Clin Rheumatol DOI 10.1007/s10067-015-3122-8
ORIGINAL ARTICLE
Variables associated to fetal microchimerism in systemic lupus erythematosus patients Greiciane Maria da Silva Florim 1 & Heloisa Cristina Caldas 1 & Erika Cristina Pavarino 2 & Eny Maria Goloni Bertollo 2 & Ida Maria Maximina Fernandes 1 & Mario Abbud-Filho 1,3,4
Received: 27 August 2015 / Revised: 23 October 2015 / Accepted: 14 November 2015 # International League of Associations for Rheumatology (ILAR) 2015
Abstract In the present study, we sought to identify the factors during the pregnancy of systemic lupus erythematosus (SLE) patients that could be linked to the presence and proliferation of male fetal cells (MFC) and the possible relation between these factors and development of lupus nephritis (LN). We evaluated 18 healthy women (control group) and 28 women affected by SLE. Genomic DNA was extracted from peripheral blood and quantified using the technique of quantitative real-time polymerase chain reaction (qPCR) for specific Y chromosome sequences. The amount of MFC was significantly higher in the SLE group compared with the controls (SLE 252±654 vs control 2.13±3.7; P=0.029). A higher amount of MFC was detected among multiparous SLE patients when compared with the control group (SLE 382±924 vs control 0.073±0.045; P=0.019). LN was associated with reduced amount of MFC (LN 95.5±338 vs control 388±827; P=0.019) especially when they have delivered their child before age 18 (LN 0.23±0.22 vs control 355±623; P=0.028). SLE patients present a higher amount of MFC, which may increase with the time since birth of the first male child. LN
patients showed an inverse correlation with MFC, suggesting that the role of the cells may be ambiguous during the various stages of development of the disease. Keywords Fetal microchimerism . Lupus nephritis . Male fetal cell . Microchimerism . Systemic lupus erythematosus
Abbreviations LN Lupus nephritis SLE Systemic lupus erythematosus FM Fetal microchimerism MFC Male fetal cell ACR American College of Rheumatology qPCR Quantitative polymerase chain reaction SRY Sex-determining region Y MCF/mL Male fetal cell per milliliter
Introduction * Mario Abbud-Filho [email protected] 1
Department of Medicine, Laboratory of Immunology and Transplantation Experimental—LITEX, Av. Brigadeiro Faria Lima 5416, 15090-000 São Jose do Rio Preto, SP, Brazil
2
Genetics and Molecular Biology Research Unit Laboratory—UPGEM, Av. Brigadeiro Faria Lima 5416, 15090-000 São Jose do Rio Preto, SP, Brazil
3
Department of Medicine, Division of Nephrology Medical School—Hospital de Base Sao Jose Rio Preto, Av. Brigadeiro Faria Lima 5416, 15090-000 São Jose do Rio Preto, SP, Brazil
4
Instituto de Urology e Nefrologia, Rua Voluntários de São Paulo, 3826, 15015-200 Sao Jose Rio Preto, SP, Brazil
Lupus nephritis (LN) is one of the most important manifestations of systemic lupus erythematosus (SLE) as it may progress to chronic renal failure [1–3]. Male microchimeric cells of fetal origin known as fetal microchimerism (FM) have been postulated to be involved in SLE pathogenesis. These cells can be detected in the mother’s blood early during pregnancy and may persist in the maternal circulation and organs for decades after delivery [4, 5]. Additionally, microchimeric male fetal cells (MFC) were reported to be twice as common in LN as in normal kidneys, suggesting that they could also play a role in LN [6]. We have previously showed that the amount of MFC in the peripheral blood of women diagnosed with SLE was
Clin Rheumatol
significantly higher when compared with those of healthy women and that these cells can proliferate over time [7]. In the present study, we sought to identify the factors during pregnancy that could be linked to the presence and proliferation of MFC and the possible relation between these factors and the development of LN.
