Rheumatology Advance Access originally published online on November 4, 2006
Rheumatology 2007 46(3):398-402; doi:10.1093/rheumatology/kel297
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Immunophenotyping of chimeric cells in localized scleroderma
1Mayo Clinic College of Medicine, Rochester, MN, 2Department of Medicine, Division of Rheumatology, 3Department of Dermatology, 4Texas Dermatology Associates, PA, Dallas, TX, and 5Department of Health Sciences Research, Division of Biostatistics, Mayo Clinic College of Medicine, Rochester, MN, USA.
Correspondence to: Ann M. Reed, MD, Mayo Clinic, 200 1st St SW, Rochester, MN 55905, USA. E-mail: reed.ann18{at}mayo.edu
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Abstract |
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Objective. Localized scleroderma causes thickening of the skin due to excessive collagen deposition. This condition has clinical and histopathological similarities to chronic graft-vs-host disease. We wanted to identify whether chimeric cells are present in the affected tissue in localized scleroderma and to further investigate the role of chimerism by immunophenotyping the chimeric cells. We hypothesize that the presence of chimerism and immunotypic chimeric cells will lend to an understanding of the pathogenesis of localized scleroderma and possible mechanisms by which chimeric cells participate in autoimmunity.
Methods. We studied skin biopsies from 18 localized scleroderma patients and compared them with concurrent biopsies from unaffected skin in a subset of patients. Skin biopsies from morphoea and linear scleroderma patients were analysed for the presence of chimeric cells using male–female (X, Y) differences. Cell surface markers (CD4, CD8, CD19/20, CD68, S100, CD14 and CD56) were determined for cell phenotyping of chimeric cells.
Results. Overall, the affected tissue contained a greater number of lymphocytic inflammatory cells. In the affected tissue, 38% of the total chimeric cells were CD68+ (dendritic cell, monocyte and macrophage marker), 29% Langerin/S100+ (dendritic cell marker), 26% CD8+ (cytotoxic T-lymphocyte marker), 20% CD19/20+ (B-lymphocyte marker), 14% CD4+ (T-helper lymphocyte) and 0% CD56+ (natural killer cell marker).
Conclusions. We report that not only are chimeric cells present in affected localized scleroderma lesions but they also are more likely to be dendritic cells and B lymphocytes suggesting a role in the pathogenesis of localized scleroderma.
KEY WORDS: Morphoea, Localized scleroderma, Chimerism, cGVHD
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Introduction |
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Localized scleroderma, including morphoea and linear scleroderma (LS), is an autoimmune disease limited primarily to the skin and sub-dermal tissue. Localized scleroderma like systemic sclerosis has skin lesions similar to what is seen in chronic graft-vs-host disease (cGVHD). Similar to other autoimmune diseases, the aetiology of localized scleroderma is unknown. It has been proposed that pregnancy-associated chimerism provides the possible link between the clinically similar aspects of systemic sclerosis, localized scleroderma and cGVHD. Support for this hypothesis includes the observation that localized scleroderma typically occurs in parous women during or in the post-childbearing years, as well as in the pediatric population, possibly as the result of maternal chimerism.
Chimeric cells are reported to occur in children with juvenile dermatomyositis and neonatal lupus syndrome-congenital heart block, while others have demonstrated chimeric cells in adults with primary biliary cirrhosis, Sjögren's syndrome, systemic lupus erythematosus (SLE), dermatomyositis, polymorphic eruption of pregnancy and systemic sclerosis or systemic scleroderma [1–7]. An increase of chimeric cells in both peripheral blood and affected tissue has been reported in systemic sclerosis. However, a comprehensive phenotype of the chimeric cells has not been clearly defined in any autoimmune disease and there are no reports of chimerism in the localized forms of scleroderma.
Because of the similarities of localized scleroderma with cGVHD, we undertook a prospective study to investigate whether chimeric cells are present in the affected skin lesions in localized scleroderma. In addition, we wanted to determine whether the chimeric cells were of a unique phenotype further defining the chimeric cells involved in the pathogenesis of localized scleroderma.
Our results include the identification of chimeric cells in both the affected and unaffected skin tissue in localized scleroderma with a greater number and distinct phenotype of the chimeric cells in the affected tissue.
