STATUS OF CELLULAR IMMUNITY IN ANEMIA OF CHRONIC DISEASES OF DIFFERENT GENESIS
Goldberg Scientific Research Institute of Pharmacology and Regenerative Medicine, Tomsk National Research Medical Center of Russian Academy of Science,
Novokuznetsk State Institute of Postgraduate Medicine, the branch of Russian Medical Academy of Continuous Professional Education, Novokuznetsk, Russia
Anemic syndrome is one of the most common hematologic disorders in patients with acute of chronical activation of the immune system after various infectious and non-infectious diseases [1, 2, 3]. According to the modern ideas, such types of anemia are defined as anemia of chronic disease (ACD) [1, 3, 4]. The rate of ACD in acute and chronical infections varies within 18-95 %, for autoimmune diseases – 8-71 %, for chronic renal diseases – 23-50 % [2, 5]. The role of anemia in such patients is often underestimated, but it often causes the unsatisfactory quality of life including easy fatigability, irritancy, sleep disorder and depression. During long period of the inflammatory process ACD can cause some difficulties for differential diagnostics of iron deficiency anemia and for further treatment.
ACD is the immune regulated condition, when bacterial lipopolysaccharides and cytokines induce the changes in iron homeostasis, disturb the production of erythropoetin and inhibit the proliferation of erythroid precursor-cells. One of the pathogenetic mechanisms of ACD is redistribution of iron into the cells of the macrophageal system, which is activated in various inflammatory (infectious and non-infectious) or tumoral processes [3]. At the same time, there are some issues concerning the participation of the individual links of immunity in the pathogenesis of ACD. Regulatory T-cells present the great interest during the recent years. They play the significant role in immune suppression and regulate T-cell homeostasis [6, 7, 8, 9].
The objective of the present study was estimation of the subpopulation composition of lymphocytes in ACD of various origin.
MATERIALS AND METHODS
The study included 276 women (age of 16-60). 79 patients were almost healthy and were included into the control group. Anemia of chronic disease was identified in 197 cases. 125 women demonstrated anemia at the background of the autoimmune diseases of connective tissue (rheumatoid arthritis), 72 – anemic of chronic diseases in bacterial infections (chronic tonsillitis, bacterial endocarditis, chronic pyelonephritis).
The research was conducted in concordance with the articles of the constitution of the Russian Federation, item No.32 of the Foundations of the Russian legislation about health protection in the population, Helsinki Declare of World Health Organization – the Recommendations for doctors dealing with biomedical research with human subjects. The study protocol was approved by the ethical committee of Novokuznetsk State Institute of Postgraduate Medicine. Before the study all participants signed the written consent confirmed by the ethical committee. The exclusion criteria were the age < 16 or > 60, other types of anemia, presence of malignant diseases, refusal from participation.
The study techniques included estimation of the peripheral link of erythron, iron metabolism and the subpopulation composition of lymphocytes. The values of the peripheral link of erythron were estimated with the hematological analyzer ADVIA 60 with assessment of the morphofunctional values of red blood cells. The microcytic anemia factor (MAF) was calculated with the formula: MAF = Hb × MCV / 100. The quantitative estimation of serum iron and total iron binding capacity (TIBC) was conducted with the ferrozine technique with use of the diagnostic tests of TECO company (USA) with subsequent calculation of latent iron-binding capacity (LIBC) and the transferrin saturation ratio (TSR). The iron volume was estimated with the level of serum ferritin (SF), which was measured with the immune enzyme technique with use of the test systems from Orgentec diagnostika (Germany). The lymphocyte subpopulations were measured with flow cytofluorometry with use of the multicolor analysis and the following combinations of the antibodies conjugate with the fluorescent colors (Becman Coulter, USA): CD8(FITC)/CD4(PE)/CD3(ICD), CD16(PC7), CD19(PC5), CD3(FITC)/CD16/56(PE), CD3(FITC)/HLA-DR(PE). The immunoregulatory index (IRI) was estimated with the formula: IRI = CD4 / CD8. Localization of T-regulatory cells was performed with the following set of the monoclonal antibodies: CD3(FITC), CD4(PС7), CD25(PС5), CD127(PE) with the flow cytometer Сytomics FC 500 (Beckman Coulter, USA). Multicolor staining and multi-staged gating allow the multiparameter analysis of the immunocompetent cells of peripheral blood with high accuracy and reliability. The sample preparation of peripheral blood was conducted with the non-washing technique with use of lysing and fixing reagents ImmunoPrep Reagent System and the automatic working station TQ-PREP (Beckman Coulter, USA). The population of lymphocytes was separated by means of heterogenic gating according to the parameters of forward scattering (FS) and side scattering (SS) which correlated with the morphological features of the cells. The histograms of FS and SS showed the easy localization of the cells of lymphocytic, monocytic and granulocytic links of differentiation, with non-overlaying their zones. It allowed using this approach for introduction of the logical limitations (gates) with the morphological signs – sizes (FS) and granularity (SS).
