Rheumatology 2001; 40: 1279-1284
© 2001 British Society for Rheumatology
Paediatric Rheumatology |
Persistent maternally derived peripheral microchimerism is associated with the juvenile idiopathic inflammatory myopathies
Paediatric Rheumatology/Series Editor: P. Woo
Division of Rheumatology, Thomas Jefferson University, Philadelphia, PA 19107,
1 Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD and
2 Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA
Abstract
Objective. Fetal cells have been demonstrated in the active lesions of adult women with systemic sclerosis. Because the juvenile idiopathic inflammatory myopathies (JIIM) share clinical and histopathological features with systemic sclerosis and graft-vs-host disease, we explored the possibility that maternal cells persist and play a role in the pathogenesis of JIIM.
Methods. DNA samples extracted from peripheral blood of 28 JIIM patients (14 females, 14 males) and 23 healthy controls were assessed for microchimerism by the HLA Cw polymerase chain reaction method. HLA Cw alleles from eight mothers and three healthy siblings of JIIM patients were also examined.
Results. A microchimeric allele was identified in 19 of 26 JIIM patients whose data were able to be interpreted, compared with two of 21 healthy controls (P<0.001). Subjects with microchimerism ranged in age from 4 to 28 yr. In eight cases in which maternal peripheral blood was available, the additional Cw allele present in the patients was confirmed to be identical to a maternal allele. Three healthy siblings of JIIM patients did not have evidence of a microchimeric Cw allele.
Conclusion. Maternal cells can persist in the peripheral blood of their children up to three decades after birth, and are found in a higher proportion in JIIM patients compared with controls. These findings, with other data, suggest that maternal cells may be involved in the immunopathogenesis of JIIM.
KEY WORDS: Microchimerism, Maternal–fetal exchange, Dermatomyositis, Polymyositis, Graft versus host reaction, Inflammatory myopathy.
The juvenile idiopathic inflammatory myopathies (JIIM) are a heterogeneous group of rare systemic connective tissue diseases characterized by chronic muscle inflammation that results in progressive proximal muscle weakness [1]. Histopathological studies demonstrate that juvenile dermatomyositis (JDM), the most common form of JIIM in childhood, is predominantly a vasculopathy that is probably mediated by B lymphocytes, macrophages and CD4+ T lymphocytes, whereas polymyositis (PM) largely involves focal muscle fibre destruction by CD8+ T lymphocytes [2]. The JIIM are thought to occur after exposure to certain environmental agents in genetically susceptible individuals, and affect girls two to three times more commonly than boys [3, 4]. HLA DRB1*0301 and the linked allele DQA1*0501 are major genetic risk factors [5], but non-MHC genes are also probably involved in the pathogenesis [6]. Although a number of infections and ultraviolet light exposure have been associated anecdotally with the onset of JIIM [1], environmental triggers have not been investigated extensively and have been difficult to identify in case-controlled studies [3].
Recent interest has focused on microchimerism as a mechanism that may initiate graft-vs-host (GVH) reactions that manifest as autoimmune diseases, particularly in adult patients with systemic sclerosis (SSc) [7, 8]. A number of clinical features of idiopathic myositis resemble those of GVH disease (GVHD), and PM has also been seen in association with GVHD following bone marrow transplantation [9]. Reed et al. [10] first hypothesized a chronic GVHD-like reaction in children with myositis in their identification of microchimeric HLA-DQA1 alleles in three JDM patients compared with two of seven unaffected siblings. We recently identified maternal chimeric cells by fluorescence in situ hybridization (FISH) in magnetically separated peripheral blood lymphocytes, as well as in the inflammatory skin and muscle lesions of several male JIIM patients [11].
In our previous study [11], the demonstration of maternal cells in the periphery and affected tissues was confined to examining male offspring. The recent development of a sensitive analysis method for HLA Cw [12], which permits the detection of microchimeric alleles regardless of gender, allowed us to investigate the frequency of chimeric DNA in the peripheral blood of a larger number of children with JIIM, healthy subjects and unaffected family members.
Patients and methods
Subjects
Twenty-eight JIIM patients (26 JDM, 1 JPM, 1 JDM/SSc overlap myositis) meeting probable or definite criteria for myositis with disease onset prior to 18 yr of age [13] and 23 healthy gender-matched controls underwent venipuncture to obtain peripheral blood cells (PBCs) for DNA extraction. JIIM patients (14 females, 14 males) had an overall mean age of 14 yr (range 4–28 yr) compared with a mean of 16.7 yr in the control group (range 10–27 yr). Ethnically, JIIM patients were diverse (16 Caucasian, 5 black, 2 Hispanic, 2 Asian, 1 Indian, 1 Caucasian/Asian, 1 Caucasian/Hispanic) whereas all the controls were Caucasian. DNA was also obtained from eight mothers and three unaffected siblings of JIIM patients. DNA was not available from additional mothers to confirm the maternal identity of microchimeric alleles, either because of loss of patient contact or parent refusal of enrolment. None of the JIIM patients or controls had a history of pregnancy or a previous blood transfusion. All subjects provided informed consent.
