Absolute count of T and B lymphocyte subsets is decreased in systemic sclerosis
© I. Holzapfel Publishers 2010
Received: 14 August 2009
Accepted: 9 September 2009
Published: 29 January 2010
Previous reports on lymphocyte subpopulations in systemic sclerosis (SSc) are conflicting. Therefore, we aimed to investigate the lymphocyte subsets in SSc patients who were not on immunosuppressive therapy.
Lymphocyte subsets were assessed in the peripheral blood of SSc patients (n = 29) and healthy controls (n = 29) using the four colour flow cytometry method. Correlation studies were also performed in order to assess the relationship between lymphocyte subsets and clinical parameters.
The absolute count of lymphocytes (P = 0.0042), CD3+ (P = 0.0014), CD4+ (P = 0.0070), CD8+ (P = 0.021), and CD19+ cells (P = 0.024) was significantly decreased in SSc patients when compared to healthy controls. CD4+/CD8+ ratio and the absolute count of CD56+ cells observed in SSc patients did not significantly differ from controls (P = 0.165; P = 0.632, respectively). There was no substantial relationship between the lymphocyte subset levels and clinical features (i.e., SSc subtype, autoantibody profiles, organ involvement), except for a significant inverse correlation of CD19+ cells and the modified Rodnan skin score (r = -0.43, P = 0.020).
Our data support previous reports indicating that subsets of T lymphocytes as well as B lymphocytes play a role in the pathogenesis of SSc.
Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition in the skin and other visceral organs. The most widely accepted classification system is that proposed by LeRoy et al. , which includes limited cutaneous SSc and diffuse cutaneous SSc, a more rapidly advancing type with more frequent visceral involvement. It was suggested that immune system changes play the major part in the development of vasculopathy and fibrosis. The presence of autoantibodies, such as DNA topoisomerase I and centromere, is a central feature of SSc. Moreover, a wide range of B and T cell abnormalities has been described in SSc. Nevertheless, previous reports on lymphocyte subpopulations in SSc are conflicting [2–13]. We here conducted a prospective study investigating lymphocyte subsets in immunosuppressant-untreated SSc patients and healthy controls.
Materials and methods
This was a prospective study on patients with SSc who were evaluated in the Department of Dermatology of the Ruhr-University Bochum (Germany) from February 2007 to July 2008. SSc was diagnosed according to the classification system proposed by LeRoy et al. . We only included patients who had no history of photo pheresis and immunosuppressive treatment (e.g., gluco corticosteroids, methotrexate, cyclophosphamide) for at least 6 months. The patients included in the study, however, had physiotherapy and/or rheological treatment. We saw to it that the control group, which included healthy individuals, did match the gender and age of the patient group. The study was conducted in the light of the declaration of Helsinki and followed a protocol approved by our institutional review board. All the patients and healthy controls were fully informed and gave their consent to participate. A complete work-up was performed in SSc patients including bodyplethysmography, thoracic high-resolution computed tomography, oesophagogastroscopy, abdominal and cardiac ultrasound, and blood collections. Skin involvement was quantified using the modified Rodnan skin score (MRSS) and was assessed clinically by palpation of the skin, as described previously .
In order to identify and determine the percentages and absolute counts of lymphocyte subsets in the peripheral blood, flow cytometric methods were used. For this purpose the following antibodies (BD Bio sciences, San Jose, CA, USA), directed against mature human lymphocyte subsets were employed: leucocytes (CD45+), T-lymphocytes (CD3+), B-lymphocytes (CD19+), helper/inducer T lymphocytes (CD3+CD4+), suppressor/cytotoxic T lymphocytes (CD3+CD8+) and natural killer lymphocytes (CD3-CD16+ and/or CD56+). The antibodies were conjugated to the following fluorescent dyes: Fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyllprotein (PerCP) and allophycocyanine (APC). Additionally, Trucount tubes™(BD Biosciences, San Jose, CA, USA) were used to determine absolute counts of lymphocytes. The FACSCalibur™cytometer (BD Biosciences, San Jose, CA, USA), equipped with 635 nm and 488 nm lasers, together with computer hardware and Multitest-software™were used to acquire and analyse the lymphocyte subsets.
Analysis of data was performed using the statistical package MedCalc Software (Mariakerke, Belgium). Normal distribution of data was confirmed by the D'Agostino-Pearson test. Data are expressed as mean ±SD and median and range. Data were analysed using the F-test for equal variances, independent t-test, Chi-square test, and Spearman correlation procedure. We constrained experiment-wise error rates due to multiple comparisons to the standard alpha (P) level of < 0.05 by the Bonferroni method.
