orchestrate various critical humoral and cellular immune functions including IgE synthesis by B cells, as well as eosinophil proliferation, recruitment, and activation, all of which serve to facilitate expression of the airway inflammatory response that characterizes the atopic asthmatic phenotype (5, 6, 7, 8). Notwithstanding this fundamental role played by T lymphocytes, together with the actions attributed to other leukocytes (e.g., mast cells, macrophages, eosinophils, etc.), certain nonbone marrow-derived cell types in the lung (e.g., airway epithelial, neurovascular, endothelial cells, fibroblasts, etc.) have also been importantly implicated in the overall pathobiology of allergic asthma (9, 10, 11, 12). Among the latter cell types, the resident airway smooth muscle (ASM)3 itself has been recently found to display a variety of proinflammatory actions in the atopic asthmatic sensitized state, including the release of different cytokines such as Th1- and Th2-type cytokines, IL-1{beta}, and others (13, 14, 15, 16, 17). Moreover, the autologous release by the ASM of these cytokines, individually or in combination, was found to act in an autocrine manner on the ASM itself to induce proasthmatic-like changes in its constrictor and relaxant responsiveness (18, 19, 20). Thus, apart from its obvious functional role as a regulator of airway caliber, given its intrinsic capability to elaborate a variety of proinflammatory cytokines, it is reasonable to hypothesize that the ASM may also serve as a potential regulator of the local airway immune response in the atopic asthmatic sensitized state. In light of the latter hypothesis, the present study examined whether, under specific conditions of cellular activation: 1) isolated ASM tissues and cultured ASM cells can directly communicate with isolated T lymphocytes; and 2) induced stimulatory reciprocal "cross-talk" between these cell types can elicit proasthmatic-like changes in ASM constrictor and relaxant responsiveness. The results provide new evidence demonstrating that reciprocal cross-talk involving ligation of specific costimulatory/cell adhesion molecules exists between activated T cells and ASM cells and that the cooperative signaling established by this intercellular communication induces proasthmatic-like changes in ASM constrictor and relaxant responsiveness.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of ASM tissue and coincubation with T lymphocytes

Fourteen adult New Zealand white rabbits were used in this study, which was approved by the Biosafety, Animal Research, and Institutional Review Board Committees of the Joseph Stokes Research Institute at Children’s Hospital of Philadelphia. The animals had no signs of respiratory disease for several weeks before the study.

To examine whether activated T lymphocytes have the capacity to induce proasthmatic changes in ASM responsiveness, anti-CD3-activated T cells (see below) were coincubated with isolated rabbit ASM tissue and examined for changes in the tissues’ agonist-mediated constrictor and relaxation responsiveness. In brief, after anesthesia with xylazine (10 mg/kg) and ketamine (50 mg/kg), rabbits were sacrificed with an overdose of pentobarbital (130 mg/kg). The tracheae were removed via open thoracotomy, cleared of loose connective tissue and epithelium, divided into eight ring segments of 6–8 mm length, and each alternative smooth muscle ring was incubated for 24 h at room temperature in either: 1) DMEM alone, or 2) DMEM in the presence of naive (resting) vs anti-CD3-activated T lymphocytes (1 x 106/ml), with and without 1 h of pretreatment with various combinations of mouse anti-human-CD40 (4 µg/ml), -CD40L (4 µg/ml), -CD80 (2 µg/ml), -CD86 (1.5 µg/ml) mAbs, or with anti-CD11a (3 µg/ml) or anti-CD54 (4 µg/ml) mAbs, in separate experiments. The incubation media were aerated with a continuous supplemental O2 mixture (95% O2/5% CO2) during the incubation phase.

Pharmacodynamic studies

After incubation of the ASM tissue samples, each segment was suspended longitudinally between stainless steel triangular supports in siliconized Harvard 20-ml organ baths (Harvard Apparatus, South Natick, MA). The lower support was secured to the base of the organ bath, and the upper support was attached via a gold chain to a Grass FT.03C force transducer (Validyne Engineering, Northridge, CA) from which isometric tension was continuously displayed on a multichannel recorder, as previously described in our laboratory (21, 22). Care was taken to place the membranous portion of the trachea between the supports to maximize the recorded tension generated by the contracting trachealis muscle. The tissues were bathed in modified Krebs-Ringer solution containing 125 mM NaCl, 14 mM NaHCO3, 4 mM KCl, 2.25 mM CaCl2·H2O, 1.46 mM MgSO4·H2O, 1.2 mM NaH2PO4·H2O, and 11 mM glucose. The baths were aerated with 5% CO2 in oxygen; a pH of 7.35–7.40 was maintained, and the organ bath temperature was held at 37°C. Passive resting tension of each ASM segment was set at 1.5–2.0 g after each tissue had been passively stretched to a tension of 8 g to optimize the resting length of each segment, as previously described (21, 22). The tissues were allowed to equilibrate in the organ baths for 45 min, at which time each tissue was primed with a 1-min exposure to 10-4 M acetylcholine (ACh). Cholinergic contractility was subsequently assessed in the ASM segments by cumulative administration of ACh in final bath concentrations ranging from 10-10 to 10-3 M. Thereafter, in separate studies, relaxation dose–response curves to isoproterenol (10-10–10-4 M) were conducted in tissues half-maximally contracted with ACh. The concentrations of ACh required to produce half-maximal contraction of the ASM segments were typically lower in the tissues exposed to anti-CD3-activated T cells, because these tissues typically exhibited enhanced constrictor sensitivity to ACh (see Results). The relaxant responses to isoproterenol were analyzed in terms of % maximal relaxation (Rmax) from the active cholinergic contraction, and sensitivity to the relaxing agent was determined as the negative logarithm of the dose of the relaxing agent producing 50% of Rmax (pD50) (i.e., geometric mean ED50 value).

Preparation and treatment of cultured ASM cells

Human airway smooth muscle (HASM) cells were derived from a 16-year-old and a 21-year-old male donor (Clonetics, San Diego, CA) who had no evidence of pulmonary disease, and the cells were cultured at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells were grown in a mixture of 5% smooth muscle basal medium (SmBM), which was supplemented with 10% FBS, insulin (5 ng/ml), epidermal growth factor (10 ng/ml; human recombinant), fibroblast growth factor (2 ng/ml; human recombinant), gentamicin (50 ng/ml), and amphotericin-B (50 ng/ml), as previously described in our laboratory (13, 22).

Before their selective treatments (see below), the HASM cells were grown to ~95% confluency, at which time the cells were starved in 10 ml of unsupplemented SmBM. The time course for starvation was 24 h, at which time the cells were incubated with either serum-free medium (SFM) alone or IgE immune complexes, comprised of 15 µg/ml IgE and of 5 µg/ml anti-IgE (goat polyclonal IgG), for various times ranging from 0 to 24 h, as previously described in our laboratory (17, 19). These experiments were performed in the absence and presence of 1 h of pretreatment with various combinations of mouse anti-human CD40 (4 µg/ml), CD40L (4 µg/ml), CD80 (2 µg/ml), CD86 (1.5 µg/ml) mAbs, or with anti-CD11a (3 µg/ml) or anti-CD54 (4 µg/ml) mAbs, in separate experiments. Each flask was washed with SFM before the incubations.