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Methods |
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Subjects
Patient samples were obtained from the Department of Dermatology at Mayo Clinic in Rochester, MN, USA, after informed consent was approved by the Mayo Clinic Institutional Review Board. Affected tissue skin biopsies were performed for diagnostic purposes, and unaffected skin biopsies were obtained for purposes of this study after informed consent was obtained. All subjects with a diagnosis of localized scleroderma were approached for consent if they were either male or a female with a known male offspring. Since the origins of the chimeric cells are proposed to occur through maternal–fetal transfer. Given the ease in detecting male–female cell differences, we chose subjects in whom passage of cells could be identified by sex differences. All male subjects and adult female with known male offspring were included. None of the patients in this study had a history of transfusion. Skin biopsies were obtained from the affected and unaffected sites at the same time. Whole blood was obtained on the same day as the biopsy. To add to our sample size, we obtained previous biopsies housed in the dermatology tissue repository.
Maternal chimerism was tested for in six males, three with morphoea and three with LS. Our study population includes three males aged 10–20 yrs (one with morphoea and two with LS) and three males aged 51–65 yrs (two with morphoea and one with LS). Fetal chimerism was tested in 12 females with morphoea whose ages ranged from 37 yrs to 79 yrs (Table 1).
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All females were known to have a male offspring and thus fetal chimerism was detected. The range of time from the birth of the last male offspring to the time of biopsy was 2–34 yrs, and from time of biopsy to the onset of symptoms was 2–48 months. Pregnancies ranged from one to four live births. Time range from onset of disease in the male subjects to biopsy was 1–18 months.
Biopsies were taken from the leading inflammatory edge of sclerotic lesions from 18 LS patients. Eight of these 18 patients also had a biopsy of their unaffected skin as control biopsy. Fresh frozen biopsies were snap frozen for use in our studies. In addition, 10 paraffin-embedded tissue samples that had been previously collected were included.
Morphoea and LS are differentiated by the area they affect. Morphoea lesions are several centimetres in diameter with circumscribed induration surrounding a waxy centre. LS is characterized by a sclerotic furrow and hypo- or hyper-pigmentation that affects the face or extremity unilaterally. The subset of localized scleroderma was determined clinically by the dermatologist and authors (R.E. and C.A.). Control skin tissue obtained was normal epithelium from a stored tissue bank. Biopsies were taken for diagnostic purposes and found to be non-inflammatory.
Tissue/blood preparation
The 16 frozen sections (eight from affected and unaffected tissues) were embedded in Optimal Cutting Temperature Media (OCT; Tissue-Tek Torrance, CA, USA) and mounted to positively charged slides fixed in acetone and dried.
Paraffin-embedded tissue was fixed in neutral-buffered formalin while frozen tissue was embedded and fixed in acetone. All tissues were sectioned on a microtome in 10 µm sections and mounted on positively charged slides (Fisher Scientific). Paraffin slides are de-paraffinized in xylene, rehydrated and washed in phosphate-buffered saline (PBS). Paraffin-embedded tissue required heat- and enzyme-induced antigen retrieval [8]. Slides were steamed in 1 mM EDTA for 20 min, rinsed in PBS and treated with pepsin (100 µg/µl, Sigma) for 10 min, and then rinsed with water.
Peripheral blood lymphocytes (PBLs) (n = 4–16 x 106) were harvested from whole blood by layering on Ficoll–Hypaque gradients. Cells were incubated in 0.075 M potassium chloride (Sigma) and fixed in Carnoy's fixative, and cells were placed drop-wise onto positively charged slides.
Detection of chimerism
We utilized fluorescent DNA probes specific to DNA satellite sequences from the X and Y chromosomes to identify the chromosomes. Following pre-treatment of paraffin slides, all samples, including frozen tissue and PBLs, were dehydrated in an ethanol series (70, 85 and 100%) for 2 min each and were air-dried. Tissue sections were cover slipped with X, Y probe solution (CEP X Spectrum Orange/Y Spectrum Green DNA Probe Kit Vysis Downers Grove, IL, USA) and denatured for 10 min and hybridized overnight, as previously described [9].
