Fetal microchimerism: an aetiological factor in primary biliary cirrhosis?

Abstract

It is now becoming clear that even normal pregnancy results in a two-way traffic of cells from maternal to fetal circulations (1Lo YMD Lo ESF Watson N Nokes L Sargent IL Thilaganathan B et al.Two-way traffic between mother and fetus biologic and clinical implications.Blood. 1996; 88: 4390-4395PubMed Google Scholar) and vice versa (2Bianchi D Zickwolf G Weil G Sylvester S Demaria M Male fetal progenitor cells persist in maternal blood for as long as 27 years.Proc Natl Acad Sci USA. 1996; 93: 705-708Crossref PubMed Scopus (1041) Google Scholar, 3Evans P Lambert N Maloney S Fuist D Moore J Nelson J Long-term fetal michrochimerism in peripheral blood mononuclear cell subsets in healthy women and women with scleroderma.Blood. 1999; 93: 2033-2037PubMed Google Scholar), the presence of small numbers of allogeneic fetal cells in the maternal system being termed fetal michrochimerism Fetal cells entering the maternal circulation, typically belong to a CD34+ haematological precursor subset, and appear to have the potential to persist for many years (4Branchi D Williams JM Sullivan LM Hanson FW Klinger KW Shuber AP PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies.Am J Hum Genet. 1996; 61: 822-829Abstract Full Text PDF Scopus (344) Google Scholar).The similarities between the patterns of tissue damage seen in graft-versus-host disease (GVHD, a parallel, albeit non-physiological scenario in which allogeneic immuno-competent cells enter the host circulation), and the connective tisue disorder scleroderma, led to the hypothesis that fetal microchimerism might, through a similar mechanism to GVHD, be giving rise to the tissue damage seen in scleroderma. The epidemiology of scleroderma, a disease predominantly affecting women and typically developing in the post-child-beating years, appeared to support this hypothesis. The hypothesis was tested by looking for the presence of fetal-derived DNA in the peripheral blood of scleroderma patients and matched controls (5Nelson JL Furst DE Maloney S Gooley T Evans PC Smith A et al.Michrochimerism and HLA-compatible relationships of pregnancy in scleroderma.Lancet. 1998; 351: 540-541Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The technical problem of distinguishing between DNA of maternal and fetal origin was solved by studying only women who had given birth to sons, thereby allowing the investigators to look, using a polymerase chain reaction (PCR)-based technique, for DNA sequences derived from the Y chromosome. Using this approach, male DNA (presumed to be from fetal-derived cells retained in the maternal circulation) was found in 4/16 (25%) controls and 10/17 (59%) scleroderma patients. Moreover, where present, male DNA was found at significantly higher levels in scleroderma patients than controls. This study demonstrated, therefore, a clear association (causal or otherwise) between the presence of male DNA in the circulation (presumed to be derived from fetal microchimerism) and scleioderma.The extension of the fetal microchimerism model to primary biliary cirrhosis (PBC) is an obvious one, given the similarities between this disease and scleroderma. PBC demonstrates similar epidemiology to scleroderma (both diseases typically affect middle-aged and older women (6Metcalf JV Bhopal RS Gray J James OFW Incidence and prevalence of primary biliary cirrhosis in the city of Newcastleupon-Tyne, England.Int J Epidemiol. 1997; 26: 830-836Crossref PubMed Scopus (158) Google Scholar)) and, as is the case with scleroderma, the classical pathological lesion (non-suppurative destructive cholangitis) shows marked similarity to one seen in GVHD (7Epstein O Thomas HC Sherlock S Primary biliary cirrhosis is a dry gland syndrome with features of chronic graft-versus-host disease.Lancet. 1980; i: 1166-1168Abstract Scopus (205) Google Scholar). Moreover, a significant clinical overlap is seen between PBC and systemic sclerosis in all its manifestations (8Jones DEJ James OFW Bassendine MF Primary biliary cirrhosis clinical and associated autoimmune features and natural history.Clin Liver Dis. 1998; 2: 265-282Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The question of the association between fetal microchimerism and PBC has been addressed in a series of recent studies (9Corpechot C Barbu V Chazouillieres O Poupon R Fetal michrochimerism in primary biliary cirrhosis.J Hepatol. 2000; 33: 696-700Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Fanning PA Jonsson JR Clouston AD Edwards-Smith C Balderson GA Macdonald GA et al.Detection of male DNA in the liver of female patients with primary biliary cirrhosis.J Hepatol. 2000; 33: 690-695Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 11Rubbia-Brandt L Philippeaux MM Chavez S Mentha G Borisch B Hadengue A FISH for Y chromosome in women with primary biliary cirrhosis lack of evidence for leucocyte michrochimerism.Hepatology. 