Micro- and tissue chimerism in physiologic, autoimmune, and transplantation settings: A meta-analysis

Micro- and Tissue Chimerism in Physiologic, Autoimmune, and Transplantation Settings: A Meta-analysis Eveline P. van Poelgeest, Hans C. van Houwelingen, Emile de Heer, Emma L. Lagaaij, Jan A. Bruijn, and Ingeborg M. Bajema We give an overview on chimerism in all its forms of appearance and discuss the methodologic complications with which this field of research is confronted. The data of 42 articles, 23 on transplantation and 19 on autoimmune disease, were entered into a meta-analysis. We found that (micro-) chimerism after solid-organ transplantation occurred significantly more often than in healthy controls (P < .001). Also, chimerism was more prevalent in patients with autoimmune diseases than in healthy controls (P < .001). Most of the criticism on transplantation studies focuses on whether the chimeric cells were not already there before the transplantation took place. The results of our meta-analysis seem to contradict this assumption. In autoimmune diseases, a hypothesis on how chimeric cells would influence the development of these diseases is still lacking. However, our results show that (micro-) chimerism occurs significantly more often in patients than in healthy controls, possibly underlining a causative relationship. © 2004 Elsevier Inc. All rights reserved.

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himerism is a phenomenon frequently discussed in the medical literature these days, especially in relation to transplantation. Some have called it an area of research “mired in controversy,”1 and much criticism has been uttered about “inappropriate methods”1 used to reach any conclusions on the presence of chimerism and its clinical relevance. The nomenclature on the subject may be confusing, largely because of the variety of circumstances in which chimerism may occur. Let us therefore briefly discuss its various forms of appearance, shown in Figure 1. In the broadest sense of the idiom, “chimeric cells” are cells derived from another organism than the organism in which they are found. Most illustrative is the example of a blood transfusion, in which donor-derived cells are transfused into a recipient. Also, in pregnancy, a chimeric state exists, more complicated than the first example because of its bidirectional cell traffic, both from mother to foetus and from foetus to mother. In

From the Departments of Pathology, Medical Statistics, and Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands. Address reprint requests to Ingeborg M. Bajema, MD, PhD, Department of Pathology, L1-Q, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands. 0955-470X/$ - see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.trre.2004.04.008

transplantation, either of solid organs or bone marrow, donor-derived peripheral cells are found in the recipient’s circulation. Most interestingly, however, recipient-derived cells have been found in the donor organ as, for example, endothelial cells of recipient origin lining the vessel walls. In situ hybridization to identify Y-chromosome-positive cells in the case of a female donor organ in a male recipient is often used.2 In autoimmune diseases, chimeric cells of male origin have been described in female patients. Their origin is unclear, however, it has been suggested that they are derived from former pregnancies. The relevance of a chimeric state in a clinic setting is largely unknown. Methodologically, it is fairly easy to identify chimeric cells in peripheral blood. This finding is sometimes called “microchimerism.” It is, however, extremely difficult to reliably identify nonperipheral chimeric cells in tissue (eg, chimeric endothelial or epithelial cells, a condition referred to as “tissue chimerism”). Double-staining methods are required to identify the chimeric cell as, for example, an epithelial or endothelial cell, and the reliability of these methods has been heavily debated.1,3 This review aims to describe the state-of-the-art on chimerism in its various forms of appearance and to discuss the methodologic complications with which this field of research is confronted. We discuss a vast number of recent publications but also refer to

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Figure 1. Induction and clinical consequences of (micro-) chimerism.

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the early literature on this subject starting in the 1970s. Finally, we present some preliminary hypotheses on the role of chimerism in transplant survival and autoimmune diseases.

