Živana Milićević

TNF Receptor-1 Is Required for the Formation of Splenic Compartments during Adult, but Not Embryonic Life

Lymphotoxin β-receptor (LTβR) and TNF receptor-1 (TNFR1) are important for the development of secondary lymphoid organs during embryonic life. The significance of LTβR and TNFR1 for the formation of lymphoid tissue during adult life is not well understood. Immunohistochemistry, morphometry, flow cytometry, and laser microdissection were used to compare wild-type, LTβR−/−, TNFR1−/− spleens with splenic tissue that has been newly formed 8 wk after avascular implantation into adult mice. During ontogeny, LTβR is sufficient to induce formation of the marginal zone, similar-sized T and B cell zones, and a mixed T/B cell zone that completely surrounded the T cell zone. Strikingly, in adult mice, the formation of splenic compartments required both LTβR and TNFR1 expression, demonstrating that the molecular requirements for lymphoid tissue formation are different during embryonic and adult life. Thus, interfering with the TNFR1 pathway offers the possibility to selectively block the formation of ectopic lymphoid tissue and at the same time to spare secondary lymphoid organs such as spleen and lymph nodes. This opens a new perspective for the treatment of autoimmune and inflammatory diseases.

Metallophilic macrophages are lacking in the thymus of lymphotoxin-β receptor-deficient mice

Lymphotoxin-β receptor (LTβR) axis plays a crucial role in development and compartmentalization of peripheral lymphatic organs. But, it is also required for the appropriate function and maintenance of structural integrity of the thymus: in LTβR-deficient animals the clonal deletion of autoreactive lymphocytes is impaired and differentiation of thymic medullary epithelial cells is disturbed. In this study, using several markers, we showed that thymic metallophilic macrophages were lacking in LTβR-deficient mice. In tumor necrosis factor receptor-I (p55)-deficient mice (which we used as positive control) thymic metallophilic cells were located, similarly as in normal mice, in the thymic cortico-medullary zone at the junction of cortex and medulla. These findings show that LTβR is necessary for maintenance of metallophilic macrophages in the thymus and provide further evidence that these cells may represent a factor involved in thymic negative selection.

Metallophilic macrophages are fully developed in the thymus of autoimmune regulator (Aire)-deficient mice

Thymic metallophilic macrophages represent a significant component in the thymus physiology. Recently, we showed their presence to be dependent on functional lymphotoxin-β receptor (LTβR) signaling pathway. However, it is unknown whether the development of metallophilic macrophages also requires the Autoimmune regulator (Aire) transcription factor, as suggested by some studies for medullary thymic epithelial cells, or perhaps the presence of Aire-expressing thymic epithelial cells themselves. Therefore, we investigated the presence of metallophilic macrophages in Aire-deficient thymus. Our study shows that the metallophilic macrophages are fully developed in the Aire-deficient thymus; their development is not regulated via Aire transcription factor and does not require the presence of Aire-expressing epithelial cells. On the contrary, in alymphoplasia (ALY) mice (deficient in nuclear factor-kappaB-inducing kinase, NIK), which we used as negative control, thymic metallophilic macrophages are completely lacking, similarly as in LTβR-deficient animals. Together, these results show that the development/maintenance of thymic metallophilic macrophages is executed via LTβR circumventing the Aire transcription factor. Thus, we shed a new light on the molecular requirements for development of these cells and also show that LTβR pathway is a common developmental regulator of metallophilic macrophages in different lymphatic organs (i.e., thymus and spleen).

Relationship between naphthol AS-D chloroacetate esterase and prostaglandin synthase

The close topographical correlation between naphthol AS-D chloroacetate esterase-positive macrophages and prostaglandin synthase-rich macrophages in the thymus of normal and cyclosporin-treated rats was observed. It seems possible that chloroacetate esterase is an enzyme related to metabolism of arachidonic acid and/or production of its active metabolites.

Role of CCL19/21 and its possible signaling through CXCR3 in development of metallophilic macrophages in the mouse thymus

We have already shown that metallophilic macrophages, which represent an important component in the thymus physiology, are lacking in lymphotoxin-β receptor-deficient mice. However, further molecular requirements for the development and correct tissue positioning of these cells are unknown. To this end, we studied a panel of mice deficient in different chemokine ligand or receptor genes. In contrast to normal mice, which have these cells localized in the thymic cortico-medullary zone (CMZ) as a distinct row positioned between the cortex and medulla, in plt/plt (paucity of lymph node T cells) mice lacking the functional CCL19/CCL21 chemokines, metallophilic macrophages are not present in the thymic tissue. Interestingly, in contrast to the CCL19/21-deficient thymus, metallophilic macrophages are present in the CCR7-deficient thymus. However, these cells are not appropriately located in the CMZ, but are mostly crowded in central parts of thymic medulla. The double staining revealed that these metallophilic macrophages are CCR7-negative and CXCR3-positive. In the CXCL13-deficient thymus the number, morphology and localization of metallophilic macrophages are normal. Thus, our study shows that CCL19/21 and its possible signaling through CXCR3 are required for the development of thymic metallophilic macrophages, whereas the CXCL13–CXCR5 signaling is not necessary.

