**3. Thymus and vanadium**

The thymus is a capsulated primary lymphoid organ to which immature peripheral T-lymphocytes, from the bone marrow, arrives to complete its maturation and immune capacitation. It is located in the mediastinum; it has two lobules that originate from the third and fourth branchial poaches. In humans the thymus is fully formed and functional at birth and it reduces its size after puberty; however, it remains functional.

Histologically, it has a connective tissue capsule that extends into the parenchyma dividing it in incomplete lobules. In each lobule medulla and cortex are well delimited. The cortex is highly basophilic when the thymus is stained with hematoxylin and eosin as a consequence of the numerous and rapidly dividing immature T-lymphocytes called thymocytes, while the medulla is less basophilic because the thymocytes density decreases. Other cells in the thymus structure are the epithelial cells, located in the cortex-cortical epithelial thymic cells (cTEC)-, the medullary epithelial thymic cells (mTEC)-in the medulla-, also dendritic cells (DCs) located in the corticomedullary zone and in the medulla in addition of widely distributed macrophages. Small spherical-shaped structures, formed by mTEC, identified as Hassall's corpuscles -are thymic unique structures; its function is to regulate the production and maturation of the regulatory-T cells (Tregs). The thymus has a hematothymic barrier constituted by the vascular face of the endothelial cells from the cortex continuous capillaries, the basal lamina from the cortex continuous capillaries and the cTEC. Its function is to prevent the contact of the circulating antigens with the cortical thymocytes [25].

### **3.1 Positive and negative thymus selection**

The maturation process from thymocytes to immunocompetent T cells depends on the cortical and medullar thymic microenvironments that is established by the cTEC, mTEC, and DC. The cTEC positively select the T-lymphocytes that recognize the major histocompatibility complex (MHC), so the selected T-lymphocytes are CD4+ or CD8+. The mTEC and the DCs are involved in negative T-cell selection and in establishing self-tolerance by eliminating through apoptosis, the lymphocytes which strongly recognize self-antigens, preventing autoreactive clones. Both processes area highly ordinated and rely on the sequential and quantitative expression of markers such as CD4, CD8, T-cell receptors (TCR1, TCR2) as well as of changes in the glycosylation of the membrane proteins on the lymphocytes in the process of maturation [26, 27].

#### **3.2 Dendritic cells**

Dendritic cells (DCs) are antigen -presenting cells (APCs) from the bone marrow. They are a heterogeneous population of cells which are identify because their surfaces biomarkers, cytokines production and location. Their progenitors could be myeloid or lymphoid. DCs are recognized by its typical dendritic morphology and by the expression of CD11c. As all of the other APCs, MHCII is expressed on its surface [28]. The differentiation and function of the DCs could be modulated by different growth factors and cytokines. There are three types: cDCs, the conventional type; pDCs, the plasmacytoid type; and mDCs, the monocyte-derived cells [29].

DCs are a bridge between innate and adaptive immune response. When they are immature its location is the skin and peripheral tissues and when an antigen is captured the cells migrate to present the antigen to the T cells. The thymus DCs and the mTEC are responsible of the thymocytes negative selection; in the medullae the single T cells (SP) interact with the DCs and with the mTEC that present them self-antigens with and immunogenic potential. The T-cell receptor (TCR) from the SP thymocytes recognizes the cells with MHC restriction and high affinity to selfantigens to eliminate them by apoptosis. This is the main mechanism to eliminate autoreactive clones and to establish central tolerance [29].

As it was previous mentioned V could damage health. Previous reports from our group demonstrated that, -in experimental model- mice that inhale V have damages in different systems including the immune system which induces changes in the cortex-medullae ratio [30, 31]. Other observed effect was in the spleen and in the humoral response [32].

Because of the morphological changes in the thymus as a consequence of the V-exposure, we decided to explore the changes in the expression of CD11c and MHCII on the DCs distribution.