Search Strategy and Selection Criteria A computerized literature search was performed in Pubmed, the NCBI web site for medical literature, for relevant human studies published in the English language between January 1966 and December 2003, containing the following MeSH terms or text words: peripheral blood microchimerism, (micro- or mixed) chimerism, pregnancy, tissue, auto-immune disease, transplantation, and (mixed) chimera. Excluded were studies in which 3 individuals or fewer were enrolled (except when multiple sites were examined in case of tissue chimerism) and studies in which chimerism was induced by stem-cell or bonemarrow transplantation. On the basis of our assumption that publication bias is of little importance in this field, no additional criteria were used. The abstracts of all potentially relevant articles were screened before full-text versions were retrieved. Data were extracted from each article, evaluated by 2 independent reviewers, and collected in a spreadsheet. A total of 42 articles, 23 on transplantation and 19 on autoimmune disease, were included in this meta-analysis. Chimerism in pregnancy had relatively few articles directly related to chimerism in transplantation and autoimmune disease; these will be discussed separately. No strict validity assessment was made; no studies were excluded from this review because of considerations pertaining to study design, combinability, control of bias, statistical analysis, sensitivity analysis, or problems of applicability. Although these are important criteria to exclude studies from meta-analyses,4 these criteria essentially apply to randomized controlled trials. Because the research on chimerism is relatively narrow at this time, we chose to take into account as many data as were available.

Pregnancy Pregnancy-related chimerism was described as early as 1979.5 The most commonly described situation is that of male cells of fetal origin circulating in women with male offspring because the sex difference makes it easy to identify these cells. It is assumed, however, that circulating fetal cells of daughters are just as likely to occur as those of sons. Fetal cells in

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the maternal circulation were reported as early as 4 weeks and 5 days after conception6 and as late as 27 years post partum.7 The reported prevalence of chimerism in pregnant women varies roughly from 60% to 100%.8,9 This variation is most likely the result of the different methods that were used to detect it: in the mentioned examples, the first study used polymerase chain reaction amplification of the SRY region, the second isolation by size of epithelial tumor cell-based fluorescent in situ hybridization. Evans et al.10 showed that in approximately 30% of women who had been pregnant, microchimerism could be detected up to 38 years later.10 This means that pregnancy may establish a low-grade chimeric state in the human female,7 which is more or less physiological. Maloney et al11 provided evidence for persistent maternal microchimerism in healthy immunocompetent subjects in an age range from 9 to 46 years old.11 It has been suggested that cells derived from the fetus not only persist in the mother’s circulation but that they also divide,7 which may have relevance to their relation to disease. A fascinating finding was recently published by Abbud et al.12 In 18 healthy women who had given birth to sons, the number of fetal DNA copies had a mean value of 2.5 (standard deviation, 4.5) within 10 years after gestational time, and this decreased to a mean value of 0.05 (standard deviation: 0.01) after 30 years. In 28 systemic lupus erythematosus (SLE) patients, the mean number of fetal DNA copies was 32.7 within 10 years after the gestational time (standard deviation, 64.8) and increased to 1,445.3 (standard deviation, 2042.9) after 30 years. This finding may indicate that the ability of chimeric cells to proliferate is associated with the development of SLE. Besides fetal cells occurring in the mother, also maternal cells may persist in their offspring, reflecting the bidirectional cell traffic between mother and fetus. That this may be related to a serious, immunologically mediated disease is shown by a correlation between chimerism and severe combined immunodeficiency. In this syndrome, maternally derived T cells lead to a graft-versus-host-like syndrome in infants.13

Autoimmune Diseases A number of studies were reported about chimerism occurring in a variety of autoimmune diseases. Primarily, patient groups consisted of women with sons, although some studies focussed on men by determining whether they had maternal DNA.14,15 In some

