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(Chest. 2005;128:2971-2977.)
© 2005 American College of Chest Physicians

Relationship of BAL and Lung Tissue CD4+ and CD8+ T Lymphocytes, and Their Ratio in Idiopathic Pulmonary Fibrosis*

Spyros A. Papiris, MD, FCCP; Androniki Kollintza, MSc; Panagiota Kitsanta, MD; George Kapotsis, MD; Marilena Karatza, PhD; Josef Milic-Emili, CM, MD, FRSC; Charis Roussos, MD, MSc, PhD, MRS and Zoe Daniil, MD

* From the Departments of Critical Care and Pulmonary Services (Drs. Papiris, Kapotsis, Roussos, and Daniil, and Mr. Kollintza), Pathology (Dr. Kitsanta), and Hematology (Dr. Karatza), National and Kapodistrian University of Athens, "Evangelismos" Hospital, Athens, Greece; and Meakins-Cristie Laboratories (Dr. Milic-Emili), McGill University, Montreal, QC, Canada.

Correspondence to: Spyros A. Papiris, MD, FCCP, Associate Professor, Department of Critical Care and Pulmonary Services, 45-47 Ipsilantou St, "Evangelismos" Hospital, GR 10675, Athens, Greece; e-mail: papiris{at}otenet.gr


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Surgical biopsy specimens have shown that T lymphocytes (TLs) infiltrate lung parenchyma in patients with idiopathic pulmonary fibrosis (IPF) and might play a pathogenetic role. BAL, a far less invasive technique, has also been used for the investigation of IPF pathogenesis. However, controversy exists whether the BAL fluid cellular profile reflects the cellular composition of the lung parenchyma.

Study objective: To compare infiltrating TLs subpopulations (CD4+, CD8+, and CD4+/CD8+ ratio) in lung tissue and BAL fluid.

Patients and methods: Immunohistochemistry was performed according to the streptavidin-biotin method on the surgical biopsy specimens of 12 untreated patients with IPF. The number of CD3+, CD4+, and CD8+ TLs was determined by observer-interactive computerized image analysis (SAMBA microscopic image processor; Meylan, France). In BAL fluid, the same TLs subpopulations were evaluated by flow cytometry.

Results: In lung tissue, CD3+ TLs accounted for a mean (± SEM) of 28.8 ± 7% of total cells, CD4+ TLs accounted for 14.5 ± 4% of total cells (50.1 ± 4% of CD3+ TLs), and CD8+ TLs accounted for 13.8 ± 4% of total cells (47.4 ± 4% of CD3+ TLs). In BAL fluid, lymphocytes accounted for 9.8 ± 2.5% of total cells, CD4+ TLs accounted for 51.8 ± 4% of CD3+ TLs, and CD8+ TLs accounted for 42.2 ± 4% of CD3+ TLs. Tissue CD4+ and CD8+ TLs (expressed as a percentage of CD3+ TLs) correlated significantly with the number of CD4+ and CD8+ TLs in BAL fluid (r = 0.846 and p = 0.001 vs r = 0.692 and p = 0.013, respectively). A significant positive correlation was also found between the mean CD4+/CD8+ ratio found in tissue and BAL fluid (1.05 ± 0.21 and 1.5 ± 0.27, respectively; r = 0.832; p = 0.01).

Conclusion: The results suggest that in patients with IPF, the TL subpopulations in BAL fluid reflect the pattern of lymphocytic infiltration in pulmonary parenchyma.