Materials and methods We evaluated 18 healthy women (control group) and 28 women followed in the nephrology/rheumatology ambulatories that fulfilled the criteria of the American College of Rheumatology (ACR) for SLE (study group) [8]. In both groups, a rigorous inclusion criteria used for inclusion in the study was to have calved at least one male child, absence of previous abortions, or blood transfusions. LN was diagnosed in 13 women based on the ACR criteria plus signs of renal abnormalities as Bactive^ semi-quantitative urinary sediment and/or renal dysfunction (proteinuria >1+, erythrocytes >5000/mL, and/or serum creatinine ≥1.5 mg/dL and/or creatinine clearance ≤80 mL/min/1.73 m2, and/or proteinuria 24 h>200 mg) observed in at least two consecutive outpatient reviews according to local clinical practice. Factors analyzed were time of SLE diagnosis, time since birth of the first male child, the number of male child, and age at the time of SLE diagnosis. The study was approved by the Institutional Ethics Committee of the medical school (number 0007/2000) in accordance with the current standards for human research, and informed consent of patients was obtained. DNA extraction and real-time quantitative polymerase chain reaction for detection of Y chromosome sequences Genomic DNA was extracted from the peripheral blood of 46 women according to the technique of Abdel-Rahman et al. [9] and was quantified by the technique of real-time quantitative polymerase chain reaction (qPCR) using a Perkin-Elmer Applied Biosystems 7700 Sequence Detector as previously described [10]. The SRY locus, which is specific for the sexdetermining gene on the Y chromosome, was used to ascertain the amount of male fetal DNA present in each maternal DNA sample. To determine the amount of MFC, a standard dilution curve using a known concentration of male genomic DNA was used. For the conversion to the amount of copies, 6.6 pg was used as described previously [11]. The result was expressed as MFC per milliliter (mL) of maternal blood, and all samples were analyzed in duplicate. The PCR for the SRY gene was performed in a volume of 25 μL containing 1 μL of maternal DNA (50 ng), 1X Universal Mastermix (PE Biosystems Warrington, Cheshire, UK), 1 pmol/ μL of each primer, and 0.5 pmol/uL probe. On each board,
amplification reactions were performed on the three controls to exclude the possibility of DNA contamination of the solutions, which were added to all reagents except template DNA. The sex-determining region Y (SRY) TaqMan system consisted of a forward primer: 5′ CGC ATT CAT CGT GTG GTC TC 3′; a reverse primer: 5′ CTC TGA GTT TCG CAT TCT GGG 3′; and a probe SRY (VIC)-5′ CGA TCA GAG GCG CAA GAT GGC TCT AG 3′-(TAMRA). For β-actin gene, the PCR reaction was also performed in a volume of 25 μL containing 1 μL of maternal DNA (50 ng), 1X Universal Mastermix (PE Biosystems Warrington, Cheshire, UK), 0.3 μM of each primer, and 0.2 mM probe. The β-actin TaqMan system consisted of β-actin (forward) primer, 5′ TCA CCC ACA CTG TGC CCA TCT ACG A 3′; a β-actin (reverse) primer, 5′ CAG CGG AAC CGC TCA TTG CCA ATG G 3′; and a probe β-actin (FAM)-5′ ACC GCC GAG ACC GCG TC (MGB-NFQ). Statistical analysis The results are given as mean±SD. A statistical analysis of the data was performed using Fisher’s exact test, Student’s t test, and Mann-Whitney test for comparison of frequencies and averages. For comparisons among means of more than two groups, ANOVA and Kruskal-Wallis test were used.
Results SLE patients and the control group were similar with regard to the age at birth of the first male child and the time since birth of the first male child. The mean age of SLE patients was 35± 10 years at the time of SLE diagnosis, and the mean follow-up time after SLE diagnosis was 6±5 years. Amount of male fetal cells per milliliter in SLE patients and control group The presence of MFC was detected in 19/28 (68 %) of women affected by SLE, while MFC was detected in 6/18 (32 %) in healthy women (controls). The amount of MFC per milliliter was significantly higher in the SLE group compared with the controls (SLE 252±654 vs control 2.13±3.7; P=0.029), suggesting a possible role for fetal microchimerism in this autoimmune disease (Table 1). MFC per milliliter and time of SLE diagnosis When the time of SLE diagnosis was stratified in periods (≤5 years, between 6 and 10 and ≥10 years), we observed differences among the amount of MFC (113.5±350, 115± 354, and 1401±1459; P=0.002), suggesting the possibility of proliferation of these cells over time (Table 1).
