Quantification of fetal microchimeric cells in clinically affected and unaffected skin of patients with systemic sclerosis

H. H. B. Sawaya, S. A. Jimenez and C. M. Artlett

Department of Medicine, Division of Rheumatology, Thomas Jefferson University, 233 S 10th Street, Rm 509 BLSB, Philadelphia, PA 19107, USA.

Correspondence to: C. M. Artlett. E-mail: Carol.Artlett{at}Jefferson.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. Fetal microchimerism has been hypothesized as a potential pathogenic mechanism for systemic sclerosis (SSc). This hypothesis was based on the clinical similarities between SSc and graft-vs-host disease and the identification of microchimeric cells in affected SSc tissues. The aim of this study was to compare the quantity of microchimeric cells in clinically affected and non-affected skin of female patients with SSc.

Methods. Fluorescence in situ hybridization (FISH) and real-time PCR were employed in paired skin biopsies obtained from clinically affected and unaffected areas from five female SSc patients with diffuse cutaneous SSc (dcSSC) and 10 healthy women. All women in the study had delivered a male fetus.

Results. FISH analysis revealed the presence of male fetal cells in 1/5 SSc patients (20.0%) compared with 0/10 healthy women (P = 0.0037), whereas quantification by real-time PCR revealed that all SSc samples were positive for male DNA compared with none of the controls. In the five patients with dcSSc, there were similar numbers of microchimeric cells in both affected and unaffected skin (P = 0.4)

Conclusion. The presence of higher numbers of microchimeric cells in clinically unaffected SSc skin, before any clinically detectable evidence of sclerotic changes, suggests that an influx of microchimeric cells may precede the development of tissue fibrosis. This provides additional support to the hypothesis that fetal microchimerism may play a role in the pathogenesis of SSc.

KEY WORDS: Microchimerism, Systemic sclerosis, Affected skin, Unaffected skin, Diffuse


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Systemic sclerosis (SSc) is an autoimmune disease of unknown aetiology characterized by dermal and internal organ fibrosis, including the lungs, kidneys, heart and gastrointestinal tract. The pathogenesis of SSc is characterized by alterations in the microvasculature, excessive extracellular matrix production and alterations in the immune system. The complex interaction between these three elements results in the development of cutaneous and visceral fibrosis.

Recently, microchimerism has been suggested as one possible contributor to the development of SSc [1–4], based on similarities in clinical features between SSc and chronic graft-vs-host disease (GVHD). The persistence of fetal microchimeric cells in the maternal circulation could induce a GVHD-like disease that manifests as SSc [1, 2, 5, 6]. If fetal cells participate in the pathogenesis of SSc, it would be expected that they are present in affected organs and tissues. As the skin is the major and, in most cases, the first clinically involved organ, it was speculated that male fetal cells should be present in the skin. Indeed, Artlett et al. were the first to identify male microchimeric cells in skin lesions of patients with SSc [1] and subsequent studies have confirmed this observation [3, 4]. However, there are no studies that have compared the presence and numbers of microchimeric cells between clinically affected and non-affected SSc skin. Thus, the objective of this study was to quantify the number of microchimeric cells in clinically affected and unaffected skin from female patients with SSc who had had at least one male child prior to the onset of SSc.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Following informed consent, five women from the Outpatient Rheumatology Clinic of the Hospital das Clinicas from São Paulo University were studied. Women selected for this study fulfilled the following criteria: they had delivered at least one male child; they had received no prior blood transfusions; they were not receiving any immunosuppressive therapy and/or D-penicillamine at the time of biopsy; and they had clinical onset of SSc after the birth of the male child. All patients fulfilled the American College of Rheumatology criteria for the classification of SSc. The mean age of the patients was 55.8 yr (range 46–68) and the mean duration of disease from first detectable evidence of cutaneous sclerosis was 2.5 yr (range 0.83–6) (Table 1). The pregnancy history obtained included the number of pregnancies, number of abortions, and age and sex of the children (Table 1). In one patient complete pregnancy information was not available, but she had had several male offspring. The skin samples from the healthy control group were obtained from the cell and tissue bank of the Division of Rheumatology, Thomas Jefferson University. The control group comprised 10 normal women from whom skin biopsies had been obtained for a previous study on osteoarthritis. All had had a male pregnancy. The mean age of the control group was 49 yr (range 25–75).


