Impact of iron deficiency anaemia on T lymphocytes & their subsets in children

Transcription

1 Indian J Med Res 124, December 2006, pp Impact of iron deficiency anaemia on T lymphocytes & their subsets in children Shalini Mullick, Usha Rusia*, Meera Sikka* & M.A. Faridi** Department of Pathology, St Johns Medical College, Bangalore, Departments of *Pathology, **Pediatrics, University College of Medical Sciences & Guru Teg Bahadur Hospital, Delhi, India Received January 19, 2006 Background & objectives: While there is evidence of an altered immune profile in iron deficiency, the precise immunoregulatory role of iron is not known. Information particulary in children who are vulnerable to iron deficiency and infection, is lacking. We undertook this study with the aim of documenting the changes in T cell subsets in children in the age group of 1 to 5 yr with iron deficiency. Methods: The levels of T lymphocytes, their CD4+ and CD8+ subsets and the CD4 : CD8 ratio were evaluated in 40 iron deficient and 30 healthy children. The impact of oral iron supplementation for three months on the same parameters was also noted in 30 children. Results: Significantly lower levels of T lymphocytes as well as CD4+ cells was observed in the iron deficient children (P<0.01 and respectively). The CD4 : CD8 ratio was also significantly lower in this group (P<0.05). Iron supplementation improved the CD4 counts significantly. Interpretation & conclusion: Our study demonstrated quantitatively altered T cell subsets in iron deficiency in children, and a relationship between the severity of haematological and immunological compromise. The clinical and epidemiological implications of this relationship have topical relevance since ID is the most common micronutrient deficiency worldwide. Key words Cell-mediated immunity - iron deficiency - T lymphocytes Anaemia is the most common nutritional disorder in the world and iron deficiency (ID) is implicated in a majority of these cases 1. While the prevalence of ID varies across the world, it is well known that children and women in the reproductive age group are especially vulnerable to ID. The significance of iron as an essential micronutrient is well recognized and a variety of systemic, non-haematological effects 647

2 648 INDIAN J MED RES, DECEMBER 2006 of iron deficiency are known 2. Both experimental, and some clinical studies have emphasized the importance of iron in the integrity of the immune system especially the innate immunity (decreased bactericidal effect and respiratory burst of neutrophils) and the cellular component system of active immunity (decreased lymphocyte proliferation and delayed hypersensitivity responses) 3,4. A recent study in pregnant women has documented significant effects of ID on cellular immune functions especially lowered levels of CD3, CD4 and CD4/CD8 ratio 5. Similarly levels of cytokines such as interleukin 2 (IL2) and IL6, have also been found to be significantly lower in iron deficiency 4,5. While some parameters of the immune system such as delayed type hypersensitivity responses 6-8 and proliferative responses of lymphocytes 9-11 have been conclusively shown to be impaired, many other indicators of the immune response have neither been evaluated nor convincingly established in iron depleted states 12,13. Though the circulating levels of total T lymphocytes and their helper and cytotoxic subsets in iron deficiency have been investigated by many groups 4,5,14-16, very few studies have been done in children 17,18. We therefore carried out this study to establish the altered status of these parameters in iron deficient preschool children, a population segment that is especially vulnerable to iron deficiency as well as infection. Further, it was also planned to evaluate the changes in these parameters if any, after correcting the iron deficiency. Material & Methods Study design: The study was conducted in the departments of pathology and paediatrics of