Original Paper Received: August 8, 2013 Accepted: March 20, 2014 Published online: August 23, 2014
Acta Haematol 2015;133:83–88 DOI: 10.1159/000362356
High Percentage of Regulatory T Cells before and after Vitamin B12 Treatment in Patients with Pernicious Anemia Satoru Watanabe a Norifumi Ide a Hatsue Ogawara a Akihiko Yokohama c Takeki Mitsui b Hiroshi Handa b Hiromi Koiso d Norifumi Tsukamoto d Takayuki Saitoh a Hirokazu Murakami a
a
Department of Laboratory Sciences, Course of Health Sciences, Gunma Graduate School of Health Sciences, Medicine and Clinical Sciences, Gunma University Graduate School of Medicine, and c Department of Blood Transfusion Service and d Oncology Center, Gunma University Hospital, Maebashi, Japan
b
Abstract Introduction: In some previous studies, vitamin B12 treatment showed immunomodulatory effects and restored the immunological abnormalities in patients with pernicious anemia (PA). In the present study, peripheral blood T cell subsets, including regulatory T cells (Tregs), were examined before and after vitamin B12 treatment in PA patients. Patients and Methods: The percentages of CD4, CD8, Th1, Th2 and Tregs were examined in 23 PA patients before vitamin B12 treatment, in 23 other PA patients after vitamin B12 treatment and in 28 healthy controls. Results: The mean percentage of CD8+ T cells was significantly higher in the control group (23.0%; 95% CI, 20.4–25.6%) than in the pre- (16.0%; 95% CI, 12.1–20.0%) and posttreatment groups (15.2%; 95% CI, 11.8–18.6%; p < 0.05). The CD4/CD8 ratio was significantly lower in the control group (2.01; 95% CI, 1.66–2.34) than in the pre- (3.45; 95% CI, 2.55–7.80) and posttreatment groups (2.97; 95% CI, 2.22–3.72; p < 0.05). There was no significant difference in the mean Th1/Th2 ratio among these groups. There were significant increases in the mean percentage of
© 2014 S. Karger AG, Basel 0001–5792/14/1331–0083$39.50/0 E-Mail [email protected] www.karger.com/aha
Tregs in the pre- (6.29%; 95% CI, 5.04–7.54%) and posttreatment groups (7.77%; 95% CI, 6.34–9.20%) compared with the control group (4.18%; 95% CI, 3.92–4.47%; p < 0.05). Conclusions: The percentage of Tregs was significantly higher in PA patients than in normal subjects, and this high Treg percentage was not different before and after vitamin B12 treatment. Other immunological alterations also did not recover after vitamin B12 treatment, so that these immunological changes appear to be the cause of PA and are not induced by vitamin B12 deficiency. © 2014 S. Karger AG, Basel
Introduction
Regulatory T cells (Tregs) play an important role in the maintenance of self-tolerance, control of autoimmunity and regulation of T cell homeostasis, and they modulate overall immune responses against infectious agents and tumor cells [1]. Natural Tregs develop during normal T cell maturation in the thymus and represent 5–10% of CD4+ cells in the peripheral blood [2]. These cells express CD4 and CD25 surface antigens, as well as CTLA-4, GITR, CD103, CD62L, CD69, CD134, CD71, CD54 and CD45RA [3]. The suppressive activity of Tregs is associHirokazu Murakami Department of Laboratory Sciences Course of Health Sciences, Gunma Graduate School of Health Sciences Showa-machi 3-39-15, Maebashi, Gunma 371-8514 (Japan) E-Mail hmura @ gunma-u.ac.jp
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Key Words CD4/CD8 ratio · Foxp3 · Pernicious anemia · Regulatory T cells · Th1/Th2 ratio · Vitamin B12
Table 1. Clinical data of the study subjects
Cases, n Gender, males/females Age, years RBC, ×1012 cells/l Hemoglobin, g/dl Hematocrit, % MCV, fl MCH, pg MCHC, g/dl WBC, ×109 cells/l Neutrophils, ×109 cells/l Lymphocytes, ×109 cells/l Platelets, ×109 cells/l
Controls
Pretreatment PA patients
Posttreatment PA patients
28 15/13 42.3 (22–65) 4.54 (4.33–4.76) 13.7 (13.2–14.2) 41.3 (40.0–42.8) 90.6 (88.9–92.4) 30.2 (29.6–30.8) 33.3 (33.0–33.5) 5.9 (5.3–6.6) 3.4 (2.9–3.9) 1.9 (1.6–2.7) 237 (215–259)
23 14/9 66.4 (36–81) 1.77 (1.57–1.98) 7.4 (6.5–8.3) 22.0 (19.4–24.5) 124.4 (120.0–129.1) 41.9 (40.6–43.3) 33.8 (33.2–34.3) 4.0 (3.4–4.7) 2.3 (1.9–2.9) 1.4 (1.2–1.6) 165 (134–196)
23 15/8 70.3 (52–94) 4.40 (4.21–4.58) 13.5 (12.8–14.2) 40.5 (38.8–42.2) 92.3 (90.8–93.7) 30.7 (30.0–31.4) 33.2 (32.8–33.8) 5.6 (5.0–6.2) 3.4 (3.0–3.9) 2.0 (1.5–2.4) 182 (154–210)
Means (95% CI).