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reports on autoimmune disease, the term “microchimerism” is used to indicate the relatively small amount of chimeric tissue cells, such as epithelial cells.16 More frequently, however, the term is used for peripheral blood chimerism to distinguish it from chimerism in tissue-derived cells. In all studies reported in this review, control groups were present: they were either healthy controls, disease controls, or a combination of these 2. Some of the studies used healthy siblings or other relatives as controls; some made a distinction between controls with and without a history of pregnancy or blood transfusions. Most of the healthy controls were tested on the occurrence of microchimerism in peripheral blood. For tissue chimerism, only few data from a few studies were available, and these were almost exclusively negative. Tissues from various organs obtained at autopsy revealed no tissue chimerism in a total of 11 female patients in 2 studies.16,17 Ten skin biopsies from normal controls in a study on systemic sclerosis were all negative.18 In this study also, 68 skin biopsies from women with osteoarthritis or from patients’ relatives were negative. The only study in which chimerism was actually found in normal tissue was about juvenile dermatomyositis, in which 2 out of 10 siblings of the patients were reported to have chimerism in muscle biopsies.15 In patient control groups a variety of diseases were present (eg, liver diseases, muscle disorders, and thyroid diseases) (Fig 2A). A minority of patients in the patient control groups had oncologic disease. Most of the material from patient control groups came from autopsies. Taking all studies together10,12,14-30 and combining micro- and tissue chimerism, the prevalence of chimerism in individuals with autoimmune diseases was 53%, and significantly more chimerism occurred in patients with autoimmune diseases than in healthy controls (P ⬍ .001, Fig 2A). Comparing various diseases (systemic sclerosis, Sjo¨gren’s syndrome, SLE, primary biliary cirrhosis, scleroderma, thyroid diseases, and myopathies), we found that the prevalence of chimerism varied from 18% to 68%, with an exceptionally low number in Sjo¨gren’s syndrome. In healthy controls, microchimerism occurred in 127 out of 411 persons (31%): the majority were women, and most of the studies used Y chromosome detection to determine the presence of microchimerism. In a minority of cases, HLA mismatches were used to detect microchimerism, and these studies sometimes incorporated men. Although microchimerism as a result of blood transfusions has been

suggested, data on whether the healthy controls had had a blood transfusion in the past were mostly lacking. In Figure 2A, 2 vertical lines indicate the prevalence of chimerism in controls: the scattered line is the result of data from all controls in all studies, incorporating microchimerism and tissue chimerism in normal controls, disease controls, relatives, and siblings. The straight line represents only the results of data on microchimerism in normal controls. For statistical analyses, the latter line was used. Within separate studies, it was concluded several times that chimerism did not occur more frequently in patients than in controls21,27 and, therefore, that it was not considered to play a role in the development of some of the diseases.23,30 In 12 articles, the authors concluded that (micro-) chimerism might be involved in the pathogenesis of autoimmune disease. In 2, the authors mentioned that, although they found that (micro-) chimerism was significantly more prevalent in disease, the possibility of it being merely “an innocent bystander” could not be ruled out.

Transplantation The goal of bone-marrow transplantation is, after myeloablation, to establish complete recipient-derived chimerism of peripheral cells. In solid-organ transplantation, circulating cells of the donor are transplanted with the organ to the recipient and remain present for some time in the recipient’s circulation, resulting in microchimerism. Microchimerism in solid-organ transplantation can be detected by immunocytochemistry or polymerase chain reaction for the Y chromosome in peripheral blood cells in the case of a male donor and a female recipient. There is extensive variation in the number of transplanted patients showing various forms of microchimerism among reported groups.31 There is even a considerable group of transplanted patients in whom microchimerism is never detected. It is likely that differences in detection methods are responsible for this variation. It has been suggested that a relatively high compatibility of HLA-DR antigens facilitates induction of microchimerism,32 suggesting that if the compatibility is low, the donor circulating cells will be attacked and destroyed by the immune system of the receiver. The idea of deliberate induction of haematopoietic cell chimerism to achieve transplantation tolerance stems from the 1950s,33 but contraindica-