University College of Medical Sciences, Delhi, India. This is a large tertiary care centre which caters to a large suburban population of northern India. A total of 203 consecutive children in the age group of 1-5 yr with anaemia who attended the haematology clinic from January 2002 to March 2003 were included in the study. The inclusion criteria were serum C-reactive protein (CRP) levels <0.6 mg/dl, no history of chronic disease, weight >80 per cent of 50th centile for that age and sex as per National Center for Health Statistics (NCHS criteria) 19. Iron deficiency anaemia was defined by the following criteria: (i) haemoglobin <110 g/l, (ii) mean corpuscular volume (MCV) <80 fl, (iii) serum ferritin <20 µg/l, and (iv) no intake of haematinics in preceding one month. Thirty healthy children of the same age group were selected from the well baby clinic in the control group. Inclusion criteria were: (i) serum CRP levels < 0.6 mg/dl; (ii) no history of chronic disease; (iii) weight >80 per cent of 50th centile for that age and sex (NCHS criteria) 19 ; (iv) absence of anaemia and iron deficiency with haemoglobin >110 g/l, MCV >80 fl, and serum ferritin >20 µg/l; and (v) no intake of haematinics in last one month. Informed consent was taken from either parent of the child. The protocol was approved by the ethical committee of the institution. Venous blood samples (8 ml) were collected following a detailed clinical examination. Blood samples were delivered in three containers as follows and two good peripheral smears made: (i) 2 ml blood was anticoagulated with disodium EDTA for processing on an automated haematology cell counter (MS-9, Melet-Scholesing laboratories, France) for a complete blood count 20. A reticulocyte count was also performed on the sample (using New Methylene Blue, Dacie) 21. (ii) 1 ml was anticoagulated with tripotassium EDTA. The sample was analyzed by flow cytometry for the levels of CD3, CD4 and CD8 lymphocytes in a FACSCount Flow-cytometer (Becton & Dickinson, USA) using monoclonal antibodies (Becton & Dickinson, USA) against these antigens 22.. CD3, CD4, and CD8 lymphocytes were enumerated. The percentages of CD4, CD8 lymphocytes were calculated from these. The CD4:CD8 ratio was also calculated in both groups. (iii) 4 ml was collected in plain glass tubes and the serum was used for the estimation of serum ferritin (Human ferritin enzyme

3 MULLICK et al: T CELL SUBSETS IN IRON DEFICIENT CHILDREN 649 immunoassay, Diagnostic Automation Inc, USA) 21 and CRP (CRP Latex agglutination, Tulip Diagnostics, India) 23. Children in the iron deficiency group were administered tablet albendazole in a single dose (200 mg for children <2 yr and 400 mg for children >2 yr). After that they were prescribed oral iron in the form of ferrous sulphate in a dose of 6 mg/kg body weight given in 2 equally divided doses for a duration of 100 ± 10 days. Compliance was assessed every fortnight by interview. The haematological and immunological parameters evaluated in each child were repeated at the completion of three months of oral iron supplementation. As no published reference ranges for T lymphocytes and their subsets in Indian children could be obtained from published literature, values obtained from the control group have been used for defining normal ranges. Statistical analysis: The control and the iron deficient group were compared for haematological and immunological parameters using the unpaired t-test. At the end of 100 ± 10 days of iron supplementation, haematological and immunological status of the 30 iron deficient children who were followed up was compared using the paired t-test. The data were analyzed using Bonferroni corrections as there were 3 comparisons and significance value used was 2 per cent. Results Of the 203 children presented with anaemia, 109 were found to have iron deficiency anaemia as the solitary pathology. Of these, 69 children were identified with one or more exclusion criteria and were