**3. Keratin-negative area/epithelium-free area**

Mesenchymal cells and fibroblasts express vimentin intermediate filament. Cortical thymocytes and epithelial cells are vimentin negative (**Figure4**), but thymic medulla shows homogenous staining pattern, indicating that keratin-positive and keratinnegative compartments cannot be distinguished (**Figure 5**). The homogenous vimentin staining of the medulla may indicate that the medullary epithelial cells express vimentin intermediate filament. Hassall's bodies are vimentin negative (**Figure 5**), like cortical epithelial cells. In the majority of vimentin-positive cells, the immunoreaction appears in the periphery of the cell cytoplasm. The nature of the medullary vimentin-positive cells is not clear, because vimentin can colocalize with other intermediate filaments, like neurofilament, cytokeratin, and desmin; therefore the anti-vimentin immunostaining used for identification of mesenchymal cells is limited [25]. Blood vessels and dendritic cells are the most significant structures of the KNA/EFA. Anti-von Willebrand factor identifies endothelial cells (**Figure6**). Transmission electron microscopy shows the organelle-rich cytoplasm of the interdigitating dendritic cell (IDC) (**Figure 7**). The IDC is located in close association with the blood vessels [2].

#### **Figure 4.**

*Anti-vimentin antibody outlines the capsule and PS. The cortical epithelial cells and T cells are negative, while all medullary cells express vimentin. C, cortex; M, medulla.*

#### **Figure 5.**

*Higher magnification of anti-vimentin-stained thymic medulla. The round-shaped Hassall's body is negative, while the medullary cells show a round-shaped-positive reaction.*

#### **Figure 6.**

*Double staining: cytokeratin (red) and von Willebrand factor (green). Blood vessels exclusively locate in the KNA/EFA.*

As mentioned above, the PVS consists of mesenchymal cells of neural crest origin; therefore, the supporting tissue of the KNA/EFA is also mesenchyme. The thickness of the PVS is used to be in one or two cell layers, but in chicken thymus, the morphometric studies show that the ratio between keratin-positive and keratin-negative fields is close to one to one [2]. Considerable size of the KNA/EFA

**11**

**Figure 8.**

*possibly Hassall's bodies.*

*Compartmentalization of Human Thymic Medulla: Facts and Hypotheses*

(**Figure 6**) suggests that its functional significance must be more than a passive transit zone for cell migration between circulation and the thymus [4, 10–12]. Thymic IDC [9] is located in the KNA/EFA [2] and contributes to T-cell selection. In nonobese mouse, autoimmune type I diabetes develops, resulting in abnormal distribution of epithelial cells and consequently giant PVS [11, 17]. After cyclosporin A (CyA) treatment of the rats, the thymic medulla disappeared, and 2 weeks after CyA treatment, the recovery of the medulla took place, but the "holes" were epithelial cell-free [5]. The occurrence and size of the KNA/EFA may be varied from species to species [2] even among the strains of rats [3]. In the CM region of BB rat thymus, the EFA has been reported, but this KNA/EFA is not as complete as in man. In the thymic medulla, the frequency of the KNA/EFA alters by age: in young Wistar rats, the occurrence of the KNA/EFA is higher than in old animals [7]. These

*Transmission electron micrograph of a thymic dendritic cell. The cell membrane is outlined. The cytoplasm is* 

changes may be related to acute [5] and/or physiological thymic involution. The thymic stromal elements develop from the endodermal epithelium and neural crest mesenchyme. Hematopoietic cells colonize the epithelial-mesenchymal anlage. In chicken embryo the KNA/EFA appears when the medulla and cortex differentiate; therefore the epithelial-mesenchymal transition [6] would create a "second" mesenchyme, besides the mesenchyme of cranial neural crest origin. Therefore, in the thymus the epithelial-mesenchymal transition may be redundant. It was difficult to identify large epithelium-free areas by transmission electron microscopy, and Foxn-1 thymic epithelial cell-specific transcription factor showed

*Thymus 5. Age 16 months: Foxn-1 antibody recognizes epithelial nuclei over the entire thymus. Density of Foxn-1-positive cells is higher in the medulla (M) than in the cortex (C). The Foxn-1-positive knots are* 