T lymphocyte isolation and activation

T lymphocytes were isolated from whole blood using the WBP1010 Acticyte-TC kit (Bioergonomics, White Bear Lake, MN). Briefly, whole blood was drawn from a peripheral vein of nonatopic/nonasthmatic healthy donors (n = 4), mixed with PrepaCyte-SC medium (Bioergonomics), thereby separating the T cells from the erythrocytes, mature granulocytes, and monocytes. The T cells were collected from the supernatant, and the residual erythrocytes were lysed. The T cells were then centrifuged, washed with PBS, and resuspended in either basal medium alone, activation medium (IGEN International, Gaithersburg, MD) containing IL-2, or in basal medium containing maximum effective concentrations of anti-CD3 ligand (determined by dose-dependent effects of neat Ab), at a cell concentration of ~1 x 106 cells/ml. The T lymphocytes were then incubated in six-well tissue culture plates for the various time points at 37°C in a 5%CO2/95% air incubator. Thereafter, the cells were washed, centrifuged, and labeled for flow cytometric studies, or further incubated with HASM cells (see below). T cell purity was confirmed by staining the cells with an anti-CD3 PE-conjugated Ab, which demonstrated >96% T cell purity and <2% of the isolated cells stained positive with anti-CD19- and anti-CD14-labeled Abs, as assessed by flow cytometry. T cell growth was measured every 24 h using a hemocytometer (Reichert, Buffalo, NY). The cells were counted by taking 10 µl of the culture, pipetting them under the coverslip, and averaging the cell counts within the two 20 x 20-µm areas.

HASM cell/T cell interaction

IL-2- and anti-CD3-activated T lymphocytes were incubated with naive HASM cells for 0–24 h and, in separate experiments, inactive T cells were incubated with HASM cells that were activated with IgE-immune complexes or exposed to SFM alone, in the absence and presence of 1 h of pretreatment with various combinations of mouse anti-human CD40 (4 µg/ml), CD40L (4 µg/ml), CD80 (2 µg/ml), CD86 (1.5 µg/ml) mAbs, or with anti-CD11a (3 µg/ml) or anti-CD54 (4 µg/ml) mAbs, in separate experiments. The media containing the HASM cells and T lymphocytes was subsequently collected and the cells washed three times with PBS. Photographs were obtained with a digital camera under a microscope to visually determine the level of adhesion between the two cell types. After removal of the T lymphocytes and media, the HASM cells were scraped off the flask with a rubber spatula. Both sets of cells were then separately prepared and stained for flow cytometric studies, as described below.

Flow cytometric analysis

Expression of CAMs/costimulatory cell surface molecules was examined in both HASM cells and T lymphocytes using a Coulter EPICS Elite flow cytometer (Coulter EPICS Division, Hialeah, FL) equipped with a 5-W argon laser operated at 488 nM and 300 mW output. Fluorescence signals were accumulated as two parameter fluorescence histograms with both % positive cells and mean channel fluorescence being recorded. As described above, the culture medium was aspirated off the cells at the 24-h time point, and the cells were prepared for flow cytometric analysis. Five milliliters of Versene (PBS lacking Ca2+ and Mg2+, with 0.2 g/L EDTA and 0.5% BSA) was added to the flasks. The cells were incubated for 15 min with this blocking buffer and transferred to an ice-cold 15-ml centrifuge tube, and passed through a 23-gauge needle to prevent aggregation. The cells were centrifuged at 1500 rpm for 2 min, resuspended in minimum PBS lacking Ca2+ and Mg2+, and stained with a primary mouse anti-human Ab specific for the IL-2R (CD25) in 1:2 dilutions of the working concentrations to measure cell activation. The cells were also stained for CD40, CD40L, CD80, CD86, CD11a, CD54, and MHC-II cell surface proteins, using mouse anti-human mAbs. After subsequent washing, FITC-labeled IgG anti-mouse secondary Ab was incubated with the cells for 1 h in a 1:250 dilution in PBS containing 0.5% BSA. The cells were also stained with mouse Abs of the identical isotypes as the primary monoclonal Abs to measure background fluorescence (i.e., mouse IgG1 and IgG2a negative controls). The stained cells were then evaluated by flow cytometry and analyzed using the Elite Immuno 4 statistical software (Coulter EPICS Division, Palo Alto, CA). Both resting and activated HASM cells and T lymphocytes were examined.

Determination of CD40, CD40L, CD80, CD86, CD54, and CD11a mRNAs expression

Total RNA was isolated from the human T lymphocytes and cultured HASM cell preparations using the modified guanidinium thiocyanate phenol-chloroform extraction method to include proteinase K (in 5% SDS) for digestion of protein in the initial RNA pellet, as previous described in our laboratory (13, 22). The concentration of each RNA sample was determined spectrophotometrically. This procedure consistently produced yields of 15–25 µg of intact RNA from each T-75 flask of HASM cells and per each tissue specimen under study. To analyze for mRNA expression of CD40, CD40L, CD80, CD86, CD54, and CD11a, we used RT-PCR and human-specific primers for these molecules. cDNA was synthesized from total RNA isolated from untreated cells and from human ASM cells passively sensitized with IgE immune complexes and anti-CD3-activated T lymphocytes. The cDNAs were primed with oligo(dT) 12–18 and extended with Superscript II reverse transcriptase (Life Technologies, Rockville, MD). The PCR was used to amplify the specific products from each cDNA reaction, based on the published sequences of the human CD40, CD40L, CD80, CD86, CD54, and CD11a genes, and included the following primer sets: CD40: 5'-primer 5'-CTGGGCTAGCGATACAGGAG-3', 3'-primer 5'-TGGATTTGGCCAACAGAAAT-3', 256-bp product; CD40L: 5'-primer 5'-AAATTGCGGCACATGTCATA-3', 3'-primer 5'-ATCTACCGGGGGACTTTAGG-3', 240-bp product; CD80: 5'-primer 5'-CATCACCATCCAAGTGTCCA-3', 3'-primer 5'-CAAAGATGGTCCGGTTCTTG-3', 255-bp product; CD86: 5'-primer 5'-CTCTCTGGTGCTGCT CCTCT-3', 3'-primer 5'-ATCTGGTGGATGCGAATCAT-3', 319-bp product; CD54: 5'-primer 5'-GAGCTGTTTGAGAACACCTC-3', 3'-primer 5'-TCACACTTCACTGTCACCTC-3', 367-bp product; CD11a: 5'-primer 5'-GTCCTCTGCTGAGCTTTACA-3', 3'-primer 5'-ATCCTTCATCCTTCCAGCAC-3', 337-bp product; and ribosomal protein L7 (RPL7): 5'-primer 5'-AAGAGGCTCTCATTTTCCTGGCTG-3', 3'-primer 5'-TCCGTTCCTCCCCATAATGTTCC-3', 157-bp product.