We have developed a CD-1 mouse inhalation experimental model to assess systemic effects of PM. We used CD-1 mice (8-weeks-old, 33 + 2 g) from the vivarium of the School of medicine. The animals were randomly placed in acrylic chambers connected to an ultra-nebulizer (UltraNeb99 Devilbiss, Somerset, Philadelphia, USA), the exposure schedule consisted of V2O5 0.02 M (Sigma, St. Louis, Missouri, USA) in saline, 1 hour twice a week for 4 weeks. The ultra-nebulizer with the size of the particle emitted being less than 5 μm (range 0.5–5 μm) at a flow rate of 10 L/ min. The concentration in the chamber was 1436 μm/m3 [30, 31]. Mice were sacrificed at the end of each exposure week, the thymus was extracted and CD11c was detected by conventional immunohistochemistry. Other thymuses were processed for cytometry for CD11c+ and MHCII+. To obtain the enough cells the Baba method

**107**

**Figure 1.**

*cells compared to the shown in (B, D).*

*The Effect of Atmospheric Pollution on the Thymus DOI: http://dx.doi.org/10.5772/intechopen.87027*

exposure progress.

was modified [33]. Briefly, mice were sacrificed, thymus were extracted, macerated and submitted to enzymic digestion to obtain a homogenate, by magnetic cell sorting (MACS) the CD11c+ and MHCII+, cells were separated. The cells from the 3rd week of exposure were selected for cytometry because previous result indicated

The observed changes in CD11c are shown in **Figure 1**. In controls more CD11c+ cells were noticed compared with the exposed mice. The findings were supported with the densitometric results (GraphPad Prism Software, V5, 2007, La Jolla, CA) that showed a diminished presence of the CD11c+ in the medullae as the time of the

Flow cytometry showed that levels of CD11c+ and MHCII+ were significantly decreased in the V-exposed mice as compared with controls. The loss was both in terms of number and in mean fluorescence intensity (MIF) values. In brief, V-inhalation decreased the thymic CD11C+ and MHCII+ cells as the exposure time increased [4]. An explanation for our findings could be the V-oxidative capacity which has been previously reported by our group and by other authors [34, 35]. Oxidative stress could affect the liberation of pro-inflammatory transcription factors by the CD11c cells. The thymus dysfunction could by in the negative selection of the T cells that, in turn, could alter the interaction of CD4 lymphocytes with the MHCII protein on the DCs. Such changes could then promote the development of autoimmune responses as suggested by Chang and indirectly by Zouali [36, 37], who hypothesized that an epigenetic effect from urban air pollutants led to altered immune cell phenotypes that, in turn, favors the development of autoimmunity.

*CD11c expression in thymus. In control and V-exposed mice, ocher color stained (arrows) cells CD11c+ were distributed mainly in the medulla (M); scanty positive cells were present in the cortex (Co). Representative photomicrographs from (A, C) control (B, D) 4-week-V-exposed. (A, C) Reveal a higher presence of CD11c+* 

that in this period of time thymus changes were more evident.

Further studies are planned to investigate this topic.

#### *The Effect of Atmospheric Pollution on the Thymus DOI: http://dx.doi.org/10.5772/intechopen.87027*

*Thymus*

maturation [26, 27].

**3.2 Dendritic cells**

cells [29].

humoral response [32].

MHCII on the DCs distribution.

**3.1 Positive and negative thymus selection**

The maturation process from thymocytes to immunocompetent T cells depends on the cortical and medullar thymic microenvironments that is established by the cTEC, mTEC, and DC. The cTEC positively select the T-lymphocytes that recognize the major histocompatibility complex (MHC), so the selected T-lymphocytes are CD4+ or CD8+. The mTEC and the DCs are involved in negative T-cell selection and in establishing self-tolerance by eliminating through apoptosis, the lymphocytes which strongly recognize self-antigens, preventing autoreactive clones. Both processes area highly ordinated and rely on the sequential and quantitative expression of markers such as CD4, CD8, T-cell receptors (TCR1, TCR2) as well as of changes in the glycosylation of the membrane proteins on the lymphocytes in the process of

Dendritic cells (DCs) are antigen -presenting cells (APCs) from the bone marrow. They are a heterogeneous population of cells which are identify because their surfaces biomarkers, cytokines production and location. Their progenitors could be myeloid or lymphoid. DCs are recognized by its typical dendritic morphology and by the expression of CD11c. As all of the other APCs, MHCII is expressed on its surface [28]. The differentiation and function of the DCs could be modulated by different growth factors and cytokines. There are three types: cDCs, the conventional type; pDCs, the plasmacytoid type; and mDCs, the monocyte-derived

