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Keywords:

  • CD4+ T lymphocytes;
  • intraepithelial lymphocytes;
  • CD103;
  • flow cytometry

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Background

The integrin CD103 is preferentially expressed on intraepithelial T lymphocytes, and cells expressing this integrin may play a regulatory role in the microenvironment of the epithelial cell layer.

Methods

The relative number of CD103+/CD4+ T cells in the bronchoalveolar lavage was significantly elevated in all patients diagnosed with interstitial lung diseases compared with patients with other non-fibrotic disorders of the lung.

Results

Analysis by flow cytometry showed that the CD103+ and the CD103 subpopulations were memory T cells based on the high expression of CD45RO+. However, the CD103+/CD4+ T cells were CD25low, CD27, CD28low, and CD62L, whereas the CD103/CD4+ T cells expressed CD25 and CD62L and were CD27high and CD28high. In addition, the CD103+/CD4+ T cells expressed significantly higher quantities of VLA-1 and CD101 than did CD103/CD4+ T cells. Reverse transcriptase polymerase chain reaction analysis of purified CD103+ and CD103 CD4+ T cells showed production of tumor necrosis factor (TNF) α-R-1 (p55), TNF-α-R-2 (p75), interferon γ, interleukin-10, and TNF-α mRNA in both subpopulations. No interleukin-4 mRNA was detected in either subpopulation.

Conclusions

CD103+/CD4+ T cells represent a T-helper 1–like subpopulation in human lungs with a distinct effector phenotype. Despite the lack of CD27 and the low CD25 and CD28 expression, these cells show a high degree of activation. These results suggest that CD103 expressing CD4 T cells in the lung are continuously activated, long-living cells. Cytometry Part B (Clin. Cytometry) 54B:19–27, 2003. © 2003 Wiley-Liss, Inc.

The epithelium represents a unique lymphoid compartment containing a distinct population of mucosal lymphocytes, the intraepithelial lymphocytes (1, 2). These T cells are nearly exclusively found in epithelial tissues, where they reside in intimate association with epithelial cells (3, 4). Most CD8+ T cells in or adjacent to the intestinal epithelium and 40–50% of the CD4+ T cells in the intestinal lamina propria express the integrin CD103 that mediates binding to E-cadherin at the basolateral side of the epithelium (5). CD103 is also expressed by lymphocytes within the bronchial epithelium, by some alveolar wall lymphocytes, and by bronchoalveolar fluid T cells (6, 7). However, in bronchoalveolar lavage fluid, the relative amount of CD103-expressing T cells is very different between CD4+ and CD8+ T cells. Most of the CD8+ T cells express this integrin independently of the disease of the lung (8). In contrast, the proportion of CD4+ T cells expressing CD103 is significantly higher in diseases associated with pulmonary fibrosis than in non-fibrotic diseases or healthy controls (8, 9). However, very little is known about the phenotype of CD103+ T cells and the conditions that lead to the striking accumulation of these cells in pulmonary fibrosis.

Activation of T cells is followed by the sequential expression of mostly well-defined activation markers. For instance, CD69 and CD25 are expressed within 24 h after activation, whereas the expression of human leukocyte antigen (HLA)–DR requires approximately 4 days. In contrast, VLA-1 and VLA-2 can be detected only several weeks after activation (10). The integrin CD103 also has been reported to be an activation marker on T cells and is expressed on the cell surface starting 2 days after in vitro activation (8). It is known that the costimulatory action of transforming growth factor (TGF) β1 and T-cell receptor–mediated signals are required for the expression of CD103. Functionally, this integrin plays an essential role as a homing receptor for epithelial tissue because CD103 knockout mice show a significant reduction in the number of intraepithelial lymphocytes (11). In addition, CD103 is critically important for the migration of CD8+ effector T cells into epithelial compartments (12).

Because the CD103+/CD4+ T lymphocyte population of the lung preferentially expands in fibrotic lung diseases, we hypothesized that this T-cell population might play a unique role in these disorders. Therefore, it was of interest to analyze the activity and surface receptor expression of these cells. We found that the CD103+ and CD103 T-cell subpopulations express a marker for memory T cells, CD45RO. To further characterize these cells, we also assessed their activity by analyzing activation markers by flow cytometry and cytokine production pattern with respect to the differentiation into T-helper 1 (Th1) or Th2 cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Antibodies and Chemicals

The antibodies used in this study including clone and company information are listed in Table 1. Phosphate buffered saline (PBS) was purchased from CellConcept (Umkirchen, Germany). PBS and fetal calf serum (FCS) were prepared with 2% FCS (Gibco Invitrogen GmbH, Karlsruhe, Germany) and used for all solutions and washes in flow cytometry. High pure RNA isolation kit was obtained from Roche Diagnostic Corporation (Mannheim, Germany), and a first-strand cDNA synthesis kit was obtained from Stratagene (Heidelberg, Germany).