Patients and Methods
Patients
Methods Measurement of Percentages of CD4 and CD8 Cells and the CD4/CD8 Ratio Whole heparinized blood was collected to measure the lymphocyte subsets using fluorescein isothiocyanate (FITC)-labeled anti-CD3, phycoerythrin (PE)-labeled anti-CD4, and CD8-PerCP (BD Pharmingen, San Diego, Calif., USA), and analysis was performed using the FACSCanto II flow cytometer (BD). Measurement of the Th1/Th2 Ratio Whole heparinized blood was incubated with 25 ng/ml phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin and 10 μg/ml brefeldin A at 37 ° C with 7% CO2 for 4 h. After treatment with fluorescence-activated cell sorter (FACS) lysing solution and FACS permeabilizing solution, cells were stained with anti-CD4-Cy-Chrome, FastImmune® FITC-labeled
Twenty-three patients with PA diagnosed at the Gunma University Hospital (pretreatment group) were included in the present study. Diagnosis was made based on medical history,
84
macrocytic anemia in peripheral blood, erythroblastosis with magaloblastic changes in bone marrow, low serum levels of vitamin B12 and clinical responsiveness to vitamin B12 therapy. None of the patients had infections or inflammation. Blood samples were obtained at the time of the initial diagnosis and before the start of any treatment. The median age of the patients was 66.4 years (range, 36–81 years), with 14 males and 9 females. Another 23 patients with PA, who were treated with vitamin B12 and had complete hematological remission, were assessed as the posttreatment group. The median age of the posttreatment group was 70.3 years (range, 52–94 years), with 15 males and 8 females. Controls were 28 healthy donors (15 males, 13 females) with a median age of 42.3 years (range, 22– 65 years). The clinical data of the study subjects are summarized in table 1. Approval for these studies was obtained from the institutional review board of the Gunma University Hospital Ethics Committee, Japan. Informed consent was obtained from the patients and controls according to the Declaration of Helsinki.
Acta Haematol 2015;133:83–88 DOI: 10.1159/000362356
Watanabe et al.
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ated with the overexpression of Foxp3, a member of the forkhead/winged helix family, which acts as a transcriptional repressor [4]. Tregs suppress CD25–CD4+ T cell proliferation on the basis of cell-cell contact and suppress immune responses by secreting immunosuppressive cytokines such as IL-10 and TGF-β [5]. Pernicious anemia (PA), an autoimmune disease, is the end stage of atrophic gastritis. The autoimmune mechanism of PA is supported by the presence of intrinsic factor and/or parietal cell autoantibodies and its frequent association with other autoimmune disorders, such as autoimmune thyroid disease, type I diabetes mellitus and vitiligo [6–9]. As a result of the deficiency in intrinsic factor, a protein that binds avidly to dietary vitamin B12 and promotes its transport to the terminal ileum for absorption, patients with PA develop a macrocytic anemia due to vitamin B12 deficiency. In some previous studies, vitamin B12 treatment showed immunomodulatory effects and restored the immunological abnormalities in PA patients [10, 11]. However, there have been no reports about the changes in Tregs and Th1/Th2 in PA. Because Tregs play an important role in modulating immune responses, the percentages of CD4, CD8, Th1, Th2 and Tregs before and after vitamin B12 treatment were examined in patients with PA.
anti-human IFN-γ and PE-labeled anti-human IL-4 (BD Biosciences, San Jose, Calif., USA) at 4 ° C for 30 min. FastImmune IgG2a FITC/IgG1 PE isotype control and mouse IgG1 PE control (Becton-Dickinson) were used as negative controls. Th subsets were assessed by three-color flow cytometry [12].
(p < 0.05)
60
(p < 0.05)
50
Statistical Analysis Analysis of data was performed using JMP6 software (SAS Institute Inc., Cary, N.C., USA). Comparisons of quantitative data were performed using a nonparametric test (unpaired Student’s t test). Values of p < 0.05 were considered significant.