Key Words: BAL • CD4+ • CD8+ • flow cytometry • idiopathic pulmonary fibrosis • immunohistochemistry • inflammation T lymphocytes


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal form of interstitial pneumonia of unknown etiology, histologically characterized by varying amounts of inflammation and fibrosis.12 A definitive diagnosis of IPF and its secure differentiation from the other interstitial pneumonias can be established only with the aid of a surgical lung biopsy.3 Surgical biopsies are also of paramount importance as a research tool. Studies of surgical biopsies have shown that, among the different inflammatory cells, T lymphocytes (TLs) appear to predominate in the lung parenchyma infiltrate of patients with IPF and might play a role in its pathogenesis.456 However, surgical biopsies are not immune to limitations such as invasiveness, which renders them not acceptable by every patient, as well as morbidity and mortality.789

BAL is a far less invasive technique that is used for research and, occasionally, for diagnostic purposes. It is of value to study the immune and inflammatory mechanisms in the lower airways, since most of the cells recovered are believed to be derived from both air spaces and lung interstitium.10 Inflammatory cells have been studied in IPF by BAL,11 and many of the conclusions regarding the role of inflammation in its pathogenesis have been drawn from these studies. The possible role of lymphocytes in the pathogenesis of IPF has received little investigation, since increases in their relative number are uncommon in BAL fluid. Therefore, early BAL studies10 have driven attention to neutrophils as well as macrophages. Investigations of BAL fluid inflammatory cells in IPF patients have shown that TLs, and among them CD8+ TLs, are prominent in patients with IPF12 and may also be associated with a worse prognosis.13

However, controversy exists as to whether the BAL fluid cellular profile reflects the cellular composition of the lung parenchyma, and no studies have evaluated subsets of lymphocytes in surgically obtained lung tissue and BAL fluid in the same IPF patients. In this study, we have compared the TL subpopulations (ie, CD4+ and CD8+), and their ratio in BAL fluid and surgical samples of 12 IPF patients. The TL subpopulations in BAL fluid and lung tissue have been studied by flow cytometry and quantitative immunohistochemistry, respectively.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
The study population consisted of 12 untreated patients with IPF (5 male patients, 7 ex-smokers, and 5 never-smokers) whose mean (± SEM) age was 60 ± 2 years. They were recruited sequentially from the respiratory outpatient clinic of the "Evangelismos" General Hospital, Athens, Greece, over a period of 3 years. The diagnosis of IPF was based on clinical findings (ie, exertional dyspnea, nonproductive cough, and fine bibasilar inspiratory crackles), pulmonary function test results (ie, restrictive pattern and impaired gas exchange), and high-resolution CT findings (ie, bibasilar reticular abnormalities with minimal ground-glass opacities consistent with the diagnosis of IPF).1 The diagnosis of IPF was confirmed by video-assisted thoracoscopic lung biopsy in all patients. A right thoracic approach was performed, and two or three samples were taken from the right lower lobe or middle lobe in the region of the greater fissure. Biopsy of the lingular tip was avoided, as changes in this area may be particularly advanced and unrepresentative. All patients experienced a normal and uncomplicated postoperative course. None of the patients included in this study had a history of environmental or occupational exposure, drug toxicity, or connective tissue disease. The study was approved by the institutional ethics committee, and informed consent was obtained from each patient.

BAL Procedure
Bronchoscopies were performed on patients under sedation by use of a flexible fiberoptic video bronchoscope wedged into a segmental bronchus of the right middle lobe. BAL was performed using 20-mL aliquots of warmed sterile normal saline solution introduced by syringe through the bronchoscopic aspiration port. A fixed volume of 120 mL of saline solution was infused sequentially. The returned fluid was extracted through the same syringe and placed on ice. BAL fluid specimens were analyzed within 2 h of their acquisition. All returned fluid was filtered through nylon sterile gauze to remove mucus and pooled, and the total volume was measured. The total cell count was evaluated on an aliquot of the pooled fluid using a Neubauer counting chamber. Cell viability was determined by the trypan blue exclusion test, and in all cases the count was higher than 90%. The BAL fluid was centrifuged at 400g at 4°C for 10 min. The cell pellet was washed twice with cold phosphate-buffered saline solution and resuspended in 4 mL of RPMI 1640 medium (Gibco; Grand Island, NY) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Gibco).

