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From the Department of Pulmonology (Drs. Bresser, Jansen, and Weller) and Clinical Immunology Laboratory (Dr. Lutter), Academic Medical Center; and Laboratory for Experimental and Clinical Immunology (Dr. Out), CLB, Sanquin Blood Supply Foundation, Amsterdam, The Netherlands.
Correspondence to: Henk M. Jansen, MD, Department of Pulmonology, Academic Medical Center, Room F4–206, PO Box 22700, 1100 DE Amsterdam, The Netherlands; e-mail: h.m.jansen{at}amc.uva.nl
Key Words: BAL • CD25 • CD27 • pulmonary fibrosis • systemic sclerosis
Systemic sclerosis is a multisystem disorder with an activated immune system playing a significant role in tissue damage. Activated T cells infiltrate perivascular inflammatory sites, skin, and interstitial tissue.1 A considerable number of patients present with impairment of pulmonary functions. In virtually all patients, the disease develops to alveolitis resulting in pulmonary fibrosis.2 The activation of T lymphocytes can be studied by measuring the expression of several cell surface molecules. In this respect, CD27 is an interesting molecule. CD27 is a member of the tumor necrosis factor-receptor family of proteins.3 The natural ligand for CD27 is CD70, and its expression is tightly related to antigen-specific activation of T cells.4 5 When there is an antigen-specific activation of T cells, the expression of CD27 decreases.5 This is in contrast to the CD25 (the chain of the receptor for interleukin-2 expression), which increases both after polyclonal and antigen-specific activation of T cells.6 Analysis of CD27 expression therefore provides the opportunity to study antigen-specific activation of T cells without necessarily knowing the nature of the antigen. We hypothesized that there is activation of T cells in the lungs of patients with systemic sclerosis long before the development of signs of pulmonary fibrosis. The analysis of T-cell activation markers on T cells from BAL fluid recovered from the patients may provide information on the nature of such activation.
Materials and Methods
A cross-sectional study of patients with systemic sclerosis without symptoms of pulmonary fibrosis (SSc; n = 11; 1 male and 10 female patients; age range, 45 to 73 years), patients with systemic sclerosis with pulmonary fibrosis (SScF; n = 10; 4 male and 6 female patients; age range, 28 to 69 years), and patients with idiopathic pulmonary fibrosis (IPF; n = 12; 8 male and 4 female patients; age range, 27 to 70 years) was performed. Systemic sclerosis was diagnosed following the criteria of the American College of Rheumatology.7 Pulmonary fibrosis in patients with SScF was detected on the basis of a high-resolution CT (HRCT) scan, a decreased vital capacity (VC; mean, 62.8 ± 3.9% predicted), and a decreased diffusion capacity of the lung for carbon monoxide (DLCO; mean, 31 ± 4.9% predicted). SSc patients showed slightly decreased DLCO (mean, 69.0 ± 5.1% predicted), a normal HRCT scan finding, and a VC > 80% of predicted. Patients with IPF had open-lung biopsy specimen-proven usual interstitial pneumonia, positive fibrotic signs on the HRCT scan, and decreased VC and DLCO values. In all patients, BAL was performed as a standard procedure in the assessment of the disease process.8 Only those patients were included who had not received immunosuppressive therapy before BAL was performed. A small, nonsmoking, healthy control group (n = 16) was included. Cell differential counts were counted on cytospin preparations, and BAL fluid cells and peripheral blood mononuclear cells were stained with fluorochrome-labeled antibodies to CD3, CD4, CD8, CD19, CD25, CD27, natural killer cells, and control antibodies. A flow cytofluorimetric assay was performed (FACScan; Becton Dickinson; Mountain View, CA).6 Statistical analysis was performed using Spearman rank test to calculate correlation between two variables, and the Mann-Whitney U test was used to evaluate differences between groups.
Results and Interpretation
The numbers of patients with a leukocyte cell count in BAL fluid
cells above the mean plus 2 SDs of that in control subjects were eight
patients (72%), six patients (60%), and nine patients (81%) with
SSc, SScF, and IPF, respectively. In BAL fluid, there were no
significant differences in the numbers of lymphocyte subpopulations:
CD3+, CD4+, CD8+, natural killer cells, and B cells. The percentage of
CD4+ T cells was higher in patients with SSc than in patients with SScF
(p < 0.03). There were no significant differences between the groups
for the mean CD4/CD8 ratios. Patients with SSc had significantly lower
CD27 expression on CD4+ T cells than patients with SScF (p < 0.001).