Phenotyping of chimeric cells
Immunophenotyping of chimeric cells involves two different techniques to allow maximum analysis. These techniques have been previously reported by Johnson et al. [8, 9]. Modifications are described in brief. The first technique involves immunoperoxidase staining followed by fluorescence in situ hybridization (FISH) for chimeric cell identification. Enzyme-labelled fluorescence (ELF; Molecular Probes, Eugene, OR, USA) is a yellow-green alkaline-phosphatase substrate that is visible under a DAPI long-pass filter (330–380 nm). This allows four fluorescent signals to be viewed simultaneously under a triple band-pass filter including dapi, texas red, FITC and ELF. Any confusion between the ELF and Spectrum Green signal can be verified by viewing under individual filters. The second step is a standard immunohistochemical streptavidin sandwich conjugation that is used to amplify an antibody of interest. Immunophenotyping by ELF followed identification of chimeric cells by FISH to conserve the integrity of the ELF precipitate. The second technique used for phenotyping was diaminobenzidene tetrachloride (DAB). This technique was used when ELF was not compatible with specific tissue antibody combinations. Standard immunohistochemical phenotyping protocols were used. Parallel negative control slides were run without primary antibody.
Antibodies of interest were used to identify cells involved in the inflammatory processing including T and B lymphocytes (NCL-CD4-368, NCL-CD8-295, NCL-CD19-2, NCL-L-CD20-L26; Novo Castra Newcastle, UK), macrophages (NCL-CD14-223, CD68 clone PG-M1; Novo Castra and Dako Carpenteria, CA), dendritic cells (CD 68 clone PG-M1, NCL-Langerin, S100; Dako and Novo Castra) and epithelial cells (Ep-CAM clone HEA125; Research Diagnostics Inc., Flanders, NJ, USA). Several antibodies were not assessed on all samples because of small tissue samples. The dendritic cell marker CD68 was used due to its duality on paraffin-embedded and frozen tissues. CD14 staining was done in conjunction with CD68+ to assure that the cells were of dendritic cell linage. CD19 and CD20 antibodies were both utilized based on the ability to stain frozen vs paraffin tissue.
All slides were read on an Olympus BX 51 microscope by a single technologist who was unaware of the status of the tissue (Olympus, Melville, NY, USA). Only non-overlapping cells with a clear view of a two orange (XX, female) or a green and orange (XY, male) signal were counted. Cells that were counted were both chimeric and those with the CD phenotype identified on the slide.
Statistical analysis
This study is descriptive and in part does not lend itself to statistical analysis. When possible, however, a direct comparison was made of the cell number and cell phenotype between the affected and non-affected tissue in the same patient and between the ‘control’ or non-localized scleroderma skin tissue. Formal comparisons between groups of the number of lymphocytes per 10X field were not performed as only two slides per group were examined. Poisson regression, accounting for overdispersion, was used to test the hypothesis that the proportion of chimeric cells with a specific immunological phenotype differs between the groups.
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Results |
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Subjects
We obtained a total of 26 biopsies from 18 unique patients with localized scleroderma. Two forms of localized scleroderma morphoea and LS are included in this study. Eight patients (seven females with morphoea and one male with LS) had two biopsies, one from an affected site and one from an unaffected site (Table 1). When biopsies from affected and unaffected tissues were obtained, they were on the same day and included the uninvolved site on the opposite side of the body from the affected lesion. This group had a same day peripheral blood sample obtained. Ten additional samples were obtained from a stored tissue bank (two males and three females with LS and five females with morphoea). Control tissue was obtained from four biopsies (one male, and three females with prior male pregnancy) of non-inflammatory normal epithelium.
Cell surface antigens on T and B lymphocytes, natural killer cells, dendritic cells and macrophages were investigated from the skin biopsies. Biopsy sample size limited the number of antigens investigated.
We identified chimerism in all 26 patient samples. Chimeric cells were present in at least one of the tissue samples from the affected areas (n = 18) as well as the unaffected areas (n = 8). The chimeric cells were seen in both mothers with male offspring fetal chimerism (n = 12) and males with maternal chimerism (n = 6). All subjects were evaluated as one group because there were no differences in the number and type of chimeric cells seen between the forms of localized scleroderma.