Results
30 20 10 0
Control
Pretreatment
Posttreatment
Fig. 1. Comparison of CD4 (□) and CD8 (■) cell percentages in
the control group, and pre- and posttreatment PA groups. Data are shown as means + 95% CI.
(p < 0.05) 5
(p < 0.05)
4 CD4/CD8 ratio
Complete Blood Cell Counts Complete blood cell counts were determined using a total blood analyzer (ADVIA 120; Siemens, Tarrytown, N.Y., USA).
Cells (%)
40
Treg Cell Measurements PE-Cyc5 anti-human CD4 monoclonal antibody (BD Pharmingen) and FITC anti-humanCD25 (BD Biosciences) were used for surface antigen staining. Mouse IgG1, κ-FITC (BD Pharmingen) was used as an isotype control. PE-conjugated anti-human Foxp3 (PCH101) and PE-conjugated rat IgG2a isotype control from eBiosciences (San Diego, Calif., USA) were used for intracellular Foxp3 staining according to the manufacturer’s instructions. Tregs were defined as CD4+CD25+Foxp3+ cells. Three-color flow-cytometric analysis was performed on a FACSCanto II flow cytometer using CellQuest software (BD Biosciences).
3 2 1
Regulatory T Cells in PA
Control
Pretreatment
Posttreatment
Fig. 2. Comparison of the CD4/CD8 ratio in the control group, and pre- and posttreatment PA groups. Data are shown as means + 95% CI.
25 20 15 10 5 0
Control
Pretreatment
Posttreatment
Fig. 3. Comparison of the Th1/Th2 ratio in the control group, and
pre- and posttreatment PA groups. Data are shown as means + 95% CI.
Acta Haematol 2015;133:83–88 DOI: 10.1159/000362356
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Th1 and Th2 Percentages and the Th1/Th2 Ratio The mean Th1/Th2 ratio was slightly higher in the pretreatment group (16.7; 95% CI, 11.2–22.3) than in the control (12.6; 95% CI, 9.4–15.9) and posttreatment groups (11.7; 95% CI, 7.7–15.6), but there was no significant difference among these groups (fig. 3).
0
Th1/Th2 ratio
CD4+ and CD8+ T Cell Percentages and the CD4/ CD8 Ratio There was no significant difference in the mean percentage of CD4+ T cells among the control group (41.0%; 95% CI, 37.9–44.1%), and pre- (43.7%; 95% CI, 36.3– 51.1%), and posttreatment groups (35.0%; 95% CI, 30.6– 39.4%; fig. 1). However, the mean percentage of CD8+ T cells was significantly higher in the control group (23.0%; 95% CI, 20.4–25.6%) than in the pre- (16.0%; 95% CI, 12.1–20.0%) and posttreatment groups (15.2%; 11.8– 18.6%; p < 0.05; fig. 1). Given the high CD8+ T cell percentage in the control group, the CD4/CD8 ratio was also significantly lower in the control group (2.01; 95% CI, 1.66–2.34) than in the pre- (3.45; 95% CI, 2.55–7.80; p < 0.05) and posttreatment groups (2.97; 95% CI, 2.22–3.72; p < 0.05; fig. 2).
Specimen_001-sample
Specimen_001-sample 105 4.07% Anti-Foxp3 PE PE-A
11.32%
Q1
Q2
103
102
102
103
Q2
Q1
102
Q4
Q3 104
105
CD25 FITC FITC-A
Fig. 4. Identification of CD25 and Foxp3-expressing CD4 cells
from peripheral blood. FACS staining of a blood sample from a PA patient for surface CD4 and CD25 and intracellular Foxp3 is shown. A gate for CD4+ T cells was set on forward scatter/CD4 dot
CD4+CD25+Foxp3+ Tregs There were significant increases in the mean percentage of Tregs in the pre- (6.29%; 95% CI, 5.04–7.54%) and posttreatment groups (7.77%; 95% CI, 6.34–9.20%) compared with the control group (4.18%; 95% CI, 3.92–4.47%; p < 0.05; fig. 4, 5).
