Other Implementation Considerations

Sickle cell disease (SCD) is characterized by significant morbidity and mortality and studies have shown that infection is the leading cause of death in Africa patients. Overwhelming infections caused by encapsulated bacteria (such as salmonella spp.) and Plasmodium falciparum are an important cause of morbidity and death in patients with sickle cell disease^37,38. The most important contributing factors to this increased susceptibility to encapsulated bacteria are: a state of functional asplenia, an opsonophagocytic defect due to abnormality in the alternative complement pathway, deficiency of specific circulating antibodies, impaired leukocyte function and loss of both humoral and cell mediated immunity.

This study assessed the cellular immunity using CD4+ T lymphocytes count, serum INF-γ and IL-4 levels in patients with sickle cell anaemia. These values were correlated with spleen sizes, in order to predict the susceptibility of HbS patients to infection.

The study population comprised 40 HbS patients in steady state and 40 HbA normal control subjects. The median age for HbS patients was 25.5 years. There were more females subjects with sickle cell anaemia than male. This was not unexpected as female patients have been shown to live longer than their male counterpart⁴⁷, and this has been reflected in the pattern of clinic attendance of HbS patients. The levels of CD4 T lymphocytes, serum IFN-γ and IL-4 were not affected by gender status both in patients and control population.

Eight (20%) of the HbS patients had autosplenectomy, which was confirmed by ultrasonography as indicated by partial or no visualization of the spleen. Twenty-six (65%) had normal splenic size on ultrasonography while splenomegaly was observed in 6 (15%) of

62

the patients. Studies have shown that most HbS patients do not have palpable spleen beyond the eighth year of life^29,34 when the spleen is expected to have undergone autosplenectomy, from repeated vaso-occlusion and infarction. However, this is not always the case with some HbS patients living in the tropics, where the spleen remains palpable in the older age group (adolescent and adult)³⁰. Factors such as high levels of foetal haemogobin, chronic malaria infection and co-inheritance of alpha thalassaemia, have been associated with the persistence of splenomegaly in older HbS patients³²

In this study, the median CD4 T lymphocytes count in both control subjects (937 cells/µL) or HbS patients (939 cells/µL) was lower than the mean CD4 T lymphocytes count reported by Koffi et a l(2003)⁸ in Cote d’Ivoire in control (1215 cells/µL) and HbS (1656 cells/µL) population but similar to the value reported in healthy Nigeria adults ( median 847 cells/µL) by Oladepo et al(2008)⁴⁸ . There was no significant difference between median values of CD4 T-cell in patients with SCA and in the controls (p=0.704). This was in accordance with previous report by Koffi et al(2003)⁸ but in contrast to the study by Kaaba et al(1989)¹²,where decreased number of CD4 cells were reported in SCA patients compared with HbA controls. Sixty-five percent of the HbS patients in this study had normal spleen size which may explain why they have similar median CD4 T-cell value compared with control. Koffi et al⁸ indicated a significant decrease in CD4 T-cell in sickle anaemia patients with asplenia when compared with those with normal spleen size. This study showed lower value of median CD4 T-cell in HbS patients with asplenia (923 cells/µL) compared with HbS patients with normal spleen size (1030 cells/µL), though not significant (p=0.539). This may be related to few number of asplenic patients. Persistence of the spleen in some HbS patients has been attributed to chronic malaria infection in Nigeria ³¹. In this study 37.5% of

63

the patients had malaria in the preceding 1year of the study and 80% had normal or enlarged spleen (6-16.4cm). The highest CD4 count value was recorded among HbS patients with normal spleen size, however there was no significant difference in the median levels of CD4 count value among the 3 groups of HbS according to the spleen size (p=0.539). This is in contrast to Koffi et al⁸ (2003) who observed significantly higher mean CD4+ T lymphocytes count among HbS patients with normal spleen size compared with those with asplenia.

Donadi and Falcao⁴⁹ queried whether the changes of lymphocyte subsets in sickle cell anaemia are due to the loss of splenic function. His study also showed that the number of total lymphocytes, T-lymphocyte subsets and B-lymphocyte subsets were increased in both splenectomised patients and sickle cell anaemia patients. It would be of interest if a subset of splenectomised patients is compared with HbS patients in Nigeria.

By secreting cytokines, CD4+ T lymphocyte influence the function of virtually all other cells of the immune system, including other T cells, B cells, macrophages, and natural killer cells^10,41,42.