Chimeric cell presence
Overall, the affected localized scleroderma tissue contained an apparent increase in lymphocytes when compared with unaffected tissue (363 lymphocytes in the affected vs 234.5 lymphocytes in the unaffected tissue per 10X field) and as compared with non-inflammatory control tissue (24 lymphocytes per 10X field). Additionally, the affected tissue contained more chimeric cells per lymphocytes present vs control tissue. Zero to 15 (median 2, mean 2.9, S.D. 3.2) chimeric cells per 10X field were identified in the affected tissue and 0–22 (median 2, mean 3.1, S.D. 0.8) in the unaffected, vs 0–1 (median 0, mean.2 S.D. 0.4) in the control tissue.
Chimeric cell phenotype
In addition to the increase in chimeric cell number, the phenotype of the chimeric cells differed between the tissues. One major difference in chimeric cell population occurred in the epithelial cells where 42% (3/7 chimeric cells) of the affected tissue chimeric cells stained for the epithelial marker (Ep-CAM) compared with 0 from the unaffected site (Fig. 1). This suggests that the localization of the chimeric cells might direct the inflammatory response in the affected tissue. Surrounding these chimeric cells were large numbers of inflammatory T cells, including both CD4 and CD8. Additionally, the chimeric B cells detected by anti-CD19/20 antibodies were increased in the affected tissue 20% (12/59) compared with 3% (1/36) in the unaffected tissue. This difference did not reach statistical significance (P = 0.27), largely due to one patient with 10 of 12 chimeric B cells detected by anti-CD19/20 antibodies.
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Dendritic cell markers represented the most abundant phenotype of chimeric cells in affected and unaffected skin tissue samples. However, the number of the dendritic chimeric cells was not different between the affected and unaffected tissues. Thirty-eight percent of the chimeric cells (0–5 per 10X field) in the localized scleroderma biopsies were CD68+ (macrophage and dendritic cell), while 48% of the chimeric cells (1–5 per 10X field) in the unaffected tissue were CD68+; P = 0.51. In identifying whether the chimeric cells were dendritic cells, we stained for S100 and Langerin, both specific dendritic cell markers. S100- and Langerin-positive chimeric cells made up 29% of all the chimeric cells (1–5 per 10X field, mean 4) in affected tissue compared with 20% (0–1 cell per 10X field, mean 1) in the unaffected tissue; P = 0.34 (Fig. 2a and b). Finally, to determine whether these chimeric dendritic cells were mature, we utilized a specific marker for dendritic cell maturation CD83. We found no chimeric cells positive for CD83 in any of the tissues.
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No differences in CD4+ chimeric cells were seen in either the affected, 14% (0–2 per 10X field), or the unaffected, 15% (0–1 per 10X field), tissue. However, CD8+ chimeric cells were 26% (range 1–11 per 10X field) in the affected tissue compared with 14% (0–3 per 10X field) in the unaffected tissue. Interestingly, when chimeric CD8+ cells were seen in the affected tissue, there was a focal accumulation of these chimeric cells transversing the epidermal–dermal junction. None of the chimeric cells were found to be natural killer cells (CD56+) or macrophages (CD14+). Chimerism was detected in 50% of LS patients’ (n = 8) peripheral blood samples.
When the affected tissue sites were compared with control or non-inflammatory epithelial biopsies, we found only 0–1 chimeric cells per 10X field. There were no CD4, CD8, CD68 or CD20 chimeric cells; the only chimeric cell phenotype seen was S100 representing immature dendritic cells (Fig. 1).
The phenotypes of the surrounding non-chimeric or host cell infiltrate in the affected tissue includes 23% CD4+, 16% CD8+, 16% CD14 +, 24% CD20+ and 14% CD68+ as a percentage of total number of inflammatory cells in a 10X field.
