Читать книгу Pathology of Genetically Engineered and Other Mutant Mice - Группа авторов - Страница 118

Among invertebrate and vertebrate animals, lymph nodes are found only in mammals. Afferent lymph vessels collect lymph fluid from organs and tissues, which allows LNs to monitor peripheral tissues for infections and tissue damage and to act as a barrier to further dissemination of potential pathogens. Specialized mesenchymal stromal cells and endothelial cells secrete chemotactic factors that control the migration of lymphocytes that enter LNs via high endothelial venules and afferent lymph vessels. In this manner, LNs play an essential role in maximizing the chances for antigen to find the rare B and T lymphocytes that express the receptors for that specific antigen. The number of lymph nodes in mice raised in a clean environment is fairly constant with about 22 LNs found in specific locations [29].

Lymph node anlagen are formed as a result of interactions between lymphoid tissue organizer (LTo) cells and lymphoid tissue inducer (LTi) cells during ED14 to ED17 [30, 31]. Secretion of CXCL13 by LTo cells recruits LTi cells released from the fetal liver. Accumulation and activation of LTi cells results in further recruitment of LTi cells in a lymphotoxin and RANK‐dependent manner followed by the recruitment of B and T lymphocytes and differentiation of stromal cells in subtypes that populate the different compartments of the lymph node. LTi cells are a type of innate lymphoid cells, whereas the LTo cells include mesenchymal stromal cells and lymphatic endothelial cells. The relative role of stromal LTo cells and lymphatic endothelial cells in the initiation of LN formation is controversial [30, 31]. Understanding the process of LN formation is clinically relevant because the same signals are thought to be involved in the generation of tertiary lymphoid tissues which can be found in cancer and chronic inflammatory lesions [32, 33].

Table 7.1 Defects in thymus development in genetically engineered mice.

Targeted mutation (gene)	Thymus	Other defects	References
Eya1	Aplasia	Absence of ears, kidneys, parathyroid aplasia, thyroid hypoplasia	[13, 14]
Foxn1	Aplasia	Hair shaft defects	[15]
Hoxa3	Aplasia	Parathyroid aplasia, thyroid hypoplasia	[16]
Pax1	Hypoplasia	Skeletal defects	[17]
Pax3	Aplasia	Cardiac defects, parathyroid aplasia, thyroid aplasia, neural tube defects	[18]
Pax9	Aplasia	Parathyroid aplasia, skeletal defects, absence of teeth	[19]
Six1	Aplasia	Absence of kidneys, parathyroid aplasia, skeletal defects	[20]
Tbx1	Aplasia	Parathyroid aplasia, cardiac defects, skeletal defects, cleft palate	[21]

Lymph nodes can be divided into the cortex, paracortex, and medulla. B cell follicles are located in the cortex and contain follicular dendritic cells, specialized stromal cells that retain immune complexes on their surface and play an important role in the affinity maturation and longevity of the humoral immune response. The paracortex is populated by T cells and dendritic cells, and contains the high endothelial venules that permit the extravasation of naïve B and T lymphocytes. The medulla is composed of medullary cords which contain macrophages and plasma cells, and medullary sinuses that converge into a single efferent lymphatic vessel. Antigens reach the lymph node via afferent lymphatics that open into the subcapsular sinus. The “floor” of the subcapsular sinus is lined by endothelial cells that limit diffusion of antigens into the underlying lymphoid tissue. At least five different types of macrophages can be identified in lymph nodes based on their localization and phenotype [34]. These are the subcapsular sinus macrophages, tingible body macrophages in germinal centers, T cell zone macrophages in the paracortex, and medullary cord and medullary sinus macrophages.

 Examination of lymph nodes: Lymph nodes vary in size and degree of lymphocyte activation based on their location and exposure to antigens. The largest lymph nodes are those that drain the intestine. Because of their small size in the mouse, it is often necessary to use a dye to visualize and identify lymph nodes. This can be readily done by injection of Evans blue or Indian ink into the tributary area of the lymph node. For example, injection of Evans blue in the footpads of the hindlegs induces accumulation of the blue dye in the popliteal and iliac lymph nodes within 30 minutes. Systemic staining of lymph nodes can be accomplished by injection of 1% pontamine sky blue in the peritoneal cavity of mice. The dye stains connective tissues blue, but after about two weeks, the staining of the connective tissues has decreased and the dye selectively accumulates in lymph nodes throughout the body. Mutations in cytokine and cytokine receptor genes or transcription factors may lead to a selective or general absence of lymph nodes (Table 7.2).For routine examination, H&E‐stained sections of formalin‐fixed lymph nodes that contain the cortex, paracortex, and medulla are adequate. It is important to recognize that multiple afferent lymph vessels enter the lymph node causing regional differences in the distribution of changes within a lymph node. Furthermore, differences in the angle at which lymph nodes are sectioned may give the wrong impression of changes in the size of the different LN compartments. Immunohistochemistry and flow cytometry can be used to identify specific lymphocyte and macrophage subpopulations.Table 7.2 Defects in the development of secondary lymphoid tissues in genetically engineered mice.CategoryTargeted geneProteinLymph nodesMucosa‐associated lymphoid tissuesSpleenReferencesPLNMLNPPNALTLDALTGCMZTNF‐superfamilyTnfTumor necrosis factor‐alpha+++++−⇓[35]LtaLymphotoxin A−−−⇓+−−[36, 37]LtbLymphotoxin B−+−⇓+−−[38, 39]LtbrLymphotoxin‐receptor−−−⇓+−−[40]Tnfsf11Receptor‐associated with NFkB‐ligand (RANKL)−−+++++[41]Tnfrsf11aReceptor‐associated with NFkB (RANK)−−+++++[42]Transcription factorsRelbRELB−−−⇓ND−−[43]Nfkb2NFKB2−+−−ND−−[43]Map3k14NFkB‐inducing kinase (NIK)−−−⇓⇓−−[44, 45]Id2Inhibitor of DNA binding 2 (ID2)−−−−+++[46]RorgRAR‐related orphan receptor gamma (RORγ)−−−−+++[47]Cbfb2Core binding factor β2 (CBFβ2)−+−−−NDND[48, 49]Chemokine (receptor)Cxcl13CXCL13⇓−⇓⇓+−+[50]Cxcr5CXCR5⇓−⇓⇓ND−+[50]