The cycling profile used was as follows: denaturation, 95°C for 1 min; annealing, 52–56°C for 1.0–1.5 min; and extension, 72°C for 1.0–1.5 min and 34–40 cycles for the CD40, CD40L, CD80, CD86, CD54, and CD11a genes, and 30 cycles for the RPL7 gene. The number of cycles was determined to be in the linear range of the individual PCR products. The PCRs for the individual products were performed using equivalent amounts of cDNA prepared from 2.5 µg of total RNA, and equal aliquots of each PCR were then run on a 1.2% agarose gel and subsequently transferred to a {zeta}-probe membrane overnight in 0.4N NaOH. After capillary transfer, the DNA was immobilized by UV-cross-linking using a Stratalinker UV Cross-linker 2400 at 120,000 mJ/cm2 (Stratagene, La Jolla, CA). Prehybridization in a Techne (Princeton, NJ) hybridization oven was conducted for 2–3 h at 42°C in 50% formaldehyde, 7% (w/v) SDS, 0.25 M NaCl, 0.12 M Na2HPO4 (pH 7.2), and 1 mM EDTA. Hybridization was for 20 h at 42°C in the same solution. The CD40, CD40L, CD80, CD86, CD54, and CD11a and RPL7 DNA levels were assayed by Southern blot analysis using 32P-labeled probes, prepared by separately pooling several RT-PCRs for the individual PCR fragments and purifying them from a 1.2% agarose gel using the Qiaex II agarose gel extraction kit (Qiagen, Chatsworth, CA). The individual PCR products were subsequently sequenced for product confirmation. Washes were as follows: 1 x 15 min in 2x SSC, 0.1% SDS; 1 x 15 min in 0.1x SSC, 0.1% SDS both at room temperature, and 2 x 15 min at 50°C in 0.1x SSC, 0.1% SDS. Southern blots were quantitated by direct measurements of radioactivity in each band using a PhosphoImager (Molecular Dynamics, Sunnyvale, CA).

Statistical analyses

Statistical analyses was performed using the two-tailed paired Student’s t test and ANOVA with multiple comparison of means, where appropriate. Values of p < 0.05 were considered significant.

Reagents

The HASM cells and SmBM were obtained from Clonetics (Walkersville, MD). The human CD40, CD40L, CD80, CD86, CD54, CD11a, and RPL7 primers were purchased from the Integrated DNA Technologies (Coralville, IA). The mouse anti-human CD40, CD40L, CD80, CD86, and MHC-IIDR mAbs were obtained from Serotec (Raleigh, NC). The CD11a, CD11b, and CD54 mAbs and the anti-mouse secondary Ab and IL-2 were purchased from R&D Systems (Minneapolis, MN). The anti-CD3 activation Ab was prepared in our laboratory from a NK3 hybridoma cell line purchased from American Type Culture Collection (Manassas, VA). ACh and isoproterenol were purchased from Sigma (St. Louis, MO). All drug concentrations are expressed as final bath concentrations. Isoproterenol and ACh were made fresh for each experiment and were dissolved in normal saline to prepare 10-4 M and 10-3 M solutions, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of activated T cells on ASM responsiveness

To assess whether activated T lymphocytes have the capacity to directly provoke changes in ASM tissue responsiveness, agonist constrictor and relaxation responses were compared in isolated rabbit ASM segments that were incubated for 24 h with vehicle alone (control) and with either resting (inactivated) T cells or T cells activated by cross-linking the TCR with anti-CD3 Ab (see Materials and Methods). As shown in Fig. 1Go, the maximal constrictor responses (Tmax) and sensitivities (pD50; i.e., -log ED50 values) to exogenously administered ACh obtained in control ({circ}) and resting T cell-exposed ({square}) ASM tissues were similar. In contrast, relative to controls, the constrictor responses to ACh were significantly enhanced in ASM that were exposed to anti-CD3-activated T cells ({blacksquare}), wherein the mean ± SE Tmax values in the control vs activated T cell-exposed tissues, amounted to 76.92 ± 3.4 vs 111.0 ± 7.45 g/g ASM weight, respectively (p < 0.01), and the corresponding pD50 values averaged 5.17 ± 0.09 vs 5.28 ± 0.14 -log M, respectively (p < 0.05).



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  		FIGURE 1. Comparison of ASM constrictor responses to ACh in rabbit tissues after 24 h of exposure to vehicle alone ({circ}), resting T cells ({square}), or anti-CD3-activated T cells ({blacksquare}). Note: Relative to tissues incubated with vehicle alone (n = 6) or with resting T cells (n = 6), both Tmax and ED50 responses to ACh were significantly enhanced (p < 0.01 and p < 0.05, respectively) in ASM samples that were coincubated with activated T lymphocytes (n = 6). In contrast, no difference was observed in ACh responses between ASMs that were incubated with inactive T cells and with vehicle alone.

 
In separate studies conducted under the same experimental conditions, during comparable levels of initial sustained ACh-induced contractions (averaging ~40% of Tmax), administration of the {beta}-adrenoceptor agonist, isoproterenol, elicited cumulative dose-dependent relaxation of the precontracted ASM segments. As shown in Fig. 2Go, whereas the Rmax responses and sensitivities (pD50 values) to isoproterenol were similar in control and resting T cell-exposed ASM tissues, the relaxation responses to isoproterenol were significantly attenuated in ASM exposed to anti-CD3-activated T cells. Accordingly, the mean ± SE Rmax values to isoproterenol amounted to 49.44 ± 8.02 vs 61.5 ± 9.5% in the activated T cell-exposed vs control ASM (p < 0.05), and the corresponding pD50 values averaged 6.14 ± 0.12 vs 6.30 ± 0.06 -log M, respectively (p < 0.05).



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  		FIGURE 2. Comparison of ASM relaxant responses to isoproterenol in rabbit tissues after 24 h of exposure to vehicle alone ({circ}), resting T cells ({square}) or anti-CD3-activated T cells ({blacksquare}). Note: Relative to tissues incubated with vehicle alone (n = 6) or with resting T cells (n = 6), both Rmax and pD50 responses to isoproterenol were significantly attenuated (p < 0.05 and p < 0.05, respectively) in ASM samples that were coincubated with activated T lymphocytes (n = 6). In contrast, no differences were observed in isoproterenol responses between ASM that were incubated with inactive T cells and with vehicle alone.

 
Expression and regulation of cell adhesion molecules (CAMs)/costimulatory molecules

Given the above evidence implicating a role for anti-CD3-activated T cells in directly inducing changes in ASM responsiveness, a series of studies were pursued to further elucidate the nature of this T cell/ASM cell interaction and to assess whether reciprocal costimulatory "cross-talk" exists among these cell types. In addressing these issues, initial experiments examined whether intercellular adhesion between T cells and cultured (near-confluent) human ASM cells is elicited after selective preactivation of either the T cells with anti-CD3 or the ASM cells with IgE immune complexes, as previously described (17, 19). Consistent with previous reports (23), when compared with inactivated T cells (Fig. 3Goa), anti-CD3-activated T cells inoculated into resting ASM cell cultures displayed (after 24 h) distinctive adhesive clustering on the surface of the resting ASM cells (Fig. 3Gob). Comparably, similar adhesive clustering was also observed after resting T cells were inoculated into cultures of ASM cells that were preactivated with IgE immune complexes (Fig. 3Goc).