The statistical analysis of the data was conducted with MS-EXCEL, MS-WORD, BIOSTAT, Version 4.03. The results of the studies were estimated with analysis of variance. Student’s test was used for estimation of reliability of the results of the study. Normal distribution was measured with Shapiro-Wilk test. The prepared data was presented as mean values (M) and error in mean (m) for each value. The critical level of significance (p) was 0.05.
RESULTS
Hypochromic microcytic anemia was identified during the examination of the values of the peripheral link of erythron in the patients with rheumatoid arthritis (RA) and in the patients with chronic infectious-inflammatory processes (table 1).
Table 1. The values of peripheral link of erythron and ferrokinetics in anemia of chronic diseases
|
Index |
Control group |
Anemia of chronic diseases in infectious-inflammatory processes |
Anemia of chronic diseases in rhematoid arthritis |
P value |
|
1 |
2 |
3 |
||
|
RBC, ×10¹²/l |
4.2 ± 0.05 |
3.2 ± 0.28 * |
3.5 ± 0.13 * |
р1-2 = 0.001 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.350 |
||||
|
HGB, g/l |
135.9 ± 3.08 |
91.0 ± 7.55 * |
103.0 ± 3.87 * |
р1-2 = 0.001 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.160 |
||||
|
HCT, % |
36.9 ± 1.1 |
25.4 ± 2.89 * |
27.0 ± 1.35 * |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.613 |
||||
|
MCV, fl |
89.2 ± 1.38 |
76.0 ± 3.88 * |
76.7 ± 2.99 * |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.891 |
||||
|
MCH, pg |
33.2 ± 0.31 |
26.8 ± 1.97 * |
27.9 ± 1.22 * |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.609 |
||||
|
MCHC, g/dl |
37.4 ± 0.55 |
34.1 ± 0.89 * |
35.2 ± 0.047 * |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.212 |
||||
|
RDW, % |
11.1 ± 0.11 |
16.5 ± 0.92 * |
15.6 ± 0.62 * |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.399 |
||||
|
MAF |
121.1 ± 2.32 |
69.2 ± 1.95 * |
78.9 ± 2.21 * |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.003 |
Note: * – reliability of differences in values as compared to the control group.
The mean volume of red blood cells and the mean volume of hemoglobin in red blood cells in the patients with ACD, RA and the infectious-inflammatory processes were significantly lower than the similar values in the healthy individuals (p = 0.000), and the level of erythrocyte anisocytosis was significantly higher as compared to the similar values in the control group (p = 0.000). Moreover, the degree of anisocytosis was most intense in the patients with ACD and the infectious-inflammatory processes (the table 1). MAF was 78.9 ± 2.21 and 69.2 ± 1.96 in the patients with ACD, RA and the infectious-inflammatory processes and 121.1 ± 2.32 in the control group (p = 0.000).
The patients with ACD with the infections-inflammatory diseases and in the autoimmune diseases of connective tissue (rheumatoid arthritis) showed the significantly lower levels of serum iron, total iron binding capacity and transferrin saturation as compared to the control group (table 2).