Lymphocyte Immunophenotyping by Flow Cytometry
Lymphocyte subsets in BAL fluid were evaluated by multiparameter analysis of leukocytes by flow cytometry. Following gentle mixing, 100 µL of 0.5 x 106 BAL fluid cells were incubated with 10 µL of monoclonal antibody at 4°C for 20 min. The following combinations of monoclonal antibodies were used: CD4-fluorescein isothiocyanate (Coulter Cyto-Stat; Beckman Coulter; Roissy, France); CD8-RD1 (Coulter Cyto-Stat; Beckman Coulter); and CD3-PECy5 (Coulter Cyto-Stat; Beckman Coulter). Following incubation, the RBCs were lysed (0.17 mmol/L NH4Cl lysis buffer), and the stained cells were washed with phosphate-buffered saline solution, collected by centrifugation, and resuspended in 1% paraformaldehyde.

The samples were analyzed using a flow cytometer (ELITE ESP; Coulter Electronics; Miami, FL), which was equipped with an argon laser providing an excitation wavelength of 488 nm. Before measurement, the optical path was adjusted (FlowChek; Beckman Coulter). The result of one half coefficient of variation was approximately < 2%. Data acquisition and analyses were performed using the Coulter Elite workstation 4.0 software (Coulter Electronics). A count cycle contained 10,000 cells. Using a combination of forward and light-scatter characteristics, the lymphocytes were identified as small cells and then were sent to a second dot-plot to discriminate CD3+ TLs. This CD3+ lymphoid population was then analyzed with respect to their CD4 and CD8 expression.1415

Histology
Lung biopsy specimens from the 12 patients were used. They were taken for diagnostic purposes and were analyzed according to the criteria of Katzenstein et al5 by our pathologist. Specimens were fixed in 4% formalin and after dehydration were embedded in paraffin. Tissue sections were oriented, serial sections of 4 µm thickness were cut, and immunohistochemistry was performed according to the streptavidin-biotin method.

Immunohistochemistry
To evaluate the lymphocyte subpopulations, lung tissues were stained with mouse monoclonal antibodies anti-pan-T cell (anti-CD3; dilution, 1:200), anti-CD4 (dilution, 1:100), and anti-CD8 (dilution, 1:40) [Dako; Glostrup, Denmark] according to the labeled streptavidin-biotin complex method. The sections were deparaffinized and rehydrated with Tris-buffered saline solution (0.005 mmol/L Tris and 0.15 mmol/L NaCl; pH, 7.6) for 10 min. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 min. Then the sections were washed in Tris-buffered saline solution and incubated with primary antibodies at appropriate dilutions for 1 h. Biotinylated antimouse IgG was used as a secondary antibody (Dako), followed by peroxidase-conjugated streptavidin (Dako). The peroxidase reaction was developed using 3,3'-diaminebenzidine tetrachloride (0.25 mg dissolved in 1 mL of 0.02% hydrogen peroxide) for 3 min.

Lung Parenchyma Computer Image Analysis
The number of positively stained cells was determined by observer-interactive computerized image analysis (SAMBA microscopic image processor; Meylan, France), the hardware and software of which have been described by Brugal et al.16 This system is fitted with a standard axioplan microscope (Carl Zeiss; Oberkochen, Germany), a color video camera (Sony Corporation; Tokyo, Japan), an image analysis processor (Matrox; Montreal, QC, Canada), and a personal computer (Pentium 2, 166-MHZ processor; Intel; Santa Clara, CA). Estimation of the SEM within 95% confidence limits required a maximum of at least 15 randomly selected high-power fields (magnification x400 with the Zeiss microscope; analysis area, approximately 110,000 µm2). The immunostaining was analyzed with cells stained a dark brown color and counterstained cells appearing as a false blue. Formal scoring (labeling index) for each antibody was then performed in one section for each paraffin block. Interobserver variability was very low (ie, < 0.03%). The results were expressed as the percentage of nuclear immunopositive surface in relation to the total nuclear surface of infiltrative cells within the tissue (labeling index), as previously described.1718 Blood vessels, connective tissue, and cartilage structures were excluded.

