Rheumatology International

, Volume 32, Issue 2, pp 317–321

Serum levels of soluble CD21 in patients with systemic sclerosis

Authors

  • Manabu Tomita
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Norihito Yazawa
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Tomohiko Kawashima
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Zenshiro Tamaki
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Ryuichi Ashida
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Hanako Ohmatsu
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Yoshihide Asano
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Makoto Sugaya
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Masahide Kubo
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Hironobu Ihn
    • Department of Dermatology and Plastic SurgeryGraduate School of Medical and Pharmaceutical Sciences, Kumamoto University
  • Kunihiko Tamaki
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
  • Shinichi Sato
    • Department of Dermatology, Faculty of MedicineUniversity of Tokyo
Original Article

DOI: 10.1007/s00296-010-1610-3

Cite this article as:
Tomita, M., Kadono, T., Yazawa, N. et al. Rheumatol Int (2012) 32: 317. doi:10.1007/s00296-010-1610-3

Abstract

Systemic sclerosis (SSc) is a systemic disorder that typically results in fibrosis of the skin and multiple internal organ systems. Although the precise mechanism is unknown, overproduction of extracellular matrix proteins, including collagens and fibronectins, and aberrant immune activation might be involved in the pathogenesis. The soluble cluster of differentiation 21 (sCD21) represents the extracellular portion of the CD21 glycoprotein that is released by shedding from the cell surfaces into plasma. sCD21 binds complement fragments and activates monocytes through binding to membrane CD23. The present study was undertaken to investigate the serum levels of sCD21 in patients with SSc. Serum sCD21 levels were reduced with age both in patients with SSc and normal controls. Serum sCD21 levels in patients with SSc were significantly decreased compared to those in control subjects. When we divided patients with SSc into limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc), patients with lcSSc had lower levels of serum sCD21 than those with dcSSc. Moreover, the prevalence of pulmonary fibrosis in the patients with dcSSc inversely correlated with serum sCD21 levels. Our finding may support the notion that B-cell activation is involved in the mechanism for pulmonary fibrosis and skin sclerosis.

Keywords

CD21Enzyme-linked immunosorbent assayPulmonary fibrosisSystemic sclerosisLimited cutaneous systemic sclerosis

Introduction

Systemic sclerosis (SSc) is a systemic autoimmune disease characterized by fibrosis of the skin and multiple internal organ systems [1]. Although the etiology of SSc is still unknown, the basic mechanism appears to involve endothelial cell injury, overproduction of extracellular matrix proteins including collagen and fibronectin, and aberrant immune activation [2].

The regulatory functions of complement are envisaged by specific recognition of pathogen bound C3 fragments by complement receptors. The end effect depends on the receptor and the cell. Five types of complement receptors are known to bind complement components, namely CD35 (CR1), CD21 (CR2), CD11b/CD18 (CR3), CD11c/CD18 (CR4), and C1q receptor. CD21 plays an important role in B cell signaling as a B-cell coreceptor. Attachment of C3 to antigens and subsequent binding to complement receptors CD21 and CD35 enhances the antigen uptake by B cells, modulates downstream events such as endosomal targeting of antigen and processing and binding of peptides to major histocompatibility complex (MHC) class I molecules [35]. On mature B cells, CD21 forms a non-covalent signal transduction complex in the plasma membrane together with the CD81, Leu-13 and the pan-B cell antigen CD19. This complex amplifies the signal transmitted through the B-cell receptor by specific antigen and thereby reduces the threshold of antigen necessary to initiate cell proliferation [6, 7].

CD21 is shed from the surface of the cell and is found circulating in plasma [810]. Soluble CD21 (sCD21) activates monocytes through binding to membrane CD23. As sCD21 could potentially bind to its ligands in plasma, the amount of sCD21 in circulation could be a modulator of immunity [11], but its role is yet unknown. The clinical significance of sCD21 is shown by higher levels in sera of patients with certain B lymphomas, EBV infection, and other lymphoblastoid tumors [1214], and by lower levels in patients with rheumatoid arthritis, systemic lupus erythematosus and Sjogren’s syndrome [15]. The serum levels of sCD21 in patients with SSc have not been previously studied.

Here, we investigated if and to what extent serum sCD21 levels are influenced by SSc. We found that the serum levels of sCD21 were significantly reduced in patients with SSc, especially limited cutaneous SSc.