1999; 30: 821-822Crossref PubMed Scopus (36) Google Scholar, 12Tanaka A Lindor K Gish R Batts K Shiratori Y Omata M et al.Fetal michrochimerism alone does not contribute to the induction of primary biliary cirrhosis.Hepatology. 1999; 30: 833-838Crossref PubMed Scopus (110) Google Scholar), two of which are published in this issue of the Journal of Hepatology (9Corpechot C Barbu V Chazouillieres O Poupon R Fetal michrochimerism in primary biliary cirrhosis.J Hepatol. 2000; 33: 696-700Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Fanning PA Jonsson JR Clouston AD Edwards-Smith C Balderson GA Macdonald GA et al.Detection of male DNA in the liver of female patients with primary biliary cirrhosis.J Hepatol. 2000; 33: 690-695Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). These studies have all used molecular biology approaches to detect the presence of Ychromosome-derived sequences in the peripheral blood and/or liver tissue of female PBC patients. In contrast to the studies performed in scleroderma, however, markedly contradictory results have been obtained in these PBC studies. It is likely that these contrasting findings result from significant differences in the experimental methodologies adopted, and the patient groups selected by the different investigators.The experimental approach adopted by Corpechot and colleagues was nested PCR for a Y-chromosome-specific sequence in DNA derived from two tissues, freshly derived peripheral blood mononuclear cells (PBMC) (from 18 PBC patients and 18 matched normal controls) and archival paraffin-embedded liver biopsy material (15 PBC patients and 25 non-matched controls with non-autoimmune chronic liver disease) (9Corpechot C Barbu V Chazouillieres O Poupon R Fetal michrochimerism in primary biliary cirrhosis.J Hepatol. 2000; 33: 696-700Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In the peripheral blood study, Y-chromosome-derived DNA was found in twice as many PBC patients (8/18 [44%]) as controls (4/18 [22%]). This difference did not reach statistical significance, although this may well have resulted simply from the small sample size. Similarly, the mean number of male cell DNA equivalents (a measure of the amount of Y-chromosome DNA present) was higher in the PBC patients (3.5±1.3 vs 1.6±1.0 cells per million), although the difference again did not reach significance. Y-chromosome DNA was, in contrast, found in liver-biopsy-derived DNA from similar numbers of PBC patients (5/15 [33%]) and chronic liver disease controls (8/25 [32%]) with similar Y-chromosome levels (1.4±0.6 vs 2.2±0.7) cells per million.Fanning and colleagues used two approaches, a nested PCR approach similar to that of Corpechot et al. (albeit with different primer pairs) for analysis of fresh PBMC from 18 PBC patients and 18 normal controls (10Fanning PA Jonsson JR Clouston AD Edwards-Smith C Balderson GA Macdonald GA et al.Detection of male DNA in the liver of female patients with primary biliary cirrhosis.J Hepatol. 2000; 33: 690-695Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and in situ hybridisation for Y-chromosome sequences using archival liver tissue (paraffin-embedded) from 19 PBC patients and 20 non-autoimmune chronic liver disease patients. In the peripheral blood study they failed to detect Y-chromosome-derived sequences in PBMC DNA from any PBC patient, and found sequences in only 1/18 controls. These findings stand in marked contrast to those of Corpechot et al. with regard to the frequency of Y-chromosome DNA sequences in both PBC patients and controls, and, strikingly, to those in other equivalent published studies in diseases other than PBC with regard to the frequency with which these sequences are seen in PBMC DNA from normal controls (5Nelson JL Furst DE Maloney S Gooley T Evans PC Smith A et al.Michrochimerism and HLA-compatible relationships of pregnancy in scleroderma.Lancet. 1998; 351: 540-541Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 13Artlett C Smith J Jiminez S Identification of fetal DNA and cells in skin lesions from women with systemic sclerosis.N Engl J Med. 1998; 1998: 1186-1191Crossref Scopus (414) Google Scholar). Although the differences between the Fanning and Corpechot studies may reflect differences in the patient populations studied (all subjects having had at least one male pregnancy in the latter cohort but not the former, for example), they are more likely to result from the methodological difficulties often encountered in studies of this nature (12Tanaka A Lindor K Gish R Batts K Shiratori Y Omata M et al.Fetal michrochimerism alone does not contribute to the induction of primary biliary cirrhosis.Hepatology. 