CD27 in patients with SSc was also lower than in healthy subjects
(p < 0.01). There was no such difference on blood T lymphocytes.
Again, in BAL fluid, the patients with SScF and IPF had CD27 expression
similar to healthy subjects. The results indicate an abnormal
antigen-specific CD4+ T-cell activation in the BAL fluid-cell
compartment of the patients with SSc. The expression of CD27 on CD8+ T
cells was heterogeneous without statistically significant differences
between the groups, and without a difference between control subjects
and any of the groups. CD25 expression on CD4+ T cells from BAL fluid
was significantly lower in SSc patients as compared to that of patients
with SScF and patients with IPF (p < 0.01 and p < 0.05,
respectively). No such difference was seen on CD8+ T cells. In patients
with SSc, both the decrease of CD27 expression and the CD25 expression
(though not significantly increased) were significantly correlated with
the impairment of DLCO percent predicted (Spearman
= - 0.78 [p < 0.02] and - 0.78 [p < 0.02],
respectively). No such correlations were observed for the patients with
SScF. The results indicate that there was specific T-cell activation on
the basis of decreased CD27 expression that is associated with low CD25
expression in SSc patients, and the reverse in the SScF patients. These
results point to active T-cell involvement in the reactions in the
lungs in SSc patients. This activation was highest (lowest CD27
expression) in the patients with a normal DLCO and appeared
to become less in patients who had impaired diffusion capacity. This
relation between T-cell activation and lung function was not found in
patients with SScF. With respect to one other marker of T-cell
activation tested, CD25, a completely different mechanism appeared.
CD25 expression was higher with increasing impairment of
DLCO. This indicates an additional mechanism of T-cell
activation operating in patients with SScF becoming increasingly
important with progression of the fibrosis. The activation of CD4+ T
cells according to decreased CD27 expression was inversely correlated
with the activation of CD4+ T cells according to CD25 expression (Fig 1 ). This correlation was statistically significant in each separate group
of patients as well as in all patients combined (SSc, Spearman ,
0.78 [p < 0.01]; SScF, Spearman , 0.78 [p < 0.02]; IPF,
Spearman , 0.66 [p < 0.03]). This suggests two mechanisms of
T-cell activation to occur: the decreased CD27 expression representing
antigen-specific activation, whereas the increased CD25 expression most
likely represents polyclonal T-cell activation, as there were no signs
of antigen-specific activation in parallel (no decrease of CD27
expression). The antigen-specific T-cell activation was prominent in
the SSc group, whereas the polyclonal activation was prominent in the
SScF group. The IPF patients showed intermediate T-cell activation
characteristics. Further flow cytofluorimetric analyses revealed
that in all patient groups, the extent of the T-cell activation as
judged by decreased CD27 expression occurred in T cells different from
those that are activated as judged by increased CD25 expression (data
not shown). This illustrates that different populations of T cells are
involved in the two mechanisms of T-cell activation. This divergent
pattern of T-cell activation in relation to developing fibrosis
challenges further analysis of T cells in the lungs of patients with
systemic sclerosis.
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In this study, we have demonstrated that a high percentage of patients with systemic sclerosis in whom there is not yet clinical evidence for pulmonary fibrosis have alveolitis and T-cell activation in the lungs as judged by the decreased expression of CD27 on CD4+ T cells in BAL fluid. This points to abnormal antigen-specific activation of T cells in the lungs of patients with systemic sclerosis.
Acknowledgements
We thank H. Dinant, Department of Clinical Immunology, Academic Medical Center, and D. Enomoto, Department of Dermatology, Academic Medical Center, for support in clinical care of the patients; K. Traanberg and A. Soede, for excellent technical assistance; and R. A. W. van Lier for stimulating discussions.
Footnotes
Abbreviations: DLCO = diffusion capacity of the lung for carbon monoxide; HRCT = high-resolution CT; IPF = idiopathic pulmonary fibrosis; SSc = systemic sclerosis without symptoms of pulmonary fibrosis; SScF = systemic sclerosis with pulmonary fibrosis; VC = vital capacity
References
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