Table 1. Antibodies Used in the Present Study
AntigenLabelaCloneIsotypeCompanyb
  • a

    APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; TC, TriColor (Caltag)

  • b

    Ancell, Bayport, MN; Beckman Coulter, Krefeld Germany; Caltag, Hamburg, Germany; Coulter, Hialeah, CA; DAKO, Buckinghamshire, UK; NatuTec GmbH, Frankfurt, Germany; Pharmingen Biosciences, Heidelberg, Germany.

CD3PE-Cy5UCHT1IgG1Coulter
CD3PE-Cy7S4.1IgG2aCaltag
CD4FITCS3.5IgG2aCaltag
CD4APCS3.5IgG2aCaltag
CD8APC3B5IgG2aCaltag
CD25FITCM-A251IgG1Pharmingen
CD27PECLB-27/1IgG2aPharmingen
CD28PEANC28.1/5D10IgG1Ancell Corp.
CD45PE-Cy5HI30IgG1Caltag
CD45APCHI30IgG1Caltag
CD45ROFITCUCHL1IgG2aDAKO
CD49a (VLA-1)FITCTS2IgG1NatuTec GmbH
CD49b (VLA-2)FITCGi9IgG1Beckmann Coulter
CD62LFITCDREG-56IgG1Caltag
CD69FITCFN50IgG1Pharmingen
CD101PEV7.1IgG1Pharmingen
CD103 (αE)FITC2G5 (HML-1)IgG2aCoulter
CD103 (αE)PEBer-ACT8IgG1DAKO
CD103 (αE)TC PE-Cy5LF61IgG1Caltag/Beckman Coulter
HLA-DRFITCTU36IgG2bCaltag

Patients

We analyzed the CD4+ T cells from a variety of fibrotic lung diseases. Table 2 summarizes the average age, the total number of patients in each group, and the proportion of females in the study, followed by a detailed description of the disease inclusion criteria.

Table 2. Disease, Average Age of Patients, and Number of Patients Analyzed
DiseaseAverage age (years)No. of patientsNo. of females
Sarcoidosis583214
Idiopathic pulmonary fibrosis54248
Hypersensitivity Pneumonitis45126
Bronchiolitis obliterans organizing pneumonia3694
Pneumoconiosis52153
Bronchial carcinoma593815
Sarcoidosis patients.

The study population consisted of 32 sarcoidosis patients with untreated sarcoidosis (mean age, 58 years; 14 females). Diagnosis was made by using the following criteria: compatible clinical history and chest x-ray; transbronchial or open lung biopsy demonstrating non-caseating epithelioid granulomas with giant cell formation and a coexisting morphology of the parenchyma with sarcoidosis; no reported history of mycobacterial, fungal, or parasitic infections; and no history of exposure to inorganic or organic material known to cause granulomatous lung diseases. All patients had active sarcoidosis with chest x-ray stage II or III and showed abnormal pulmonary functions with reduced lung volumes and impairment in diffusing capacity. Patients had high serum angiotensin-converting-enzyme levels and a CD4/CD8 ratio exceeding 5.0.

Idiopathic pulmonary fibrosis (IPF) patients.

The study population consisted of 24 patients with untreated IPF (mean age, 54 years; eight females). The diagnosis of IPF was determined according to the criteria of the International Consensus Statement. A definite diagnosis required an open or video-assisted thoracoscopic surgery (VATS) lung biopsy showing histologic features of usual interstitial pneumonia. Patients also showed abnormal pulmonary function test, impaired gas exchange, and characteristic abnormalities on conventional or high-resolution computed tomography (13).

Hypersensitivity pneumonitis (HP) patients.