Metallophilic macrophages of the rodent thymus

For a very long time, we studied the metallophilic macrophages of the rodent thymus and in this review our results on morphological, histochemical, enzymehistochemical, immunohistochemical, ultrastructural and functional features of these cells, as well as the molecular regulation of their development, will be presented. Furthermore, the differences between species will also be discussed and the comparisons with similar/related cell types (metallophilic macrophages in the marginal sinus of the spleen, subcapsular sinus of the lymph nodes and germinal centers of secondary lymphoid follicles) will be made. Metallophilic macrophages are strategically positioned in the thymic cortico-medullary zone and are very likely to be involved in: (i) the metabolism, synthesis and production of bioactive lipids, most likely arachidonic acid metabolites, based on their histochemical and enzymehistochemical features, and (ii) the process of negative selection that occurs in the thymus, based on their ultrastructural features and their reactivity after the application of toxic or immunosuppressive/immunomodulatory agents. Taken together, their phenotypic and functional features strongly suggest that metallophilic macrophages play a significant role in the thymic physiology.

Lipopolysaccharide-induced in vivo activation of follicular dendritic cells is tumor necrosis factor receptor-1 independent.

It is well recognized that tumor necrosis factor receptor‐1 (TNFR1) signaling pathway (with lymphotoxin‐β receptor) is of critical importance for the development, activation, and clustering of follicular dendritic cells (FDCs) within the lymphoid follicles. However, further information on the molecular control of these processes is very sparse. Here, we show that intravenous application of lipopolysaccharide induces the clear and prominent morphological signs of FDC development and activation in vivo, which is independent of TNFR1 pathway. Anat Rec, 2012.

Stereologic analysis of tissue compartments of gunshot-injured and blunt-injured spleen.

The spleen is composed of several tissue compartments and the respective histoquantitative data are essential for complete understanding of immune or pathological processes in this organ. The aim of our study was to determine and compare the stereologic parameters of all tissue compartments of the gunshot-injured and blunt-injured human spleen. The model-based stereology with point-counting method was utilized to study the volume densities of red pulp, perifollicular zone, marginal zone, white pulp (follicles and periarteriolar lymphoid sheath), and connective tissue. The areal numerical density (the number of follicles per mm2 of tissue section), the numerical density (the number of follicles per mm3 of tissue) of lymphoid follicles and the mean follicle diameter were also determined. Our study provides stereological parameters for all tissue compartments of the human spleen. No morphometric differences were registered between tissue compartments of the blunt-injured and gunshot-injured spleen. As the gunshot-injured spleen was taken as presumably unstimulated in immunological regard, our results suggest that both gunshot-injured and blunt-injured organs may be used as models of the normal human spleen.

Lipopolysaccharide induces tumor necrosis factor receptor-1 independent relocation of lymphocytes from the red pulp of the mouse spleen

It is well known that bacterial lipopolysaccharide (LPS) induces migration of several cellular populations within the spleen. However, there are no data about the impact of LPS on B and T lymphocytes present in the red pulp. Therefore, we used an experimental model in which we tested the effects of intravenously injected LPS on the molecular, cellular and structural changes of the spleen, with special reference to the red pulp lymphocytes. We discovered that LPS induced a massive relocation of B and T lymphocytes from the splenic red pulp, which was independent of the tumor necrosis factor receptor-1 signaling axis. Early after LPS treatment, quantitative real-time PCR analysis revealed the elevated levels of mRNA encoding numerous chemokines and proinflammatory cytokines (XCL1, CXCL9, CXCL10, CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, TNFα and LTα) which affect the navigation and activities of B and T lymphocytes in the lymphoid tissues. An extreme increase in mRNA levels for CCL20 was detected in the white pulp of the LPS-treated mice. The CCL20-expressing cells were localized in the PALS. Some smaller CCL20-expressing cells were evenly dispersed in the B cell zone. Thus, our study provides new knowledge of how microbial products could be involved in shaping the structure of lymphatic organs.

Remodeling of extracellular matrix of the lamina propria in the uninvolved human rectal mucosa 10 and 20 cm away from the malignant tumor

In recent years, it has been demonstrated that malignancy arises and advances through the molecular interplay between tumor cells and non-malignant elements of the tumor stroma, that is, fibroblasts and extracellular matrix. However, in contrast to the mounting evidence about the role of tumor stroma in the genesis and progression of the malignant disease, there are very few data regarding the uninvolved stromal tissue in the remote surrounding of the tumor. Using the objective morphometric approach in patients with adenocarcinoma, we demonstrate the remodeling of extracellular matrix of the lamina propria in the uninvolved rectal mucosa 10 and 20 cm away from the neoplasm. We show that the representation of basic extracellular matrix constituents (reticular and collagen fibers and ground substance) is decreased. Also, the diameter of empty spaces that appear within the extracellular matrix of the lamina propria is increased. These spaces do not represent the blood or lymphatic vessel elements. Very likely, they reflect the development of tissue edema in the remote, uninvolved lamina propria of the mucosa in patients with the malignant tumor of the rectum. We hypothesize that the remodeling of extracellular matrix in lamina propria of the rectal mucosa may increase its stiffness, modulating the mechano-signal transduction, and thus promote the progression of the malignant disease.