Clin Rheumatol Table 1 Amount of male fetal cells (MFC) in peripheral blood of women with SLE and controls Pregnancy history features MFC/mL
SLE
P value (SLE)
255±654a
–
(n=28)
6–10 >10
2.13±3.7b
–
6(32 %)
– 0.002
–
– – –
1401±1459
(n=3) – MFC and age at birth of the first male child (years) ≤18
178±451 (n=8)
19–25
294±920c (n=10) 270±522 (n=10)
1
0.027
382±924e
0.073±0.045f
(n=11)
(n=5)
SLE patients showed an increased amount of MFC with time since birth of the first male child. SLE patients who delivered the first child more than 15 years ago were at approximately tenfold amount of MFC than those who delivered 10 years ago or less and almost triple the amount MFC when delivery occurred between 11 and 15 years ago (>15 years, 395±860 vs 140±382; 11–15 vs ≤10 years, 33±65; P=0.003; Table 1). Gestational history of SLE patients, lupus nephritis, and MFC per milliliter
– NS
MFC per milliliter and time since birth of the first male child
–
–
113.5±350 (n=12) 115±354 (n=13)
P value (control)
(n=28)
Presence of MFC 19(68 %) – [n(%)] MFC and time of SLE diagnosis (years) ≤5
Control
of MFC detected in the SLE patients when compared with the control group (SLE 382±924 vs control 0.073±0.045; P=0.019; Table 1).
Data are expressed as mean±standard deviation. a vs b (P=0.029); c vs d (P=0.012); e vs f (P=0.019) MFC male fetal cell, SLE systemic lupus erythematosus, NS not significant
MFC per milliliter, age at birth of the first male child, and number of male pregnancies No woman in the control group had a male pregnancy before the age of 18 years, and statistical analysis within the group did not detect any differences on the amount of MFC among women whose male pregnancies occurred after 18 years of age. Comparison between groups showed a higher amount of MFC in SLE patients when the male pregnancy was in the range 19– 25 years (P=0.012, Table 1). The number of male pregnancies did not affect the amount of MFC in primiparous or multiparous SLE patients. However, in the control group, a significant decrease in the amount of MFC was noted among women with more than one male pregnancy (Table 1; P=0.027). Yet, multiparity seemed to differently affect the two groups, increasing the amount of MFC in SLE patients and decreasing in the control group, with a higher amount
In the group of 28 patients with SLE, 13 were diagnosed with LN and the amount of MFC was significantly higher among patients without LN when compared with those with LN (without LN 388±827 vs with LN 95.5±338; P=0.019). There were no differences with regard to the number of male pregnancies and amount of MFC between SLE patients with and without LN, although it is noticeable that multiparous women without LN present a greater amount of MFC (Table 2). MFC per milliliter, LN, and age at birth of the first male child SLE patients that had delivered their child before age 18 and with LN presented a significantly lower amount of MFC than those without LN (with LN 0.23±0.22 vs without LN 355± 623; P=0.028; Table 2). Table 2 Amount of MFC in peripheral blood of SLE patients, number of male pregnancies, and age at birth of first male child Characteristic
SLE patients With LN (N=13)
MFC/mL 95.5±338* Number of male pregnancies 1 155±430 (N=8) >1 0.126±0.10 (N=5) Age at birth of first male child ≤18 years old 0.23±0.22** (N=4) >18 years old 138±406 (N=9) Data are expressed as mean±standard deviation LN lupus nephritis *P=0.019; **P=0.028
SLE patients Without LN (N=15) 388±827* 179±420 (N=9) 700±1200 (N=6) 355±623** (N=4) 400±917 (N=9)
Clin Rheumatol
Discussion The association between fetal microchimerism and SLE is of increasing interest, although there are still few experimental and clinical data suggesting that MFC would have a role in the development of SLE [12]. MFC was first reported to be increased in association with systemic sclerosis, and since that time, it has been found in patients with thyroid disease, primary biliary cirrhosis, and SLE and, in many cases, also in healthy women [13, 14]. In a preliminary brief report, we found that 68 % of SLE patients had significant higher amounts of MFC in their peripheral blood, detected by the Y chromosome sequences, compared with 32 % in the healthy control group [7]. As the factors involved in the occurrence of microchimerism are largely unexplored, in the present manuscript, we extended our previous observations and sought to determine which variables could be associated with the presence of MFC and to establish possible associations between parity and disease, especially in those SLE patients with lupus nephritis. Overall, the present study reinforces that fetal microchimerism, expressed by a higher amount of MFC in the peripheral blood of SLE patients, may have an important role in the SLE pathogenesis. The different findings observed by other authors could be possibly attributed to assay sensitivity to detect the microchimeric cells, race, or disease severity [15–17]. In examining whether pregnancy history would have an impact in the development of microchimerism, we did not find a correlation between parity and amount of MFC. However, different from Kremer Hovinga et al. [18] who evaluated the effect of parity among chimeric and nonchimeric women, we observed a trend toward a positive