This study population consisted of 12 patients with untreated HP (mean age, 45 years; six females). The diagnosis required the reported antigen exposure, dyspnea on exertion, inspiratory crackles, lymphocytosis in bronchoalveolar lavage (BAL), facultatively recurrent febrile episodes, infiltrates on chest radiographs, decreased carbon monoxide diffusion in the lung (DLCO), precipitating antibodies to farmer lung antigens, granulomas on transbronchial lung biopsy, and improvement with contact avoidance (14).

Bronchiolitis obliterans organizing pneumonia (BOOP) patients.

This study population consisted of nine patients with untreated BOOP (mean age, 36 years; four females). Diagnosis of BOOP required exclusion of any other condition that might provoke the clinical feature such as community acquired pneumonia, an unproductive cough, breathlessness with exertion, and a combination of BAL lymphocytosis (>25%), CD4+/CD8+ ratio less than 0.9 combined with foamy macrophages (20%), neutrophils (>5%), or eosinophils (2–25%). Diagnosis was accepted when lung biopsies demonstrated the typical histopathologic pattern, such as excessive proliferation of granulation tissue within small airways and alveolar ducts and chronic inflammation in surrounding alveoli (15).

Pneumoconiosis patients.

This study population consisted of 15 patients with untreated pneumoconiosis (mean age, 52 years; three females). The diagnosis was based on clinical signs such as cough and breathlessness, the documented occupational exposure to a known hazardous dust, the presence of characteristic radiographic changes, and exclusion of other interstitial lung diseases (16).

Bronchial carcinoma patients.

This study population consisted of 38 patients with bronchial carcinoma (mean age, 59 years; 15 females). Bronchogenic carcinoma was verified histologically with transbronchial lung biopsy or surgical resection. Typing and grading of tumors was performed according to the standards of the Union Internationale Contre le Cancer. None of the subjects showed sings of pneumonia or fibrosis (17).

Flow Cytometry and Cell Sorting

Flow cytometry was performed according to standard protocols. BAL fluid was filtered through three layers of gauze and then centrifuged at 280g for 10 min. The cells were resuspended in PBS containing 2% FCS (PBS-FCS), counted, and 105 cells were used for cytospin preparation (Shandon, Berlin, Germany). Cytospins were stained with May-Grünwald-Giemsa, and 200 cells were differentiated on a microscope (Zeiss, Jena, Germany). Between 105 and 5 × 105 BAL T cells per staining were labeled with anti–CD25 and fluorescein isothiocyanate (FITC), anti–CD69-FITC, anti–HLA-DR-FITC, anti–CD45RO-FITC, CD62L-FITC, anti–VLA-1-FITC, or anti–VLA-2-FITC with anti–CD103 and phycoerythrin (PE), anti–CD3-PE-Cy5, and anti–CD4 and allophycocyanin (APC). In addition, T cells were stained with anti–CD103-FITC (CD103 = αE) and anti–CD27-PE, anti–CD28-PE, or anti–CD101-PE, anti–CD3-PE-Cy5, and anti–CD4-APC. Alternatively, anti–CD8-APC or anti–γδ–T-cell–APC antibodies replaced the anti–CD4-APC antibody. Analysis was done on a FACSCalibur (BD Biosciences, Heidelberg, Germany), with scatter gates set on the lymphocyte population on forward scatter versus side scatter (SSC). Further, gating was done on SSC versus CD3+ and SSC versus CD4+, CD8+ or γδ T-cell population. Sorting of the T-cell subpopulations was done by staining with anti–CD4-FITC, anti–CD103-PE, anti–CD45-PE-Cy5, and anti–CD3-PE-Cy7. Two populations, CD103+/CD4+ and CD103/CD4+ T cells, were sorted with a MoFlo high-speed cell-sorter (Cytomation, Fort Collins, CO), and RNA extraction was performed immediately after sorting.

Total RNA Extraction and Reverse Transcription

Sorted CD4+ T cells (1–2 × 105) were lysed in lysis buffer followed by RNA extraction according to the standard protocol of the High-Pure RNA isolation kit (Roche, Ingelheim, Germany). RNA was isolated and immediately subjected to reverse transcription. For reverse transcription, purified RNA was supplemented with oligo(dT) primer followed by buffer, RNase block, dNTPs, and MMLV reverse transcriptase (Stratagene).