Discussion
It is well known that PA is induced by immunological mechanisms. These autoimmune mechanisms are supported by the presence of some autoantibodies in patients with PA. The most prevalent is the parietal cell antibody, which is observed in approximately 84% of PA patients. Intrinsic factor antibody is also detected in the serum of approximately 60% of PA patients. In addition, PA is frequently accompanied by several other autoimmune diseases, such as autoimmune thyroid diseases, type I diabetes mellitus and vitiligo [7]. Wodzinski et al. [13] reported that the CD4/CD8 ratio was significantly higher in PA patients with intrinsic factor antibody than in those who lacked the antibody, and they proposed that alterations in the T cell subsets may be associated with PA. Two other investigators showed a decrease in CD8 cells and an increase in the CD4/CD8 ratio, as well as recovery of these alterations after vitamin B12 treatment. Based on the recovery of immune alterations after vitamin B12 treatment, 86
103
Q4
Q3
a
104
Acta Haematol 2015;133:83–88 DOI: 10.1159/000362356
102
b
103
104
105
CD25 FITC FITC-A
plots to identify the desired subset. In a second dot plot, coexpression of CD25 and Foxp3 was assessed in comparison to the control isotype staining. Data are expressed as dot plots in a control subject (a) and a patient with PA before treatment (b).
(p < 0.05)
10 (p < 0.05)
8 6 4 2 0
Control
Pretreatment
Posttreatment
Fig. 5. Comparison of the percentages of CD4+CD25+Foxp3+ Tregs in the control group, and pre- and posttreatment PA groups. Data are shown as means + 95% CI.
they concluded that immunological alterations were not the primary event in patients with PA, but they were induced by vitamin B12 deficiency [10, 11]. In addition to these reports, Wada et al. [14] reported that serum C3 content decreased in vitamin B12-deficient rats and recovered with methylcobalamin administration. Funada et al. [15] reported that Th2 polarization was observed in vitamin B12-deficient mice. These reports proposed that vitamin B12 may have some immune-modulatory effects in animals and humans. Watanabe et al.
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104
CD4+CD25+Foxp3+ Tregs (%)
Anti-Foxp3 PE PE-A
105
In the present study, the percentage of CD4 T cells in patients with PA was in the normal range; however, the percentage of CD8 T cells was lower than in normal controls, which translated to a higher CD4/CD8 ratio compared with normal controls. These data were similar to the three previous reports, but these changes were not normalized after vitamin B12 treatment. Given these results, the changes in CD8 and the CD4/CD8 ratio are primary events and related to PA. As described above, Wada et al. [14] and Funada et al. [15] reported Th2 polarization in vitamin B12-deficient animals. However, there was no significant difference in the percentages of Th1 and Th2 cells and the Th1/Th2 ratio between normal controls and PA patients in the present study. A controversial age-related increase in Tregs has been described [16]. Rosenkranz et al. [17] reported that the frequency of Tregs (CD4+Foxp3+) increased with aging, and Gregg et al. [18] also reported an increase in peripheral blood CD4+CD25high regulatory T cells associated with aging. Conversely, others reported no correlation between the number of circulating CD4+CD25high Tregs and age [19]. We have already reported that there was no significant difference in the mean number of Tregs between subjects 40 years [20]. Therefore, the age difference between the control group and the patient groups did not appear to affect the evaluation of Tregs in the PA patients. In the present study, the percentage of Tregs was significantly higher in PA patients than in normal controls, and this high Treg percentage was not different before and
after vitamin B12 treatment. Thus, the change in Tregs is the primary event and related to PA. It is generally accepted that Tregs are elevated in the peripheral blood and target organs of patients with autoimmune diseases. Alonso et al. [21] reported that the percentage of peripheral blood Tregs was significantly higher in patients with type I diabetes mellitus and chronic atrophic gastritis than in healthy controls, and they suggested that Tregs were stimulated in patients with combined autoimmune diseases in an ineffectual attempt to control the autoimmune response. Wang et al. [22] reported transient expression of Foxp3 in human nonregulatory CD4+ T cells after activation. According to these reports, the presence of ineffectual Tregs and/or transient expression of Foxp3 in patients with PA is also possible. In conclusion, the immunological alterations did not recover after vitamin B12 treatment in the present study, suggesting that these immunological changes seem to be caused by PA and not induced by vitamin B12 deficiency. Acknowledgments The authors would like to thank the medical and nursing staff of the Department of Hematology at the Gunma University Hospital for providing samples and clinical data.
Disclosure Statement The authors state that there are no conflicts of interest relevant to this paper that might bias their work.