 Lymphoid hypoplasia: Mutations that affect the production of lymphocytes in the primary lymphoid tissues result in greatly decreased numbers of lymphocytes in the lymph nodes and other secondary lymphoid tissues. Null mutations of Foxn1 selectively affect the generation of T cells in the thymus and cause a marked decrease of lymphocytes in the paracortex of the lymph nodes (Figure 7.3). Mutations of the Rag1, Rag2, and Prkdc genes result in a lack of B and T cells, and the lymph nodes are largely devoid of lymphocytes (Figure 7.3). Il2rg mutations affect the production of lymphocytes as well as other immune cells. NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice have small rudimentary lymph nodes (Figure 7.3).

 Reactive lymph node: As a physiologic response to antigenic stimulation, e.g. by injection of a vaccine or an infection, draining lymph nodes undergo a rapid increase in size as a result of increased blood supply, increased lymphocyte influx, and reduced egress of lymphocytes from the lymph node. Chemokines that reach the lymph node via afferent lymph vessels induce hypertrophy of high endothelial venules in the paracortex and increased extravasation of lymphocytes. This is followed by proliferation of lymphocytes resulting in expansion of the paracortex and increased size of follicles. The follicles are comprised of a germinal center surrounded by a mantle zone of recirculating resting B lymphocytes. The germinal center has a dark zone in which B cells undergo proliferation and a light zone in which B cells with high affinity receptors are selected and other B cells undergo apoptosis. Upon prolonged stimulation of lymph nodes, there may be a marked accumulation of plasma cells in the medullary cords.

 Aging‐associated changes: A detailed study of aging changes in the inguinal lymph node of C57BL/6J mice revealed a decrease of the percentage of T cells and increase of B cells in lymph nodes of old mice [51]. B cell follicles were less well defined, and there was a reduced number of follicular dendritic cells. There was an increased number of subcapsular sinus, medullary sinus, and medullary cord macrophages.

 Amyloidosis. Accumulation of amyloid occurs most commonly in mesenteric lymph nodes. Amyloid appears as pale eosinophilic amorphous material in H&E‐stained sections (Figure 7.4). Amyloid deposits first in the subcapsular sinus and may expand into the paracortex.

 Sinus dilatation: Dilatation of the medullary sinuses is a common finding in the mesenteric and mediastinal lymph nodes of old mice. The sinuses contain proteinaceous fluid, lymphocytes, and macrophages.

 Sinus erythrocytosis: The presence of erythrocytes in the subcapsular and medullary sinuses may be the result of antemortem hemorrhage in the tributary area or be caused by tissue manipulation and euthanasia especially in the bronchial and mediastinal lymph nodes when the mice are euthanized by CO2 asphyxiation. In chronic hemorrhage, erythrocyte and hemosiderin‐laden macrophages are typically present.

 Angiomatous hyperplasia: This is a common finding in the mesenteric lymph nodes of old mice characterized by an increased number of endothelium‐lined spaces in the cortex and medulla. The vessels may contain proteinaceous fluid and erythrocytes. There is no evidence that this lesion will progress to a neoplasm.

 Pigmentation: Accumulation of hemosiderin and ceroid‐lipofuscin is common in sinusoidal macrophages that drain areas of hemorrhage and inflammation. Melanin may be observed in skin‐draining lymph nodes of black mice.

 Macrophage hyperplasia: Diffuse or focal increases of the different macrophage populations may occur in lymph nodes as a result of influx from the blood or draining area or as a result of proliferation. A diffuse accumulation of subcapsular and medullary sinus macrophages is commonly referred to as sinus histiocytosis. The macrophages may contain pigment, red blood cells, or material injected in the tributary area such as mineral or oil from vaccine adjuvants (Figure 7.4).