Previously we found higher frequency and number of microchimeric cells in the peripheral blood and muscle tissue of JIIM patients compared to noninflammatory or healthy control subjects, suggesting that microchimeric cells may play a role in the pathogenesis of JIIM [4, 20]. One limitation in those studies was that we did not phenotype the microchimeric cells in the tissues; however, we did find microchimeric cells in the CD4+ and CD8+ lineages in peripheral blood . The current study further investigated microchimeric cells in JIIM, compared with inflammatory but nonautoimmune muscle disease (MD) and with noninflammatory control muscle and examined the immunophenotypes of microchimeric cells in muscle tissue. Our goals were to determine whether microchimeric cells play a pathogenic role in disease and/or whether they are involved in tissue repair. Persistent microchimeric cells have been found in patients with other inflammatory diseases, suggesting that these cells might be recruited nonspecifically to sites of inflammation [7–10, 13, 14].
Compared with our prior study, we found a similar frequency of JIIM muscle tissue samples containing microchimeric cells and a similar overall quantity of microchimeric cells in JIIM muscle tissue . Here, we also saw a similar concentration of microchimeric cells in MD tissues, suggesting that this finding is not specific to autoimmune diseases but may be generally associated with overt inflammatory muscle disease. This suggests that the number of inflammatory microchimeric cells recruited to the site of inflammation does not depend on the total number of inflammatory cells present. MD is a genetic disease caused by mutations in dystrophin inducing muscle fiber damage, with resultant muscle necrosis and tissue inflammation , whereas the cause of JIIM is unknown but presumed to result primarily from an autoimmune-mediated destruction of myofibers and muscle capillaries . In both diseases, tissue destruction is mediated by T and B cells, and in JDM by dendritic cells [22–24]. We were surprised to find microchimeric cells also in a high proportion of control muscle tissues, in contrast to our prior study ; however, in the prior study, only one muscle section was examined, whereas in the present study, ten sections from each patient’s muscle tissue were examined. Microchimeric cells were not observed in every tissue section from a given patient and thus overall were considered to be a rare event.
To characterize the phenotype of microchimeric cells in JIIM and MD, we stained muscle tissue for various immunophenotypes. Overall, we detected few microchimeric cells within inflammatory phenotypes, and we did not find microchimeric cells in B cell or dendritic cell lineages, which are thought to be important in JIIM pathogenesis. Of the T cell immunophenotypes, generally the concentration of microchimeric cells was greater in MD tissues than in noninflammatory controls, and typically T cells or activated T cells were not elevated in JIIM muscle tissue. Microchimeric cells of a given phenotype did not occur more frequently than their autologous counterparts in JIIM or MD muscle tissue, compared to all microchimeric or immunophenotyped autologous cells. In terms of location, the endomysial and perivascular regions of the muscle showed similar numbers of microchimeric cells among the three groups. However, MD and JPM tissues were more likely to have microchimeric cells in the perimysium compared with controls. It is this sole finding that weakly suggests that microchimeric cells might play a pathogenic or reparative role.
We also found microchimeric cells in noninflammatory tissues and in myofibers, not only in infiltrating inflammatory cells, suggesting that these cells are resident in tissues regardless of inflammation. Tissues with maternal microchimeric cells include autoimmune and nonautoimmune thyroid , pancreas in type I diabetes , neonatal lupus heart muscle , tonsils and adenoids , cutaneous inflammatory diseases  and inflammatory bowel disease . Normal tissues with maternal microchimeric cells include heart , tonsils/adenoids , skin diseases  and inflammatory bowel disease ; albeit at lower levels than diseased tissue of the same type, suggesting that a threshold number of microchimeric cells might be necessary to drive disease. However, in some instances microchimeric cells were found at comparable numbers in healthy tissues .