Statistical Analysis
All data were expressed as the arithmetic mean ± SEM. Correlation coefficients were calculated using the Spearman rank method. A p value of < 0.05 was considered to be statistically significant. Analysis was performed using a statistical software package (SAS; SAS Institute; Cary, NC).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In lung tissue, the mean TL (CD3+) cell population was 28.8 ± 7% of the total number of cells. The mean CD4+ subpopulation was 14.5 ± 4% of the total number of cells, while the CD8+ subpopulation was 13.8 ± 4% of the total number of cells. In BAL fluid, the mean total number of cells per milliliter of recovered BAL fluid was 128 ± 17 x 103, and TLs were 9.8 ± 2.5% of the total number cells.

Tissue and BAL lymphocyte subsets expressed as percentage of TLs (CD3+ cells) are shown in Table 1 . A greater dispersion of the data was observed in the percentage of CD4+ and CD8+ TLs found in the BAL fluid than in the biopsy specimens, but there was a significant correlation for each subset between the two specimens (Fig 1, 2 ) A significant positive correlation was also found between the CD4+/CD8+ ratios in tissue and BAL fluid (Fig 3 ).


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  		Table 1.. The Subpopulations of Lung Tissue and BAL Fluid TLs and Their Respective Correlations*

 


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  		Figure 1.. Correlation between the percentage of CD4+ TLs in lung tissue and BAL fluid. The p value was derived from the Spearman rank correlation coefficient.

 


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  		Figure 2.. Correlation between the percentage of CD8+ TLs in lung tissue and BAL fluid. The p value was derived from the Spearman rank correlation coefficient.

 


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  		Figure 3.. Correlation between CD4+/CD8+ ratios in lung tissue and BAL fluid. The p value was derived from the Spearman rank correlation coefficient.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that in patients with IPF the TL subpopulations (ie, CD4+, CD8+, and CD4+/CD8+ ratio) recovered by BAL, as evaluated by flow cytometry, were closely correlated with the corresponding values found in lung tissue, as evaluated by quantitative immunohistochemistry. This implies that further investigations of their phenotypic and functional characteristics by means of the safer and less invasive BAL technique should provide valid insights regarding their role in the pathogenesis of the IPF.

In IPF, histologic damage progressively leads to defects in respiratory mechanics and gas exchange, and manifests clinically with exertional dyspnea ending in respiratory failure.1 Histologically, IPF is characterized by the coexistence of a patchy chronic inflammatory infiltrate with an abnormal extracellular matrix deposition, foci of fibroblasts, and alveolar collapse. This histologic picture of usual interstitial pneumonia is a prerequisite for diagnosis, but other less aggressive forms of histologic damage, such as nonspecific interstitial pneumonia (NSIP), may coexist in different lung surgical specimens from the same IPF patient.19 However, although inflammation and fibrosis coexist in IPF patients, the role of inflammation in the pathogenesis of fibrosis is debatable.20 Few studies6212223 have attempted to characterize the chronic inflammatory infiltrate using immunohistologic analysis in order to define also the TL subpopulations in lung tissue. From these studies, it has been shown that both TL subsets, CD4+ and CD8+, infiltrate altered lung tissue. The potential role of the TL subsets in the pathogenesis of IPF has largely not been investigated, but it has been postulated24 that the type of inflammatory process may modulate tissue injury. From animal studies,2526 it has been shown that TLs might play a role in the initiation and evolution of pulmonary fibrosis through their ability to secrete fibrogenic cytokines.