References
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1 Bluestone JA, Abbas AK: Natural versus adaptive regulatory T cells. Nat Rev Immunol 2003;3:253–257. 2 Sakaguchi S: Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005;6:345–352. 3 Belkaid Y, Rouse BT: Natural regulatory T cells in infectious disease. Nat Immunol 2005; 6:353–360. 4 Fontenot JD, Rudensky AY: A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nat Immunol 2005;6:331–337. 5 Von Boehmer H: Mechanisms of suppression by suppressor T cells. Nat Immunol 2005; 6: 338–344. 6 Carmel R, Spencer CA: Clinical and subclinical thyroid disorders associated with pernicious anemia. Observations on abnormal thy-
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14 Wada M, Kawata T, Yamada K, Funada U, Kuwamori M, Endo M, Tanaka N, Tadokoro T, Maekawa A: Serum C3 content in vitamin B(12)-deficient rats. Int J Vitam Nutr Res 1998;68:94–97. 15 Funada U, Wada M, Kawata T, Mori K, Tamai H, Isshiki T, Onoda J, Tanaka N, Tadokoro T, Maekawa A: Vitamin B-12-deficiency affects immunoglobulin production and cytokine levels in mice. Int J Vitam Nutr Res 2001; 71: 60–65. 16 Dejaco C, Duftner C, Schirmer M: Are regulatory T-cells linked with aging? Exp Gerontol 2006;41:339–345.
Acta Haematol 2015;133:83–88 DOI: 10.1159/000362356
High Percentage of Regulatory T Cells before and after Vitamin B12 Treatment in Patients with Pernicious Anemia Satoru Watanabe a Norifumi Ide a Hatsue Ogawara a Akihiko Yokohama c Takeki Mitsui b Hiroshi Handa b Hiromi Koiso d Norifumi Tsukamoto d Takayuki Saitoh a Hirokazu Murakami a
a
Department of Laboratory Sciences, Course of Health Sciences, Gunma Graduate School of Health Sciences, Medicine and Clinical Sciences, Gunma University Graduate School of Medicine, and c Department of Blood Transfusion Service and d Oncology Center, Gunma University Hospital, Maebashi, Japan
b
Abstract Introduction: In some previous studies, vitamin B12 treatment showed immunomodulatory effects and restored the immunological abnormalities in patients with pernicious anemia (PA). In the present study, peripheral blood T cell subsets, including regulatory T cells (Tregs), were examined before and after vitamin B12 treatment in PA patients. Patients and Methods: The percentages of CD4, CD8, Th1, Th2 and Tregs were examined in 23 PA patients before vitamin B12 treatment, in 23 other PA patients after vitamin B12 treatment and in 28 healthy controls. Results: The mean percentage of CD8+ T cells was significantly higher in the control group (23.0%; 95% CI, 20.4–25.6%) than in the pre- (16.0%; 95% CI, 12.1–20.0%) and posttreatment groups (15.2%; 95% CI, 11.8–18.6%; p < 0.05). The CD4/CD8 ratio was significantly lower in the control group (2.01; 95% CI, 1.66–2.34) than in the pre- (3.45; 95% CI, 2.55–7.80) and posttreatment groups (2.97; 95% CI, 2.22–3.72; p < 0.05). There was no significant difference in the mean Th1/Th2 ratio among these groups. There were significant increases in the mean percentage of
© 2014 S. Karger AG, Basel 0001–5792/14/1331–0083$39.50/0 E-Mail [email protected] www.karger.com/aha
Tregs in the pre- (6.29%; 95% CI, 5.04–7.54%) and posttreatment groups (7.77%; 95% CI, 6.34–9.20%) compared with the control group (4.18%; 95% CI, 3.92–4.47%; p < 0.05). Conclusions: The percentage of Tregs was significantly higher in PA patients than in normal subjects, and this high Treg percentage was not different before and after vitamin B12 treatment. Other immunological alterations also did not recover after vitamin B12 treatment, so that these immunological changes appear to be the cause of PA and are not induced by vitamin B12 deficiency. © 2014 S. Karger AG, Basel
Introduction
Regulatory T cells (Tregs) play an important role in the maintenance of self-tolerance, control of autoimmunity and regulation of T cell homeostasis, and they modulate overall immune responses against infectious agents and tumor cells [1]. Natural Tregs develop during normal T cell maturation in the thymus and represent 5–10% of CD4+ cells in the peripheral blood [2]. These cells express CD4 and CD25 surface antigens, as well as CTLA-4, GITR, CD103, CD62L, CD69, CD134, CD71, CD54 and CD45RA [3]. The suppressive activity of Tregs is associHirokazu Murakami Department of Laboratory Sciences Course of Health Sciences, Gunma Graduate School of Health Sciences Showa-machi 3-39-15, Maebashi, Gunma 371-8514 (Japan) E-Mail hmura @ gunma-u.ac.jp
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Key Words CD4/CD8 ratio · Foxp3 · Pernicious anemia · Regulatory T cells · Th1/Th2 ratio · Vitamin B12