excluded. The remaining 40 children had iron deficiency anaemia as per our inclusion criteria. Of these, 21 had haemoglobin <90 g/l. Thirty healthy asymptomatic children selected from the well baby clinic served as the control group. The iron deficient and control group were comparable with respect to the age and sex distribution. The mean haemoglobin, RBC count, red cell indices and serum ferritin were significantly lower in the iron deficient group. The total leukocyte count and absolute lymphocyte counts were comparable (Table I). Table I. Haematological parameters in iron deficient and control groups Control (n=30) Iron deficient (n=40) Haemoglobin (g/l) ± 0.79 ( ) 85.2 ± 2.02** (33-109) RBC (x /l) 4.69 ± 0.31 ( ) 4.29 ± 0.68* ( ) PCV (l/l) 38.1 ± 1.8 ( ) ± 5.72** ( ) MCV (fl) ± 3.4 ( ) ± 9.46** ( ) MCH (pg) ± 1.75 ( ) ± 4.21** ( ) MCHC (g/dl) 31.8 ± 1.33 ( ) ± 3.28** ( ) RDW 8.03 ± 0.7 ( ) ± 1.41** ( ) TLC x 10 9 /l ± 2.56 ( ) ± 3.93 ( ) Lymphocyte X10 9 /l 5.34 ± 1.45 ( ) 6.02 ± 2.14 ( ) Platelet X10 9 /l ± ( ) ± 163.1** (87-929) Reticulocyte (%) 2.07 ± 0.65 ( ) 2.09 ± 1.15 ( ) Serum ferritin (µ/l) ± (24-80) 4.38 ± 1.31** ( ) Values are mean ± SD (range). P*<0.005, **0.001 compared to controls RBC, red blood cells; PCV, packed cell volume; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, MCH concentration; RDW, red cell distribution width; TLC, total leukocyte count

4 650 INDIAN J MED RES, DECEMBER 2006 The mean value of CD3 lymphocytes in the iron deficient group was significantly lower (P<0.010) than the control group. Anaemic children with haemoglobin (Hb) less than 90 g/l (n=21) had mean CD3 count of /µl. The mean CD4+ counts were significantly lower (P<0.002) in iron deficient children as compared to controls. The mean values of CD4 counts was still lower in children with Hb<90.0 g/l, being /µl. Similarly the mean percentage of CD 4+ lymphocytes in iron deficient group was lower compared to controls. Children with haemoglobin <90 g/l had mean CD4 percentage of Although no statistically significant difference in CD8+ counts was seen between iron deficient children and controls, the percentage of CD 8 positive lymphocytes was slightly higher in iron deficient children. In iron deficient children with haemoglobin less than 90 g/l, the mean CD8 count (1333/µl)and percentage (43.31%) were significantly higher. The iron deficient children recorded a significantly lower mean CD4:CD8 ratio (P<0.05) than the control group. In children with haemoglobin < 90.0 g/l, the CD4: 8 ratio was still lower at 1.3 (P<0.01) (Table II). Of the initial 40 iron deficient children included in the study, only 30 were available for re-evaluation at the end of three months of oral iron supplementation. Haemoglobin, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and MCHC increased significantly (P<0.001) after completion of iron therapy. All cases recorded a rise in haemoglobin, which ranged from 2.0 to 49.0 g/l. The mean change in haemoglobin was 25.3 g/l. Twenty nine of the 30 children recorded a rise in haemoglobin, which was more than 5 g/l; 20 of the 30 children became non anaemic at the end of the study, i.e., with haemoglobin >110 g/l. The red cell distribution width (RDW) and platelet counts decreased significantly. A significant rise (P<0.001) in reticulocyte count was observed after iron supplementation. The total leukocyte and absolute lymphocyte counts remained similar. Though the mean value of serum ferritin increased the change was not statistically significant (Table III). The mean CD3 count rose significantly (P<0.05) after iron supplementation and was comparable to the mean value in the control group. At the end of three months of oral iron supplementation, 23 of the 30 (76.7%) children recorded an increase in the levels of CD 3 positive lymphocytes. The 20 children who had Hb ³ 110g/l after iron supplementation recorded a mean CD3+ count of 3434/µl, which was very close to the value of the control group. The 