relation between the amount of MFC and multiparity in SLE patients, whereas a trend toward an inverse correlation occurred in healthy women. We believe that by excluding all other forms of acquired microchimerism as abortions and transfusions and by correlating quantitatively the amount of MFC with the number of male pregnancies, we could detect such gestational effect (Table 1). Also, it was reported that the risk of developing autoimmune disease was lower when pregnancy occurred at a young age [19]. In this regard, our results did not show any correlation between MFC and age of SLE patients at birth of the first male child, although healthy women who gave birth after age of 25 years presented a tenfold increase in their microchimeric cells. Interestingly, women who gave birth to their first child at age 19 to 25 years old had significantly higher amounts of MFC when compared with the control group at the same age range. These results suggest that male pregnancies did affect differently the two groups of women and, indeed, could modulate the development and evolution of disease. Mosca et al. quantified the male genomic DNA of 22 SLE patients and found no significant difference in the amount of
male fetal cells in this group compared with that in the control group. However, in the group of patients with lupus nephritis, significantly more male fetal cells were detected [17]. Our results differ from these findings in two ways: (1) our patients with SLE had higher amount of MFC than the healthy control group and (2) patients with LN curiously had significantly lower amount of MFC than patients without renal injury. Assuming that the presence of LN is a severe form of SLE, our findings suggest that the greater amount of MFC observed in patients without nephritis might suggest a Bprotective^ role played by the microchimeric cells as a result of their plasticity [20].
Conclusions In conclusion, our data demonstrate that the amount of MFC in peripheral blood is higher in SLE patients than in healthy women, and it does increase with the time since SLE diagnosis and time since birth of the first male child, suggesting that in SLE patients, MFC do proliferate with time but decrease or tend to disappear in healthy women. Young patients developing LN presented with a lower amount of MFC; therefore, it is possible that these cells might provide some protection against the LN in some point of its development. Authors’ contributions GMSF and IMMF collected and assembled data, performed data analysis and interpretation, and helped to prepare the manuscript. HCC performed data analysis and interpretation, provided administrative support, and helped to prepare the manuscript. ECP and EMGB provided technical support and helped to prepare the manuscript. MAF was responsible for study conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript. All authors read and approved the final manuscript. Compliance with ethical standards The study was approved by the Institutional Ethics Committee of the medical school (number 0007/2000) in accordance with the current standards for human research, and informed consent of patients was obtained. Disclosures None.
References 1. 2.
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Riemekasten G, Hahn BH (2005) Key autoantigens in SLE. Rheumatology 44:975–982 Jacobsen S, Petersen J, Ullman S, Junker P, Voss A, Rasmussen JM et al (1998) A multicenter study of systemic lupus erythematosus in 513 Danish patients. I: disease manifestations and analyses of clinical subsets. Clin Rheumatol 17:468–477 Cervera R, Khamasthta MA, Font J, Sebastiani GD, Gil A, Lavilla P et al (1993) Systemic lupus erythematosus: clinical and immunological patterns of disease expression in a cohort of 1000 patients. Medicine 72:113–124
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Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA (1996) Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci U S A 93:705–708 Lee TH, Paglieroni T, Ohto H, Holland PV, Busch MP (1999) Survival of donor leukocyte subpopulations in immunocompetent transfusion recipients: frequent long-term microchimerism in severe trauma patients. Blood 93:3127–3139 Kremer Hovinga IC, Koopmans M, Baelde HJ, van der Wal AM, Sijpkens YW, de Heer E et al (2006) Chimerism occurs twice as often in lupus nephritis as in normal kidneys. Arthritis Rheum 54: 2944–2950 Abbud Filho M, Pavarino-Bertelli EC, Alvarenga MP, Fernandes IM, Toledo RA, Tajara EH et al (2002) Systemic lupus erythematosus and microchimerism in autoimmunity. Transplant Proc 34: 2951–2952 Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF et al (1992) The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 25:1271 Abdel-Rahman SZ, Nouraldeen AM, Ahmed AE (1994) Molecular interaction of [2,3-14C] acrylonitrile with DNA in gastric tissue of rat. J Biochem Toxicol 9:191–198 Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986 Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM et al (1998) Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 62:768–775 Sarkar K, Miller FW (2004) Possible roles and determinants of microchimerism in autoimmune and other disorders. Autoimmun Rev 3:454–463