Multiplex Polymerase Chain Reaction (PCR)

We used the multiplex PCR proinflammatory and Th1/Th2 kits from Biosource to analyze multiple mRNA expression in the target cells (Biosource International, Solingen, Germany). Complementary DNA was mixed with buffer, primer mixture, Taq DNA polymerase, and dNTP. PCR began with denaturing at 96°C for 1 min followed by a 4-min annealing step at 60°C. Thirty-three cycles were performed with denaturing at 96°C for 1 min and annealing at 60°C for 2.5 min each. For the final step, the mixture was incubated at 72°C for 10 min followed by 5 min at 25°C. PCR products were analyzed by 1.6% agarose gel electrophoresis and visualized after staining with ethidium bromide using a gel-detection system from Biostep (Jahnsdorf, Germany).

Statistical Analysis

Statistical analysis of the results was performed on a PC using SPSS 10.0 for Windows. Differences between different populations were analyzed with the Wilcoxon signed rank test, and differences with P ≤ 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Table 3 lists the BAL cells used for sorting and analysis of mRNA expression of CD103+ and CD103 CD4+ T cells and the physiologic data obtained from these patients.

Table 3. BAL CD4+ T Cells Used for mRNA Analysis of Pro-Inflammatory and Th1/Th2 Cytokines and Receptorsa
BAL no.% CD103+b% LymphcDiseaseFEV1VCFEVI/VCDLCO
  • a

    BAL, bronchoalveolar lavage; DLCO, carbon monoxide transfer factor; FEV1, forced expiratory volume in 1 s; HP, hypersensitivity pneumonitis; IPF, idiopathic pulmonary fibrosis; n.p., not performed; VC, vital capacity.

  • b

    Proportion of CD103+ CD4+ T lymphocytes within the CD45+ CD3+ CD4+ T-cell population.

  • c

    Proportion of lymphocytes within the total BAL cell population.

B-11482.277.0HP86%94%93%91%
B-11514.822.0Sarcoidosis II78%89%95%98%
B-18356.874.0IPF75%74%106%46%
B-20051.626.5Pleuritis tuberculosa38.3%34.8%101%n.p.
B-20736.219.3Collagen vascular disease53%49%101%78.5%
B-21737.643.0Sarcoidosis I126%133%99%88%
B-21964.338.0IPF95%97%102%57%

CD103 Expression on T-Lymphocyte Subpopulations in the Lung

The integrin CD103 is expressed on intraepithelial lymphocytes in the lung. There was, however, a significant difference between the BAL T-cell subpopulations in patients without pneumonia or pulmonary fibrosis, with most of the CD8+ T lymphocytes expressing this integrin, but fewer than 10% of the CD4+ T cells expressing it (Fig. 1, row 1). Patients with inflammatory lung diseases showed a significantly increased proportion of CD103+/CD4+ T cells compared with the group without pneumonia or pulmonary fibrosis (Fig. 1, row 2). However, patients with interstitial lung diseases showed an even higher relative number of BAL CD4+ T cells expressing CD103 (Fig. 1, row 3). The BAL γδ T cells also showed an increased proportion expressing CD103 in fibrotic lung diseases compared with the group without pneumonia or pulmonary fibrosis (Fig. 1, column 3). However, this difference was not statistically significant. Likewise, no significant change was observed in the CD8+ T-cell population after comparing the different study populations (Fig. 1, column 2). Table 4 summarizes these data for several patients in each group. Only the proportion of CD103+/CD4+ T cells was significantly different between the three groups of patients.

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Figure 1. Expression of CD103 on bronchoalveolar lavage CD4, CD8, and γδ T cells. Row 1 shows a set of representative histograms of the integrin CD103 expression on CD4 (left), CD8 (middle), and γδ (right) cells of a patient without pneumonia or fibrosis (bronchial carcinoma). Row 2 shows representative histograms of the expression of CD103 on T cells from a patient with pneumonia. Row 3 shows a set of representative histograms from a patient with pulmonary fibrosis. The percentage given in each histogram indicates the proportion of CD103+ cells within the analyzed T cell population gated on side scatter (SSC) versus CD3 and SSC versus CD45.

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Table 4. Proportion of CD103+ T Cells in T-Cell Subpopulationsa
 %CD103+ CD4n%CD103+ CD8n%CD103+ γδn
  • a

    Proportion of CD103+ T cells ± standard deviation; n, number of patients in the group.