Patterns of cytokine expression regulate lymphocyte effector mechanisms^41,42. Human Th1 cells within the CD4 T cells population produce the following cytokine IFN-γ, TNF-β, and IL-2, while the Th2 clones produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13. Th1 cytokines are associated with cell-mediated inflammatory responses, whereas Th2 cytokines are associated with strong antibody and allergic responses. Cytokines from Th1 cells inhibits the actions of Th2 cells and vice-versa, thus an immune response becomes polarized to a predominant Th1 or Th2 type.

The prototype of human Th1 cytokines is IFN-γ, they promote the production of IgG2a, opsonizing and compliment-fixing antibodies, macrophage activation, antibody –dependent

64

cell-mediated cytotoxity, and delayed type hypersensitivity. In this study, IFN-γ was significantly higher among HbS patients (median 8.6pg/mL) than control (5.58pg/mL), (p=0.04) which might be accounted for by subclinical chronic inflammatory state in HbS patients. However, there was no significant difference in IFN-γ level in patients that have had pneumonic infectious complication within a year preceding the study. The elevated value obtained in these HbS patients might also be from other sources of interferon gamma such as CD8 T lymphocytes as the study by Koffi et al (2003)⁸ showed that numbers of CD8 T lymphocytes were significantly increased in HbS patients . This was in agreement with Pathare et al (2004)⁵⁰ but in contrast to report obtained by Raghupathy et al (2000)⁵¹ and Taylor et al (1999)⁵² who found that there was no significant difference in the IFN-γ level between HbS and HbA individuals.

IFN-γ level was found to be significantly higher in female HbS patients as compared to their female HbA counterparts. However, IFN-γ value was not affected by gender status both in HbS patients and control population. This was in agreement with Raghupathy et al (2000)⁵¹ who found that there was no gender bias on the IFN-γ level. Karpuzoglu-Sahin et al (2001)⁵³ found that estrogen increases the production IFN-γ. The increase in IFN-γ level in female HbS patients in this study may be due to the disease couple with hormonal factor. Study by Miller et al (1990)⁵⁴ found that IFN-γ significantly decreases synthesis of HbF. One of the survival factors which the female HbS patients have over the male HbS patients has been identified as increase in HbF level. It has been shown by miller et al (1990)⁵⁴ that IFN-γ suppresses the production of HbF. The rise in IFN-γ level and degree of negative influence on the production of HbF has to be further evaluated in HbS patients. Marcal et al (2007)⁵⁵ recorded significantly higher IFN-γ in HbS patients and implied that this may contribute to

65

inflammation and tissue damage in these patients. IFN-γ level was highest in HbS group with asplenia compared with HbS group with normal spleen and splenomegaly who had similar values. However this difference did not reach stastical significance, perhaps a larger population of patients will resolve this issue. However, there was a significantly higher IFN-γ in HbS with asplenia when compared with the control population (p=0.025). There was a negative relationship (r=0.023) between spleen size and IFN-γ level though not statistically significant (p=0.887).

The prototype of human Th2 cytokines is IL-4; they provide optimal help for humoral immune responses, including IgG1 and IgE switching and mucosal immunity, stimulation of mast cells, eosinophil growth and differentiation, and IgA synthesis. HbS patients (6.74pg/mL) in this study had similar IL-4 values with the HbA individuals (7.67pg/mL).

This is in agreement with Pathare et al (2004)⁵⁰ and Musa et al (2010)⁴⁵ who found that there was no significant difference in the levels of IL-4 level in HbS and HbA individuals in Oman and Nigerian population respectively. Taylor et al (1997)⁵⁶ found significantly higher level of IL-4 in HbS patients than the HbA control subjects in America.

IL-4 is important in antibody production, in view of the fact that in this study there was no difference in the levels of IL-4 between HbS patients and HbA controls, this could explain the finding by Olaniyi et al⁵⁷ on similar values of immunoglobulin classes in HbS patients and HbA individuals. As regards influence of gender in this work there was no stastically significant difference in both HbS patients and HbA controls which is in agreement with Raghupathy et al (2000)⁵¹. There was a negative relationship (r=0.179) between splenic size and IL-4 in HbS patients however this difference was not significant (p=0.269).

66

The commonest causes of infectious complication warranting hospital admission in the University College Hospital Ibadan include malaria (37.5%) and Pneumonia (15%) in HbS adult patients.