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  		FIGURE 3. Representative experiment demonstrating cell-to-cell surface adhesion between cultured HASM cells and isolated peripheral blood T lymphocytes. Note: As shown in b, relative to resting T cells (a), 24 h of exposure of naive human ASM cells to anti-CD3-activated T cells produced notable adhesion between the activated T cells and ASM cells. Comparable adhesion formation was also observed when resting T cells were exposed to IgE-immune complex-activated human ASM cells (c). Experiments were performed in triplicate.

 
Because, apart from T cells and other leukocytes, ASM cells were recently shown to also express certain CAMs on their cell surface (22, 23, 24), in light of the above observations, we next examined whether specific CAMs and other costimulatory molecules implicated in intercellular immune responses are expressed in ASM and modulated in the presence of ASM cell activation under the above experimental conditions. mRNA and cell surface protein expression of ICAM-1 (CD54), its endogenous counterreceptor ligand, LFA-1 (CD11a/CD18), as well as the "accessory" costimulatory molecules, CD40, CD40L, CD80, and CD86, was first examined in cultured HASM cells in the absence and presence of activation with exogenously administered IgE immune complexes. Using RT-PCR for mRNA analysis, ASM cells were found to constitutively express mRNAs for CD40, CD40L, CD80, and CD86, as well as for CD54 and CD11a, under control conditions (Fig. 4Go, left). When activated by the administration of IgE immune complexes, with exception to unaltered CD11a expression, these mRNA signals were markedly up-regulated at 3 h and remained enhanced at 24 h (Fig. 4Go, right). In contrast, mRNA expression of the constitutively expressed RPL7 was unaffected, and the signal was similar in intensity under both treatment conditions. In separate studies, qualitatively similar up-regulation of mRNA expression of the above CAMs/costimulatory molecules was also detected in T cells that were activated by CD3 engagement (data not shown).



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  		FIGURE 4. mRNAs expression of the costimulatory molecules, CD40, CD40L, CD80, and CD86; the ICAM, CD54, and its natural counterligand, CD11a, in human ASM cells in the absence (left) and presence (right) of 0, 3, and 24 h of exposure to IgE immune complexes. Note: Human ASM cells express mRNAs for the CD40, CD40L, CD80, CD86, CD54, and CD11a molecules. Moreover, apart from unaltered CD11a expression, the mRNA expression signals for these molecules were notably enhanced (range: 2- to 5-fold) in the IgE immune complex-activated HASM cells at 3 h, and were sustained for 24 h (right). Experiments were performed in triplicate.

 
In accordance with the above results, using flow cytometric analysis, we found that cell surface expression of the above CAMs/costimulatory molecules was also detected in ASM cells under resting conditions (Fig. 5Go, left) and that, with the exception of CD11a, expression of these molecules was distinctly enhanced at 24 h after activation of the cells with IgE immune complexes (Fig. 5Go, right). Accordingly, as displayed in Fig. 5Go, increases in both the percentage of cells positively stained for these proteins and their respective mean fluorescence intensity (MFI) values were observed. Relative to the resting condition, the increases in the percentage of positive staining for CD40, CD40L, CD80, and CD86 amounted to 2.1-, 9.2-, 5.6-, and 4.4-fold, respectively, and the corresponding changes in MFI amounted to 1.3-, 0.95-, 2.1-, and 2.2-fold, respectively. As further depicted in Fig. 5Go, >95% of ASM cells also stained positively for the MHC class II Ag, HLA-DR, under resting conditions, and the MFI for HLA-DR expression was enhanced by 33% in the presence of ASM cell activation.



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  		FIGURE 5. Flow cytometric analysis of HASM cell surface expression of the costimulatory molecules CD40, CD40L, CD80, and CD86 (A), the ICAM, CD54, its natural counterligand, CD11a, and the MHC, MHC-II (HLA-DR) (B), in the absence (left) and presence (right) of 24 h of exposure to IgE immune complexes. Data show the percentage of positive cells and MFI values relative to isotype control Abs (left histogram). Note: Although the percentage of positive cell surface expression levels of CD40, CD40L, CD80, CD86, and CD54 were low in the resting HASM cells (range: 3–8%), in contrast to unaltered CD11a expression, cell surface expression of CD40, CD40L, CD80, CD86, and CD54 was notably up-regulated (range: 2- to 15-fold) in the IgE immune complex-activated cells at 24 h. Experiments were performed in triplicate.

 
Bi-directional activation by stimulated ASM and T cells

In view of the above results, we next examined for related evidence of reciprocal activation between ASM cells and T lymphocytes. In these flow cytometric experiments, using cell surface expression of CD25 (IL-2R protein) as a marker for cell "activation," we found that CD25 was notably enhanced both in anti-CD3-activated T cells (Fig. 6GoA) and in ASM cells that were stimulated with IgE immune complexes (Fig. 6GoB), with increases in the percentage of positive staining for CD25 in the activated T cells and ASM cells amounting to ~4-fold and 3-fold, respectively. Moreover, as demonstrated by the representative experiments in Fig. 7Go, when resting T cells were exposed to ASM cells preactivated with IgE immune complexes, their CD25 protein expression was also markedly increased by ~4-fold in the percentage of positive staining (Fig. 7GoA) and, comparably, when naive ASM cells were exposed to anti-CD3-activated T cells, the ASM cells also displayed up-regulated expression of CD25 (~4-fold increase in the percentage of positive staining), whereas exposure to resting T cells had no effect (Fig. 7GoB). Finally, in association with these observed changes in CD25 expression, exposure of resting ASM cells to anti-CD3-activated T cells was also found to up-regulate the expression of CD40, CD86, and CD54 on the surface of the naive ASM cells (Fig. 8Go).



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  		FIGURE 6. Cell surface expression of the "activation marker," CD25, in isolated peripheral blood T lymphocytes and cultured HASM cells in the absence (left) and presence (right) of cell activation (see Materials and Methods). Data show the percentage of positive cells and MFI values relative to isotype control Abs (left histogram). Note: Expression of CD25 was markedly enhanced (range: 3- to 5-fold) in the anti-CD3-activated T lymphocytes, and comparable induction in CD25 expression was also observed in HASM cells that were exposed to IgE immune complexes. Experiments were performed in triplicate.

 


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  		FIGURE 7. A, Cell surface expression of the activation marker, CD25, in isolated peripheral blood T lymphocytes after exposure of the cells to resting (left) and IgE immune complex-activated (right) human ASM cells. B, Cell surface expression of CD25 in human ASM cells after exposure of the cells to resting (left) and anti-CD3-activated (right) T cells. Data show the percentage of positive cells and MFI values relative to isotype control Abs (left histogram). Note: CD25 expression in T lymphocytes that were exposed to activated human ASM cells was markedly enhanced (range: 3- to 4-fold), and comparable induction in CD25 expression was also observed in human ASM cells that were exposed to activated T lymphocytes. Experiments were performed in triplicate.