10 children who remained anaemic even after iron supplementation recorded a mean CD3+ count of /µl. The mean CD 4 lymphocyte values increased significantly (P<0.01) after therapy and approached the control level (Table IV). The CD 4 lymphocyte level rose in 25 of the 30 children Table II. T lymphocytes and their subsets in control and iron deficient groups Control (n=30) Iron deficient (n=40) CD 3+ count (cells/µl) ± ( ) ± ** ( ) CD 4+ count (cells/µl) ± ( ) ± *** ( ) CD 4+ percentage ± 8.85 ( ) ± 7.05 ( ) CD 8+ count (cells/µl) ± ( ) ± ( ) CD 8+ percentage 39.2 ± 8.58 ( ) ± 8.32 ( ) CD4 : CD8 ratio 1.59 ± 0.41 ( ) 1.4 ± 0.4* ( ) Values are mean ± SD (range). P*<0.05, **0.01, ***<0.002 compared to controls

5 MULLICK et al: T CELL SUBSETS IN IRON DEFICIENT CHILDREN 651 (83.3%). The value remained unchanged in one case and decreased slightly in 4 cases. When the post therapy group was analysed according to the haemoglobin levels, it was observed that the children who had Hb <110 g/l even after iron supplementation had a mean CD4 count of /µl, while those who were not anaemic had a mean CD4 count of / µl. The mean percentage of CD 4+ cells increased to per cent after therapy and was similar to the value in the control group (58.48%). Mean CD8 lymphocytes did not show significant difference after therapy although there was a slight decline in the mean CD8 percentage. The mean CD 4: CD 8 ratio improved with therapy and approached that of the control group (Table IV). Discussion Our observations that the iron deficient children had a significantly lower mean level of CD3 lymphocytes as compared to the control group were consistent with earlier reports 6,9. Kemhali et al 24 quantified the percentage of T lymphocytes and found it significantly lower in iron deficient compared to control children. Berger et al 17 evaluated ID children and found that the number as well as percentage of mature T cells were lower as compared to controls. In this study, the CD3 counts were shown to be correlated with free erythrocyte protoporphyrin (FEP), MCV, Hb, tranferrin saturation and serum iron. Among Table III. Haematological parameters before and after iron supplementation Before iron supplementation After iron supplementation Haemoglobin (g/l) 86.1 ± 0.21 (33-109) ± 0.16** (71-129) RBC count (x /l) 4.65 ± 0.65 ( ) 4.66 ± 0.5 ( ) PCV (l/l) ± 5.29 ( ) ± 4.44** ( ) MCV (fl) ± 9.63 ( ) ± 6.62** ( ) MCH (pg) ± 4.42 ( ) ± 3.56** ( ) MCHC (g/dl) ± 3.48 ( ) ± 1.99** ( ) RDW 10.6 ± 1.44 ( ) 9.55 ± 1.4** ( ) Platelet count (x 10 9 /l) ± ( ) ± * ( ) Reticulocyte % 2.08 ± 1.18 ( ) 5 ± 1.67** ( ) Serum ferritin (µg/l) 4.68 ± 1.36 ( ) 7.61 ± 3.13 ( ) Values are mean ± SD (range). (n=30). P*<0.005, ** <0.001 compared to before iron supplementation Abbreviations as in Table I Table IV. T lymphocytes and their subsets before and after iron supplementation Before iron supplementation After iron supplementation CD 3+ count (cells/µl) ± ( ) ± * ( ) CD 4+ count (cells/µl) ± ( ) 1900 ± ** ( ) CD 4+ percentage ± 7.85 ( ) ± 6.89* ( ) CD 8+ count (cells/µl) ± ( ) ± ( ) CD 8+ percentage ± 8.99 ( ) ± 6.26 ( ) CD4 : CD8 ratio 1.42 ± 0.43 ( ) 1.49 ± 0.36 ( ) Values are mean ± SD (range). (n=30). P*<0.05, **<0.01

6 652 INDIAN J MED RES, DECEMBER 2006 studies carried out on adults with iron deficiency lower levels of CD3 positive lymphocytes have been reported Our findings on lower CD3 lymphocytes in iron deficiency are consistent with these authors. Moreover, with greater degree of anaemia (Hb <90 g/l) the CD3 levels were further decreased. We also observed a significantly lower level of CD4 lymphocytes in the iron deficient children. Thibault et al 18 reported a qualitative rather than a quantitative defect in cell mediated immunity (CMI) in 3-36 month old children. They observed decreased in vitro IL-2 production by lymphocytes of iron deficient children; while the numbers of lymphocytes were noted to be similar in iron deplete and control group. However, the