*DOI: http://dx.doi.org/10.5772/intechopen.88588*

*rich in organelles and electron-dense granules.*

**Figure 7.**

*Compartmentalization of Human Thymic Medulla: Facts and Hypotheses DOI: http://dx.doi.org/10.5772/intechopen.88588*

**Figure 7.**

*Thymus*

**Figure 4.**

**10**

**Figure 6.**

**Figure 5.**

*KNA/EFA.*

*Double staining: cytokeratin (red) and von Willebrand factor (green). Blood vessels exclusively locate in the* 

*Higher magnification of anti-vimentin-stained thymic medulla. The round-shaped Hassall's body is negative,* 

*Anti-vimentin antibody outlines the capsule and PS. The cortical epithelial cells and T cells are negative, while* 

*all medullary cells express vimentin. C, cortex; M, medulla.*

*while the medullary cells show a round-shaped-positive reaction.*

As mentioned above, the PVS consists of mesenchymal cells of neural crest origin; therefore, the supporting tissue of the KNA/EFA is also mesenchyme. The thickness of the PVS is used to be in one or two cell layers, but in chicken thymus, the morphometric studies show that the ratio between keratin-positive and keratin-negative fields is close to one to one [2]. Considerable size of the KNA/EFA

*Transmission electron micrograph of a thymic dendritic cell. The cell membrane is outlined. The cytoplasm is rich in organelles and electron-dense granules.*

(**Figure 6**) suggests that its functional significance must be more than a passive transit zone for cell migration between circulation and the thymus [4, 10–12]. Thymic IDC [9] is located in the KNA/EFA [2] and contributes to T-cell selection. In nonobese mouse, autoimmune type I diabetes develops, resulting in abnormal distribution of epithelial cells and consequently giant PVS [11, 17]. After cyclosporin A (CyA) treatment of the rats, the thymic medulla disappeared, and 2 weeks after CyA treatment, the recovery of the medulla took place, but the "holes" were epithelial cell-free [5]. The occurrence and size of the KNA/EFA may be varied from species to species [2] even among the strains of rats [3]. In the CM region of BB rat thymus, the EFA has been reported, but this KNA/EFA is not as complete as in man. In the thymic medulla, the frequency of the KNA/EFA alters by age: in young Wistar rats, the occurrence of the KNA/EFA is higher than in old animals [7]. These changes may be related to acute [5] and/or physiological thymic involution.

The thymic stromal elements develop from the endodermal epithelium and neural crest mesenchyme. Hematopoietic cells colonize the epithelial-mesenchymal anlage. In chicken embryo the KNA/EFA appears when the medulla and cortex differentiate; therefore the epithelial-mesenchymal transition [6] would create a "second" mesenchyme, besides the mesenchyme of cranial neural crest origin. Therefore, in the thymus the epithelial-mesenchymal transition may be redundant.

It was difficult to identify large epithelium-free areas by transmission electron microscopy, and Foxn-1 thymic epithelial cell-specific transcription factor showed

#### **Figure 8.**

*Thymus 5. Age 16 months: Foxn-1 antibody recognizes epithelial nuclei over the entire thymus. Density of Foxn-1-positive cells is higher in the medulla (M) than in the cortex (C). The Foxn-1-positive knots are possibly Hassall's bodies.*

positive cells in the KNA/EFA (**Figure 8**). Foxn-1 expression in the medullary KNA/ EFA is a puzzle. In early embryogenesis, Foxn-1 expresses in several mesenchymal and epithelial cells [27]; therefore one of the possibilities for solving the puzzle is that Foxn-1 is maintained in the KNA/EFA after thymus development. The other possibility is that the thymic epithelial cells induce Foxn-1 expression in mesenchymal cells of cranial neural crest origin [28]. Removal of perithymic mesenchyme at ED12 or culture of purified ED14 epithelial cells alone resulted in a threefold reduction in the bromodeoxyuridine incorporation by keratin-positive cells. Proliferation of thymic epithelial cells in the early thymus is regulated by signals from mesenchyme [29]. These mesenchymal cells produce fibroblast growth factors 7 and 10, which stimulate epithelial cell proliferation [20, 30, 31], but differentiation requires Foxn-1 [32]. The KNA/EFA [2, 9, 11] consists of mesenchyme; therefore the term EFA seems to be more appropriate than the KNA that we used [2].