 


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  		FIGURE 8. Cell surface expression of the CAMs/costimulatory molecules, CD40, CD86, and CD54 in human ASM cells that were exposed to anti-CD3-activated T cells for 24 h. Histograms for isotype control Abs are shown on left. Note: Exposure of the resting human ASM cells to activated T lymphocytes resulted in markedly enhanced expression of the CD40, CD86, and CD54 cell surface molecules (range: 2- to 10-fold). Experiments were performed in triplicate.

 
In addressing the potential mechanism(s) underlying the above evidence of T cell/ASM cell reciprocal activation, extended studies demonstrated that this phenomenon is primarily attributed to an induced coligation between specific cell surface CAMs/costimulatory molecules expressed in these cell types. Accordingly, as shown by representative experiments in Fig. 9Go, anti-CD3-activated T cell-induced up-regulation of CD25, CD40, CD86, and CD54 expression in naive ASM cells was found to be completely inhibited by pretreating the resting ASM cells with either the combination of neutralizing mAbs directed against CD40 and CD86 (i.e., anti-CD40/CD86 mAbs) (Fig. 9Go, middle) or anti-CD11a mAb alone (Fig. 9Go, right). Comparably, pretreatment of resting T cells with either anti-CD40/CD86 mAbs or anti-CD11a mAb alone was also found to completely prevent their up-regulated expression of CD25 and the above CAMs/costimulatory molecules in response to inoculation into ASM cell cultures preactivated with IgE immune complexes (data not shown). Finally, in concert with these results, pretreatment of naive ASM cells with either anti-CD40/CD86 mAbs or anti-CD11a mAb was further found to abrogate the adhesion of anti-CD3-activated T cells after their inoculation into the ASM cell cultures (Fig. 10Go).



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  		FIGURE 9. Cell surface expression of the activation marker, CD25 (A), and the costimulatory/adhesion molecules, CD40, CD86, and CD54 (B) in human ASM cells that were exposed to anti-CD3-activated T lymphocytes for 24 h in the absence (left) and presence of 1 h of pretreatment with the combination of anti-CD40 and anti-CD86 mAbs (middle), and with the anti-CD11a mAb alone (right). Histograms for isotype control Abs are shown on left. Note: Both anti-CD11a alone and the combination of anti-CD40 and anti-CD86 completely abrogated the induced up-regulated cell surface expression signals of CD25, CD40, CD86, and CD54 in ASM cells that were exposed to activated T cells. Experiments were performed in triplicate.

 


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  		FIGURE 10. Representative experiment demonstrating cell-to-cell adhesion between human ASM cells and anti-CD3-activated T lymphocytes. Note: Relative to exposure to resting T cells (a), 24 h of exposure of human ASM cells to activated T lymphocytes resulted in notably enhanced cell surface adhesion between the activated T cells and ASM cells (b). In contrast, 1 h of pretreatment of cells with the combination of anti-CD40 and anti-CD86 mAbs (c) or with anti-CD11a mAb alone (d), largely prevented cell-to-cell adhesion. Experiments were performed in triplicate.

 
Role of CAMs/costimulatory molecules in mediating T cell-induced changes in ASM responsiveness

The above results implicating CAM/costimulatory molecules in ASM cell/T cell reciprocal activation raised the consideration that coligation of these molecules with their endogenous counter-receptor ligands may be responsible for eliciting the observed changes in ASM responsiveness induced by exposure of naive ASM tissues to activated T cells (Fig. 1Go). In addressing this possibility, ASM constrictor responses to ACh and relaxation responses to isoproterenol were separately examined in tissues exposed for 24 h to resting and anti-CD3-activated T cells in the absence and presence of pretreatment of the tissues with either anti-CD40/CD86 mAbs or anti-CD11a mAb. As shown in Fig. 11Go, pretreatment with either anti-CD40/CD86 mAbs (Fig. 11Goa) or anti-CD11a mAb (Fig. 11Gob) prevented the induction of increased constrictor responsiveness of ASM tissues exposed to anti-CD3-activated T cells. Similarly, pretreatment with either anti-CD40/CD86 mAbs (Fig. 12GoA) or anti-CD11a mAb (Fig. 12GoB) also largely abrogated the induction of impaired relaxation responsiveness to isoproterenol in ASM exposed to the activated T cells. In accordance with these observations, extended experiments further demonstrated that pretreatment of ASM tissues with a neutralizing mAb directed against ICAM-1, the endogenous counterreceptor ligand for CD11a/CD18, also completely prevented the changes in agonist constrictor and relaxant responsiveness induced by exposure of naive ASM to anti-CD3-activated T cells (data not shown). In contrast, pretreatment of naive ASM with a mAb directed against the related {beta}2 integrin, Mac-1 (i.e., anti-CD11b mAb), had no preventative effect on activated T cell-induced changes in ASM responsiveness (data not shown).



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  		FIGURE 11. Comparison of ASM constrictor responses to ACh in rabbit tracheal tissues after 24 h of exposure to resting T cells ({circ}) or to anti-CD3-activated T cells in the absence ({blacksquare}) and presence ({square}) of pretreatment with anti-CD40 and anti-CD86 mAbs combined (A), or with anti-CD11a mAb alone (B). Note: Relative to tissues incubated with resting T cells, both the Tmax and ED50 responses to ACh were significantly enhanced (p < 0.01 and p < 0.05, respectively) in ASM segments that were exposed to activated T cells, whereas the latter effects were primarily prevented by pretreating the ASM tissues with either the combination of anti-CD40 and CD86 mAbs (A) or anti-CD11a mAb alone (B).

 


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  		FIGURE 12. Comparison of ASM relaxant responses to isoproterenol in rabbit tracheal tissues after 24 h of exposure to resting T cells ({circ}) or to anti-CD3-activated T cells in the absence ({blacksquare}) and presence ({square}) of pretreatment with anti-CD40 and anti-CD86 mAbs combined (A), or with anti-CD11a mAb alone (B). Note: Relative to tissues incubated with resting T cells, both the Rmax and pD50 responses to isoproterenol were significantly attenuated (p < 0.05 and p < 0.05, respectively) in ASM segments that were exposed to activated T cells, whereas the latter effects were primarily prevented by pretreating the ASM tissues with either the combination of anti-CD40 and CD86 mAbs (A) or anti-CD11a mAb alone (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Bronchial asthma is characterized by exaggerated agonist-induced bronchoconstriction, attenuated {beta}-adrenoceptor-mediated airway relaxation and airway inflammation, the latter principally involving infiltration of the airways with T lymphocytes, mast cells, and eosinophils (1, 2, 3, 4, 5, 6, 7, 8, 25, 26, 27, 28). Although the mechanistic interplay between airway inflammation and the associated changes in ASM responsiveness remains to be fully characterized, the wealth of evidence accumulated to date suggests that the altered airway responsiveness in asthma is primarily attributed to the actions of various cytokines, which are primarily produced by infiltrating CD4+ T lymphocytes expressing the Th2 profile of cytokine release (1, 2, 3, 4, 5, 6, 7, 8, 27, 28). In keeping with this notion, the observed changes in ASM responsiveness in asthmatic individuals and in animal models of allergic asthma have been associated with elevated bronchoalveolar lavage fluid and serum levels of the Th2-type cytokines, notably IL-4, IL-5, IL-13, and GM-CSF (15, 16, 27, 28, 29, 30). Moreover, relative to nonatopic, nonasthmatic subjects, mononuclear cells isolated from peripheral blood and bronchoalveolar lavage fluid samples obtained from atopic asthmatic patients have also been found to display enhanced production of these Th2-type cytokines when these cells are activated in vitro, whereas their profile of Th1-type cytokine expression is suppressed (30, 31). Thus, although multiple cell types are known to actively participate in the proinflammatory reaction in allergic asthma, it is the general consensus that CD4+ T lymphocytes play a critical role in regulating both the initiation and perpetuation of the net airway inflammatory response in this disease (1, 2, 3, 31, 32).