entire spectrum of iron deficiency was not covered as only children with haemoglobin ranging from g/l were included. All children in our study were screened for chronic disorders and infection and none had CRP levels > 0.6 mg/dl. While the authors 18 have specified that the depressed IL-2 production persisted even after exclusion of children with laboratory signs of inflammation, it is not clear if the lymphocyte analysis was also subject to this exclusion and thus the presence of inflammation as a confounding factor cannot be conclusively ruled out. In our study, the levels of CD 8 lymphocytes were similar in the iron deficient and control groups as also reported by others 17,18. However, Santos & Falcao 14 have observed that while the number of CD 8 cells in ID was unaltered, there was an increase in percentage of CD8 cells. The difference between the mean CD4 : CD8 ratio in the iron deficient group and control group in our study was found to be statistically significant. Reports by other authors regarding the CD4 : CD8 ratio in IDA are variable 14,16,17. An interesting observation in our study was the lower mean values of CD3 and CD4 lymphocytes within the iron deficiency group when children with Hb<90 g/l and those with Hb between g/l were analyzed separately. We observed a trend of lower CD3 and CD4 cells with increasing severity of iron deficiency anaemia. The mean values of CD3 and CD4 lymphocytes in the former group were lower than the mean value in ID children whole. However, these differences did not reach to statistical significance, which may be due to small number of children. Administration of oral iron supplements resulted in a significant improvement in the haematological status of the children. Thus, prevalence of anaemia decreased and the mean haemoglobin improved. Iron supplementation was associated with an increase in CD3 lymphocytes. This indicates that a greater haematological recovery may be needed for an immunological improvement. Similar improvement in CD3 counts have been reported by others 14,16,18. The CD4 lymphocyte level as well as the percentage of CD4 cells was found to rise significantly after therapy with oral iron supplementation. Similar findings have been reported by others 14, The level and percentages of CD8 lymphocytes remained largely unchanged after therapy, showing only a slight decrease. It is important to emphasize that immunological alterations observed in one study may or may not be observed in another, if strict criteria with respect to age groups, infection and malnutrition are not adhered to. Therefore, careful selection of study groups and ensuring adequate comparability are essential for reliable results. Also, a larger number of children, belonging to well defined age groups and different geographic areas need to be evaluated for these parameters in iron deficiency for a conclusive interpretation, especially since the normal values for lymphocytes and lymphocyte subsets have a wide range, and cut-off values are difficult to establish. Analysis of more detailed immunological parameters such as helper T cell subsets may further elucidate the mechanism of lowered immunological competence in iron deficiency. Some recent studies 5,24

7 MULLICK et al: T CELL SUBSETS IN IRON DEFICIENT CHILDREN 653 suggest that cell mediated immunity as well as activity of various cytokines involved in immunogenic mechanisms are influenced by IDA. In conclusion, lower CD3 and CD4 lymphocyte levels and the CD4 : CD8 ratio in iron deficiency in children may contribute to the decreased CMI in iron deficiency. The degree of the anaemia probably influenced the extent of immunological compromise. Increased CD3 and CD4 lymphocytes at the end of iron supplementation period supports the positive effect of iron CMI. Acknowledgment The authors acknowledge the help and co-operation of Dr Usha Baweja and her team at the National Institute of Communicable Diseases, Delhi, India, for making