1999; 30: 833-838Crossref PubMed Scopus (110) Google Scholar). The similarity in control data between the Corpechot study and previous described series suggest that the findings of this study are more likely to reflect the true occurrence of Y-chromosome sequences in PBMC DNA from PBC patients.The data become even more complex when we address the question of the presence of Y-chromosome-derived DNA sequences in liver tissue. Fanning and colleagues identified Y-chromosome DNA sequences in liver tissue from 8/19 [42%] PBC patients using an in situ hybridisation approach. Such sequences were absent from liver tissue from 20 patients with other forms of non-autoimmune chronic liver disease. One advantage of the in situ hybridisation approach is that morphometric analysis can also be performed, allowing detailing of where within the liver the DNA sequences of interest are found. The Y-chromosome-derived sequences present in PBC patients were all contained within nucleated cells located in the hepatic sinusoids and portal tracts (but not the inflammatory infiltrate surrounding damaged bile ducts). Y-chromosome-containing cells were relatively sparse in all cases (a mean of 0 2 positive cells per mm2 of biopsy tissue). Interestingly, all Y-chromosome-positive cells were HLA DR negative with the exception of a single cell in one biopsy. None of the cells expressed CD34.How can we reconcile the apparently contradictory findings of these two studies of liver tissue (Fanning et al. having shown male cells in a significant proportion of PBC patients but no controls, whilst Corpechot et al. showed male cells in an equal minority of PBC patients and controls)? Furthermore, how can we reconcile the findings of both these studies with previous reports describing the equal presence of Y-chromosome DNA in the liver tissue of a majority of PBC patients and controls (using a nested PCR approach (12Tanaka A Lindor K Gish R Batts K Shiratori Y Omata M et al.Fetal michrochimerism alone does not contribute to the induction of primary biliary cirrhosis.Hepatology. 1999; 30: 833-838Crossref PubMed Scopus (110) Google Scholar)) and the absence of Y-chromosome DNA in the liver of a small number of PBC patients (using an in situ hybridisation approach (11Rubbia-Brandt L Philippeaux MM Chavez S Mentha G Borisch B Hadengue A FISH for Y chromosome in women with primary biliary cirrhosis lack of evidence for leucocyte michrochimerism.Hepatology. 1999; 30: 821-822Crossref PubMed Scopus (36) Google Scholar))?The differences between the two in situ hybridisation studies are most likely to result simply from the small numbers of PBC patients who had carried a male fetus included in the Rubbia-Brandt study (n=5 only). The contrasting findings of the two studies using nested PCR approaches could result from either (or both) of two important differences in the experimental protocols adopted (9Corpechot C Barbu V Chazouillieres O Poupon R Fetal michrochimerism in primary biliary cirrhosis.J Hepatol. 2000; 33: 696-700Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12Tanaka A Lindor K Gish R Batts K Shiratori Y Omata M et al.Fetal michrochimerism alone does not contribute to the induction of primary biliary cirrhosis.Hepatology. 1999; 30: 833-838Crossref PubMed Scopus (110) Google Scholar). The first difference relates to the populations studied. In the Tanaka study the majority of subjects in both the PBC patient and control groups had received blood transfusion at a point prior to the study. In the Corpechot study patients who had undergone blood transfusion at any stage were excluded. Blood transfusion from a male donor is an obvious potentially confounding source of any male cells found to be be retained in the liver. The second difference was with regard to the detection system used for PCR product. Tanaka et al adopted highly sensitive WAVE technology, whilst in the Corpechot study a more conventional gel electrophoresis and Western blotting approach was used. The use of WAVE technology may have resulted in increased sensitivity for detection of product, with a resulting increase in the frequency with which Y-chromosome DNA could be detected in both patients and controls.How might we account for the differences in the findings of the Corpechot and Fanning studies? It is inevitably difficult to compare the findings of studies carried out at different times in different laboratories on different patient groups using very different experimental approaches. The apparent absence of male DNA in the control subjects in the Fanning study may have resulted from as simple a factor as the relatively small cross-sectional areas of liver tissue available for study, the use of which could have resulted in low frequencies of male cells being missed in control liver.What conclusions can be drawn from these studies and what, if anything do they tell us about the pathogenesis of PBC? One reasonable conclusion would be that retention of allogeneic cells within the liver appears to occur in an appreciable proportion of PBC patients with a history of pregnancy. Careful exclusion of patients with a history of blood transfusion, or who were themselves part of a di-zygotic twin pair (placental sharing in this