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Discussion |
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This is the first report to demonstrate chimeric cells in the skin of subjects with LS and morphoea. This study is unique because we were able to obtain controls from unaffected contralateral region from the affected patients. While dendritic cells appear throughout the skin in all samples, we identified epithelial and dendritic cells as the greatest proportion of chimeric cells in LS patients. This finding is not surprising, as skin dendritic cells have been previously reported [10]. Langerhans cells, specialized dendritic cells in the skin, are specialized antigen-presenting cells that continually sample their microenvironment within the epidermis. Other skin-specific epithelial chimeric cells are also seen. This is most exciting since it suggests that primary tissue chimeric cells could be the focal point for an auto-inflammatory response of a cGVHD. Surrounding these epithelial chimeric cells are large accumulations of inflammatory T cells both CD4 and CD8 forming layers of cells in this region—again a possible explanation for the regionality of this disease. We also identified other antigen-presenting cells in the form of CD19/20-positive B lymphocytes present in greater percentages in affected 20 vs unaffected 3 tissues. The B chimeric cells were also intimately involved in the inflammatory cell accumulations. These accumulations of cells resemble the lymphoid aggregate reported in other autoimmune disease processes; however, further evaluation is needed to confirm this finding.
Chimeric cells are universal throughout the skin tissue in localized scleroderma; however, they are more concentrated in the affected areas at greater than expected rates even when accounting for the increase in inflammatory cells. Well over half the chimeric cells were epithelial cells demonstrating that not only are the chimeric cells haematopoietic in origin but they are primary-organ-derived as well. This also brings up the possible theory that the organ-specific chimeric cells are the nidus for inflammatory response and thus the reason for inflammation in the skin.
Chimerism is described as the presence of foreign, non-self cells or DNA in an individual. Chimerism is acquired through a myriad of routes. Excluding organ and tissue recipients, the most common transfer of cells occurs between mother and child in utero. Fetal chimerism is the transfer of fetal cells to mother, and maternal chimerism is the transfer of cells from mother to fetus. Other possible sources of chimerism include, but are not limited, to twin–twin and child–mother–future offspring. Transfer of cells is thought to occur as early as in the first trimester of pregnancy, and therefore transfer could occur before miscarriages and abortions. Therefore, the presence of fetal chimerism is not exclusively limited to parous women. Earlier studies have shown chimeric cells to persist in recipients for decades [11]. The presence of chimeric cells in scleroderma patients is hypothesized to play a role in the clinical similarities between scleroderma and cGVHD.
Previous studies of chimerism have used both non-quantitative and quantitative methods, making it difficult to make comparisons between studies. Most studies of fetal–maternal chimerism focus on fetal chimerism, reporting between 36 and 70% (non-quantitative) of female patients with autoimmune diseases, with at least one male pregnancy, have evidence of male DNA (Y) or male staining cells [3, 12]. The quantity of fetal chimerism in women with autoimmune disease bearing at least one male pregnancy is 2.7 times and 27.5 times greater in tissues and blood, respectively, vs controls [12]. Few studies exist focusing on maternal chimerism. One study identified maternal chimiersm in 72% of patients with systemic sclerosis vs 22% of controls; however, the quantity of chimeric cells did not differ [12]. Our previous unpublished control data has suggested an insignificant likelihood to observe female cells in male offspring vs male cells in a mother. Differences in number of chimeric cells between patients with fetal and maternal chimerism may exist; however, these difference were not apparent in our study. Previous studies have shown increased numbers of chimeric cells in formalin-fixed paraffin-embedded (FFPE) tissue due to formalin fixation. Since our study included both FFPE and frozen tissue, we investigated a difference and found none.
cGVHD is an iatrogenic form of chimerism resulting from an organ or tissue transplant that is reported in 30–50% of recipients. In this disease, lymphocytes extravasate from donor tissue to the recipient and remain. The presence of foreign lymphocytes mounts an immune response against the host. Intriguingly, consequences of this immune response mimic symptoms of scleroderma including sclerotic skin, contractures, hair loss, ulcerations, myositis, sicca syndrome and gastrointestinal and pulmonary manifestations.
Chimerism is found in an increasing number of autoimmune diseases; however, the presence of these cells does not explain their role in disease. This is the first study that implicates specific chimeric cell phenotypes in an autoimmune disease. Future experiments underway include the investigation of chemokines produced by the chimeric cells. This will allow us to directly hypothesize a role for these cells in the disease. Further work is underway to see if other autoimmune diseases also have a skewing of the chimeric cells presence.
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These studies were supported by the Mayo Foundation. We are very appreciative of superior technical support from Heidi Hermes and Anne Stevens (Fred Hutchinson Cancer Research Center, Seattle WA, USA).
The authors have declared no conflicts of interest.
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References |
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