**5. Immunological dysfunction: Effect on antigen presentation**

Interestingly, thalassemias have been clinically associated with an increased risk of recurrent bacterial infections [70–87]. This is most evident in under-developed nations where sanitary and medical facilities are most lacking. Despite the clinical evidence of recurrent bacterial infections in thalassemic patients, the biological events underlying this finding are unclear. This confusion arises as a natural consequence of the heterogeneity of the microbial disease itself, the patients age, the state of splenic function, the frequency of transfusion, the degree of similarity between the patient and the blood donor pool, the nutritional status of the patient (e.g., United States versus Thailand) and whether one is looking at humoral or cell-mediated immunity [70–87].

In general, studies on the humoral (i.e., immunoglobulin-based) immunity of thalassemic patients suggest that this arm of the immune system is 'relatively' normal. These studies have indicated normal to elevated levels of IgG, IgA, and IgM but decreased levels of Factor B, C3, and C4 (perhaps due to consumption via oxidatively damaged β thalassemic cells). Reflective of this normality, and consequent to the extensive oxidant injury to the thalassemic cells, circulating immune complexes and an elevated risk of autoimmune hemolytic anemia have been described in β thalassemia intermedia and major patients. Serum fractions from these patients also exhibited increased amounts of C1q-precipitable immune complexes. In contrast, as suggested by the clinically described recurrent bacterial infections, cell-mediated immunity is highly suspect in the thalassemic patient (and sickle cell patients). The few direct studies on cell-mediated immunity in thalassemic patients were, typically, enumeration of the mononuclear cell populations (T cells, B cells, NK cells and monocytes). In general, these studies suggest normal cell numbers but a skewed distribution of the CD4+ to CD8+ T cell ratio. The altered ratio was characterized by a relative depression in CD4+ T cells (i.e., helper T cells) and NK (Natural Killer) cells and a relative rise in CD8+ (cytotoxic and suppressor) T cells that increased linearly with the number of units transfused. However, very

**79**

**Figure 8.**

ity; O2

*Model Human β Thalassemic Erythrocytes: Effect of Unpaired Purified α-Hemoglobin Chains…*

few functional studies have been done in thalassemic patients to answer the ques-

Previous studies have suggested that increased bioavailable iron in transfused patients might facilitate the growth of organisms in which iron is a limiting nutrient (i.e., most bacteria). Other studies have implicated the loss of splenic function. While both of these factors may indeed play important roles in recurrent bacterial infections, they may not offer a complete explanation. In addition to thalassemia, a number of other diseases and trauma scenarios are characterized by recurrent bacterial infections (e.g., malaria and burn injury) suggestive of impaired cellmediated immunity. Interestingly, a common characteristic of all these conditions is erythrophagocytosis. Previous studies have demonstrated that phagocytic uptake of IgG-coated and oxidatively stressed RBC resulted in a transient depression of further macrophage phagocytosis, decreased respiratory burst (i.e., NADPH-oxidase activ-

<sup>−</sup> production), and impaired killing of bacteria [88–93]. Interestingly, in the case of *Plasmodium falciparum*-infected RBC, only phagocytosis of mature (trophozoite), but not immature (ring stage), stages had an inhibitory effect on monocyte function. Importantly, a major difference between the mature and immature malarial infected RBC is the presence of malarial pigment (hemozoin), an iron/heme rich degradation product of parasite hemoglobin catabolism. Τhe heme- and iron-rich membranes of the β thalassemic RBC, which we have previously documented [22], may function in a manner analogous to malarial pigment or iron salts and impair cell-mediated immunity—primarily at the level of the APC but potentially extending to the T cell level. Some data from thalassemic patients support the hypothesis for impairment of the T cell response. For example, patients with thalassemia intermedia have been reported to have diminished T cell mitogen responses when their serum

tion: *Why are β thalassemic patients at risk of recurrent bacterial infections?*

iron and ferritin were higher than 200 and 600 μg/dl, respectively [94].