Notwithstanding the crucial role played by T lymphocyte and other airway infiltrating leukocytes in atopic asthma, emerging new evidence demonstrates that various cytokines, including those of the Th1 and Th2 phenotypes, are also expressed by certain nonbone marrow-derived airway structural cells, such as bronchial epithelial cells and microvascular endothelial cells (9, 10, 11, 12), as well as by the ASM cell itself (13, 14, 15, 18). In this regard, apart from its intrinsic role as a regulator of airway caliber, given its extended capacity to elaborate a variety of cytokines, it is reasonable to speculate that the ASM may also serve as a regulator of the local airway immune response in atopic asthma. In light of this consideration, and given the well-established importance of T lymphocyte activation in the overall pathobiology of atopic asthma, this study examined the potential mechanistic interplay between activated T cells and ASM cells in the regulation of changes in ASM responsiveness. The results provide new evidence demonstrating that reciprocal cross-talk involving ligation of specific CAMS/costimulatory molecules exists between activated T cells and ASM cells and that the cooperative signaling established by this intercellular communication induces proasthmatic-like phenotypic changes in ASM constrictor and relaxant responsiveness.

To our knowledge, this study is the first to demonstrate that anti-CD3-activated T lymphocytes have the capacity to directly elicit proasthmatic-like changes in agonist-induced ASM responsiveness, including increased ASM contractility to ACh (Fig. 1Go) and impaired {beta}-adrenoceptor-mediated ASM relaxation (Fig. 2Go). To address the mechanism underlying this phenomenon, a series of studies were conducted to examine for evidence of direct T cell/ASM cell interaction and to further assess whether reciprocal costimulatory "cross-talk" exists among these cell types. As shown in Fig. 3Go, intercellular adhesion was elicited between anti-CD3-activated T cells and HASM cells. The finding of induced adhesion between activated T cells and naive ASM cells in culture (Fig. 3Gob) is consistent with previous reports (23, 33), and the present results further demonstrate that such intercellular adhesion is similarly elicited by exposure of resting T cells to cultured ASM cells preactivated with IgE immune complexes (Fig. 3Goc). In this context, it should be noted that our rationale for using IgE immune complexes to initially selectively activate the ASM cells was based on our previous studies demonstrating that ASM cells express the low affinity receptor to IgE, Fc{epsilon}RII (CD23), and that exposure of the ASM cells to IgE (and most notably to IgE immune complexes) effectively stimulates the cells via binding and activation of Fc{epsilon}RII on the ASM cell surface (17, 19). The efficacy of this approach to activate the ASM cells was further evidenced in the present study, wherein the results demonstrated that treatment of the cells with IgE immune complexes evoked up-regulation of their cell surface expression of various CAMs/costimulatory molecules (Figs. 4Go and 5Go).

Constitutive expression of specific CAMs on the surface of ASM cells has been previously reported (6, 22, 23), including ICAM-1 (CD54), LFA-1 (CD11a/CD18), and VCAM-1 (6, 22, 23, 24). Moreover, we recently identified that ICAM-1 expression in ASM cells and tissue is up-regulated after inoculation of the ASM with rhinovirus-16 (22) or exposure of the ASM to atopic asthmatic serum (24). In contrast, neither experimental condition was found to be associated with altered expression of LFA-1 (22, 24). Our present results largely concur with these recent findings and demonstrate that, whereas CD54 mRNA and surface protein are up-regulated in ASM cells treated with IgE immune complexes, expression of CD11a is essentially unaltered (Figs. 4Go and 5Go). The present observations provide additional new evidence demonstrating that the intercellular adhesion/costimulatory molecules, CD40, CD40L, CD80, and CD86, as well as the MHC class II Ag, HLA-DR, are also constitutively expressed by ASM cells and that the expression of these molecules is up-regulated after exposure of the ASM cells to IgE immune complexes (Figs. 4Go and 5Go). Although the latter molecules are known to be characteristically expressed on the surface of various inflammatory cells and professional APCs, the observations herein support the concept that ASM cells possess the cell surface molecular machinery for potential coligation with inflammatory cells involved in local (peripheral tissue) immune responses. Indeed, in this context, identification of the above CAMs/costimulatory molecules on the surface of ASM cells, together with their observed up-regulated expression in the ASM activated state, suggested a potential mechanistic role for these molecules in ASM cell/T cell coactivation, as discussed below.

Up-regulated expression of the cell activation marker, CD25, was independently elicited in anti-CD3-activated T cells and in ASM cells stimulated by IgE immune complexes (Fig. 6Go). Moreover, the results demonstrated that CD25 expression (Fig. 7Go), together with cell surface expression of CD40, CD86, and CD54, was also up-regulated in resting T cells and ASM cells when either cell type was coincubated with its preactivated counterpart (Fig. 8Go). Thus, both cell types exhibited evidence of reciprocal activation, supporting the concept that there exists a dynamic interaction involving bi-directional signaling between these cell types. Additionally, because the observed stimulatory effects of this reciprocal activation on CD25 expression in both T cells and ASM cells (Fig. 7Go) were roughly comparable in magnitude to those elicited by preactivation of either cell type independently (Fig. 6Go), the results suggest that the phenomenon of T cell/ASM cell reciprocal activation represents a potent mechanism of intercellular communication. In considering these observations, it is relevant to note that previous studies have reported that vascular smooth muscle cells can also activate both allogenic and HLA-matched T cells, as reflected by induced expression of CD25 and release of IL-2 by the T cells (34, 35). Although, in the latter studies, the induced T cell activation was not accompanied by enhanced T cell proliferation (detected by [3H]thymidine incorporation), due to arrest of the T cells in the G1 phase of the cell cycle, these data suggested that the vascular smooth muscle cells were capable of processing and presenting Ag to T cells (34, 35). Our results herein fundamentally concur with this concept in demonstrating that ASM cells express the above collection of CAMs/costimulatory molecules and, by virtue of their high constitutive expression of the MHC class II protein, HLA-DR, the possibility is raised that ASM cells also have the capacity to act as APCs. In this respect, Ag presentation to both autologous and allogeneic T cells by nonprofessional APCs has been clearly demonstrated in other parenchymal cell types, including keratinocytes, fibroblasts, epithelial cells, and endothelial cells (36, 37, 38). However, with respect to smooth muscle cells, although several studies have implicated such cells as potential APCs (39, 40), other reports have suggested that smooth muscle cells, including ASM cells (23), may inhibit the process of T cell activation via the release of a putative inhibitory factor (e.g., cytokine), resulting in inhibition of the progression of activated T cell clones through the cell cycle, despite their increased expression of CD25 and release of IL-2 (34, 35). Thus, evidence defining the role of smooth muscle cells as APCs is, at present, somewhat inconclusive, and this issue remains to be systematically investigated.