Detection of microchimerism in peripheral blood by HLA Cw analysis
HLA Cw analysis was performed with 12 µg extracted DNA by a sequence-specific primer method according to Artlett et al. [12]; each sample was tested once. The method used 23 polymerase chain reaction (PCR) reactions, which analysed 17 HLA Cw alleles. Ethidium bromide staining following electrophoresis in 1% agarose gels identified the resulting PCR products. The two intense bands of the PCR products were assigned as the inherited genotype, whereas a weaker third band was classified as the microchimeric allele.
FISH of magnetically separated PBCs
Cells from five male JIIM patients and three healthy controls were sorted magnetically for CD4+ and CD8+ populations (antibodies from Dako, Carpinteria, CA, USA), according to the method of Artlett [7, 11], and were used to confirm the HLA Cw findings.
Statistical analysis
All laboratory studies were performed blinded to the subjects' diagnoses and clinical information. Results from the patients and controls were compared using the 
2 test with Yates' correction or the Fisher exact test. The Fisher exact test was used to investigate the effects of immunosuppressive therapies on microchimerism status.
Logistic regression was used to examine the relationship between microchimerism status and methotrexate (MTX) dose and the relationship between microchimerism and age.
Results
Microchimerism in peripheral blood based on HLA Cw typing
Using HLA Cw typing, microchimeric Cw alleles were found in 19 of 26 (73%) JIIM patients compared with two of 20 (10%) healthy controls (P<0.001, 2=15.7, Yates corrected), based on the detection of a third, fainter allele in these subjects (Table 1
). In addition, two patients and three controls were homozygous for HLA Cw. Although a fainter allele was not detected in these subjects, microchimerism cannot be confirmed or excluded and they were not included in the statistical analyses. The controls were not exactly age-matched to the patients (10–27 and 4–28 yr respectively). We therefore performed additional statistical analyses in which we did not include JIIM patients who were under the age of 10 yr (n=5). We found that microchimeric Cw alleles were present in 15 of 21 (71%) JIIM patients >10 yr of age (P<0.001, 2=13.5, Yates corrected).
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Only 6% of the microchimeric alleles in the JIIM patients were HLA Cw12, in contrast to a higher than expected frequency of HLA Cw12 in microchimeric adult SSc patients [12]. Twenty-eight per cent of JIIM patients had an HLA Cw4 allele compared with 4% of the controls (P=0.02). These results may not be representative of the general population; the frequency of HLA Cw4 is approximately 20% in the US Caucasian population [14].
Ethnicity and gender did not influence whether or not an individual had microchimerism. We found in the patients that 10 of 16 Caucasians were positive for microchimerism compared with nine of 12 non-Caucasians (P=0.6). In addition, we found that nine of 14 males with JIIM had microchimerism compared with eight of 14 female JIIM patients (P=0.4).
Treatment with prednisone had no apparent effect on microchimerism status: 11 of 18 patients receiving prednisone were positive for microchimeric Cw alleles compared with seven of nine patients receiving no corticosteroid therapy (P=0.67). Interestingly, patients receiving MTX were less likely to have a microchimeric Cw allele (three of eight patients) compared with patients not obtaining MTX (15 of 17, P=0.017). Although the number of patients studied was small and the dose range of MTX was narrow (0–0.45 mg/kg per week), there was also an inverse relationship between the dose of MTX and the presence of microchimerism (odds ratio=0.0004, P=0.015). There was no association between age of patients and controls and the presence of microchimerism (odds ratio=0.93, P=0.20). There was also no relationship between the presence of microchimerism and the gender of the JIIM patients or control subjects: 11 of 22 (50%) males were positive for microchimeric Cw alleles and 10 of 25 (40%) females were microchimeric. Analysis of PBLs by FISH and PCR of the HLA Cw gene demonstrated the same results for microchimerism status in the five patients and three controls tested by both methods.
The mothers of eight JIIM patients with evidence of microchimeric Cw alleles were also HLA Cw-typed, and the additional allele present in the patients was confirmed to be identical to a maternal Cw allele in all cases tested (Table 2). Of interest, three healthy siblings of JIIM patients, of whom one was a dizygotic twin with a fused placenta, did not have evidence of a microchimeric allele (Table 2). Figure 1 displays an example of HLA Cw analysis from one family in which a 14-yr-old boy with JDM had evidence of three distinct Cw alleles, one of which was a less intense microchimeric allele, which was confirmed to be maternal in origin. In contrast, the patient's 16-yr-old healthy brother had two Cw alleles and did not demonstrate a microchimeric allele.
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Discussion
The results presented here confirm that a childhood-onset autoimmune disease, JIIM, can be associated with the presence of maternal DNA in the peripheral blood. The increased frequency of maternal microchimeric alleles in both female and male patients compared with healthy controls and the absence of maternal chimerism in unaffected siblings of patients extend our prior findings of microchimeric cells detected in peripheral blood lymphocytes and affected tissues of JIIM patients [11] and, in combination, support a role for microchimerism in the pathogenesis of JIIM. These findings also extend previously published studies, which suggested that the transplacental passage of fetal cells contributes to the induction of autoimmune disease in multiparous women [7, 8] by demonstrating that maternal lymphocyte trafficking to the offspring may be involved in juvenile myositis.