Table 2. The values of ferrokinetics in anemia of chronic diseases of different origin
|
Index |
Control group |
Anemia of chronic diseases in infectious-inflammatory processes |
Anemia of chronic diseases in rhematoid arthritis |
P value |
|
1 |
2 |
3 |
||
|
Serum iron, mcM/l |
20.4 ± 1.02 |
11.1 ± 1.9 * |
9.5 ± 1.0 * |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.472 |
||||
|
TIBC, mcM/l |
65.7 ± 1.83 |
53.2 ± 4.5 * |
49.7 ± 6.56 * |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.653 |
||||
|
LIBC, mcM/l |
44.5 ± 1.87 |
42.9 ± 4.38 |
38.4 ± 8.02 |
р1-2 = 0.734 |
|
р1-3 = 0.460 |
||||
|
р2-3 = 0.624 |
||||
|
Transferrin saturation ratio, % |
32.3 ± 1.84 |
14.9 ± 2.73 * |
16.7 ± 2.02 * |
р1-2 = 0.000 |
|
р1-3 = 0.002 |
||||
|
р2-3 = 0.608 |
||||
|
SF, ng/ml |
33.55 ± 2.59 |
178.6 ± 75.52 |
238.4 ± 64.16 |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.548 |
Note: * – reliability of differences in values as compared to the control group
Therefore, the values of ferrokinetics and the erythron condition in the infectious-inflammatory processes and the autoimmune diseases of connective tissue demonstrate the functional deficiency of iron, when iron is blocked in macrophages and erythron incurs the iron deficiency that is indicated by hypochromic and monocytic pattern of anemia.
The results of the examination of the immune status in anemia with rheumatoid arthritis showed the decrease in the total amount of CD3+СD19-lymphocytes in peripheral blood by means of the population of CD3+CD4+-cells as compared to the healthy individuals (p = 0.000). The average level of CD3+CD4+ was 40.8 ± 2.46 % in the patients with RA and anemia and it was significantly lower than the similar value in the healthy individuals – 52.2 ± 1.65 % (p = 0.000). The average level of cytotoxic CD3+CD8+-cells was 27.6 ± 2.82 % in the patients with RA and anemia and it did not differ significantly from the values in the healthy individuals (p = 0.667). The immune regulatory index (IRI) was 1.46 ± 0.05 and was lower as compared to the control group (p = 0.000). The estimation of CD3-CD(16+/56+)-lymphocytes showed the decrease in in the level of this population in peripheral blood as compared to the healthy individuals (p = 0.009) (table 3).
Table 3. The subpopulation composition of peripheral blood lymphocytes in anemia of chronic diseases of different origin
|
Value |
Control group |
Anemia of chronic diseases in rhematoid arthritis |
Anemia of chronic diseases in infectious-inflammatory processes |
P value |
|
1 |
2 |
3 |
||
|
Leukocytes, ×109/l |
6.7 ± 0.31 |
5.9 ± 0.38 * |
6.9 ± 0.28 |
р1-2 = 0.000 |
|
р1-3 = 0.450 |
||||
|
р2-3 = 0.000 |
||||
|
Lymphocytes, % |
34.1 ± 0.82 |
31.4 ± 2.22 |
27.7 ± 0.97* |
р1-2 = 0.979 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.13 |
||||
|
Lymphocytes, ×109/l |
2.3 ± 0.09 |
1.9 ± 0.1* |
1.9 ± 0.22 |
р1-2 = 0.003 |
|
р1-3 = 0.083 |
||||
|
р2-3 = 0.8 |
||||
|
CD3+CD19-, % |
73.6 ± 1.22 |
69.9 ± 1.69 |
72.7 ± 1.11 |
р1-2 = 0.072 |
|
р1-3 = 0.598 |
||||
|
р2-3 = 0.183 |
||||
|
CD3+CD19-, ×109/l |