No fibrosis, no pneumonia8.5 ± 3.63859.8 ± 21.93545.3 ± 24.928
Pneumonia, no fibrosis21.7 ± 7.11671.5 ± 14.41660.9 ± 24.315
Fibrosis57.8 ± 11.41676.4 ± 16.91572.8 ± 14.616

Expression of CD25, CD27, CD62L, CD28, VLA-1, CD101, CD45RO, HLA-DR, and CD69 on CD4+ T Lymphocytes in the BAL of Patients With Interstitial Lung Diseases

Further, we analyzed the phenotype of CD4+ T lymphocytes characterized by the expression or absence of CD103+. In contrast to the CD103 CD4+ T lymphocyte population, hardly any CD103+ cells expressed CD25 (interleukin-2 [IL-2] receptor), CD27, or CD62L (Fig. 2, top row). The expression of CD28 differed significantly, which was much lower on CD103+ cells than on CD103 cells, whereas the expressions of VLA-1 and CD101 were higher on the CD103+ T-cell population (Fig. 2, middle row). Hardly any difference between the two CD4+ T-cell populations was seen with regard to the expressions of CD45RO, HLA-DR, and CD69 (Fig. 2, bottom row). The data are summarized in Table 5, which also shows that a slightly higher proportion of the CD103+ CD4+ T cells expressed VLA-2. This difference between these two populations was found in all patients irrespective of the underlying form of pulmonary disorder.

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Figure 2. Expression of different surface markers on CD103+ and CD4 T cells. Expressions of CD25, CD27, and CD62L (top), CD28, CD49a, and CD101 (middle), and CD45RO, HLA-DR, and CD69 (bottom) responses, from left to right, on CD103+ and CD103 CD4+ bronchoalveolar lavage (BAL) T cells. Dot plots show analysis of one representative BAL (BAL 183 or BAL 209). The cells were gated on the lymphocyte population and the expressions of CD3 and CD4, and differentiation was based on the expression of CD103. The fluorescence of the CD103 antibody (x axes) was chosen based on the availability of the antibody shown (y axes).

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Table 5. Expression of Various Activation Markers and Receptors on the Surface of CD103+ and CD103 CD4+ T Cells in the Bronchoalveolar Lavage of Patients With Various Lung Diseases
Markerna%CD103+b ± S.D.a%CD103 ± S.D.P*
  • a

    n, number of samples analyzed; S.D., standard deviation.

  • b

    proportion of CD103+ T cells within the CD4+ T-cell population.

  • *

    Wilcoxon signed rank test was used to calculate statistical differences.

CD25414.9 ± 1.315.2 ± 1.4<0.001
CD45RO2898.6 ± 0.397.1 ± 0.80.034
HLA-DR3612.0 ± 1.88.4 ± 1.30.002
CD693669.4 ± 3.949.3 ± 4.2<0.001
CD27195.9 ± 1.138.3 ± 3.6<0.001
CD282522.1 ± 3.377.9 ± 2.50.004
CD62L163.3 ± 0.923.1 ± 3.7<0.001
VLA-13639.7 ± 4.317.7 ± 3.1<0.001
VLA-2115.7 ± 1.31.9 ± 0.50.015
CD1013475.0 ± 4.640.5 ± 4.6<0.001

Analysis of Inflammatory Cytokine mRNA Expression of CD103+ and CD103 BAL CD4+ T Cells

The accumulation of CD103+ CD4+ T cells in interstitial lung diseases suggested their involvement in the pathogenesis of the disease. Therefore, we analyzed the transcriptional activity of these cells for a variety of pro-inflammatory cytokines and receptors. As shown in Figure 3, the activation pattern of the purified CD103+and CD103 T cells was very similar, with high mRNA production for tumor necrosis factor receptor 1 (TNF-R1; p55) and TNF-R2 (p75) and for the IL-6 receptor mRNA. Messenger RNA for TNF-α and IL-6 was also detected, with no difference between the CD103+ and CD103 populations. However, TNF-α mRNA was not detectable in BALs 183 and 219. Both patients were diagnosed with IPF and showed a high amount of CD103+ CD4+ T cells.