In this study, we found that 10% of healthy male and nulliparous female controls had evidence for a maternal HLA Cw allele by PCR of their PBCs 13–18 yr after birth (Table 1
). The prevalence and extent of microchimerism of maternal origin in the peripheral blood of these healthy subjects appeared to be substantially lower than the microchimerism of fetal origin found in the peripheral blood of healthy multiparous women [8, 12]. The reasons for these differences remain unclear, but may relate partly to limitations of the Cw PCR assay. The Cw PCR method requires the presence of a third, fainter allele to establish microchimerism, and therefore may lead to some false-negative results, particularly in cases when subjects could be microchimeric with two Cw alleles, depending on the maternal haplotype. This methodological limitation, however, should not result in more false negatives in the control subjects than in the patients, and the concordance of microchimerism status in eight patients and controls using two different methods, the Cw PCR method and FISH of PBCs [11], is additionally reassuring. The low percentage of microchimerism in healthy control subjects may also result from the fact that children have had only one opportunity to acquire chimeric cells, which is in utero, in contrast to adult women, who have exposures to chimeric cells both in utero and during their own pregnancies [7, 8]. It is also possible that there is less cross-placental transfer of cells from mother to infant than vice versa, as in our previous study 28% of adult control subjects were positive for a microchimeric HLA Cw allele [12]. We speculate that activation of the immune system via environmental stimuli may result in preferential expansion of microchimeric cell populations and thereby increase the likelihood of detection of a microchimeric allele [15, 16]. Alternatively, the detection of microchimeric cells may be a time-dependent phenomenon due to the relatively slow proliferation of these cells [15].
Of interest, we found that MTX therapy was associated with less microchimerism in JIIM patients. Although this finding requires confirmation in larger studies, it suggests that some patients may be microchimeric at the onset of their disease, and that immunosuppressive therapies may diminish the number of microchimeric cells below the limit of detection. Investigations of serial samples from additional patients should address this issue, and careful assessment of immunosuppressive therapies should be incorporated into all future investigations of microchimerism.
There has been much less investigation of cells crossing the placenta from mother to fetus than from fetus to mother in healthy pregnancies [17]. The prevalence of microchimerism seen in the peripheral blood of our control subjects also appeared to be lower than that seen in studies of samples taken from younger subjects. Studies performed in the third trimester found that all fetal blood samples contained maternal cells, and intrapartum 14–42% of cord bloods sampled had maternal cells [17, 18]. Evaluation of two healthy male controls (mean age 25 yr) detected no chimeric cells by FISH of a cytospin preparation of whole peripheral blood mononuclear cells [18].
The fate of the cells transferred from the maternal to the fetal circulation (or vice versa) is not known. The published data suggest that the number of maternally derived chimeric cells may be highest at birth and decline with age, although there have been no serial studies to confirm this. Among the possible mechanisms that may explain this finding are that the chimeric cells are actively eliminated post-partum by the neonate, and that many maternal cells are short-lived (such as B cells) [19] or are sequestered (as has been shown previously in studies of bone marrow, spleen and liver [20]) and escape detection in the peripheral circulation [21].
The consequences of the intermixing of cells allogeneic to the child are unknown, but probably depend on the alloantigens they express, on the antigen-presenting capacity of the cells, on the number and types of environmental exposures over time, and on the response of the neonatal immune system to their presence. Tolerance to non-inherited maternal antigens has been reported in 50% of individuals undergoing kidney transplantation, and maternal cells entering the bloodstream of the fetus might be responsible for the induction of non-responsiveness [22]. Alternatively, maternal cells may undergo selection in the periphery and become tolerant to fetal antigens, or they may act as veto cells, inactivating host T cells that recognize them [22].
This study complements and confirms our previous findings [11], and supports the hypothesis that cells of maternal origin may be involved in the pathogenesis of juvenile myositis and potentially in other paediatric autoimmune diseases. It is becoming increasingly clear that, under certain circumstances, maternal lymphocytes gain access to the fetal circulation and are not eliminated by the developing neonatal and childhood immune system. The critical question that must now be addressed is whether these microchimeric cells actually induce or sustain pathological immune processes.
Appendix
Childhood Myositis Heterogeneity Collaborative Study Group Members contributing patient material to this study: B. Adams, M. Borzy, G. Cawkwell, J. Eggert, R. Fuhlbrigge, V. Garwood, S. George, E. Goldmuntz, D. Goldsmith, H. Haftel, M. Henrickson, G. Higgins, I. Katona, C. Lindsley, E. Love, M. Lundberg, K. Madson, S. R. Mitchell, D. Person, L. Ray, R. Rivas-Chacon, D. Sherry, S. Vogelgesang, C. Wallace and P. White.
Acknowledgments
The authors thank Drs S. Epstein and A. Rosenberg for critical review of the manuscript and valuable discussions, and members of the Childhood Myositis Heterogeneity Collaborative Study Group Members, who contributed patient material to this study.
Notes
Correspondence to: C. M. Artlett, Division of Rheumatology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA. 
* A complete list of members is given in the Appendix.
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