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Adams KM, Nelson JL (2004) Microchimerism: an investigative frontier in autoimmunity and transplantation. JAMA 291:1127– 1131 14. Stevens AM, McDonnell WM, Mullarkey ME, Pang JM, Leisenring W, Nelson JL (2004) Liver biopsies from human females contain male hepatocytes in the absence of transplantation. Lab Invest 84:1603–1609 15. Miyashita Y, Ono M, Ono M, Ueki H, Kurasawa K (2000) Y chromosome microchimerism in rheumatic autoimmune disease. Ann Rheum Dis 64:605–612 16. Gannagé M, Amoura Z, Lantz O, Piette JC, Caillat-Zucman S (2002) Fetomaternal microchimerism in connective tissue diseases. Eur J Immunol 32:3405–3413 17. Mosca M, Curcio M, Lapi S, Valentini G, D’Angelo S, Rizzo G, Bombardieri S (2003) Correlations of Y chromosome microchimerism with disease activity in patients with SLE: analysis of preliminary data. Ann Rheum Dis 62:651–654 18. Kremer Hovinga IC, Koopmans M, Grootscholten C, van der Wal AM, Bijl M, Derksen RH et al (2008) Pregnancy, chimerism and lupus nephritis: a multi-centre study. Lupus 17:541–547 19. Launay D, Hebbar M, Hatron PY, Michon-Pasturel U, Queyrel V, Hachulla E et al (2001) Relationship between parity and clinical and biological features in patients with systemic sclerosis. J Rheumatol 28:509–513 20. Wang Y1, Iwatani H, Ito T, Horimoto N, Yamato M, Matsui I et al (2004) Fetal cells in mother rats contribute to the remodeling of liver and kidney after injury. Biochem Biophys Res Commun 325:961–967
ORIGINAL ARTICLE
Variables associated to fetal microchimerism in systemic lupus erythematosus patients Greiciane Maria da Silva Florim 1 & Heloisa Cristina Caldas 1 & Erika Cristina Pavarino 2 & Eny Maria Goloni Bertollo 2 & Ida Maria Maximina Fernandes 1 & Mario Abbud-Filho 1,3,4
Received: 27 August 2015 / Revised: 23 October 2015 / Accepted: 14 November 2015 # International League of Associations for Rheumatology (ILAR) 2015
Abstract In the present study, we sought to identify the factors during the pregnancy of systemic lupus erythematosus (SLE) patients that could be linked to the presence and proliferation of male fetal cells (MFC) and the possible relation between these factors and development of lupus nephritis (LN). We evaluated 18 healthy women (control group) and 28 women affected by SLE. Genomic DNA was extracted from peripheral blood and quantified using the technique of quantitative real-time polymerase chain reaction (qPCR) for specific Y chromosome sequences. The amount of MFC was significantly higher in the SLE group compared with the controls (SLE 252±654 vs control 2.13±3.7; P=0.029). A higher amount of MFC was detected among multiparous SLE patients when compared with the control group (SLE 382±924 vs control 0.073±0.045; P=0.019). LN was associated with reduced amount of MFC (LN 95.5±338 vs control 388±827; P=0.019) especially when they have delivered their child before age 18 (LN 0.23±0.22 vs control 355±623; P=0.028). SLE patients present a higher amount of MFC, which may increase with the time since birth of the first male child. LN
patients showed an inverse correlation with MFC, suggesting that the role of the cells may be ambiguous during the various stages of development of the disease. Keywords Fetal microchimerism . Lupus nephritis . Male fetal cell . Microchimerism . Systemic lupus erythematosus
Abbreviations LN Lupus nephritis SLE Systemic lupus erythematosus FM Fetal microchimerism MFC Male fetal cell ACR American College of Rheumatology qPCR Quantitative polymerase chain reaction SRY Sex-determining region Y MCF/mL Male fetal cell per milliliter
Introduction * Mario Abbud-Filho [email protected] 1
Department of Medicine, Laboratory of Immunology and Transplantation Experimental—LITEX, Av. Brigadeiro Faria Lima 5416, 15090-000 São Jose do Rio Preto, SP, Brazil
2
Genetics and Molecular Biology Research Unit Laboratory—UPGEM, Av. Brigadeiro Faria Lima 5416, 15090-000 São Jose do Rio Preto, SP, Brazil
3
Department of Medicine, Division of Nephrology Medical School—Hospital de Base Sao Jose Rio Preto, Av. Brigadeiro Faria Lima 5416, 15090-000 São Jose do Rio Preto, SP, Brazil
4
Instituto de Urology e Nefrologia, Rua Voluntários de São Paulo, 3826, 15015-200 Sao Jose Rio Preto, SP, Brazil
Lupus nephritis (LN) is one of the most important manifestations of systemic lupus erythematosus (SLE) as it may progress to chronic renal failure [1–3]. Male microchimeric cells of fetal origin known as fetal microchimerism (FM) have been postulated to be involved in SLE pathogenesis. These cells can be detected in the mother’s blood early during pregnancy and may persist in the maternal circulation and organs for decades after delivery [4, 5]. Additionally, microchimeric male fetal cells (MFC) were reported to be twice as common in LN as in normal kidneys, suggesting that they could also play a role in LN [6]. We have previously showed that the amount of MFC in the peripheral blood of women diagnosed with SLE was
Clin Rheumatol
significantly higher when compared with those of healthy women and that these cells can proliferate over time [7]. In the present study, we sought to identify the factors during pregnancy that could be linked to the presence and proliferation of MFC and the possible relation between these factors and the development of LN.