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Figure 3. Multiplex polymerase chain reaction for inflammatory cytokines. Multiplex reverse transcriptase polymerase chain analysis of isolated bronchoalveolar lavage (BAL) CD103+ and CD103 CD4+ T cells from patients with different lung diseases (Table 2). The base-pair marker (lane M) is a 100-bp ladder with 1,000 bp at the top. The expected bands of the product (lanes C) are Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (921 bp), tumor necrosis factor α (TNF-α; 680 bp), granulocyte-macrophage colony-stimulating factor receptor (GMCSF-R; 607 bp), interleukin-1β (IL-1β; 555 bp), tumor necrosis factor receptor 1 (TNF-R1; 490 bp), granulocyte-macrophage colony-stimulating factor (GMCSF; 424 bp), interleukin-6 (IL-6; 360 bp), interleukin-6 receptor (IL-6-R; 300 bp), and tumor necrosis factor receptor 2 (TNF-R2; 220 bp). The BAL numbers at the top of the lanes correspond to the BAL numbers in Table 3. Lanes marked with plus signs represent CD103+ CD4+ T cells.

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Th1/Th2 Cytokine Pattern of CD103+ and CD103 BAL CD4+ T Lymphocytes

Analysis of the Th1/Th2 cytokine pattern in the purified CD103+ and CD103 T lymphocytes showled strong interferon-γ (IFN-γ) and weak IL-10 mRNA expression. In parallel, no IL-4 mRNA could be detected (Fig. 4). IL-2 mRNA was detected only in cells from patients diagnosed with sarcoidosis, which may relate to the expansion of the CD4+ T cells in this disease. Aside from this difference, there was no disparity between the analyzed patient groups. In addition, no difference was observed between the CD103+ and CD103 CD4+ T lymphocytes.

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Figure 4. T-helper 1/2 cytokine mRNA analysis of bronchoalveolar lavage (BAL) CD103+ and CD103 CD4+ T cells from patients with different lung diseases (Table 2). The expected bands of the product (lanes C) are GAPDH (921 bp), interleukin (IL)–4 (525 bp), IL-2 (425 bp), interferon-γ (IFN-γ; 292 bp), and IL-10 (223 bp).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The integrin CD103 functions to promote T-cell migration into the epithelium and is the requisite homing molecule of T lymphocytes to the epithelium (11, 12). Although it is expressed on only a small proportion of blood T cells, it is induced upon activation of the T cells in the presence of transforming growth factor (TGF) β1 (8, 18). In previous work we reported that the regulation of CD103 expression differs between CD4+ and CD8+ T cells. Moreover, we and others reported that the proportion of CD103+ T cells in the BAL within the CD4+ T-cell population increases significantly in interstitial lung diseases (8, 9).

The data presented show that the CD103+ and CD103 CD4+ BAL T-lymphocyte subpopulations are highly activated CD69+ and CD45RO+ T lymphocytes. There were, however, some remarkable differences. CD25, CD27, and CD62L were almost only expressed on CD103 in comparison with CD103+ CD4+ T cells. CD28 expression was high on CD103 but low on CD103+ T cells. In contrast, CD101, VLA-1, and VLA-2 were expressed on CD103+ and significantly less on CD103 T cells. Despite these differences, the CD103+ and CD103 CD4+ BAL T-cell populations showed characteristics of Th1 cells, with IFN-γ and no IL-4 mRNA production. In addition to the production of IFN-γ mRNA, we detected IL-10 mRNA. These data propose that the CD103+ CD4+ T lymphocytes are a phenotypic distinct memory or effector T-cell population with respect to the expression of this integrin and with respect to the expression of receptors characteristic for naive or activated CD4+ T lymphocytes.

During the response to antigen, the numbers of activated cells increases rapidly. This expansion is driven by engagement of T-cell receptors and costimulatory proteins such as CD28, CD27, CD134, 4-1BB, LFA-1, and CD2 on the surface of antigen-engaged T cells (19). Activated antigen-specific T cells are short-lived, and death of these massively expanded activated cells probably serves to control inflammation. However, a small proportion of the activated T cells differentiated into memory T cells characterized by the expression of CD45RO. In the present study the CD103 and the CD103+ were CD45RO+, which classifies them as memory cells. It has been described that memory cells can be distinguished by their expression of CD62L and CCR7. CD62LhiCCR7+ are central memory cells and CD62LloCCR7 are effector memory cells (20, 21). The expression of CD62L was low on the CD103 CD4+ T-cell population and nearly absent on the CD103+ T cells. This finding and the one that both cell populations produce mRNA for IFN-γ and no IL-2 suggest that both cell populations are effector memory cells. However, we did not analyze the expression of CCR7.