scenario being another theoretical source for allogeneic cells) leads us to the inevitable conclusion that these cells are derived from a fetus carried at some point in the past. This phenomenon would appear not, however, to be exclusive to PBC patients, being seen in a proportion of non-PBC liver patients. These observations give rise to two questions. First what is the physiological role of the retention of fetal cells in this way, and, second, does this leave any role for fetal microchimerism in the pathogenesis of PBC?The fact that retention of fetal cells occurs in normal liver raises fascinating questions regarding a potential role for this process in the state of immune tolerance to the fetus seen in pregnancy. It has been appreciated for many years that administration of antigen via the portal vein results in the development of immune tolerance (14Nakano Y Monden M Valdevia LA Gotoh M Tono T Mori T Permanent acceptance of liver allografts by intraportal injection of donor spleen cells in rats.Surgery. 1992; 111: 668-676PubMed Google Scholar). Moreover, the phenomenon of oral tolerance (the induction of a tolerising response to antigen administered via the gastrointestinal tract), which is presumed to be a protective pathway to prevent the induction of potentially harmful responses to dietary antigens, can be by-passed by surgical porto-systemic shunting (15Yang R Liu Q Grosfeld JL Pescovitz MD Intestinal drainage through the liver is a pre-requisite for oral tolerance induction.J Paediatr Surg. 1994; 29: 1525-1529Google Scholar). Taken together, these observations suggest that exposure to antigen within the liver can result in the active induction of immune tolerance. Were this to occur in pregnancy, to fetal cells lodging in the liver, then this may represent a mechanism whereby immune tolerance to the semi-allogeneic fetus is induced and maintained.If fetal microchimerism plays any role in the pathogenesis of PBC, it is clearly not obligatory for disease development. In all the studies discussed, significant numbers of PBC patients lacked evidence of microchimerism. Moreover, the disease clearly occurs in both men and nulliparous women in whom the process cannot have occurred. Tellingly, the numbers of pregnancies reported by PBC patients also appears to be the same as for controls in a recently reported case control study (16Howel D Fischbacher CM Bhopal RS Gray J Metcalf JV James OFW An exploratory population-based case-control study of primary biliary cirrhosis.Hepatology. 2000; 31: 1055-1060Crossref PubMed Scopus (119) Google Scholar), suggesting no association between numbers of pregnancies (which might be considered to be an incremental risk factor) and disease. Conversely, the fact that fetal microchimerism is seen in a proportion of normal individuals suggests that the process is not, in isolation, sufficient to induce the disease. Presumably, any susceptibility to the induction of PBC resulting from the presence of fetal microchimerism results in expression of the disease only in individuals exposed to other additional environmental or genetic risk factors.The inevitable conclusion of these studies is that any connection between fetal microchimerism and PBC is a complex one. One hypothesis is that the clinical entity we recognise as PBC in fact represents a “final common pathway” of tissue damage triggered by possibly quite distinct aetiological processes. It may be that fetal microchimerism (in an appropriately susceptible host) is one such trigger. Is there any evidence to support this view? Intriguingly, Corpechot and colleagues showed that the prevalence of antinuclear antibodies is significantly higher in PBC patients showing evidence of fetal microchimerism. Moreover, the presence of anti-centromere antibodies (ACA, associated with the CREST variant of systemic sclerosis (17Fritzler MJ, Kinsella TD The CREST syndrome a distinct serologic entity with anti-centromere antibodies Am J Med 69 520-6Google Scholar) which shows a significant association/overlap with PBC (8Jones DEJ James OFW Bassendine MF Primary biliary cirrhosis clinical and associated autoimmune features and natural history.Clin Liver Dis. 1998; 2: 265-282Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 18Powell FC Schroeter AL Dickson ER Primary biliary cirrhosis and the CREST syndrome a report of 22 cases.Q J Med. 1987; 62: 75-82PubMed Google Scholar)) was also associated with the presence of fetal microchimerism. The implication of this (as yet unconfirmed) observation is that fetal microchimerism may act as a trigger for disease in the subgroup of patients with anti-nuclear activity (ANA) and ACA.In summary, therefore, these studies suggest that fetal microchimerism is not a simple “answer” to PBC. They may, however, if the observation regarding the selective associations with disease subgroups is confirmed, serve an even more interesting purpose in helping to change our long-standing view of the disease as a single entity. It is now becoming clear that even normal pregnancy results in a two-way traffic of cells from maternal to fetal circulations (1Lo YMD Lo ESF Watson N Nokes L Sargent IL Thilaganathan B et al.Two-way traffic between mother and fetus biologic and clinical implications.Blood. 