Materials and methods

Patients

Serum samples were obtained from 53 Japanese patients with SSc (45 females, 8 males; age mean ± SD, 50.4 ± 20.8) who fulfilled the preliminary criteria for SSc proposed by American College of Rheumatology (formerly the American Rheumatism Association) [16]. The serum samples were obtained at the patients’ first visit after obtaining informed consent prior to the administration of therapies including oral corticosteroids. These patients were grouped into limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc) according to the classification system proposed by LeRoy et al. [2]. Twenty-eight patients had lcSSc, and the other 25 patients had dcSSc. Forty-nine age- and sex-matched healthy subjects (40 females and 9 males; age, 49.9 ± 18.2) were also included in this study. Clinical or laboratory data were obtained at the same time with serum sampling.

The involvement of organ systems with SSc was defined as described by Steen et al. [17], with some modifications. Pulmonary fibrosis was defined by bibasilar fibrosis on chest radiography and high resolution computed tomography. Esophageal involvement is defined by hypomotility observed by barium radiography or gastrocamera. Heart involvement is defined by the occurrence of pericarditis, congestive heart failure, or arrhythmias requiring treatment. Kidney involvement was defined by the experience of an episode of renal crisis due to malignant hypertension or rapidly progressive renal failure. Joint involvement is defined by the presence of inflammatory polyarthralgias or arthritis. Antinuclear antibodies were detected by indirect immunofluorescence by using HEp-2 cells as substrates. Specific antibodies including anti-topoisomerase I antibodies, anti-U1RNP antibodies and anti-centromere antibodies were detected using enzyme-linked immunosorbent assay (ELISA).

Measurement of serum sCD21 levels

The serum levels of sCD21 were determined using sandwich ELISA kits obtained from Euroclone Life Sciences (Pero, Italy), according to the manufacture’s protocol. In brief, a diluted serum sample (100 μl) was added to each well coated with anti-human CD21 antibodies. Biotin-conjugated anti-CD21 monoclonal antibodies were added as well. After incubation for 1 h and several washings, streptavidin-conjugated horse radish peroxidase was added to each well and incubated for 30 min. Toluidine methylene blue substrate solution was added and incubated for 20 min, and stop solution was added to terminate the peroxidase reaction. The absorbance at 450 nm was measured using a spectrometer. The concentration of sCD21 in a sample was determined by interpolation from a standard curve.

Statistics

The results were expressed as the means ± standard deviation (means ± SD). For statistical analyses, Mann–Whitney’s U-tests were performed for the comparison of means, and the chi-square test and the Fisher’s exact test were used for categorical comparisons of data. P values of less than 0.05 were considered to be significant.

Results

Serum sCD21 levels in healthy volunteers and patients with SSc

We analyzed sCD21 levels in sera of 49 healthy volunteers of different ages who consented to our research. In accordance with the previous report [18], sCD21 concentration was lower in older individuals (data not shown). The mean of sCD21 concentrations in healthy individuals was 99.3 ± 45.7 ng/ml. While serum levels of sCD21 were not significantly different between genders (the mean of sCD21 in female; 103.0 ± 49.9 ng/ml, male; 87.8 ± 27.7 ng/ml), an age-dependent decrease was found in both male and female control groups (data not shown).

Serum samples from 53 patients with SSc were tested for sCD21 concentrations by ELISA. Soluble CD21 concentrations in patients with SSc were reduced with age (data not shown). The mean of sCD21 concentrations in patients with SSc was 80.4 ± 53.8 ng/ml. While the serum levels of sCD21 were not significantly different between genders (the mean of sCD21 of female; 82.1 ± 55.3 ng/ml, male; 70.7 ± 46.4 ng/ml), an age-dependent decrease was found in both male and female control groups (data not shown). Serum sCD21 concentrations were found to be significantly lower in patients with SSc than in normal controls (P = 0.036; Fig. 1). Means of age were almost equal, and there were no significant differences of age distribution between patients with SSc and normal controls (data not shown).
https://static-content.springer.com/image/art%3A10.1007%2Fs00296-010-1610-3/MediaObjects/296_2010_1610_Fig1_HTML.gif
Fig. 1

Comparison of serum sCD21 levels between patients with SSc and controls. Serum from patients with SSc (n = 53) and controls (n = 49) were tested. Patients with SSc were subdivided into lcSSc (n = 28) and dcSSc (n = 25)

We further analyzed serum sCD21 levels in patients with SSc by dividing them into lcSSc and dcSSc. Serum sCD21 concentrations in lcSSc were significantly lower than those of normal controls (P = 0.000018) or dcSSc (P = 0.010; Fig. 1) (mean in lcSSc, 60.1 ± 31.6 ng/ml; mean in dcSSc, 102.4 ± 64.8 ng/ml). No significant difference was observed between normal controls and dcSSc (P = 0.72; Fig. 1). Similar to controls, serum sCD21 levels were reduced with age in both lcSSc and dcSSc. There were no significant differences of age distribution between lcSSc, dcSSc, and normal controls (age mean in lcSSc, 54.3 ± 19.4; age mean in dcSSc, 52.9 ± 12.1).