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Figure 2A. Prevalence of (micro-) chimerism in autoimmune disease. Depicted are the prevalences of (micro-) chimerism (markers) and the 95% confidence intervals (lines) of all manuscripts individually. The scattered reference line at .26 represents the prevalence of microchimerism in healthy controls; the reference line at .31 represents the prevalence of chimerism in the controls of all manuscripts. Based on the assumption of heterogeneity between blood and tissue chimerism, the mean prevalences are .48 and .59, respectively; based on the assumption of homogeneity, the mean prevalence is .53. As can be seen, chimerism is more prevalent in individuals with autoimmune disease than in controls.

tions such as the risk of graft-versus-host disease have so far stood in the way of its clinical application.34 There have been recent reports suggesting an association between microchimerism and the induction of tolerance.35,36 In a meta-analysis of Sahota et al,37 microchimerism in solid-organ transplantation was associated with a higher incidence of acute rejection for heart, lung, and kidney transplants and, contrastively, with a lower incidence for liver transplants. However, other studies showed that the presence of microchimerism is not related to allograft tolerance, rejection, or to clinical outcome in the broadest sense of the word.38 We have proclaimed that microchimerism would not be an unexpected finding in a transplanted patient, but what about tissue chimerism? In the early 1960s, Medawar suggested that, in time, the endothelium of the donor organ would become “owned” by the recipient. He linked this idea to a concept known as graft adaptation, a condition of the donor organ characterized by a low degree of susceptibility to rejection. The first description of graft ad-

aptation stems from the work of Woodruff in 1950. A passage of his description of the term illustrates its essence: “. . . homo-transplants become less vulnerable as time goes on, and, after a certain critical period, are capable of surviving in the face of a high degree of immunity in the recipient, which they would not have been able to withstand earlier in their life history.”39 Medawar suggested that graft adaptation could be induced by focal replacement of graft endothelial cells by recipient cells, reducing the foreign antigen load.40 Why would the endothelium be replaced? Probably because its original lining of host origin is injured (eg, by rejection, ischemia, or cyclosporin toxicity). In 1965, Medawar wrote that “’a study of the cellular dynamics of the vascular endothelium is a matter of some urgency; it should not be impossibly difficult, by using for example fluorescent antibodies, to find out who—donor or host—owns the endothelium of a long-lived graft.”41 Two attempts were made in the 1970s by Williams et al42 and Sinclair.43 These 2 studies described endothelial chimerism in only a minority of kidney grafts, studied for the presence of

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Figure 2B. Prevalence of (micro-) chimerism after solid-organ transplantation. Depicted are the prevalences of (micro-) chimerism (markers) and the 95% confidence intervals (lines) of all manuscripts individually. The scattered reference line at .26 represents the prevalence of microchimerism in healthy controls; the reference line at .31 represents the prevalence of chimerism in the controls of all manuscripts. Based on the assumption of heterogeneity between blood and tissue chimerism, the mean prevalences are .63 and .59, respectively; based on the assumption of homogeneity, the mean prevalence is .61. As can be seen, chimerism is more prevalent in individuals after transplantation than in controls.

sex-chromatin bodies. One of the conclusions was that endothelial repopulation of organ grafts is likely to be rare or nonexistent, another that endothelial repopulation probably occurs after severe tissue injury, and that it cannot explain the phenomenon of graft adaptation. After these 2 studies, the discussion on chimerism in transplanted organs grew quiet until 2001, when we showed the presence of recipient’s endothelial cells in a kidney graft by the following 3 different techniques based on donor-recipient mismatches: (1) in situ hybridization for the Y chromosome in case of a gender mismatch; (2) immunohistochemical stainings for blood groups in case of a donor O kidney in a blood group A or B recipient; and (3) immunohistochemical stainings for various HLA antigens, known to be present in the recipient and absent in the donor. A correlation appeared to be present between the amount of endothelial chimerism in peritubular capillaries and the presence of vascular rejection. Next to chimeric endothelial cells, chimeric tubular epithelial cells in kidney transplants were recently described.44 Also in other transplanted organs, a variety of cells were reported to be chimeric includ-