Materials and methods We evaluated 18 healthy women (control group) and 28 women followed in the nephrology/rheumatology ambulatories that fulfilled the criteria of the American College of Rheumatology (ACR) for SLE (study group) [8]. In both groups, a rigorous inclusion criteria used for inclusion in the study was to have calved at least one male child, absence of previous abortions, or blood transfusions. LN was diagnosed in 13 women based on the ACR criteria plus signs of renal abnormalities as Bactive^ semi-quantitative urinary sediment and/or renal dysfunction (proteinuria >1+, erythrocytes >5000/mL, and/or serum creatinine ≥1.5 mg/dL and/or creatinine clearance ≤80 mL/min/1.73 m2, and/or proteinuria 24 h>200 mg) observed in at least two consecutive outpatient reviews according to local clinical practice. Factors analyzed were time of SLE diagnosis, time since birth of the first male child, the number of male child, and age at the time of SLE diagnosis. The study was approved by the Institutional Ethics Committee of the medical school (number 0007/2000) in accordance with the current standards for human research, and informed consent of patients was obtained. DNA extraction and real-time quantitative polymerase chain reaction for detection of Y chromosome sequences Genomic DNA was extracted from the peripheral blood of 46 women according to the technique of Abdel-Rahman et al. [9] and was quantified by the technique of real-time quantitative polymerase chain reaction (qPCR) using a Perkin-Elmer Applied Biosystems 7700 Sequence Detector as previously described [10]. The SRY locus, which is specific for the sexdetermining gene on the Y chromosome, was used to ascertain the amount of male fetal DNA present in each maternal DNA sample. To determine the amount of MFC, a standard dilution curve using a known concentration of male genomic DNA was used. For the conversion to the amount of copies, 6.6 pg was used as described previously [11]. The result was expressed as MFC per milliliter (mL) of maternal blood, and all samples were analyzed in duplicate. The PCR for the SRY gene was performed in a volume of 25 μL containing 1 μL of maternal DNA (50 ng), 1X Universal Mastermix (PE Biosystems Warrington, Cheshire, UK), 1 pmol/ μL of each primer, and 0.5 pmol/uL probe. On each board,
amplification reactions were performed on the three controls to exclude the possibility of DNA contamination of the solutions, which were added to all reagents except template DNA. The sex-determining region Y (SRY) TaqMan system consisted of a forward primer: 5′ CGC ATT CAT CGT GTG GTC TC 3′; a reverse primer: 5′ CTC TGA GTT TCG CAT TCT GGG 3′; and a probe SRY (VIC)-5′ CGA TCA GAG GCG CAA GAT GGC TCT AG 3′-(TAMRA). For β-actin gene, the PCR reaction was also performed in a volume of 25 μL containing 1 μL of maternal DNA (50 ng), 1X Universal Mastermix (PE Biosystems Warrington, Cheshire, UK), 0.3 μM of each primer, and 0.2 mM probe. The β-actin TaqMan system consisted of β-actin (forward) primer, 5′ TCA CCC ACA CTG TGC CCA TCT ACG A 3′; a β-actin (reverse) primer, 5′ CAG CGG AAC CGC TCA TTG CCA ATG G 3′; and a probe β-actin (FAM)-5′ ACC GCC GAG ACC GCG TC (MGB-NFQ). Statistical analysis The results are given as mean±SD. A statistical analysis of the data was performed using Fisher’s exact test, Student’s t test, and Mann-Whitney test for comparison of frequencies and averages. For comparisons among means of more than two groups, ANOVA and Kruskal-Wallis test were used.