Table 1. Clinical data of the study subjects
Cases, n Gender, males/females Age, years RBC, ×1012 cells/l Hemoglobin, g/dl Hematocrit, % MCV, fl MCH, pg MCHC, g/dl WBC, ×109 cells/l Neutrophils, ×109 cells/l Lymphocytes, ×109 cells/l Platelets, ×109 cells/l
Controls
Pretreatment PA patients
Posttreatment PA patients
28 15/13 42.3 (22–65) 4.54 (4.33–4.76) 13.7 (13.2–14.2) 41.3 (40.0–42.8) 90.6 (88.9–92.4) 30.2 (29.6–30.8) 33.3 (33.0–33.5) 5.9 (5.3–6.6) 3.4 (2.9–3.9) 1.9 (1.6–2.7) 237 (215–259)
23 14/9 66.4 (36–81) 1.77 (1.57–1.98) 7.4 (6.5–8.3) 22.0 (19.4–24.5) 124.4 (120.0–129.1) 41.9 (40.6–43.3) 33.8 (33.2–34.3) 4.0 (3.4–4.7) 2.3 (1.9–2.9) 1.4 (1.2–1.6) 165 (134–196)
23 15/8 70.3 (52–94) 4.40 (4.21–4.58) 13.5 (12.8–14.2) 40.5 (38.8–42.2) 92.3 (90.8–93.7) 30.7 (30.0–31.4) 33.2 (32.8–33.8) 5.6 (5.0–6.2) 3.4 (3.0–3.9) 2.0 (1.5–2.4) 182 (154–210)
Means (95% CI).
Patients and Methods
Patients
Methods Measurement of Percentages of CD4 and CD8 Cells and the CD4/CD8 Ratio Whole heparinized blood was collected to measure the lymphocyte subsets using fluorescein isothiocyanate (FITC)-labeled anti-CD3, phycoerythrin (PE)-labeled anti-CD4, and CD8-PerCP (BD Pharmingen, San Diego, Calif., USA), and analysis was performed using the FACSCanto II flow cytometer (BD). Measurement of the Th1/Th2 Ratio Whole heparinized blood was incubated with 25 ng/ml phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin and 10 μg/ml brefeldin A at 37 ° C with 7% CO2 for 4 h. After treatment with fluorescence-activated cell sorter (FACS) lysing solution and FACS permeabilizing solution, cells were stained with anti-CD4-Cy-Chrome, FastImmune® FITC-labeled
Twenty-three patients with PA diagnosed at the Gunma University Hospital (pretreatment group) were included in the present study. Diagnosis was made based on medical history,
84
macrocytic anemia in peripheral blood, erythroblastosis with magaloblastic changes in bone marrow, low serum levels of vitamin B12 and clinical responsiveness to vitamin B12 therapy. None of the patients had infections or inflammation. Blood samples were obtained at the time of the initial diagnosis and before the start of any treatment. The median age of the patients was 66.4 years (range, 36–81 years), with 14 males and 9 females. Another 23 patients with PA, who were treated with vitamin B12 and had complete hematological remission, were assessed as the posttreatment group. The median age of the posttreatment group was 70.3 years (range, 52–94 years), with 15 males and 8 females. Controls were 28 healthy donors (15 males, 13 females) with a median age of 42.3 years (range, 22– 65 years). The clinical data of the study subjects are summarized in table 1. Approval for these studies was obtained from the institutional review board of the Gunma University Hospital Ethics Committee, Japan. Informed consent was obtained from the patients and controls according to the Declaration of Helsinki.
Acta Haematol 2015;133:83–88 DOI: 10.1159/000362356
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ated with the overexpression of Foxp3, a member of the forkhead/winged helix family, which acts as a transcriptional repressor [4]. Tregs suppress CD25–CD4+ T cell proliferation on the basis of cell-cell contact and suppress immune responses by secreting immunosuppressive cytokines such as IL-10 and TGF-β [5]. Pernicious anemia (PA), an autoimmune disease, is the end stage of atrophic gastritis. The autoimmune mechanism of PA is supported by the presence of intrinsic factor and/or parietal cell autoantibodies and its frequent association with other autoimmune disorders, such as autoimmune thyroid disease, type I diabetes mellitus and vitiligo [6–9]. As a result of the deficiency in intrinsic factor, a protein that binds avidly to dietary vitamin B12 and promotes its transport to the terminal ileum for absorption, patients with PA develop a macrocytic anemia due to vitamin B12 deficiency. In some previous studies, vitamin B12 treatment showed immunomodulatory effects and restored the immunological abnormalities in PA patients [10, 11]. However, there have been no reports about the changes in Tregs and Th1/Th2 in PA. Because Tregs play an important role in modulating immune responses, the percentages of CD4, CD8, Th1, Th2 and Tregs before and after vitamin B12 treatment were examined in patients with PA.
anti-human IFN-γ and PE-labeled anti-human IL-4 (BD Biosciences, San Jose, Calif., USA) at 4 ° C for 30 min. FastImmune IgG2a FITC/IgG1 PE isotype control and mouse IgG1 PE control (Becton-Dickinson) were used as negative controls. Th subsets were assessed by three-color flow cytometry [12].