ing endothelium, duct epithelium, and hepatocytes in the liver45,46 and cardiomyocytes and smooth muscle cells in the heart.2,47 We have found 1 study searching for tissue chimerism in transplanted lungs with negative results in all 12 patients.48 In Figure 2B, we have listed all transplantation studies on chimerism that have appeared so far in the literature.2,31,32,35,38,42-59 None of these studies incorporated control groups. For statistical analyses, we used the reference line of the control groups in the autoimmune diseases for comparison. It is easily seen that in most of the studies, chimerism is found more extensively in transplanted organs than in controls, and for the whole group, the prevalence of chimerism (61%) differed significantly from the healthy controls (P ⬍ .001). The exceptions are mostly old studies that used less advanced methods to detect chimerism than nowadays are available. We have tried to find out whether the incidence of chimerism would be higher in some transplanted organs than in others. However, the numbers of transplanted organs from the studies reported so far are small; a statistical analysis is therefore not applicable.

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Discussion Chimerism is a phenomenon that can occur in various conditions (Fig 1). Practically all studies in this review have aimed to establish the presence of chimeric cells in transplantation or autoimmune settings. Thus far, the question about the role of chimerism both in the pathogenesis of autoimmune diseases and in the induction of graft tolerance is far from being answered. In transplantation, it is clear that if tissue chimerism is present, its appearance is limited to a small percentage of cells in the graft. It is conceivable that, for example, chimeric endothelial cells are the result of a physiologic repair process by which tissue injury in the graft is resolved by recipient stem cells. This means that tissue chimerism primarily is a consequence of previous hits to the graft. These previous hits, such as ischemia and rejection episodes, are known to be of relevance for graft survival. Therefore, if “graft injury” is measured with the amount of chimerism as a parameter, the data may well show that the amount of chimerism has prognostic relevance. However, given the small amount of chimeric cells detected in grafts, it almost seems unlikely that they play a significant role in the immunologic processes mediating graft survival. This may be different in the case of autoimmune diseases, in which relatively high numbers of chimeric cells were reported. Moreover, in patients with autoimmune diseases the source of the chimeric cells seems more elusive than in transplanted patients. Whereas chimerism in peripheral blood cells (called “microchimerism”) is fairly easy and reliably detectable, it is extremely difficult to identify a chimeric cell of tissue origin, such as an epithelial or an endothelial cell, with more than moderate certainty. The identification of these chimeric cells lies at the basis of hypotheses about recipient stem cells repairing damage in a solid donor organ transplant. The circulating recipient cells, which of course occur in large amounts in the solid-donor organ, are a large confounder in all of these methods, be it in situ hybridization of the Y chromosome in a femalederived donor organ placed in a male recipient or immunohistochemical techniques that detect either blood group antigens or HLA-types for which donor and recipient do not match. Identifying the chimeric cells on consecutive slides such as endothelial or epithelial cells by immunohistochemical stainings and at the same time excluding CD45 positive leucocytes is possible, but every consecutive slide may