Results SLE patients and the control group were similar with regard to the age at birth of the first male child and the time since birth of the first male child. The mean age of SLE patients was 35± 10 years at the time of SLE diagnosis, and the mean follow-up time after SLE diagnosis was 6±5 years. Amount of male fetal cells per milliliter in SLE patients and control group The presence of MFC was detected in 19/28 (68 %) of women affected by SLE, while MFC was detected in 6/18 (32 %) in healthy women (controls). The amount of MFC per milliliter was significantly higher in the SLE group compared with the controls (SLE 252±654 vs control 2.13±3.7; P=0.029), suggesting a possible role for fetal microchimerism in this autoimmune disease (Table 1). MFC per milliliter and time of SLE diagnosis When the time of SLE diagnosis was stratified in periods (≤5 years, between 6 and 10 and ≥10 years), we observed differences among the amount of MFC (113.5±350, 115± 354, and 1401±1459; P=0.002), suggesting the possibility of proliferation of these cells over time (Table 1).
Clin Rheumatol Table 1 Amount of male fetal cells (MFC) in peripheral blood of women with SLE and controls Pregnancy history features MFC/mL
SLE
P value (SLE)
255±654a
–
(n=28)
6–10 >10
2.13±3.7b
–
6(32 %)
– 0.002
–
– – –
1401±1459
(n=3) – MFC and age at birth of the first male child (years) ≤18
178±451 (n=8)
19–25
294±920c (n=10) 270±522 (n=10)
1
0.027
382±924e
0.073±0.045f
(n=11)
(n=5)
SLE patients showed an increased amount of MFC with time since birth of the first male child. SLE patients who delivered the first child more than 15 years ago were at approximately tenfold amount of MFC than those who delivered 10 years ago or less and almost triple the amount MFC when delivery occurred between 11 and 15 years ago (>15 years, 395±860 vs 140±382; 11–15 vs ≤10 years, 33±65; P=0.003; Table 1). Gestational history of SLE patients, lupus nephritis, and MFC per milliliter
– NS
MFC per milliliter and time since birth of the first male child
–
–
113.5±350 (n=12) 115±354 (n=13)
P value (control)
(n=28)
Presence of MFC 19(68 %) – [n(%)] MFC and time of SLE diagnosis (years) ≤5
Control
of MFC detected in the SLE patients when compared with the control group (SLE 382±924 vs control 0.073±0.045; P=0.019; Table 1).
Data are expressed as mean±standard deviation. a vs b (P=0.029); c vs d (P=0.012); e vs f (P=0.019) MFC male fetal cell, SLE systemic lupus erythematosus, NS not significant
MFC per milliliter, age at birth of the first male child, and number of male pregnancies No woman in the control group had a male pregnancy before the age of 18 years, and statistical analysis within the group did not detect any differences on the amount of MFC among women whose male pregnancies occurred after 18 years of age. Comparison between groups showed a higher amount of MFC in SLE patients when the male pregnancy was in the range 19– 25 years (P=0.012, Table 1). The number of male pregnancies did not affect the amount of MFC in primiparous or multiparous SLE patients. However, in the control group, a significant decrease in the amount of MFC was noted among women with more than one male pregnancy (Table 1; P=0.027). Yet, multiparity seemed to differently affect the two groups, increasing the amount of MFC in SLE patients and decreasing in the control group, with a higher amount
In the group of 28 patients with SLE, 13 were diagnosed with LN and the amount of MFC was significantly higher among patients without LN when compared with those with LN (without LN 388±827 vs with LN 95.5±338; P=0.019). There were no differences with regard to the number of male pregnancies and amount of MFC between SLE patients with and without LN, although it is noticeable that multiparous women without LN present a greater amount of MFC (Table 2). MFC per milliliter, LN, and age at birth of the first male child SLE patients that had delivered their child before age 18 and with LN presented a significantly lower amount of MFC than those without LN (with LN 0.23±0.22 vs without LN 355± 623; P=0.028; Table 2). Table 2 Amount of MFC in peripheral blood of SLE patients, number of male pregnancies, and age at birth of first male child Characteristic
SLE patients With LN (N=13)
MFC/mL 95.5±338* Number of male pregnancies 1 155±430 (N=8) >1 0.126±0.10 (N=5) Age at birth of first male child ≤18 years old 0.23±0.22** (N=4) >18 years old 138±406 (N=9) Data are expressed as mean±standard deviation LN lupus nephritis *P=0.019; **P=0.028
SLE patients Without LN (N=15) 388±827* 179±420 (N=9) 700±1200 (N=6) 355±623** (N=4) 400±917 (N=9)
Clin Rheumatol