One hypothesis proposed by other investigators is that microchimeric cells might mediate tissue repair via microchimeric stem cells. Initially we stained for muscle fibers, but this staining procedure interfered with subsequent FISH. Therefore, we counted the numbers of microchimeric nuclei that were apparently myofibers, based on their morphological position and shape within the muscle fiber, and compared them to the numbers of autologous myofiber nuclei. We found significantly more microchimeric muscle nuclei in MD than JIIM muscle, but there were no significant differences between JIIM and controls. However, each disease class had significantly fewer microchimeric myonuclei than autologous muscle nuclei. One caveat is that we do not know how many myofibers were regenerated from autologous progenitors during the disease process , and this study was not designed to determine that. Overall, these results suggest that microchimeric myofibers are no more frequent than microchimeric inflammatory cells in diseased tissues, and that noninflammatory tissue resident in microchimeric myofiber nuclei are present at higher levels than observed in JIIM. This result suggests that microchimeric cells are not enriched in muscle tissue in any of these disease conditions, and that they do not mediate tissue repair at an augmented rate compared to autologous myofibers.
We stained one phenotype per FISH analysis for microchimeric cells in the tissue, but we did not stain for all cell phenotypes in the tissues. Consequently, we may have missed a microchimeric cell phenotype(s) that was noninflammatory, including muscle stem cells. We did not analyze all inflammatory phenotypes, such as macrophages, that are frequent in the inflammatory infiltrates of JIIM and MD muscle [22, 23], and some microchimeric cells did not match any of the phenotypes analyzed. The number of inflammatory cells in the JIIM samples was comparable to previously published data , and this suggests that the immunophenotyping was not underrepresenting inflammatory cell phenotypes. Furthermore, although we did not observe many differences in the phenotypic numbers of microchimeric cells between disease groups, only one or a small number of pathogenic microchimeric cells might be sufficient to cause disease; however, we believe this is unlikely as microchimeric cells were also present in the muscle tissue of noninflammatory controls. In addition, we did not examine the functionality of the microchimeric cells phenotyped and although microchimeric cells were found in inflammatory cells of the controls, they may have been quiescent, whereas in the inflammatory muscle diseases they may be activated, indicated by different cytokine profiles . We did, however, investigate T cell activation markers, such as Class II, CD25, and memory T cell subsets, and did not detect more microchimeric cells in JIIM muscle compared to noninflammatory controls, and only MD had significantly more CD25 microchimeric cells. Finally, our sample sizes were relatively small and the study may have been underpowered to detect differences between groups.
Murine studies suggest that maternal microchimeric cells are able to manipulate the fetal immune system and promote the development of various Regulatory T cells (Tregs) that are tolerant toward the noninherited maternal antigens . Additional studies using inbred mice have also reported the trafficking of alloreactive T cells in offspring and that the maternal cells can influence the fetal response to targeting specific tissues . These studies were elegantly performed in mice, and whether this same phenomenon occurs to the same extent during human gestation leading to increased risk for autoimmune diseases is yet to be proved. Currently, no studies have been performed to determine whether maternal microchimerism of cells that carry noninherited shared epitopes place the JIIM cohort at a greater risk for disease, like they apparently do for rheumatoid arthritis . HLA-DRB1*0301 in linkage with HLA-DQA1*0501 confers the highest susceptibility for JIIM; however, in contrast to previous studies , we found that HLA-DQA*0501 was not significantly associated with microchimerism . Maternal microchimeric cell transfer is a frequent occurrence during fetal development , and these cells most likely distribute throughout all the tissues and become embedded in the bone marrow with the ability to differentiate into inflammatory cells or stromal cells upon cell damage. Thus, the presence of maternal microchimeric cells in tissues is not surprising in light of the high number of normal samples that were positive in our study and in other studies [6, 37]. Our data support the findings of Ye et al., who also reported their controls being positive for maternal microchimerism . The presence of microchimeric cells in the control tissues without inflammation suggests that they are present either from fetal development if stromal in nature, or are inflammatory cells trafficking through the tissue. In contrast with Ye et al., we found the presence of some microchimeric T cells, albeit at very low percentages in our JDM samples. However, the direct role of maternal microchimeric cells in human disease is still not clear and against the complex genetic background of humans may mean that the direct role of these cells in autoimmunity may never be conclusively demonstrated.