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TABLE 1. Pregnancy history and of patients with SSc included in the study

 
Skin biopsies
Skin punch biopsies (6 mm in diameter, two from each site) were obtained from areas that were clinically assessed as being affected and unaffected, by clinical examination and palpation. The skin samples were immediately frozen in liquid nitrogen until required. In the control group, the skin biopsies were taken from the back. Five-micrometre sections were obtained from the skin biopsies using a cryostat and stuck onto Super Frost Plus slides (Fisher, Pittsburgh, PA, USA) and fixed in acetone at 4°C for 5 min. The slides were then stored at –70°C until required. Before immunofluorescence and fluorescence in situ hybridization (FISH) were performed, one slide from each biopsy was stained with haematoxylin and eosin to verify the histological appearance of the tissue and the presence of inflammatory cells. A second slide was also stained with Masson's trichrome and assessed for collagen content, and a third slide was analysed by FISH.

Fluorescence in situ hybridization
Slides were washed in phosphate-buffered saline and denatured at 73°C for 5–9 min in 70% formamide/2 x standard saline citrate (SSC), then dehydrated in ethanol 70, 80 and 100% for 1 min. DNA probes specific for the alpha satellite region of the X and Y chromosomes were purchased from Vysis. The X-chromosome probe was labelled with SpectrumRedTM and the Y-chromosome probe with SpectrumGreenTM. The sections were incubated at 42°C overnight with 5 µl of probe. After hybridization, the sections were washed in 0.4 x SSC at 73°C for 2 min to remove unbound probe, rinsed in 0.1% NP-40/2x SSC, and allowed to air-dry. The slides were counterstained with DAPI, viewed with an epifluorescence microscope, and photographed. The entire surface of tissue present on the slides from each of the five patients corresponding to skin sections from clinically affected and unaffected areas was analysed and the number of male cells was determined by counting the nuclei which had one green signal and one red signal, corresponding to the X (red) and Y (green) chromosomes. The total number of cells in each slide was corrected for the area (mm2) of the tissue evaluated. The area of tissue analysed was calculated with the Image J 1.26t program (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov.ij).

Quantification of Y chromosome by real-time PCR
DNA was extracted from 4 mm pieces of skin using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA). The DNA bound to the column was eluted in 100 µl of buffer. The recovered DNA was quantified at 260 nM with a RNA–DNA spectrophotometer (Pharmacia, Piscataway, NJ, USA). Quantification of the Y chromosome in skin samples was performed with real-time PCR according to the previously published methods of Zhong et al. and Hahn et al. 2000 [7, 8]. The number of copies of the Y chromosome obtained was normalized to 100 µg of DNA in each sample. The number of male cells/100 µg DNA was determined by assuming that 6.6 pg of DNA corresponded to one cell.

Statistical analysis
A contingency table was used for the evaluation of the PCR and FISH results. A paired t test or the Mann–Whitney rank sum test was used to compare the numbers of microchimeric cells, calculated from the amount of DNA extracted from biopsies using the GraphPad InStat statistical program (GraphPad Software, San Diego, CA, USA). The results are expressed as median with interquartile range (IQR).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Quantification of Y chromosome in skin samples
Biopsy samples were analysed by real-time PCR to quantify the number of male microchimeric cells/100 µg DNA extracted from the lesions. Y-chromosome sequences were amplified in all samples. In patients with dcSSc, similar numbers of microchimeric cells were found in the affected and unaffected biopsies 4.73 cells/100 µg DNA; (IQR 3.76–5.63) vs 12.49 cells/100 µg DNA (IQR 3.47–13.55). These differences were not found to be statistically significant (P = 0.4).

Fluorescence in situ hybridization (FISH) of skin samples
The presence of male cells was confirmed by FISH in the affected and unaffected tissues. Male cells were identified by the presence of one Y chromosome (green signal) and one X chromosome (red signal) in the nuclei (Figs 1 and 2; these figures may be viewed in colour at Rheumatology Online). The results are shown in Table 2. Male cells were detected in biopsies from 4/5 patients with SSc (80.0%) compared with 0/10 in samples from healthy women (P = 0.0037, odds ratio = 63.0, IQR 2.1–1862.6).



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FIG. 1. Fluorescence in situ hybridization (FISH) specific for the centromeric region of X chromosome (red signal) (this figure may be viewed in colour at Rheumatology Online) in a biopsy of affected skin from an SSc patient. Nuclei were counterstained with DAPI and viewed with a Nikon epi-fluorescent microscope at 1000X magnification as described in Materials and methods.

 


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FIG. 2. Fluorescence in situ hybridization (FISH) specific for the centromeric region of X chromosome (red signal) and Y chromosome (green signal) (this figure may be viewed in colour at Rheumatology Online) in a biopsy of affected skin from an SSc patient. Nuclei were counterstained with DAPI and viewed with a Nikon epi-fluorescent microscope at 1000X magnification as described in Materials and methods.