1996; 88: 4390-4395PubMed Google Scholar) and vice versa (2Bianchi D Zickwolf G Weil G Sylvester S Demaria M Male fetal progenitor cells persist in maternal blood for as long as 27 years.Proc Natl Acad Sci USA. 1996; 93: 705-708Crossref PubMed Scopus (1041) Google Scholar, 3Evans P Lambert N Maloney S Fuist D Moore J Nelson J Long-term fetal michrochimerism in peripheral blood mononuclear cell subsets in healthy women and women with scleroderma.Blood. 1999; 93: 2033-2037PubMed Google Scholar), the presence of small numbers of allogeneic fetal cells in the maternal system being termed fetal michrochimerism Fetal cells entering the maternal circulation, typically belong to a CD34+ haematological precursor subset, and appear to have the potential to persist for many years (4Branchi D Williams JM Sullivan LM Hanson FW Klinger KW Shuber AP PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies.Am J Hum Genet. 1996; 61: 822-829Abstract Full Text PDF Scopus (344) Google Scholar). The similarities between the patterns of tissue damage seen in graft-versus-host disease (GVHD, a parallel, albeit non-physiological scenario in which allogeneic immuno-competent cells enter the host circulation), and the connective tisue disorder scleroderma, led to the hypothesis that fetal microchimerism might, through a similar mechanism to GVHD, be giving rise to the tissue damage seen in scleroderma. The epidemiology of scleroderma, a disease predominantly affecting women and typically developing in the post-child-beating years, appeared to support this hypothesis. The hypothesis was tested by looking for the presence of fetal-derived DNA in the peripheral blood of scleroderma patients and matched controls (5Nelson JL Furst DE Maloney S Gooley T Evans PC Smith A et al.Michrochimerism and HLA-compatible relationships of pregnancy in scleroderma.Lancet. 1998; 351: 540-541Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The technical problem of distinguishing between DNA of maternal and fetal origin was solved by studying only women who had given birth to sons, thereby allowing the investigators to look, using a polymerase chain reaction (PCR)-based technique, for DNA sequences derived from the Y chromosome. Using this approach, male DNA (presumed to be from fetal-derived cells retained in the maternal circulation) was found in 4/16 (25%) controls and 10/17 (59%) scleroderma patients. Moreover, where present, male DNA was found at significantly higher levels in scleroderma patients than controls. This study demonstrated, therefore, a clear association (causal or otherwise) between the presence of male DNA in the circulation (presumed to be derived from fetal microchimerism) and scleioderma. The extension of the fetal microchimerism model to primary biliary cirrhosis (PBC) is an obvious one, given the similarities between this disease and scleroderma. PBC demonstrates similar epidemiology to scleroderma (both diseases typically affect middle-aged and older women (6Metcalf JV Bhopal RS Gray J James OFW Incidence and prevalence of primary biliary cirrhosis in the city of Newcastleupon-Tyne, England.Int J Epidemiol. 1997; 26: 830-836Crossref PubMed Scopus (158) Google Scholar)) and, as is the case with scleroderma, the classical pathological lesion (non-suppurative destructive cholangitis) shows marked similarity to one seen in GVHD (7Epstein O Thomas HC Sherlock S Primary biliary cirrhosis is a dry gland syndrome with features of chronic graft-versus-host disease.Lancet. 1980; i: 1166-1168Abstract Scopus (205) Google Scholar). Moreover, a significant clinical overlap is seen between PBC and systemic sclerosis in all its manifestations (8Jones DEJ James OFW Bassendine MF Primary biliary cirrhosis clinical and associated autoimmune features and natural history.Clin Liver Dis. 1998; 2: 265-282Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The question of the association between fetal microchimerism and PBC has been addressed in a series of recent studies (9Corpechot C Barbu V Chazouillieres O Poupon R Fetal michrochimerism in primary biliary cirrhosis.J Hepatol. 2000; 33: 696-700Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Fanning PA Jonsson JR Clouston AD Edwards-Smith C Balderson GA Macdonald GA et al.Detection of male DNA in the liver of female patients with primary biliary cirrhosis.J Hepatol. 2000; 33: 690-695Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 11Rubbia-Brandt L Philippeaux MM Chavez S Mentha G Borisch B Hadengue A FISH for Y chromosome in women with primary biliary cirrhosis lack of evidence for leucocyte michrochimerism.Hepatology. 