1.7 ± 0.06 |
1.3 ± 0.06* |
1.4 ± 0.05* |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.527 |
||||
|
CD3+CD4+, % |
52.2 ± 1.65 |
40.8 ± 2.46* |
44.5 ± 1.27* |
р1-2 = 0.000 |
|
р1-3 = 0.001 |
||||
|
р2-3 = 0.168 |
||||
|
CD3+CD4+, ×109/l |
1.2 ± 0.07 |
0.8 ± 0.06* |
0.8 ± 0.05* |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.954 |
||||
|
CD3+CD8+, % |
26.3 ± 1.74 |
27.6 ± 2.82 |
27.5 ± 1.11 |
р1-2 = 0.667 |
|
р1-3 = 0.613 |
||||
|
р2-3 = 0.967 |
||||
|
CD3+CD8+, ×109/l |
0.6 ± 0.05 |
0.5 ± 0.04 |
0.5 ± 0.02 |
р1-2 = 0.257 |
|
р1-3 = 0.178 |
||||
|
р2-3 = 0.827 |
||||
|
CD3+HLA-DR+, % |
3.1 ± 0.9 |
10.4 ± 1.97* |
15 ± 0.62* |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.013 |
||||
|
CD3+HLA-DR+, ×109/l |
0.1 ± 0.01 |
0.2 ± 0.02*◊ |
0.3 ± 0.04*◊ |
р1-2 = 0.000 |
|
р1-3 = 0.000 |
||||
|
р2-3 = 0.014 |
||||
|
CD3-CD16+56+, % |
9.8 ± 0.87 |
6.6 ± 0.6*◊ |
13.4 ± 0.92* |
р1-2 = 0.009 |
|
р1-3 = 0.008 |
||||
|
р2-3 = 0.000 |
||||
|
CD3-CD16+56+, ×109/l |
0.2 ± 0.03 |
0.1 ± 0.01*◊ |
0.3 ± 0.02 |
р1-2 = 0.01 |
|
р1-3 = 0.474 |
||||
|
р2-3 = 0.000 |
||||
|
CD3-CD19+, % |
9.6 ± 0.65 |
9.3 ± 0.79 |
9.0 ± 0.46 |
р1-2 = 0.734 |
|
р1-3 = 0.498 |
||||
|
р2-3 = 0.772 |
||||
|
CD3-CD19+, ×109/l |
0.2 ± 0.04 |
0.2 ± 0.03 |
0.2 ± 0.01 |
р1-2 = 0.473 |
|
р1-3 = 0.446 |
||||
|
р2-3 = 1.00 |
||||
|
IRI |
1.9 ± 0.08 |
1.5 ± 0.05* |
1.8 ± 0.13 |
р1-2 = 0.000 |
|
р1-3 = 0.678 |
||||
|
р2-3 = 0.01 |
Note: * – reliability of differences in values as compared to the control group; ◊ - reliability of differences in persons with anemia of chronic diseases in RA as compared to the values in persons with anemia of chronic diseases in infectious-inflammatory processes
Patients with anemia of chronic disease and the infectious-inflammatory processes demonstrate the disbalance in the system of T-lymphocytes. The decrease in the absolute amount of CD3+CD19-lymphocytes (as compared to the healthy individuals) was associated with the significant reduction of lymphocytes expressing CD4+-antigen, whereas the level of CD3+CD8+-lymphocytes and IRI did not change significantly and did not vary from the healthy individuals. The absolute amount of CD3-CD(16+/56+)-lymphocytes did not vary significantly in the patients with anemia and the infectious-inflammatory processes as compared to the healthy individuals (table 3).
The intensity of the immune response is directly associated with activity of various subpopulations of immune competent cells. The amount of T-lymphocytes expressing the activation marker HLA-DR increased in the patients with ACD in rheumatoid arthritis and in the infectious-inflammatory processes (table 3).
Our examinations show that the highest increase in CD3+HLA-DR+-lymphocytes was found in the patients with anemia of chronic disease with the infectious-inflammatory processes (0.3 ± 0.04 × 109/l vs. 0.2 × 109/l in the patients with ACD and RA, p = 0.014). The increased expression of HLA-DR molecules shows the intensity and activity of the inflammatory process and it distinguishes ACD in the infectious-inflammatory processes from ACD in RA.