available the facility of flowcytometric analysis, and the University Grants Commission, New Delhi, India, for financial assistance. References 1. UNICEF/UNO/WHO/MI. Preventing iron deficiency in women and children. Technical consensus on key issues. International Nutrition Foundation, Buston, MA; 1999 p Hercberg S, Galan P. Biochemical effects of iron depletion. Acta Pediatr Scand 1989; 361 (Suppl) : Beard JL. Iron biology in immune function, muscle metabolism, and neuronal functioning. J Nutr 2001; 131 (Suppl 2) : 568s-80s. 4. Ekiz C, Agaoglu L, Karakas Z, Gurel N, Yalcin I. The effect of iron deficiency anemia on the function of the immune system. Hematol J 2005; 5 : Tang YM, Chen XZ, Li GR, Zhou RH, Ning H, Yan H. Effects of iron deficiency anemia on immunity and infectious disease in pregnant women. Wei Sheng Yan Jiu 2006; 35 : Chandra RK, Saraya AK. Impaired immunocompetence associated with iron deficiency. J Pediatr 1975; 86 : MacDougall LG, Anderson R, McNab GM, Katz J. The immune response in iron deficient children: Impaired cellular defense mechanisms with altered humoral components. J Pediatr 1975; 86 : Krantman HJ, Young SR, Ank BJ, O Donell MC, Rachelefsky GS, Steihm RE. Immune function in pure iron deficiency. Am J Dis Child 1982; 136 : Srikantia SG, Sivaprasad J, Bhaskaram C, Krishnamachari KAVR. Anemia and immune response. Lancet 1976; 3 : Kuvibidila S, Dardene M, Savino W, Lepault F. Influence of iron-deficiency anemia on selected thymus functions in mice: thymulin biological activity, T cell subsets and thymocyte proliferation. Am J Clin Nutr 1990; 51 : Van Heerden C, Oosthuizen R, VanWyk R, Prinsloo P, Anderson R. Evaluation of neutrophil and lymphocyte function in subjects with iron deficiency. SA Med J 1981; 89 : Beger J, Dyck JL, Galan P, Aplogan A, Schneider D, Traissac P, et al. Effect of daily iron supplementation on iron status, cell mediated immunity and incidence of infections in 6 to 36 month old Togolese children. Eur J Clin Nutr 2001; 54 : Walter T, Aredondo S, Arevalo M, Stekel A. Effect of iron therapy on phagocytosis and bactericidal activity in neutrophils of iron deficient infants. Am J Clin Nutr 1986; 44 : Santos PC, Falcao RP. Decreased lymphocyte subsets and K cell activity in iron deficiency anemia. Acta Hematol 1990; 84 : Zimmer JP, Garza C, Heller ME, Butte N, Goldman AS. Postpartum maternal blood helper T (CD 3+, CD4+) and cytotoxic (CD 3+ CD 8+) cells: correlations with iron status, parity, supplement use and lactational status. Am J Clin Nutr 1998; 67 : Luraschi A, Borgotti P, Gioria A, Fedeli P. Determination of lymphocyte subpopulations, defined with monoclonal antibodies, in patients with iron deficiency anemia. Minerva Med 1991; 82 : Berger J, Schneider D, Dyck JL, Joseph A, Aplogan A, Galan P, et al. Iron deficiency, cell mediated immunity and infection among 6-36 month old children living in rural Togo. Nutr Res 1992; 12 : Thibault H, Galan P, Selz F, Preziosi P, Olivier C, Badoul J, et al. The immune response in iron deficient young children: effect of iron supplementation on cell mediated immunity. Eur J Pediatr 1993; 152 : Ghai OP. Essential pediatrics. 5 th ed. New Delhi: Interprint; 2001.

8 654 INDIAN J MED RES, DECEMBER Perkins LS. Wintrobe s clinical hematology. 10 th ed. USA. Lippincott: Williams and Wilkins; 2001 p Lewis SM, Bain BJ, Bates I. Practical hematology, 9 th ed. London: Churchill Livingstone; Marti GE, Stetler-Stevenson M, Bleesing JJH, Fleisher TA. Introduction to flow cytometry. Sem Hematol 2001; 38 : Kind CRH, Pepys MB. The role of C-reactive protein (CRP) measurement in clinical practice. Int Med 1984; 5 : Kemhali AS, Babacan E, Cavdar AO. Cell mediated immune responses in children with iron deficiency and children with combined iron and zinc deficiency. Nutr Res 1988; 8 : Reprint requests: Dr Usha Rusia, Professor, Department of Pathology, University College of Medical Sciences & GTB Hospital, Dilshad Garden, Delhi , India sprusia@hotmail.com