Inflammatory cells including subpopulations of TLs have been also studied by BAL, and many of the conclusions regarding the role of inflammation in interstitial lung disorders have been drawn from these studies.10 The possible role of lymphocytes in the pathogenesis of IPF traditionally received little investigation, since an increase in their number is an uncommon finding in BAL samples and very few studies have investigated TL subpopulations in BAL fluid.1213 However, from these studies it has been shown that the number of CD8+ TLs is increased in BAL fluid in IPF patients12 and that this may also be associated with a worse prognosis, implicating some role in the pathogenesis of fibrosis from this subset of TLs.13

TLs and their phenotypic and functional characteristics have been more extensively studied in patients with scleroderma fibrosis.272829 IPF and scleroderma fibrosis are two fibroses with different prognoses.30 This might be related to the fact that a less aggressive form of fibrosing interstitial pneumonia, NSIP, develops in most patients with scleroderma fibrosis.31 Indeed, studies32 that compared the prognosis of patients with idiopathic NSIP to that of patients with the usual interstitial pneumonia type/IPF have clearly shown that NSIP presents a far better prognosis than the latter type. Some studies using BAL in scleroderma patients have shown that a subset of patients who present with > 15% lymphocytes in BAL fluid,27 have activated, long-lived CD8+ T cells,29 or produce type 2 cytokines (ie, interleukin-4 and interleukin-5) by the CD8+ TLs28 present with a more aggressive form of interstitial pneumonia. Hence, TLs, and in particular the CD8+ subset, may be associated with progressive fibrosis in scleroderma that resembles IPF. Analogously, CD8+ TLs have been associated with a worse prognosis in IPF patients,13 implying that CD8+ TLs may have some role in the pathogenesis of both fibroses.

Nevertheless, controversy exists whether the BAL fluid cellular profile reflects the cellular composition of the lung parenchyma in patients with interstitial pneumonias. Some studies have shown correlations for all or at least some of the cell populations studied between BAL and tissue biopsy samples in the same patients with IPF,2333 while others have not.34 Discrepancies might be explained, at least in part, in the following ways: the selection of patients; different methodologies; and the fact that older studies might have included a mixed population (ie, patients with IPF, patients with NSIP, and patients with pulmonary fibrosis associated with collagen vascular disorders) since there were not yet defined strict criteria for these entities.3

To the best of our knowledge, no studies have evaluated TL subpopulations in both BAL and surgically obtained lung tissue samples by flow cytometry and quantitative immunohistochemistry, respectively, in IPF patients. This might be related to the fact that reliable cell counts in tissue were not feasible with past technologies. In interstitial disorders, surgical lung biopsies may be required when less invasive modalities fail to produce a diagnosis. For the diagnosis of IPF, clinicophysiologic and roentgenographic criteria are actually clearly established.3 As patients undergoing surgical lung biopsies are not immune to significant morbidity and mortality, especially IPF patients,789 the necessity for surgical lung biopsies has become less imperative, leading as well to the lower availability of lung tissue for research purposes. Our observations that in IPF patients the TL subpopulations (ie, CD4+ and CD8+ TLs), recovered by BAL, as well as the CD4+/CD8+ ratio relate to those in lung tissue indicate that by the use of a far less invasive technique of BAL, reliable information can be derived regarding the pathogenetic role of TL subpopulations in IPF.

The scatter of our data and the differences observed in the individual values for the TL subpopulations studied might be related to the fact that we used different techniques to sample lung involvement by the disease. Indeed, the technique of BAL samples inflammatory cells from a more extensive, and almost always different part of the lung than the histologic sections of biopsy specimens.35 In conclusion, our results suggest that in IPF patients the TL subpopulations in BAL fluid reflect the pattern of cellular infiltration in the lung parenchyma, and that the further investigation of their phenotypic and functional characteristics may lead to valid insights regarding their role in the pathogenesis of the IPF.


   Footnotes
 
Abbreviations: IPF = idiopathic pulmonary fibrosis; NSIP = nonspecific interstitial pneumonia; TL = T lymphocyte

This research was supported by the "Thorax" Foundation, Athens, Greece.

Received for publication January 13, 2005. Accepted for publication March 21, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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