 

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TABLE 2. Number of microchimeric cells in affected and unaffected skin

 
In affected skin, 1/5 samples were found to be positive by FISH, whereas in the unaffected skin 4/5 were positive (Table 2). Although there was no statistically significant difference, there was a tendency for a greater number of microchimeric cells/mm2 in unaffected skin (median 1.5 cells/mm2, IQR 0.3–1.6) when compared with affected skin (median 0 cells/mm2, IQR 0–0).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The presence of male microchimeric cells was analysed by FISH analysis and quantified by real-time PCR of DNA extracted from involved and uninvolved skin biopsies from five women with SSc. In affected skin, male microchimeric cells were identified by FISH in one of five patients with SSc. However, FISH is a relatively insensitive technique, and these results represent only a very small portion of the whole skin sample. In contrast, all of the samples were positive by real-time PCR. None of the patients had received a transfusion or organ transplant, but all had a male child. Therefore, these microchimeric cells were most likely derived from a previous male pregnancy. In one patient, male cells were not documented by FISH; however, the samples were positive for male DNA when examined by PCR. In agreement with results from others, microchimeric cells were not identified in skin samples from the normal women [1, 3]. The present study, however, shows for the first time that microchimeric cells were also present in uninvolved skin biopsies of women with SSc. Recently, some of the pathogenic mechanisms responsible for the alterations observed in patients with SSc have been elucidated. An increased number of CD4+ T cells has been found in SSc skin lesions and affected organs in the early phase of the disease. This observation suggests that cytokines secreted by T cells and/or other cells from the immune system play an important role in the pathogenesis of SSc [9]. In a recent study, Scaletti et al. [4] generated T-cell clones from the peripheral blood and skin biopsies from women with SSc of recent onset and from peripheral blood from three healthy control women. All subjects had at least one male child. They observed an increased number of T-cell clones obtained from SSc patients, which proliferated in response to autologous T cells when compared with clones obtained from the healthy women. They also demonstrated that 18% of the reactive clones from the SSc samples and 9% of the clones from healthy women were positive for the Y chromosome. Furthermore, they demonstrated that the clones from the SSc patients produced higher levels of the profibrotic cytokine IL-4, whereas the normal clones did not. This observation suggests that male fetal cells are present in the circulation and/or skin of women with SSc, are reactive against maternal major histocompatibility complex antigens, and display a TH2-oriented profile [4].

The presence of microchimeric cells in the tissues demonstrated in this study confirms previous findings by us and others [1, 3, 4]. Microchimeric cells were detected by FISH in one of five individuals analysed; however, by PCR quantification, male cells were detected in all samples. In the present study, it was also observed that clinically uninvolved SSc skin contained microchimeric cells. This finding reinforces the potential role of microchimeric cells in SSc lesions. We identified microchimeric cells in a woman approximately 50 yr after the birth of her son. It is conceivable that microchimeric cells, once established in a person, remain for the life of that individual. We believe that it is not sufficient to have microchimeric cells present, as it appears to be a frequent phenomenon in women who have carried fetuses. However, we believe that a second event (bacterial, viral or chemical) is necessary to activate the microchimeric cells, which subsequently move to the lesions. Similarly, in men or nulliparous women with SSc, it is possible that microchimeric cells are maternal in origin.

In the present study, the selection of anatomical areas for the skin biopsies was based on clinical assessment of the skin, but the unaffected skin may not necessarily become indurated over time. However, the haematoxylin/eosin and trichrome staining confirmed the presence of an increased thickness of the dermis and of the number of thicker collagen fibres in the affected tissue sections from patients with SSc when compared with unaffected tissue sections. Nevertheless, it is recognized that clinical assessment is not conclusive, as demonstrated by the presence of inflammatory and fibrotic alterations in affected and non-affected skin samples of patients with SSc [10]. These observations suggest that vascular and fibrotic abnormalities at the tissue level precede the clinical evidence of the disease. In agreement with these observations, we found in the present study that the clinically unaffected skin contained inflammatory cells, although the number of cells was lower than that observed in the affected skin. The lower number of microchimeric cells in affected skin compared with unaffected skin may be accounted for by the increased number of inflammatory cells in affected skin, as the cells were normalized to 100 µg of DNA. It is not possible to conclude whether microchimeric cells are involved in the early events that cause disease merely by their presence in the unaffected skin; however, the absence of microchimeric cells in tissues from healthy individuals gives support to the possible role that they have in the development of disease.

The present study adds substantial support to the hypothesis that microchimeric cells may be involved in the pathogenesis of SSc, as microchimeric cells were identified in SSc skin even before the tissue displayed clinically or histopathologically detectable evidence of sclerotic changes


    Acknowledgments
 
This work was supported by National Institutes of Health grant AR 45399 (to CMA) and the CAPES Foundation (to HHBS).

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 19 November 2003; revised version accepted 24 March 2004.