1999; 30: 821-822Crossref PubMed Scopus (36) Google Scholar, 12Tanaka A Lindor K Gish R Batts K Shiratori Y Omata M et al.Fetal michrochimerism alone does not contribute to the induction of primary biliary cirrhosis.Hepatology. 1999; 30: 833-838Crossref PubMed Scopus (110) Google Scholar), two of which are published in this issue of the Journal of Hepatology (9Corpechot C Barbu V Chazouillieres O Poupon R Fetal michrochimerism in primary biliary cirrhosis.J Hepatol. 2000; 33: 696-700Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 10Fanning PA Jonsson JR Clouston AD Edwards-Smith C Balderson GA Macdonald GA et al.Detection of male DNA in the liver of female patients with primary biliary cirrhosis.J Hepatol. 2000; 33: 690-695Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). These studies have all used molecular biology approaches to detect the presence of Ychromosome-derived sequences in the peripheral blood and/or liver tissue of female PBC patients. In contrast to the studies performed in scleroderma, however, markedly contradictory results have been obtained in these PBC studies. It is likely that these contrasting findings result from significant differences in the experimental methodologies adopted, and the patient groups selected by the different investigators. The experimental approach adopted by Corpechot and colleagues was nested PCR for a Y-chromosome-specific sequence in DNA derived from two tissues, freshly derived peripheral blood mononuclear cells (PBMC) (from 18 PBC patients and 18 matched normal controls) and archival paraffin-embedded liver biopsy material (15 PBC patients and 25 non-matched controls with non-autoimmune chronic liver disease) (9Corpechot C Barbu V Chazouillieres O Poupon R Fetal michrochimerism in primary biliary cirrhosis.J Hepatol. 2000; 33: 696-700Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In the peripheral blood study, Y-chromosome-derived DNA was found in twice as many PBC patients (8/18 [44%]) as controls (4/18 [22%]). This difference did not reach statistical significance, although this may well have resulted simply from the small sample size. Similarly, the mean number of male cell DNA equivalents (a measure of the amount of Y-chromosome DNA present) was higher in the PBC patients (3.5±1.3 vs 1.6±1.0 cells per million), although the difference again did not reach significance. Y-chromosome DNA was, in contrast, found in liver-biopsy-derived DNA from similar numbers of PBC patients (5/15 [33%]) and chronic liver disease controls (8/25 [32%]) with similar Y-chromosome levels (1.4±0.6 vs 2.2±0.7) cells per million. Fanning and colleagues used two approaches, a nested PCR approach similar to that of Corpechot et al. (albeit with different primer pairs) for analysis of fresh PBMC from 18 PBC patients and 18 normal controls (10Fanning PA Jonsson JR Clouston AD Edwards-Smith C Balderson GA Macdonald GA et al.Detection of male DNA in the liver of female patients with primary biliary cirrhosis.J Hepatol. 2000; 33: 690-695Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and in situ hybridisation for Y-chromosome sequences using archival liver tissue (paraffin-embedded) from 19 PBC patients and 20 non-autoimmune chronic liver disease patients. In the peripheral blood study they failed to detect Y-chromosome-derived sequences in PBMC DNA from any PBC patient, and found sequences in only 1/18 controls. These findings stand in marked contrast to those of Corpechot et al. with regard to the frequency of Y-chromosome DNA sequences in both PBC patients and controls, and, strikingly, to those in other equivalent published studies in diseases other than PBC with regard to the frequency with which these sequences are seen in PBMC DNA from normal controls (5Nelson JL Furst DE Maloney S Gooley T Evans PC Smith A et al.Michrochimerism and HLA-compatible relationships of pregnancy in scleroderma.Lancet. 1998; 351: 540-541Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 13Artlett C Smith J Jiminez S Identification of fetal DNA and cells in skin lesions from women with systemic sclerosis.N Engl J Med. 1998; 1998: 1186-1191Crossref Scopus (414) Google Scholar). Although the differences between the Fanning and Corpechot studies may reflect differences in the patient populations studied (all subjects having had at least one male pregnancy in the latter cohort but not the former, for example), they are more likely to result from the methodological difficulties often encountered in studies of this nature (12Tanaka A Lindor K Gish R Batts K Shiratori Y Omata M et al.Fetal michrochimerism alone does not contribute to the induction of primary biliary cirrhosis.Hepatology. 