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).
Mucosa‐Associated Lymphoid Tissues
The mucosa‐associated lymphoid tissues are composed of the inductive sites for adaptive immune responses in the intestinal and upper respiratory tract. In the mouse, they include the Peyer's patches, solitary lymphoid follicles, and colonic lymphoid patches in the intestine, and the nasopharynx‐associated lymphoid tissue (NALT) and lacrimal duct‐associated lymphoid tissue (LDALT) in the nose (Figure 7.5). Mice do not have tonsils. Mucosa‐associated lymphoid organs do not have afferent lymphatics, and antigens gain access to the lymphoid tissue from the mucosal surface via specialized epithelial cells (microfold or M cells). In addition, dendritic cells may extend dendritic processes between epithelial cells to sample the lumen of the intestinal and respiratory tract. M cells are derived from epithelial stem cells. Mature M cells are characterized electron microscopically by short, irregular microvilli and a thin brush border, and express glycoprotein 2 as a distinct surface marker [52]. The basolateral membrane of M cells is invaginated forming a pocket that often contains lymphocytes, macrophages, and dendritic cells. Antigens taken up by transcytosis through M cells are processed by dendritic cells and activate B and T cells in the underlying lymphoid tissue.
Examination of mucosa‐associated lymphoid tissues: Peyer's patches can be observed and counted macroscopically in adult mice. However, for a detailed investigation of lymphoid tissues in the small intestine, the intestine should be cut in two pieces, and each piece rolled up in a cassette as a “Swiss roll”. Serial longitudinal sections allow for a thorough examination of the intestine. The large intestine is first opened longitudinally to empty the contents and then rolled up in a cassette. To examine the development of Peyer's patches in late‐stage embryos and newborn pups, whole mount immunohistochemistry can be performed [53]. Microscopic examination of the NALT and LDALT requires cross sections of the nose after fixation and decalcification of the head. A cross section just rostral of the first molar will include the NALT.Figure