Discussion The association between fetal microchimerism and SLE is of increasing interest, although there are still few experimental and clinical data suggesting that MFC would have a role in the development of SLE [12]. MFC was first reported to be increased in association with systemic sclerosis, and since that time, it has been found in patients with thyroid disease, primary biliary cirrhosis, and SLE and, in many cases, also in healthy women [13, 14]. In a preliminary brief report, we found that 68 % of SLE patients had significant higher amounts of MFC in their peripheral blood, detected by the Y chromosome sequences, compared with 32 % in the healthy control group [7]. As the factors involved in the occurrence of microchimerism are largely unexplored, in the present manuscript, we extended our previous observations and sought to determine which variables could be associated with the presence of MFC and to establish possible associations between parity and disease, especially in those SLE patients with lupus nephritis. Overall, the present study reinforces that fetal microchimerism, expressed by a higher amount of MFC in the peripheral blood of SLE patients, may have an important role in the SLE pathogenesis. The different findings observed by other authors could be possibly attributed to assay sensitivity to detect the microchimeric cells, race, or disease severity [15–17]. In examining whether pregnancy history would have an impact in the development of microchimerism, we did not find a correlation between parity and amount of MFC. However, different from Kremer Hovinga et al. [18] who evaluated the effect of parity among chimeric and nonchimeric women, we observed a trend toward a positive relation between the amount of MFC and multiparity in SLE patients, whereas a trend toward an inverse correlation occurred in healthy women. We believe that by excluding all other forms of acquired microchimerism as abortions and transfusions and by correlating quantitatively the amount of MFC with the number of male pregnancies, we could detect such gestational effect (Table 1). Also, it was reported that the risk of developing autoimmune disease was lower when pregnancy occurred at a young age [19]. In this regard, our results did not show any correlation between MFC and age of SLE patients at birth of the first male child, although healthy women who gave birth after age of 25 years presented a tenfold increase in their microchimeric cells. Interestingly, women who gave birth to their first child at age 19 to 25 years old had significantly higher amounts of MFC when compared with the control group at the same age range. These results suggest that male pregnancies did affect differently the two groups of women and, indeed, could modulate the development and evolution of disease. Mosca et al. quantified the male genomic DNA of 22 SLE patients and found no significant difference in the amount of
male fetal cells in this group compared with that in the control group. However, in the group of patients with lupus nephritis, significantly more male fetal cells were detected [17]. Our results differ from these findings in two ways: (1) our patients with SLE had higher amount of MFC than the healthy control group and (2) patients with LN curiously had significantly lower amount of MFC than patients without renal injury. Assuming that the presence of LN is a severe form of SLE, our findings suggest that the greater amount of MFC observed in patients without nephritis might suggest a Bprotective^ role played by the microchimeric cells as a result of their plasticity [20].
Conclusions In conclusion, our data demonstrate that the amount of MFC in peripheral blood is higher in SLE patients than in healthy women, and it does increase with the time since SLE diagnosis and time since birth of the first male child, suggesting that in SLE patients, MFC do proliferate with time but decrease or tend to disappear in healthy women. Young patients developing LN presented with a lower amount of MFC; therefore, it is possible that these cells might provide some protection against the LN in some point of its development. Authors’ contributions GMSF and IMMF collected and assembled data, performed data analysis and interpretation, and helped to prepare the manuscript. HCC performed data analysis and interpretation, provided administrative support, and helped to prepare the manuscript. ECP and EMGB provided technical support and helped to prepare the manuscript. MAF was responsible for study conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript. All authors read and approved the final manuscript. Compliance with ethical standards The study was approved by the Institutional Ethics Committee of the medical school (number 0007/2000) in accordance with the current standards for human research, and informed consent of patients was obtained. Disclosures None.
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