Main clinical and laboratory features of 29 patients with systemic sclerosis (SSc)
Age (mean ± SD)
56.3 ± 12.9
SSc type (limited/diffuse)
Duration of disease (median, months)
42 (1 - 390)
Raynaud phenomenon (no/yes)
7 (2 - 24)
Renal involvement (no/yes)
Lung involvement (no/yes)
Oesophagus involvement (no/yes)
Antinuclear antibodies on HEp-2 cell line
Peripheral blood lymphocyte subsets in patients with systemic sclerosis (SSc) and healthy controls
SSc (n = 29)
Controls (n = 29)
1676 ± 602
2273 ± 561
1193 ± 474
1688 ± 424
71 ± 7
74 ± 5
808 ± 279
1115 ± 346
49 ± 9
49 ± 8
2.3 ± 1.2
2.8 ± 1.6
363 ± 256
570 ± 214
21 ± 7
25 ± 8
197 ± 89
297 ± 136
12 ± 5
13 ± 4
265 ± 153
247 ± 114
16 ± 8
11 ± 4
In the present study, we found that SSc patients, who were not on immunosuppressive treatment, had significantly reduced numbers of lymphocytes and its subsets including CD3+, CD4+, CD8+, and CD19 cells. However, the percentages of lymphocyte subsets investigated were very similar to those observed in healthy controls. In accordance with our results, Ercole et al.  recently found that SSc patients had similar percentages of CD3+, CD4+, and CD8+ cells and CD4+/CD8+ cell ratio when compared to matched normal controls. Ingegnoli et al.  found that absolute counts of CD4+CD25+ and CD8+ lymphocytes are reduced in SSc patients. On the other hand, Artlett et al.  reported that the numbers of CD4+ and CD8+ cells were found to be significantly higher in SSc patients than in controls. However, Fiocco et al.  observed that patients with SSc showed increased CD4+CD26+ and CD4+CD25+ absolute numbers and percentages and decreased CD8+CD29+ percentages compared with controls.
Natural killer cells are large granular lymphocytes easily identified morphologically by the presence of azurophil granules in their cytoplasm and they commonly express certain cell surface markers such as CD56+ which is a homophilic adhesion molecule that belongs to the immunoglobulin superfamily. Natural killer cells mediate antigen presentation and secrete immune modulator cytokines like interferon, colony stimulating factor - these functions suggested the involvement of natural killer cells in the pathophysiology of SSc. Reduction of natural killer cells has been described by Riccieri et al. . Interestingly, Mitsuo et al.  observed that the frequency as well as the absolute number of CD161+CD8+ cells, which are closely related to CD8+ natural killer cells, were decreased in the peripheral blood of SSc patients.
Presence of autoantibodies and hypergammaglobulinaemia support the role of humoral immunity. CD19+ is a cell surface marker of B lymphocytes. We observed significantly reduced levels of CD19+ cells which inversely correlated with the clinical score of SSc patients. Sato et al.  found that CD19 is over-expressed in naive B cells and, to a greater extent, in memory B cells in the blood SSc patients. This CD19 overexpression was specific to SSc, since SLE patients exhibited significantly down-regulated CD19 expression. Sato et al.  suggested that the CD19 overexpression in SSc memory B cells induces both the activated phenotype of memory B cells and augmented IgG production. However, Majone et al.  recently reported on increased apoptosis in circulating lymphocyte cultures of anti-RNA polymerase III positive patients with SSc. Indeed, this might be a possible mechanism to explain the reduced lymphocyte counts in SSc patients.
In conclusion, the aforementioned data on lymphocyte subsets in SSc are conflicting very likely due to different methodologies particularly with regard to patient selection, pre-treatments, assays, and sample sizes. Although the relations between the lymphocyte subpopulations investigated was not different from healthy controls, we found a significant reduction of absolute numbers of lymphocytes and CD3+, CD4+, CD8+, and CD19+ cells SSc patients. Interestingly, the absolute number of CD19+ cells appears to inversely correlate with the severity of cutaneous involvement. Hence, the CD19 count may be a potential biomarker for the severity of skin involvement. Taken together, our data support previous reports indicating that subsets of T lymphocytes as well as B lymphocytes play a role in the pathogenesis of SSc.