In addressing the potential mechanism underlying T cell/ASM cell reciprocal activation, our extended present observations demonstrated that responses related to this phenomenon were completely abrogated by pretreating either cell type with anti-CD40/CD86 mAbs or with anti-CD11a mAb (Figs. 9Go and 10Go). Thus, the results suggest that induced molecular interactions involving coligation of CD40, CD86, and LFA-1 with their respective CAMs/counterreceptor ligands are events that fundamentally underlie T cell/ASM cell adhesion and reciprocal activation. Moreover, in this respect, to the extent that anti-CD40/CD86 mAbs and anti-CD11a mAb also abrogated the proasthmatic-like changes in ASM tissue responsiveness elicited by exposure of the tissues to activated T cells (Figs. 11Go and 12Go), the results further imply that the mechanism of T cell-induced changes in ASM responsiveness is also fundamentally dependent on intercellular coligation involving these CAMs/costimulatory molecules. In this connection, it should be noted that molecular coligation involving T cells and other cell types is a process typically characterized by the evoked release of various cytokines (2, 3, 4, 5, 30, 31, 32, 41, 42, 43). In this regard, with respect to ASM activation, we recently identified that the elicitation of proasthmatic-like changes in agonist responsiveness (i.e., as depicted herein) in ASM tissues exposed to atopic asthmatic serum is primarily initiated by IL-5 release, and that the latter is attributed to ICAM-1/LFA-1 coligation involving the atopic-sensitized ASM (13, 14, 24). Thus, in light of these recent findings, it is reasonable to speculate that our present observed changes in agonist responsiveness elicited in ASM tissues exposed to anti-CD3-activated T cells were also mechanistically coupled, at least in part, to an induced release of IL-5. This possibility, together with the potential release of other proinflammatory cytokines/mediators in the process of ASM cell/T cell interaction, remains to be systematically evaluated. Finally, to the extent that the present observations demonstrate that ASM cells can effectively reciprocally activate T cells, the possibility is raised that this phenomenon rendered a state of positive feedback in the process of ASM/T cell interaction that served to amplify our observed changes in ASM responsiveness. Clearly, this provocative consideration also warrants future investigation.

In conclusion, the present study examined the role and mechanism of T cell interaction with ASM in eliciting changes in ASM responsiveness. The results provide new evidence demonstrating that stimulated T cells and ASM cells exhibit bi-directional activation that involves induced up-regulated expression and coligation of specific CAMs/costimulatory molecules implicated in peripheral tissue immune responses and Ag presentation. Moreover, this cooperative intercellular signaling mediates the induction of proasthmatic-like changes in ASM responsiveness. Taken together, these finding provide certain new insights into the overall proinflammatory mechanism of T cell/ASM cell interaction in the pathobiology of atopic asthma.


   Acknowledgments
 
We thank J. Grunstein and S. Chuang for their expert technical assistance.


   Footnotes
 
1 This work was supported by National Heart, Lung, and Blood Institute Grants HL-59906, HL-31467, and HL-58245. Back

2 Address correspondence and reprint requests to Dr. Hakon Hakonarson, Division of Pulmonary Medicine, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, 34th Street and Civic Center Boulevard, Philadelphia, PA 19104. Back

3 Abbreviations used in this paper: ASM, airway smooth muscle; ACh, acetylcholine; Rmax, maximal relaxation; HASM, human ASM; SmBM, smooth muscle basal medium; SFM, serum-free medium; Tmax, maximal constrictor response; MFI, mean fluorescence intensity; CAM, cell adhesion molecule; RPL7, ribosomal protein L7. Back