(p < 0.05)
60
(p < 0.05)
50
Statistical Analysis Analysis of data was performed using JMP6 software (SAS Institute Inc., Cary, N.C., USA). Comparisons of quantitative data were performed using a nonparametric test (unpaired Student’s t test). Values of p < 0.05 were considered significant.
Results
30 20 10 0
Control
Pretreatment
Posttreatment
Fig. 1. Comparison of CD4 (□) and CD8 (■) cell percentages in
the control group, and pre- and posttreatment PA groups. Data are shown as means + 95% CI.
(p < 0.05) 5
(p < 0.05)
4 CD4/CD8 ratio
Complete Blood Cell Counts Complete blood cell counts were determined using a total blood analyzer (ADVIA 120; Siemens, Tarrytown, N.Y., USA).
Cells (%)
40
Treg Cell Measurements PE-Cyc5 anti-human CD4 monoclonal antibody (BD Pharmingen) and FITC anti-humanCD25 (BD Biosciences) were used for surface antigen staining. Mouse IgG1, κ-FITC (BD Pharmingen) was used as an isotype control. PE-conjugated anti-human Foxp3 (PCH101) and PE-conjugated rat IgG2a isotype control from eBiosciences (San Diego, Calif., USA) were used for intracellular Foxp3 staining according to the manufacturer’s instructions. Tregs were defined as CD4+CD25+Foxp3+ cells. Three-color flow-cytometric analysis was performed on a FACSCanto II flow cytometer using CellQuest software (BD Biosciences).
3 2 1
Regulatory T Cells in PA
Control
Pretreatment
Posttreatment
Fig. 2. Comparison of the CD4/CD8 ratio in the control group, and pre- and posttreatment PA groups. Data are shown as means + 95% CI.
25 20 15 10 5 0
Control
Pretreatment
Posttreatment
Fig. 3. Comparison of the Th1/Th2 ratio in the control group, and
pre- and posttreatment PA groups. Data are shown as means + 95% CI.
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Th1 and Th2 Percentages and the Th1/Th2 Ratio The mean Th1/Th2 ratio was slightly higher in the pretreatment group (16.7; 95% CI, 11.2–22.3) than in the control (12.6; 95% CI, 9.4–15.9) and posttreatment groups (11.7; 95% CI, 7.7–15.6), but there was no significant difference among these groups (fig. 3).
0
Th1/Th2 ratio
CD4+ and CD8+ T Cell Percentages and the CD4/ CD8 Ratio There was no significant difference in the mean percentage of CD4+ T cells among the control group (41.0%; 95% CI, 37.9–44.1%), and pre- (43.7%; 95% CI, 36.3– 51.1%), and posttreatment groups (35.0%; 95% CI, 30.6– 39.4%; fig. 1). However, the mean percentage of CD8+ T cells was significantly higher in the control group (23.0%; 95% CI, 20.4–25.6%) than in the pre- (16.0%; 95% CI, 12.1–20.0%) and posttreatment groups (15.2%; 11.8– 18.6%; p < 0.05; fig. 1). Given the high CD8+ T cell percentage in the control group, the CD4/CD8 ratio was also significantly lower in the control group (2.01; 95% CI, 1.66–2.34) than in the pre- (3.45; 95% CI, 2.55–7.80; p < 0.05) and posttreatment groups (2.97; 95% CI, 2.22–3.72; p < 0.05; fig. 2).
Specimen_001-sample
Specimen_001-sample 105 4.07% Anti-Foxp3 PE PE-A
11.32%
Q1
Q2
103
102
102
103
Q2
Q1
102
Q4
Q3 104
105
CD25 FITC FITC-A
Fig. 4. Identification of CD25 and Foxp3-expressing CD4 cells
from peripheral blood. FACS staining of a blood sample from a PA patient for surface CD4 and CD25 and intracellular Foxp3 is shown. A gate for CD4+ T cells was set on forward scatter/CD4 dot
CD4+CD25+Foxp3+ Tregs There were significant increases in the mean percentage of Tregs in the pre- (6.29%; 95% CI, 5.04–7.54%) and posttreatment groups (7.77%; 95% CI, 6.34–9.20%) compared with the control group (4.18%; 95% CI, 3.92–4.47%; p < 0.05; fig. 4, 5).