DCs are a bridge between innate and adaptive immune response. When they are immature its location is the skin and peripheral tissues and when an antigen is captured the cells migrate to present the antigen to the T cells. The thymus DCs and the mTEC are responsible of the thymocytes negative selection; in the medullae the single T cells (SP) interact with the DCs and with the mTEC that present them self-antigens with and immunogenic potential. The T-cell receptor (TCR) from the SP thymocytes recognizes the cells with MHC restriction and high affinity to selfantigens to eliminate them by apoptosis. This is the main mechanism to eliminate

As it was previous mentioned V could damage health. Previous reports from our group demonstrated that, -in experimental model- mice that inhale V have damages in different systems including the immune system which induces changes in the cortex-medullae ratio [30, 31]. Other observed effect was in the spleen and in the

Because of the morphological changes in the thymus as a consequence of the V-exposure, we decided to explore the changes in the expression of CD11c and

We have developed a CD-1 mouse inhalation experimental model to assess systemic effects of PM. We used CD-1 mice (8-weeks-old, 33 + 2 g) from the vivarium of the School of medicine. The animals were randomly placed in acrylic chambers connected to an ultra-nebulizer (UltraNeb99 Devilbiss, Somerset, Philadelphia, USA), the exposure schedule consisted of V2O5 0.02 M (Sigma, St. Louis, Missouri, USA) in saline, 1 hour twice a week for 4 weeks. The ultra-nebulizer with the size of the particle emitted being less than 5 μm (range 0.5–5 μm) at a flow rate of 10 L/

ficed at the end of each exposure week, the thymus was extracted and CD11c was detected by conventional immunohistochemistry. Other thymuses were processed for cytometry for CD11c+ and MHCII+. To obtain the enough cells the Baba method

[30, 31]. Mice were sacri-

autoreactive clones and to establish central tolerance [29].

min. The concentration in the chamber was 1436 μm/m3

**106**

was modified [33]. Briefly, mice were sacrificed, thymus were extracted, macerated and submitted to enzymic digestion to obtain a homogenate, by magnetic cell sorting (MACS) the CD11c+ and MHCII+, cells were separated. The cells from the 3rd week of exposure were selected for cytometry because previous result indicated that in this period of time thymus changes were more evident.

The observed changes in CD11c are shown in **Figure 1**. In controls more CD11c+ cells were noticed compared with the exposed mice. The findings were supported with the densitometric results (GraphPad Prism Software, V5, 2007, La Jolla, CA) that showed a diminished presence of the CD11c+ in the medullae as the time of the exposure progress.

Flow cytometry showed that levels of CD11c+ and MHCII+ were significantly decreased in the V-exposed mice as compared with controls. The loss was both in terms of number and in mean fluorescence intensity (MIF) values. In brief, V-inhalation decreased the thymic CD11C+ and MHCII+ cells as the exposure time increased [4]. An explanation for our findings could be the V-oxidative capacity which has been previously reported by our group and by other authors [34, 35]. Oxidative stress could affect the liberation of pro-inflammatory transcription factors by the CD11c cells. The thymus dysfunction could by in the negative selection of the T cells that, in turn, could alter the interaction of CD4 lymphocytes with the MHCII protein on the DCs. Such changes could then promote the development of autoimmune responses as suggested by Chang and indirectly by Zouali [36, 37], who hypothesized that an epigenetic effect from urban air pollutants led to altered immune cell phenotypes that, in turn, favors the development of autoimmunity. Further studies are planned to investigate this topic.

#### **Figure 1.**

*CD11c expression in thymus. In control and V-exposed mice, ocher color stained (arrows) cells CD11c+ were distributed mainly in the medulla (M); scanty positive cells were present in the cortex (Co). Representative photomicrographs from (A, C) control (B, D) 4-week-V-exposed. (A, C) Reveal a higher presence of CD11c+ cells compared to the shown in (B, D).*