Received for publication June 21, 2000. Accepted for publication September 27, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hamelmann, E., U. Wahn, E. W. Gelfand. 1999. Role of the Th2 cytokines in the development of allergen-induced airway inflammation and hyperresponsiveness. Int. Arch. Allergy Immunol. 118:90.[Medline]
  2. Robinson, D. S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A. M. Bentley, C. Corrigan, S. R. Durham, A. B. Kay. 1992. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 326:298.[Abstract]
  3. Walker, C., E. Bode, L. Boer, T. T. Hansel, K. Blaser, J. C. Virchow. 1992. Allergic and non-allergic asthmatics have distinct pattern of T-cell activation and cytokine production in peripheral blood and bronchoalveolar lavage. Am. Rev. Respir. Dis. 146:109.[Medline]
  4. Romagnani, S.. 1991. Human TH1 and TH2 subsets: doubt no more. Immunol. Today 12:256.[Medline]
  5. Kotsimbos, A. T., Q. Hamid. 1997. IL-5 and IL-5 receptor in asthma. Mem. Inst. Oswaldo Cruz 2:75.
  6. Coffman, R. L., B. W. Seymour, D. A. Lebman, D. D. Hiraki, J. A. Christiansen, B. Shrader, H. M. Cherwinski, H. F. Savelkoul, F. D. Finkelman, M. W. Bond, et al 1988. The role of helper T cell products in mouse B cell differentiation and isotype regulation. Immunol. Rev. 102:5.[Medline]
  7. Holgate, S. T.. 1999. The epidemic of allergy and asthma. Nature 402:B2.[Medline]
  8. Beasley, R., W. R. Roche, J. A. Roberts, S. T. Holgate. 1989. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am. Rev. Respir. Dis. 139:806.[Medline]
  9. Kelley, J.. 1990. Cytokines of the lung. Am. Rev. Respir. Dis. 141:765.[Medline]
  10. Stadnyk, A. W.. 1994. Cytokine production by epithelial cells. FASEB J. 8:1041.[Abstract]
  11. Jordana, M., B. Sarnstrand, P. J. Sime, I. Ramis. 1994. Immune-inflammatory functions of fibroblasts. Eur. Respir. J. 7:2212.[Abstract]
  12. Mantovani, A., F. Bussolino, E. Dejana. 1992. Cytokine regulation of endothelial cell function. FASEB J. 6:2591.[Abstract]
  13. Hakonarson, H., N. Maskeri, C. Carter, M. M. Grunstein. 1999. Regulation of TH1- and TH2-type cytokines expression and action in atopic asthmatic sensitized airway smooth muscle. J. Clin. Invest. 103:1077.[Medline]
  14. Hakonarson, H., N. Maskeri, C. Carter, S. Chuang, M. M. Grunstein. 1999. Autocrine interaction between IL-5 and IL-1{beta} mediates altered responsiveness of atopic asthmatic sensitized airway smooth muscle. J. Clin. Invest. 104:657.[Medline]
  15. Barnes, P. J., K. F. Chung, C. P. Page. 1998. Inflammatory mediators and asthma. Pharmacol. Rev. 40:49.[Medline]
  16. Litchfield, T. M., T. H. Lee. 1992. Asthma: cells and cytokines. J. Asthma 29:181.[Medline]
  17. Hakonarson, H., C. Carter, C. Kim, M. M. Grunstein. 1999. Altered expression and action of the low-affinity IgE receptor Fc{epsilon}RII (CD23) in asthmatic airway smooth muscle. J Allergy Clin. Immunol. 104:575.[Medline]
  18. Hakonarson, H., D. J. Herrick, P. Gonzalez-Serrano, M. M. Grunstein. 1997. Autocrine role of interleukin 1{beta} in altered responsiveness of atopic asthmatic sensitized airway smooth muscle. J. Clin. Invest. 99:117.[Medline]
  19. Hakonarson, H., M. M. Grunstein. 1998. Autologously up-regulated Fc receptor expression and action in airway smooth muscle mediates its altered responsiveness in the atopic asthmatic sensitized state. Proc. Natl. Acad. Sci. USA 95:5257.[Abstract/Free Full Text]
  20. Hakonarson, H., C. Carter, N. Maskeri, R. L. Hodinka, M. M. Grunstein. 1999. Rhinovirus-mediated changes in airway smooth muscle responsiveness: induced autocrine role of interleukin-1{beta}. Am. J. Physiol. 277:L13.[Abstract/Free Full Text]
  21. Hakonarson, H., D. J. Herrick, M. M. Grunstein. 1995. Mechanism of impaired {beta}-adrenoceptor responsiveness in atopic sensitized airway smooth muscle. Am. J. Physiol. 269:L645.[Abstract/Free Full Text]
  22. Hakonarson, H., N. Maskeri, C. Carter, R. L. Hodinka, D. Campbell, M. M. Grunstein. 1998. Mechanism of rhinovirus-induced changes in airway smooth muscle responsiveness. J. Clin. Invest. 102:1732.[Medline]
  23. Lazaar, A. L., H. E. Reitz, Jr R. A. Panettiery, S. P. Peters, E. Pure. 1997. Antigen receptor-stimulated pheripheral blood and bronchoalveolar lavage-derived T cells induce MHC class II and ICAM-1 expression of human airway smooth muscle. Am. J. Respir. Cell. Mol. Biol. 16:38.[Abstract]
  24. Grunstein, M. M., H. Hakonarson, N. Maskeri, C. Kim, S. Chuang. 2000. Intrinsic ICAM-1/LFA-1 activation mediates altered responsiveness of atopic asthmatic airway smooth muscle. Am. J. Physiol. 278:L1154.[Abstract/Free Full Text]
  25. Goldie, R. G., D. Spina, P. J. Henry, K. M. Lulich, J. W. Paterson. 1986. In vitro responsiveness of human asthmatic bronchus to carbachol, histamine, {beta}-adrenoceptor agonists and theophylline. Br. J. Clin. Pharmacol. 22:669.[Medline]
  26. Jr McFadden, E. R.. 1994. Asthma: morphologic-physiologic interactions. Am. J. Respir. Crit. Care Med. 150:523.
  27. Kay, A. B.. 1991. Asthma and inflammation. J. Allergy Clin. Immunol. 87:893.[Medline]
  28. Holt, P. G., C. Macaubas, P. A. Stumbles, P. D. Sly. 1999. The role of allergy in the development of asthma. Nature 402:B12.[Medline]
  29. Aalberts, R., H. F. Kauffman, B. Vrugt, et al 1993. Allergen-induced recruitment of inflammatory cells in lavage 3 and 24 h after allergen challenge in allergic asthmatic lungs. Chest 103:1178.[Abstract/Free Full Text]
  30. Chung, K. F., P. J. Barnes. Cytokines in asthma.. 1999. Thorax 54:825.[Free Full Text]
  31. Lee, S. C., Z. H. Jaffar, K. S. Wan, S. T. Holgate, K. Roberts. 1999. Regulation of pulmonary T cell responses to inhaled antigen: role of TH1 and TH2-mediated inflammation. J. Immunol. 162:6867.[Abstract/Free Full Text]
  32. Stanciu, L. A., R. Djukanovic. 1998. Role of ICAM-1 on T-cells in the pathogenesis of asthma. Eur. Respir. J. 11:949.[Abstract]
  33. Lazaar, A. L., S. M. Albelda, J. M. Pilewski, B. Brennan, E. Pure, Jr R. A. Panettieri. 1994. T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis. J. Exp. Med. 180:807.[Abstract/Free Full Text]
  34. Murray, A. G., P. Libby, J. S. Pober. 1995. Human vascular smooth muscle cells poorly co-stimulate and actively inhibit allogeneic CD4+ T cell proliferation in vitro. J. Immunol. 154:151.[Abstract]
  35. Suttles, J., R. W. Miller, C. F. Moyer. 1995. T cell-vascular smooth muscle cell interactions: antigen-specific activation and cell cycle blockade of T helper clones by cloned vascular smooth muscle cells. Exp. Cell Res. 218:331.[Medline]
  36. Savage, C. O. S., C. C. W. Hughes, B. W. McIntyre, J. K. Picard, J. S. Pober. 1993. Human CD4+ T cells proliferate to HLA-DR+ allogeneic vascular endothelium. Transplantation 56:128.[Medline]
  37. Nickoloff, B. J., R. S. Mitra, J. Green, X. Zheng, Y. Shimizu, C. Thompson, L. A. Turka. 1993. Accessory cell function of ketratinocytes for superantigens: dependence on lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. J. Immunol. 150:2148.[Abstract]
  38. Cunningham, A. C., J. G. Zhang, J. V. Moy, S. Ali, J. A. Kirby. 1997. A comparison of the antigen-presenting capabilities of class II MHC expressing human lung epithelial and ECs. Immunology 91:458.[Medline]
  39. Hansson, G. K., L. Jonasson, J. Holm, M. M. Clowest, A. W. Clowes. 1988. Gamma-interferon regulates vascular smooth muscle proliferation and 1a antigen expression in vivo and in vitro. Circ. Res. 63:712.[Abstract/Free Full Text]
  40. Fabry, Z., M. M. Waldschmidt, L. van Dyk, S. A. Moore, M. N. Hart. 1990. Activation of CD4+ lymphocytes by syngeneic brain microvascular smooth muscle cells. J. Immunol. 145:1099.[Abstract]
  41. Schroth, M. K.. 1996. Adhesion molecules in asthma and allergy. Immunol. Allergy Clin. North Am. 16:643.
  42. Calderson, E., R. F. Lockey. 1992. A possible role for adhesion molecules in asthma. J Allergy Clin. Immunol. 90:852.[Medline]
  43. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221.[Abstract/Free Full Text]



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