1999; 30: 833-838Crossref PubMed Scopus (110) Google Scholar). The similarity in control data between the Corpechot study and previous described series suggest that the findings of this study are more likely to reflect the true occurrence of Y-chromosome sequences in PBMC DNA from PBC patients. The data become even more complex when we address the question of the presence of Y-chromosome-derived DNA sequences in liver tissue. Fanning and colleagues identified Y-chromosome DNA sequences in liver tissue from 8/19 [42%] PBC patients using an in situ hybridisation approach. Such sequences were absent from liver tissue from 20 patients with other forms of non-autoimmune chronic liver disease. One advantage of the in situ hybridisation approach is that morphometric analysis can also be performed, allowing detailing of where within the liver the DNA sequences of interest are found. The Y-chromosome-derived sequences present in PBC patients were all contained within nucleated cells located in the hepatic sinusoids and portal tracts (but not the inflammatory infiltrate surrounding damaged bile ducts). Y-chromosome-containing cells were relatively sparse in all cases (a mean of 0 2 positive cells per mm2 of biopsy tissue). Interestingly, all Y-chromosome-positive cells were HLA DR negative with the exception of a single cell in one biopsy. None of the cells expressed CD34. How can we reconcile the apparently contradictory findings of these two studies of liver tissue (Fanning et al. having shown male cells in a significant proportion of PBC patients but no controls, whilst Corpechot et al. showed male cells in an equal minority of PBC patients and controls)? Furthermore, how can we reconcile the findings of both these studies with previous reports describing the equal presence of Y-chromosome DNA in the liver tissue of a majority of PBC patients and controls (using a nested PCR approach (12Tanaka A Lindor K Gish R Batts K Shiratori Y Omata M et al.Fetal michrochimerism alone does not contribute to the induction of primary biliary cirrhosis.Hepatology. 1999; 30: 833-838Crossref PubMed Scopus (110) Google Scholar)) and the absence of Y-chromosome DNA in the liver of a small number of PBC patients (using an in situ hybridisation approach (11Rubbia-Brandt L Philippeaux MM Chavez S Mentha G Borisch B Hadengue A FISH for Y chromosome in women with primary biliary cirrhosis lack of evidence for leucocyte michrochimerism.Hepatology. 1999; 30: 821-822Crossref PubMed Scopus (36) Google Scholar))? The differences between the two in situ hybridisation studies are most likely to result simply from the small numbers of PBC patients who had carried a male fetus included in the Rubbia-Brandt study (n=5 only). The contrasting findings of the two studies using nested PCR approaches could result from either (or both) of two important differences in the experimental protocols adopted (9Corpechot C Barbu V Chazouillieres O Poupon R Fetal michrochimerism in primary biliary cirrhosis.J Hepatol. 2000; 33: 696-700Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12Tanaka A Lindor K Gish R Batts K Shiratori Y Omata M et al.Fetal michrochimerism alone does not contribute to the induction of primary biliary cirrhosis.Hepatology. 1999; 30: 833-838Crossref PubMed Scopus (110) Google Scholar). The first difference relates to the populations studied. In the Tanaka study the majority of subjects in both the PBC patient and control groups had received blood transfusion at a point prior to the study. In the Corpechot study patients who had undergone blood transfusion at any stage were excluded. Blood transfusion from a male donor is an obvious potentially confounding source of any male cells found to be be retained in the liver. The second difference was with regard to the detection system used for PCR product. Tanaka et al adopted highly sensitive WAVE technology, whilst in the Corpechot study a more conventional gel electrophoresis and Western blotting approach was used. The use of WAVE technology may have resulted in increased sensitivity for detection of product, with a resulting increase in the frequency with which Y-chromosome DNA could be detected in both patients and controls. How might we account for the differences in the findings of the Corpechot and Fanning studies? It is inevitably difficult to compare the findings of studies carried out at different times in different laboratories on different patient groups using very different experimental approaches. The apparent absence of male DNA in the control subjects in the Fanning study may have resulted from as simple a factor as the relatively small cross-sectional areas of liver tissue available for study, the use of which could have resulted in low frequencies of male cells being missed in control liver. What conclusions can be drawn from these studies and what, if anything do they tell us about the pathogenesis of PBC? One reasonable conclusion would be that retention of allogeneic cells within the liver appears to occur in an appreciable proportion of PBC patients with a history of pregnancy. Careful exclusion of patients with a history of blood transfusion, or who were themselves part of a di-zygotic twin pair (placental sharing in this scenario being another theoretical source