Discussion
It is well known that PA is induced by immunological mechanisms. These autoimmune mechanisms are supported by the presence of some autoantibodies in patients with PA. The most prevalent is the parietal cell antibody, which is observed in approximately 84% of PA patients. Intrinsic factor antibody is also detected in the serum of approximately 60% of PA patients. In addition, PA is frequently accompanied by several other autoimmune diseases, such as autoimmune thyroid diseases, type I diabetes mellitus and vitiligo [7]. Wodzinski et al. [13] reported that the CD4/CD8 ratio was significantly higher in PA patients with intrinsic factor antibody than in those who lacked the antibody, and they proposed that alterations in the T cell subsets may be associated with PA. Two other investigators showed a decrease in CD8 cells and an increase in the CD4/CD8 ratio, as well as recovery of these alterations after vitamin B12 treatment. Based on the recovery of immune alterations after vitamin B12 treatment, 86
103
Q4
Q3
a
104
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102
b
103
104
105
CD25 FITC FITC-A
plots to identify the desired subset. In a second dot plot, coexpression of CD25 and Foxp3 was assessed in comparison to the control isotype staining. Data are expressed as dot plots in a control subject (a) and a patient with PA before treatment (b).
(p < 0.05)
10 (p < 0.05)
8 6 4 2 0
Control
Pretreatment
Posttreatment
Fig. 5. Comparison of the percentages of CD4+CD25+Foxp3+ Tregs in the control group, and pre- and posttreatment PA groups. Data are shown as means + 95% CI.
they concluded that immunological alterations were not the primary event in patients with PA, but they were induced by vitamin B12 deficiency [10, 11]. In addition to these reports, Wada et al. [14] reported that serum C3 content decreased in vitamin B12-deficient rats and recovered with methylcobalamin administration. Funada et al. [15] reported that Th2 polarization was observed in vitamin B12-deficient mice. These reports proposed that vitamin B12 may have some immune-modulatory effects in animals and humans. Watanabe et al.
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104
CD4+CD25+Foxp3+ Tregs (%)
Anti-Foxp3 PE PE-A
105
In the present study, the percentage of CD4 T cells in patients with PA was in the normal range; however, the percentage of CD8 T cells was lower than in normal controls, which translated to a higher CD4/CD8 ratio compared with normal controls. These data were similar to the three previous reports, but these changes were not normalized after vitamin B12 treatment. Given these results, the changes in CD8 and the CD4/CD8 ratio are primary events and related to PA. As described above, Wada et al. [14] and Funada et al. [15] reported Th2 polarization in vitamin B12-deficient animals. However, there was no significant difference in the percentages of Th1 and Th2 cells and the Th1/Th2 ratio between normal controls and PA patients in the present study. A controversial age-related increase in Tregs has been described [16]. Rosenkranz et al. [17] reported that the frequency of Tregs (CD4+Foxp3+) increased with aging, and Gregg et al. [18] also reported an increase in peripheral blood CD4+CD25high regulatory T cells associated with aging. Conversely, others reported no correlation between the number of circulating CD4+CD25high Tregs and age [19]. We have already reported that there was no significant difference in the mean number of Tregs between subjects 40 years [20]. Therefore, the age difference between the control group and the patient groups did not appear to affect the evaluation of Tregs in the PA patients. In the present study, the percentage of Tregs was significantly higher in PA patients than in normal controls, and this high Treg percentage was not different before and
after vitamin B12 treatment. Thus, the change in Tregs is the primary event and related to PA. It is generally accepted that Tregs are elevated in the peripheral blood and target organs of patients with autoimmune diseases. Alonso et al. [21] reported that the percentage of peripheral blood Tregs was significantly higher in patients with type I diabetes mellitus and chronic atrophic gastritis than in healthy controls, and they suggested that Tregs were stimulated in patients with combined autoimmune diseases in an ineffectual attempt to control the autoimmune response. Wang et al. [22] reported transient expression of Foxp3 in human nonregulatory CD4+ T cells after activation. According to these reports, the presence of ineffectual Tregs and/or transient expression of Foxp3 in patients with PA is also possible. In conclusion, the immunological alterations did not recover after vitamin B12 treatment in the present study, suggesting that these immunological changes seem to be caused by PA and not induced by vitamin B12 deficiency. Acknowledgments The authors would like to thank the medical and nursing staff of the Department of Hematology at the Gunma University Hospital for providing samples and clinical data.
Disclosure Statement The authors state that there are no conflicts of interest relevant to this paper that might bias their work.
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