- Leroy EC, Black C, Fleischmajer R, Jablonska S, Krieg T, et al.: Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J Rheumatol 1988, 15: 202–205.PubMed
- Melendro EI, Saldate C, Rivero SJ, Alarcon-Segovia D: T-cell subpopulations in the peripheral blood of patients with connective tissue diseases as determined by flow cytometry using monoclonal antibodies. Clin Immunol Immunopathol 1983, 27: 340–347. 10.1016/0090-1229(83)90086-7PubMedView Article
- Dalkiliç E, Dilek K, Güllülü M, Yavuz M, Karakoç Y, Yurtkuran M, et al.: Lymphocyte phenotypes in systemic sclerosis. Ann Rheum Dis 1999, 58: 719–720.PubMed CentralPubMedView Article
- Kahan A, Kahan A, Picard F, Menkès CJ, Amor B: Abnormalities of T lymphocyte subsets in systemic sclerosis demonstrated with anti-CD45RA and anti-CD29 monoclonal antibodies. Ann Rheum Dis 1991, 50: 354–358. 10.1136/ard.50.6.354PubMed CentralPubMedView Article
- Gorla R, Airò P, Malagoli A, Carella G, Prati E, Brugnoni D, Franceschini F, Cattaneo R: CD4+ and CD8+ subsets: naive and memory cells in the peripheral blood of patients with systemic sclerosis. Clin Rheumatol 1994, 13: 83–87. 10.1007/BF02229871PubMedView Article
- Fiocco U, Rosada M, Cozzi L, Ortolani C, De Silvestro G, Ruffatti A, Cozzi E, Gallo C, Todesco S: Early phenotypic activation of circulating helper memory T cells in scleroderma: correlation with disease activity. Ann Rheum Dis 1993, 52: 272–277. 10.1136/ard.52.4.272PubMed CentralPubMedView Article
- Ercole LP, Malvezzi M, Boaretti AC, Utiyama SR, Rachid A: Analysis of lymphocyte subpopulations in systemic sclerosis. J Investig Allergol Clin Immunol 2003, 13: 87–93.PubMed
- Ingegnoli F, Trabattoni D, Saresella M, Fantini F, Clerici M: Distinct immune profiles characterize patients with diffuse or limited systemic sclerosis. Clin Immunol 2003, 108: 21–28. 10.1016/S1521-6616(03)00062-7PubMedView Article
- Artlett CM, Cox LA, Ramos RC, Dennis TN, Fortunato RA, Hummers LK, Jimenez SA, Smith JB: Increased microchimeric CD4+ T lymphocytes in peripheral blood from women with systemic sclerosis. Clin Immunol 2002, 103: 303–308. 10.1006/clim.2002.5222PubMedView Article
- Tsuji-Yamada J, Nakazawa M, Minami M, Sasaki T: Increased frequency of interleukin 4 producing CD4+ and CD8+ cells in peripheral blood from patients with systemic sclerosis. J Rheumatol 2001, 28: 1252–1258.PubMed
- Riccieri V, Parisi G, Spadaro A, Scrivo R, Barone F, Moretti T, Bernardini G, Strom R, Taccari E, Valesini G: Reduced circulating natural killer T cells and gamma/delta T cells in patients with systemic sclerosis. J Rheumatol 2005, 32: 283–286.PubMed
- Mitsuo A, Morimoto S, Nakiri Y, Suzuki J, Kaneko H, Tokano Y, Tsuda H, Takasaki Y, Hashimoto H: Decreased CD161+CD8+ T cells in the peripheral blood of patients suffering from rheumatic diseases. Rheumatology (Oxford) 2006, 45: 1477–1484. 10.1093/rheumatology/kel119View Article
- Sato S, Fujimoto M, Hasegawa M, Takehara K: Altered blood B lymphocyte homeostasis in systemic sclerosis: expanded naive B cells and diminished but activated memory B cells. Arthritis Rheum 2004, 50: 1918–1927. 10.1002/art.20274PubMedView Article
- Balbir-Gurman A, Denton CP, Nichols B, Knight CJ, Nahir AM, et al.: Non-invasive measurement of biomechanical skin properties in systemic sclerosis. Ann Rheum Dis 2002, 61: 237–241. 10.1136/ard.61.3.237PubMed CentralPubMedView Article
- Majone F, Olivieri S, Cozzi F, Montaldi A, Tonello M, Visentin MS, Ciprian L, Ruffatti A: Increased apoptosis in circulating lymphocyte cultures of anti-RNA polymerase III positive patients with systemic sclerosis. Rheumatol Int 2008, in press.