The examination of the levels of lymphocytes identified the significant decrease in the amount of T-lymphocytes with the phenotype of T-reg cells (CD3+ CD4+CD25bright, CD127dim-to-neg) in the patients with ACD at the background of RA. The level of T-reg was 1.2 ± 0.08 % in the patients with RA and anemia and it was significantly lower than the similar value in the healthy individuals (3.9 ± 0.06 %, p = 0.000) (table 4).
Table 4. The level of T-regulatory cells with phenotype CD3+ CD4+CD25bright, CD127dim-to-neg in peripheral blood in anemia of chronic diseases of different origin
|
Index |
Control group |
Anemia of chronic diseases in rhematoid arthritis |
Anemia of chronic diseases in infectious-inflammatory processes |
P value |
| 1 | 2 | 3 | ||
|
Leukocytes, ×109/l |
6.7 ± 0.31 | 5.9 ± 0.38 * | 6.9 ± 0.28 | р1-2 = 0.000 |
| р1-3 = 0.450 | ||||
| р2-3 = 0.000 | ||||
|
Lymphocytes, % |
34.1 ± 0.82 | 31.4 ± 2.22 | 27.7 ± 0.97* | р1-2 = 0.979 |
| р1-3 = 0.000 | ||||
| р2-3 = 0.13 | ||||
| ЛLymphocytes, ×109/l | 2.3 ± 0.09 | 1.9 ± 0.1* | 1.9 ± 0.22 | р1-2 = 0.003 |
| р1-3 = 0.083 | ||||
| р2-3 = 0.8 | ||||
|
Т-reg CD3+ CD4+CD25bright, CD127dim-to-neg,% |
3.9 ± 0.06 | 1.2 ± 0.08*◊ | 4.2 ± 0.11* | р1-2 = 0.000 |
| р1-3 = 0.000 | ||||
| р2-3 = 0.000 | ||||
|
Т-reg CD3+ CD4+CD25bright, CD127dim-to-neg×109/l |
0.1 ± 0.002 | 0.02 ± 0.003*◊ | 0.06 ± 0.001 | р1-2 = 0.000 |
| р1-3 = 1.000 | ||||
| р2-3 = 0.000 |
Note: * – reliability of differences in values as compared to the control group; ◊ - reliability of differences in persons with anemia of chronic diseases in RA as compared to the values in persons with anemia of chronic diseases in infectious-inflammatory processes
At the modern stage, T-reg cells are considered as the main immune regulatory cells, which are able to suppress the immune response types mediated by Th1- and Th2-lymphocytes [2, 6]. The significant decrease in the amount of CD3+ CD4+ CD25bright and CD127dim-to-neg-lymphocytes in the patients with ACD and RA shows the activity of the immune inflammatory process in rheumatoid arthritis that is associated with disappearance or attenuation of the restricting function of T-reg cells in relation to the autospecific clones of T-cells.
There are some findings about the role of T-reg lymphocytes in development of autotolerance and suppression of excessive inflammatory response in various autoimmune diseases [6, 10]. Our results concerning the decrease in T-reg cells in the patients with ACD at the background of RA show the significance of T-reg in development of the immune inflammatory process in RA and in development of anemic syndrome.
The level of T-regulatory lymphocytes tended to increase in the patients with anemic syndrome in the infectious-inflammatory diseases, but the absolute amount did not vary significantly from the healthy individuals.
CONCLUSION
The patients with anemia of chronic disease demonstrate the evident changes in the cellular link of immunity that testified the disbalance in the system of T-cells in combination with suppressed level of natural (mediated by NK-cells) cytotoxicity in the patients with rheumatoid arthritis. The level of regulatory T-cells, which are important for suppressing excessive inflammatory response and autotolerance, significantly decreases in ACD at the background of RA. It can be the predictor of development of anemic syndrome.
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