for allogeneic cells) leads us to the inevitable conclusion that these cells are derived from a fetus carried at some point in the past. This phenomenon would appear not, however, to be exclusive to PBC patients, being seen in a proportion of non-PBC liver patients. These observations give rise to two questions. First what is the physiological role of the retention of fetal cells in this way, and, second, does this leave any role for fetal microchimerism in the pathogenesis of PBC? The fact that retention of fetal cells occurs in normal liver raises fascinating questions regarding a potential role for this process in the state of immune tolerance to the fetus seen in pregnancy. It has been appreciated for many years that administration of antigen via the portal vein results in the development of immune tolerance (14Nakano Y Monden M Valdevia LA Gotoh M Tono T Mori T Permanent acceptance of liver allografts by intraportal injection of donor spleen cells in rats.Surgery. 1992; 111: 668-676PubMed Google Scholar). Moreover, the phenomenon of oral tolerance (the induction of a tolerising response to antigen administered via the gastrointestinal tract), which is presumed to be a protective pathway to prevent the induction of potentially harmful responses to dietary antigens, can be by-passed by surgical porto-systemic shunting (15Yang R Liu Q Grosfeld JL Pescovitz MD Intestinal drainage through the liver is a pre-requisite for oral tolerance induction.J Paediatr Surg. 1994; 29: 1525-1529Google Scholar). Taken together, these observations suggest that exposure to antigen within the liver can result in the active induction of immune tolerance. Were this to occur in pregnancy, to fetal cells lodging in the liver, then this may represent a mechanism whereby immune tolerance to the semi-allogeneic fetus is induced and maintained. If fetal microchimerism plays any role in the pathogenesis of PBC, it is clearly not obligatory for disease development. In all the studies discussed, significant numbers of PBC patients lacked evidence of microchimerism. Moreover, the disease clearly occurs in both men and nulliparous women in whom the process cannot have occurred. Tellingly, the numbers of pregnancies reported by PBC patients also appears to be the same as for controls in a recently reported case control study (16Howel D Fischbacher CM Bhopal RS Gray J Metcalf JV James OFW An exploratory population-based case-control study of primary biliary cirrhosis.Hepatology. 2000; 31: 1055-1060Crossref PubMed Scopus (119) Google Scholar), suggesting no association between numbers of pregnancies (which might be considered to be an incremental risk factor) and disease. Conversely, the fact that fetal microchimerism is seen in a proportion of normal individuals suggests that the process is not, in isolation, sufficient to induce the disease. Presumably, any susceptibility to the induction of PBC resulting from the presence of fetal microchimerism results in expression of the disease only in individuals exposed to other additional environmental or genetic risk factors. The inevitable conclusion of these studies is that any connection between fetal microchimerism and PBC is a complex one. One hypothesis is that the clinical entity we recognise as PBC in fact represents a “final common pathway” of tissue damage triggered by possibly quite distinct aetiological processes. It may be that fetal microchimerism (in an appropriately susceptible host) is one such trigger. Is there any evidence to support this view? Intriguingly, Corpechot and colleagues showed that the prevalence of antinuclear antibodies is significantly higher in PBC patients showing evidence of fetal microchimerism. Moreover, the presence of anti-centromere antibodies (ACA, associated with the CREST variant of systemic sclerosis (17Fritzler MJ, Kinsella TD The CREST syndrome a distinct serologic entity with anti-centromere antibodies Am J Med 69 520-6Google Scholar) which shows a significant association/overlap with PBC (8Jones DEJ James OFW Bassendine MF Primary biliary cirrhosis clinical and associated autoimmune features and natural history.Clin Liver Dis. 1998; 2: 265-282Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 18Powell FC Schroeter AL Dickson ER Primary biliary cirrhosis and the CREST syndrome a report of 22 cases.Q J Med. 1987; 62: 75-82PubMed Google Scholar)) was also associated with the presence of fetal microchimerism. The implication of this (as yet unconfirmed) observation is that fetal microchimerism may act as a trigger for disease in the subgroup of patients with anti-nuclear activity (ANA) and ACA. In summary, therefore, these studies suggest that fetal microchimerism is not a simple “answer” to PBC. They may, however, if the observation regarding the selective associations with disease subgroups is confirmed, serve an even more interesting purpose in helping to change our long-standing view of the disease as a single entity.