Hence, injury arising from the iron-GSH pathway can result in (any combination of) RBC opsonization by endogenous antibodies, phosphatidylserine (PS) exposure, protein clustering, sublytic levels of complement binding, and/or loss of cellular deformability (**Figure 7**) that leads to the removal of the damaged β thalassemic cells from the circulatory system by components of the mononuclear phagocytic system (MPS). Erythrophagocytosis can occur within the spleen (if present and functioning), liver (Kupffer cells) or the microvasculature itself when

*Antigen processing and presentation is inhibited by oxidized RBC and hemin. (A) Antigen presentation of TT and SM was inhibited by the erythrophagocytosis of oxidized RBC. Normal RBC had no inhibitory effect. The* 

*concentrations. Results shown are of a representative experiment with quadruplicate samples. (B, C) Heme pretreatment of PBMC (2 h at 37°C) dramatically inhibits the proliferative response to tetanus toxoid (B) and*  S. mutans *(C). Interestingly, the ability of PBMC to respond to intact bacteria is more significantly blunted than is the response to tetanus toxoid at intermediate hemin concentrations (50 μM). Results shown are of a* 

*H-thymidine incorporation in proliferating* 

 *per 200 μl. Antigens were diluted in Aim V and added at the indicated* 

 *PBMC per 200 μl.* 

*efficacy of antigen processing and/or presentation was assessed by 3*

*representative experiment with quadruplicate samples.*

*Final RBC concentration was 8 × 106*

*T cells. PBMC were resuspended in Aim V media at a final concentration of 2.5 × 105*

*DOI: http://dx.doi.org/10.5772/intechopen.90288*

#### *Model Human β Thalassemic Erythrocytes: Effect of Unpaired Purified α-Hemoglobin Chains… DOI: http://dx.doi.org/10.5772/intechopen.90288*

few functional studies have been done in thalassemic patients to answer the question: *Why are β thalassemic patients at risk of recurrent bacterial infections?*

Previous studies have suggested that increased bioavailable iron in transfused patients might facilitate the growth of organisms in which iron is a limiting nutrient (i.e., most bacteria). Other studies have implicated the loss of splenic function. While both of these factors may indeed play important roles in recurrent bacterial infections, they may not offer a complete explanation. In addition to thalassemia, a number of other diseases and trauma scenarios are characterized by recurrent bacterial infections (e.g., malaria and burn injury) suggestive of impaired cellmediated immunity. Interestingly, a common characteristic of all these conditions is erythrophagocytosis. Previous studies have demonstrated that phagocytic uptake of IgG-coated and oxidatively stressed RBC resulted in a transient depression of further macrophage phagocytosis, decreased respiratory burst (i.e., NADPH-oxidase activity; O2 <sup>−</sup> production), and impaired killing of bacteria [88–93]. Interestingly, in the case of *Plasmodium falciparum*-infected RBC, only phagocytosis of mature (trophozoite), but not immature (ring stage), stages had an inhibitory effect on monocyte function. Importantly, a major difference between the mature and immature malarial infected RBC is the presence of malarial pigment (hemozoin), an iron/heme rich degradation product of parasite hemoglobin catabolism. Τhe heme- and iron-rich membranes of the β thalassemic RBC, which we have previously documented [22], may function in a manner analogous to malarial pigment or iron salts and impair cell-mediated immunity—primarily at the level of the APC but potentially extending to the T cell level. Some data from thalassemic patients support the hypothesis for impairment of the T cell response. For example, patients with thalassemia intermedia have been reported to have diminished T cell mitogen responses when their serum iron and ferritin were higher than 200 and 600 μg/dl, respectively [94].

Hence, injury arising from the iron-GSH pathway can result in (any combination of) RBC opsonization by endogenous antibodies, phosphatidylserine (PS) exposure, protein clustering, sublytic levels of complement binding, and/or loss of cellular deformability (**Figure 7**) that leads to the removal of the damaged β thalassemic cells from the circulatory system by components of the mononuclear phagocytic system (MPS). Erythrophagocytosis can occur within the spleen (if present and functioning), liver (Kupffer cells) or the microvasculature itself when

#### **Figure 8.**

*Beta Thalassemia*

ektacytometric analysis of the model β thalassemic RBC shows a significant loss of cellular deformability induced by shear stress (e.g., large vessels). Moreover, cell transit analysis of these cells (analogous to capillary deformation) showed a very significant loss of deformability in the model β thalassemic cells as reflected by the

Consequent to the loss of deformability and immune recognition (e.g., Kupffer cells of the liver and, potentially, antibodies), the circulatory survival of β thalassemic RBC is impacted. As demonstrated in **Figure 7A**, model β thalassemic RBC (Blood group A) exhibited enhanced immune recognition and phagocytosis by autologous monocytes when compared to control cells from the same donor. Indeed, the level of phagocytosis was similar to that of the anti-A opsonized positive control RBC. The loss of deformability and enhanced immune recognition both contribute to decreased *in vivo* survival. This was demonstrated using mice transfused with model β thalassemic murine cells (mouse RBC + human α-chains) in which the transfused RBC exhibited a dramatic reduction in the circulatory lifespan (**Figure 7B**). The role of α-chain mediated oxidation was supported by the finding that lightly oxidized (phenazine methosulphate treated; 50 μM) murine cells showed similar circulatory dynamics. These results are comparable to that observed in humans where, consequent to the α-chain driven oxidation, β thalassemic RBC have a very short circulatory lifespan (7–14 days depending on spleen status)

very large and significant increase in transit time (**Figure 6C**).