Serum sCD21 levels are decreased in dcSSc with pulmonary fibrosis

The clinical and laboratory findings were compared between SSc patients with low (less than mean–SD) sCD21 levels and those with normal levels (Table 1). For most of clinical and laboratory findings such as sex, disease duration, the prevalence of esophageal, heart, kidney, or joint involvement, no significant difference was observed between the patients with decreased serum sCD21 levels and those with normal levels. However, the prevalence of pulmonary fibrosis (PF) was high in dcSSc patients with low serum sCD21 levels (63.6% of patients with low sCD21 levels and 40.5% of patients with normal sCD21 levels; Table 1).
Table 1

Clinical and laboratory features of patients with SSc categorized by soluble CD21 levels

 

Patients with lower Scd21 levels (n = 11)

Patients with normal Scd21 levels (n = 42)

Male/female

2/9

6/36

Age, years (mean ± SD)

49.1 ± 22.7

55.5 ± 9.8

Disease duration, years (mean ± SD)

7.38 ± 9.82

6.91 ± 7.13

Number (%) with

 dcSSc

45.5%

47.6%

 lcSSc

54.5%

52.4%

Nail fold bleeding

36.4%

40.5%

Calcinosis

0.0%

11.9%

Diffuse pigmentation

36.4%

54.8%

Pulmonary fibrosis

*63.6%

40.5%

Esophageal involvement

9.1%

33.3%

Heart involvement

0.0%

9.5%

Kidney involvement

0.0%

2.4%

Joint involvement

18.2%

19.1%

Anti-topoisomerase I antibody

54.5%

28.6%

Anti centromere antibody

45.5%

31.0%

Anti U1RNP antibody

0.0%

14.%

*  P = 0.05 versus SSc patients with normal sCD21 levels

We also subdivided patients with dcSSc into those with PF and those without PF. Serum sCD21 levels in dcSSc patients with PF were significantly lower than those without PF (P = 0.021; Fig. 2).
https://static-content.springer.com/image/art%3A10.1007%2Fs00296-010-1610-3/MediaObjects/296_2010_1610_Fig2_HTML.gif
Fig. 2

Comparison of sCD21 levels between dcSSc with pulmonary fibrosis (PF) and those without PF

Discussion

In patients with SSc, serum sCD21 levels were significantly lower than those in controls, and serum sCD21 levels were decreased in patients with lcSSc. Among patients with dcSSc, sCD21 levels were decreased in those with PF.

The levels of sCD21 decline significantly with age in healthy individuals [18]. Early in life, there is a large generation of cells emerging from the bone marrow into the periphery, including CD21 expressing B cells [19]. Those hematopoietic cells may cause higher serum sCD21 levels in younger individuals and this is similarly true for patients with SSc.

One of the characteristics of SSc is polyclonal B-cell hyperactivity, cytokine release and hypergammaglobulinemia, with antinuclear antibodies detected in >90% of patients [20, 21]. B cell-associated gene transcripts are increased in SSc lesional skin compared with normal skin, suggesting the local involvement of B cells [22], and CD21 expression is increased on B cells from patients with SSc [23]. Moreover, the total number of blood B cells is expanded in patients with SSc [24]. Contrary to our expectation, serum sCD21 levels in patients with SSc were decreased irrespective of age and gender. There are several reports of reduced serum sCD21 levels in other autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, Sjogren’s syndrome [15, 18], antiphospholipid syndrome [25], and pregnancy [26]. There are several possible reasons for this reduction. The first is that the elevated concentrations of protease inhibitors, such as the tissue inhibitors of matrix metalloproteinases or protease nexin-1, could prevent shedding of CD21 from the lymphocyte surface by inhibiting unknown protease [2731]. The second is an increased amount of complement activation products (e.g. C3f-des-arginine), which could bind to sCD21, leading to increased clearance from plasma [3235]. Several studies have reported abnormal complement activation and subendothelial deposition of immune complexes in patients with SSc [3639].

Our finding that serum sCD21 levels are decreased in lcSSc and decreased in dcSSc with pulmonary fibrosis may support the notion that B-cell activation is involved in PF and skin sclerosis, although the exact mechanism is yet to be defined.

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© Springer-Verlag 2010