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differ substantially enough from the previous slide to make this sort of evidence only circumstantial. Double labeling on the same slide may be used as well, but the blurriness of the markers may give a relative but not absolute certainty about the origin of the chimeric cell. And this is not the end of it because the next important issue is: was this cell not already present in the donor organ before transplantation? Most studies on transplant chimerism focus on the presence of Y chromosomes in female donor organs transplanted into male recipients. As reported in this review, during pregnancy fetal-cell microchimerism occurs, which may last for as long as 27 years after delivery.7 It is therefore that some of the criticism on studies dealing with chimerism in transplanted organs focuses on the question whether the chimeric cells in female donor transplants were not already there before transplantation. In the nontransplant setting, chimeric tissue cells have been found in diseases such as systemic sclerosis,17,18 primary biliary cirrhosis,21,29,30 and autoimmune thyroid disease.19,24 It is assumed that in these female patients, chimeric cells are derived from previous pregnancies of sons. It is still uncertain if the presence of these cells influences the development of disease, especially because they are occasionally (although in lesser amounts) found in normal controls, where they are also considered to be derived from previous pregnancies. The real evidence for these hypotheses has to be sought in DNA testing. This would make an end to the discussion that is currently centred on the uncertainty of the origin of the chimeric cells. In 2002, Kleeberger et al46 described how chimeric cells in transplanted livers were isolated by laser capture microdissection, after which a short tandem repeat analysis was performed, proving that these cells were recipient-derived. This is a methodologically challenging technique because in most transplanted organs only a small proportion of cells are chimeric. Nevertheless, an effort should be made to perform a similar experiment in tissue biopsies from female patients with autoimmune diseases to find out whether it can be proven that these cells are from their offspring. But this is a methodologic question. What is largely unknown until now is why chimeric cells, wherever they come from, would induce disease. The results of our meta-analysis show that, overall, the incidence of chimerism is higher in autoimmune disease than in controls. However, because the frequency of chimeric cells in all conditions is low, it cannot be excluded that we are focusing on an epi-

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phenomenon. In the transplantation setting, it is likely that chimeric cells are the product of tissue repair by recipient stem cells. In the case of endothelial cells, chimerism could be the end product of maintenance angiogenesis, a physiologic process by which the vessel wall lining is repaired by bonemarrow-derived cells in the case of damage.60,61 So far, however, no relation has been found between the amounts of chimeric cells in transplanted organs with graft prognosis. We are still a long way from finding out whether Medawar’s suggestion of chimerism as a pathway to graft tolerance is correct.

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50. Fogt F, Beyser KH, Poremba C, et al: Recipient-derived hepatocytes in liver transplants: A rare event in sex- mismatched transplants. Hepatology 2002, 36:173 51. Grimm PC, Nickerson P, Jeffery J, et al: Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renal-allograft rejection. N Engl J Med 2001, 345:93 52. Koolbergen DR, Hazekamp MG, Kurvers M, et al: Tissue chimerism in human cryopreserved homograft valve explants demonstrated by in situ hybridization. Ann Thorac Surg 1998, 66:S225 53. Lagaaij EL, Cramer-Knijnenburg GF, van Kemenade FJ, et al: Endothelial cell chimerism after renal transplantation and vascular rejection. Lancet 2001, 357:33 54. Lo YM, Tein MS, Pang CC, et al: Presence of donor-specific DNA in plasma of kidney and liver-transplant recipients. Lancet 1998, 351:1329 55. Matsushita K, Sakamoto K, Sakamaki T, et al: Microchimerism in renal transplant recipients correlates with better HLADRB1 matched status. Transplant Proc 1997, 29:2290 56. Sakamoto K, Matsushita K, Sakamaki T, et al: Peripheral blood microchimerism does not correlate with the state of graft acceptance in HLA-DRB1 mismatched renal transplant recipients. Transplant Proc 1998, 30:7 57. Spriewald BM, Wassmuth R, Carl HD, et al: Microchimerism after liver transplantation: Prevalence and methodological aspects of detection. Transplantation 1998, 66:77 58. Starzl TE, Demetris AJ, Trucco M, et al: Systemic chimerism in human female recipients of male livers. Lancet 1992, 340:876 59. Wang J, Liu J, Guan D, et al: The study of peripheral blood microchimerism in kidney transplantation. Transplant Proc 2001, 33:177 60. Gunsilius E, Duba HC, Petzer AL, et al: Evidence from a leukaemia model for maintenance of vascular endothelium by bone-marrow-derived endothelial cells. Lancet 2000, 355:1688 61. Gunsilius E, Petzer AL, Duba HC, et al: Circulating endothelial cells after transplantation. Lancet 2001, 357:1449