**5. Immunological dysfunction: Effect on antigen presentation**

Interestingly, thalassemias have been clinically associated with an increased risk of recurrent bacterial infections [70–87]. This is most evident in under-developed nations where sanitary and medical facilities are most lacking. Despite the clinical evidence of recurrent bacterial infections in thalassemic patients, the biological events underlying this finding are unclear. This confusion arises as a natural consequence of the heterogeneity of the microbial disease itself, the patients age, the state of splenic function, the frequency of transfusion, the degree of similarity between the patient and the blood donor pool, the nutritional status of the patient (e.g., United States versus Thailand) and whether one is looking at humoral or cell-mediated immunity

In general, studies on the humoral (i.e., immunoglobulin-based) immunity of thalassemic patients suggest that this arm of the immune system is 'relatively' normal. These studies have indicated normal to elevated levels of IgG, IgA, and IgM but decreased levels of Factor B, C3, and C4 (perhaps due to consumption via oxidatively damaged β thalassemic cells). Reflective of this normality, and consequent to the extensive oxidant injury to the thalassemic cells, circulating immune complexes and an elevated risk of autoimmune hemolytic anemia have been described in β thalassemia intermedia and major patients. Serum fractions from these patients also exhibited increased amounts of C1q-precipitable immune complexes. In contrast, as suggested by the clinically described recurrent bacterial infections, cell-mediated immunity is highly suspect in the thalassemic patient (and sickle cell patients). The few direct studies on cell-mediated immunity in thalassemic patients were, typically, enumeration of the mononuclear cell populations (T cells, B cells, NK cells and monocytes). In general, these studies suggest normal

T cells that increased linearly with the number of units transfused. However, very

to CD8+

T cell ratio. The altered

T cells (i.e., helper T cells)

(cytotoxic and suppressor)

compared to the 120 days of a normal RBC.

cell numbers but a skewed distribution of the CD4+

ratio was characterized by a relative depression in CD4+

and NK (Natural Killer) cells and a relative rise in CD8+

**78**

[70–87].

*Antigen processing and presentation is inhibited by oxidized RBC and hemin. (A) Antigen presentation of TT and SM was inhibited by the erythrophagocytosis of oxidized RBC. Normal RBC had no inhibitory effect. The efficacy of antigen processing and/or presentation was assessed by 3 H-thymidine incorporation in proliferating T cells. PBMC were resuspended in Aim V media at a final concentration of 2.5 × 105 PBMC per 200 μl. Final RBC concentration was 8 × 106 per 200 μl. Antigens were diluted in Aim V and added at the indicated concentrations. Results shown are of a representative experiment with quadruplicate samples. (B, C) Heme pretreatment of PBMC (2 h at 37°C) dramatically inhibits the proliferative response to tetanus toxoid (B) and*  S. mutans *(C). Interestingly, the ability of PBMC to respond to intact bacteria is more significantly blunted than is the response to tetanus toxoid at intermediate hemin concentrations (50 μM). Results shown are of a representative experiment with quadruplicate samples.*

non-deformable RBC are trapped and then cleared by circulating macrophages. Regardless of the location of removal, erythrophagocytosis results in impaired MPS function. As shown in **Figure 8A**, antigen presentation of purified tetanus toxoid (TT; a peptide) or fixed, intact, *S. mutans* (SM; an intact bacteria) by normal human antigen presenting cells (APC; blood monocytes) was dramatically, and differentially, affected by the presence of either control (unoxidized) or oxidized (50 μM PMS as per **Figure 7**) human RBC. As shown, oxidized RBC prevented successful antigen presentation to human T cells while normal RBC showed no detrimental effects. Further experimentation demonstrated that the inhibitory effect was due to heme/iron. As shown in **Figure 8B, C**, direct addition of hemin to the APC impaired successful antigen presentation of both tetanus toxoid and Strep. mutans in a dose dependent manner.