Peripheral “memory” CD45RO+ human CD4+ T cells express CD28 (22). In this study the BAL “memory” CD45RO+ CD4+ T cells varied with respect to CD28 expression and allowed separation into two groups of cells: one that expressed CD28 but not CD103 and another that expressed low CD28 and high CD103. Low expression of CD28 has been described on CD4+ T cells (23). The significance of this finding is not known. However, T cells from CD28-deficient mice showed a reduced proliferative response to antigen in vitro (24). Therefore, CD103+ CD28low T cells may represent a differentiated population of low proliferation rate. This possibility would be in agreement with the notion that memory T lymphocytes are slowly but continuously dividing cells (19). However, we did not teste the proliferative capacity of the BAL CD4+ T cells, and the relative expansion in fibrotic lung diseases remains to be analyzed. CD101 and CD103 have been described as costimulatory molecules for the activation of T cells. High expression of these molecules therefore may substitute for the lack of CD27 and CD28. It has been shown that the antibodies 2E7 and M290 against CD103 act as costimulatory signals with anti–T-cell receptor antibodies to increase the lytic machinery of intraepithelial CD8+ cells (25). This finding was confirmed by the experiments of Sarnacki et al. who described a strong synergistic effect of CD3-induced activation and CD103 crosslinking on the proliferative response of intraepithelial lymphocytes (26, 27). In addition, the molecule CD101 seems to be a strong costimulatory molecule for T cells (28). A gene that predicts a seven-immunoglobulin domain chain-like structure encodes CD101. It has restricted expression predominantly on mucosal T lymphocytes and appears to have a costimulatory function of special relevance for CD28 T cells and for mucosal lymphocytes. The expressions of CD101 and CD103 may explain the strong activation status of the CD103+ T cells in the absence of CD25, CD27, and CD28. More experiments are required to identify the difference in activity between the CD103+ and CD103 T lymphocytes.

Another finding of the study was that the CD103+/CD4+ T cells express VLA-1 and VLA-2, activation markers expressed several weeks after activation (10). These molecules are expressed after prolonged activation of T cells and indicate the presence of “older” or differentiated cells. This is supported by the observation that repeated activation induces the loss of CD28 (29). Therefore, CD103+/CD4+ T cells appear to participate in fibrogenic inflammation and may have developed as consequence of recurring contact with activation signals such as cytokines or other triggers. However, it is not clear whether these cells represent pro-inflammatory or protective cells. Data from work with experimental colitis indicated that the pro-inflammatory component might be dominating (30). In contrast to this finding, CD103-deficient mice showed increased skin inflammation, suggesting a protective role of these cells (31). Aside from the phenotypic differences between CD103+ and CD103 CD4+ T lymphocytes, we did not identify factors that distinguish between these two populations, even when cells obtained from non-fibrotic conditions were analyzed. Both subpopulations showed expression of mRNA for inflammatory cytokines and receptors. There were, however, differences between some diseases with regard to the CD103+ and CD103 CD4+ T lymphocytes. We found IL-2 mRNA only in patients with sarcoidosis, and TNF-α mRNA was absent in IPF patients. But the small number of patients does not allow generalizing this observation.

In addition to the loss of CD25 and CD28 expression, the CD103+ CD4+ T cells in the BAL fluid did not express CD27. It has been reported that loss of CD27 expression is irreversible and represents terminal effector T-cell differentiation (32, 33). This observation may suggest that the CD103+ CD4+ T lymphocytes are terminally differentiated effector T cells. Therefore, the increase in CD103+/CD4+ T cells may be a consequence of expansion and accumulation of effector CD4+ T cells that might be involved in the process of lung fibrosis. However, the CD103 CD4+ T-cell population also showed signs of high activation and expresses CD27. Consistent with the work from Ludvikson et al. (30), this finding suggests that CD103+ T lymphocytes are effector cells and CD103 comprise the memory cell population. More data are required to confirm this hypothesis because we could not identify differences between the CD103+ and CD103 populations with respect to pro-inflammatory parameters.

In summary, the results described in this paper demonstrate that the CD103 and the CD103+ BAL CD4+ T-cell populations are highly active cells. The CD103+ population displays a distinct phenotype of a continuously activated long-living cell that may exert effector function and may be distinct from the CD103 subpopulation. Newly discovered markers may provide an opportunity to distinguish memory from effector T cells (34, 35). Although detection of these cells may provide a diagnostic marker when interstitial lung diseases are expected, their functional involvement in this process warrants further investigation.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED