Abstract
Lymph nodes function as filters of tissues and tissue fluids and are sites of origin and production of lymphocytes for normal physiological functions. As part of this normal function, they react to both endogenous and exogenous substances with a variety of specific morphological and functional responses. Lesions can be both proliferative and nonproliferative, and can be treatment-related or not. The histological evaluation of lymph nodes is necessary in order to understand the immunotoxic effects of chemicals with the resulting data providing an important component of human risk assessment. It is the challenge of the toxicologic pathologist to interpret the pathology data within the complete clinical evaluation of the entire animal. Daily insults, ageing and toxins can alter the normal histology and primary function of lymph nodes. Therefore it is important to distinguish and differentiate lesions that occur naturally during normal development and ageing from those that are induced by xenobiotics. To achieve this goal, comparison with strain- age- and sex-matched controls is crucial.
Introduction
The lymph nodes are organized lymphoid organs that contain lymphocytes within a fine reticular stroma. The structures within a lymph node include the capsule, subcapsular sinus, cortex (B cell zone with follicles and germinal centers), paracortex (T cell zone), medullary sinuses, medullary cords and hilus. In order to ensure that all of these components are evaluated, particular attention should be paid to tissue collection and orientation. Lymph nodes should routinely be examined grossly and microscopically since they may reflect lesions in organs and tissues they drain. Moreover, special attention should be given to lymph nodes that are more likely to be exposed to a test compound (Haley et al., 2005; Ruehl-Fehlert et al., 2005). The bronchial and mediastinal lymph nodes should be examined for compounds administered via inhalation whereas orally administered compounds would most likely affect the mandibular (superficial cervical) and mesenteric lymph nodes. For dermal or subcutaneous exposures, the most proximal draining peripheral node or nodes (auricular, axillary, inguinal and popliteal) should be examined.
The range of normal histological findings within groups of lymph nodes due to normal immune functions should also be considered. For example, sinus histiocytosis is a normal finding in mesenteric lymph nodes and the macrophages may contain endogenous pigment (hemosiderin, lipofuscin) or exogenous pigments reflecting antigen uptake from the digestive tract. The mandibular lymph nodes are exposed to antigens from the oropharyngeal region and will typically have well-developed secondary follicles and considerable numbers of plasma cells within the medullary cords. For systemic effects of compounds, examination of lymph nodes distal to the site of application may provide additional information. Unless draining a site of application, the popliteal and axillary lymph nodes are often not stimulated and can give information on the resting state of the lymph node. However these lymph nodes can be highly variable, small and difficult to adequately sample. It is also useful to have knowledge of the regions drained by specific nodes for determining the origin of metastatic neoplasms. Detailed information on the location of specific lymph nodes and patterns of lymphatic drainage in the rat are described by Tilney (1971) and Sainte-Marie et al. (1982). Information on anatomy and nomenclature of murine lymph nodes is provided by Van den Broeck et al. (2006). Finally, a careful and detailed examination of the lymph nodes may give valuable clues to the possible mechanism of action of the test material. For a thorough description of the normal structure, function and histology of the lymph node, refer to the article by Willard-Mack (2006). For detailed information on the enhanced histopathological evaluation of the lymph node, refer to the article by Elmore (2006). The following figures and descriptions illustrate and discuss some of the typical lesions observed in lymph nodes.
Lymphoid Necrosis
Lymphoid necrosis may either be focal, multifocal or diffuse within a lymph node and there can be differences in the presence and severity of lymphoid necrosis between lymph nodes in the same animal, depending on the inciting factor and the effectiveness of the immune response. Lymphocyte necrosis is characterized by cell swelling with chromatin clumping, karyorrhexis or karyolysis and in more severe lesions, abundant eosinophilic cellular debris (Figures 1A–C). Necrosis is frequently accompanied by inflammatory cells including neutrophils and phagocytic macrophages with intracytoplasmic cellular debris (Figures 1D–E). This form of necrosis should be distinguished from apoptosis in which there is individual cell death characterized by cell shrinkage, nuclear pyknosis and fragmentation with apoptotic bodies and tingible body macrophages. This type of cell death normally occurs within the germinal centers of secondary follicles where it is an important homeostatic mechanism. Apoptosis can also be produced by a variety of injurious stimuli when given at low doses, but these same stimuli may be capable of producing necrosis at higher doses. Examples are heat, irradiation, hypoxia and cytotoxic cancer drugs (i.e., cyclophosphamide). Dexamethasone, a glucocorticoid, can also cause lymphocyte apoptosis in the lymph node, but the thymus is the more sensitive organ (Elmore, 2006).
Lymphoid depletion or atrophy is the sequela to chronic necrosis or apoptosis. It can occur in any lymph node and there can be differences in the presence and severity of lymphoid depletion between lymph nodes in the same animal. Lymphoid depletion is typically characterized by a decrease in the number and size of follicles with few to no germinal centers and/or depletion of paracortical lymphocytes (Figure 1F). With a depletion of paracortical lymphocytes, the stromal cells may become more prominent (Figure 1G).
Massive necrosis of the lymph nodes is uncommon and may be induced by obstruction of the blood flow (infarction). This lesion is characterized by diffuse coagulation necrosis with loss of cell nuclei but, early on, with well preserved cell outlines. Caseous necrosis is also an uncommon lesion but can be produced by infection with tuberculosis organisms and fungi in nonhuman primates. It can also be seen in the centers of rapidly growing neoplasms. Grossly, there is semisolid, gray to pale yellow tissue. Microscopically, there is an amorphous mass of granular eosinophilic material with no cell outlines as well as no identifiable nuclei.
Lymphatic Sinus Ectasia
Lymphatic sinus ectasia can involve both the medullary and subcapsular sinuses. Diffuse sinus ectasia is typically associated with lymphoid atrophy. This lesion can be found in control animals, especially in the mesenteric and mediastinal lymph nodes of ageing mice. Figure 2 is an example of medullary sinus ectasia. This lesion is characterized by the presence of dilated or cystic sinuses lined by lymphendothelium and filled with pale eosinophilic/amphophilic material (presumably lymph) that has a delicate lacy appearance (Figure 2B). A few lymphocytes, plasma cells and macrophages can be found admixed with the lymph.
Synonymous names for this lesion are lymphangiectasia, lymphatic cysts, cystic lymphatic ectasia and sinus dilatation. A group of pathologists that are associated with the National Toxicology Program were surveyed for the preferred terminology for this lesion. The 2 favored terms were “lymphatic sinus ectasia” and “lymphangiectasia.” Lymphatic sinus ectasia more clearly defines the lesion location.
Vascular Lesions
When defining vascular lesions in the lymph node, various terminologies can be used, each with a very specific definition. Angiectasis is defined in
Intrasinusoidal erythrocytes (sinus erythrocytosis) can result from a lymph node draining a region of hemorrhage. This can also be an artifact that results from euthanasia or tissue dissection during necropsy, especially in the bronchial or mediastinal lymph nodes. The trimming and sectioning of lymph node tissue can also dislodge erythrocytes from congested blood vessels, which can then appear in the sinusoids or in the perinodal region (Figures 3A–B). Lymph nodes draining sites of hemorrhage can also have intrasinusoidal erythrocytes but, depending on chronicity, they can be accompanied by variable numbers of hemosiderin-laden macrophages, erythrophagocytosis and inflammatory cells (Figures 3C–D).
Lesions of lymph node vascular angiectasis can be difficult to differentiate from moderate to marked sinus erythrocytosis because dilated blood vessels can resemble dilated and blood-filled lymphatic vessels. Both are lined by flattened cells and both occur throughout the lymph node. In trying to resolve this issue, a more holistic diagnostic approach could be taken. For example, evaluation of the other organ systems for congestion could rule in/out blood vessel congestion as a possible diagnosis. A survey for regions of hemorrhage in the drainage field for the node could help to rule in/out sinus erythrocytosis. Figures 3E–H are examples of blood-filled vascular spaces that would require this type of holistic approach to more clearly define the lesion.
A wide range of vascular lesions in treated animals can include sinus congestion, sinus erythrocytosis, nodal and perinodal angiectasia with congestion, hemorrhage within the nodal parenchyma, perinodal vascular proliferation, and perivascular hemorrhage (Figures 3I–P). The diagnosis of treatment-related vascular lesions in the lymph node should be made with consideration of the animal’s overall health status and the presence or absence of vascular lesions in other organs and tissues. A survey of lesion terminology for the two lymph nodes in Figures 3M–P was obtained from a group of pathologists. Without any prior knowledge of treatment-related lesions and pathogenesis of the lymph node lesions in these mice, there was disagreement as to the most appropriate terminology. Sinus erythrocytosis and congestion emerged as the two favored terms. However, most agreed that information on other organs, potential areas of hemorrhage in the drainage field and time from death to necropsy were needed in order to make an accurate diagnosis.
Telangiectasia and angiomatosis are two terms that would not typically be used to define the lesions of vascular ectasia in the rodent lymph node because in human medicine they are terms that define neoplastic lesions. Telangiectasia is defined as “an abnormal dilatation of capillary vessels and arterioles that often forms an angioma” and angiomatosis is defined as “a diseased state of the vessels with the formation of multiple angiomas.” An angioma is considered a tumor that is made up of chiefly blood or lymph vessels. However, when blood-filled vessels are present in the lymph node, it is important to distinguish them from vascular tumors such as hemangiomas and hemangiosarcomas.
Pigment
Pigment is a common finding within the cytoplasm of sinusoid macrophages in both control and treated animals. The most common pigments are hemosiderin and ceroid/lipofusin. Hemosiderin is an iron-containing golden brown granular material and macrophages containing this pigment are most likely found within the medullary cords and lymphatic sinuses of nodes with sinus erythrocytosis. Lipofuscin is also a golden brown, finely granular pigment but it is derived chiefly from the breakdown products of lipids, usually those derived from cell membranes. Ceroid is a variant of lipofuscin that is acid-fast and autofluorescent. The numbers of hemosiderin- or ceroid/lipofuscin-laden macrophages can be increased with associated macrophage hyperplasia in nodes draining various lesions (inflammatory, necrotic, neoplastic, etc.). These pigments are difficult to distinguish from each other with a conventional hematoxylin and eosin stain. In order to differentiate the two, an iron stain (Perl’s iron stain, Prussian blue reaction) can be used to stain hemosiderin blue. A variety of stains and methods can be used to identify ceroid/lipofuscin including Sudan Black B, Schmorl’s reaction, Oil red O, carbol lipofuscin stain, Periodic acid-Schiff, Ziehl-Neelsen acid fast stain, autofluorescence or the lysosomal acid phosphatase and esterase stains. Melanin is another endogenous pigment that can be found within lymph nodes. This is a normal finding in black-skinned mice and is not considered a lesion. For positive identification of melanin, Schmorl’s method can be used which stains the melanin granules blue-green. DOPA-oxidase is an enzyme histochemical method that can also be used and is very specific for melanin.
A variety of inhaled, ingested, injected, and topically applied chemicals can induce sinus histiocytosis in associated lymph nodes with macrophages that contain inert or insoluble pigmented test substance (Goginpath et al., 1987). Tail tattoo pigment, which is inert and non-polarizable, can sometimes be found as aggregates of scattered brown/black material in the lymph nodes adjacent to the tattoo. Regional lymph nodes draining test article application sites should always be inspected for the presence of exogenous pigments (Figures 4A–B) but the parent compound, or its metabolite, can sometimes localize in specific tissues in the body (Figures 4C–F).
Amyloidosis
With the light microscope and hematoxylin and eosin stain, amyloid appears as amorphous, eosinophilic and hyalinized extracellular material. With large accumulations, amyloid will encroach on adjacent tissue causing pressure atrophy. Congo red is the most common stain used to evaluate amyloid, imparting an apple-green birefringence when polarized. Amyloidosis occurs in a number of strains of mice and other rodents, but is not seen in the lymph nodes of rats. Amyloidosis occurs in a low incidence in most mouse strains but in CD1 mice, there appears to be a genetic predisposition (Frith and Chandra, 1991). Lymph node amyloidosis is more prevalent in female CD1 mice and the degree of amyloidosis increases with age with 20–30% of the lymph nodes affected by 24 months of age. In these mice, amyloid deposition occurs in a variety of tissues, including the lymph node. The mesenteric lymph node is most commonly affected and amyloid predominately accumulates within the subcapsular sinuses with progressive extension into the paracortical areas of the lymph node (Figures 5A–D). Early lesions typically occur in the periphery of the node.
Lymphadenitis
Inflammatory cells can be found in lymph nodes draining sites of inflammation, necrosis, neoplasia, etc. or they can be the result of the administration of an irritating test compound. Inflammatory cells can also be present within a lymph node in response to primary lymphocyte necrosis. The type of lymphadenitis can vary depending on the inciting factor (foreign body, bacteria, etc.) and the response can vary from acute to granulomatous (Figure 6). In acute lymphadenitis, neutrophils and immature myeloid cells can be found within the sinuses and medullary cords. Lymphoid hyperplasia or atrophy may also occur in association with nodal inflammation. Inflammatory infiltrates should be distinguished from conditions of extramedullary hematopoiesis (EMH) (Figures 6F–J) and granulocytic leukemia.
With EMH, there is typically a mixture of megakaryocytes and other hematopoietic elements whereas granulocytic leukemia will have high numbers of immature myeloid cells with multiple organ involvement. The term “chronic lymphadenitis” and “granulomatous lymphadenitis” should be reserved for lymph nodes with chronic abscesses or granulomatous lesions that partially or completely efface the normal nodal architecture as opposed to increased numbers of histiocytes within the subcapsular and medullary sinuses (sinus histiocytosis) (Figures 6K–R). Abscesses may be acute or chronic and are characterized by a central region of necrosis associated with predominately neutrophils. In the later stages, they are surrounded by variable amounts of granulation tissue which can progress to fibrous connective tissue (Figures 6K–M). Pyogranulomatous lymphadenitis is characterized by an abundance of neutrophils and macrophages partially or completely effacing the normal nodal architecture.
Lymphocyte Hyperplasia
The description of reactive hyperplastic lesions, including lymphocyte hyperplasia, in rodent lymph nodes has been described (Ward, 1990). In normal rodents, lymphocyte hyperplasia may be evident to varying degrees depending on the location of the lymph node, health status of the animal, age of the animal, and plane of section of the node. Mesenteric lymph nodes, in particular, may show a wide variation in degree of reactive lymphocyte hyperplasia between animals due to stimulation by antigens in the intestinal tract. If an increase in lymphocytes is suspected to be treatment-related, then this potential for variability underscores the need to compare with control tissues. If a treatment-related effect is suspected, then enhanced histopathology may be performed to more clearly define the nature and degree of this lesion (Elmore, 2006).
Lymphocyte hyperplasia can involve both the B-cell-rich follicles and the T-cell-rich paracortex and can be indicative of a humeral or cell-mediated response, respectively (Figure 7). Lymphoid hyperplasia is generally a reactive or immune response and is not considered to be a preneoplastic lesion in the lymph node. Stimulated (reactive) follicles, also called secondary follicles, are usually larger than the unstimulated primary follicles and will have a paler staining germinal center with large lymphoblasts and increased numbers of apoptotic lymphocytes and tingible body macrophages. The mantle zone surrounding the germinal center is composed of small to medium-sized darker staining B lymphocytes. Hyperplastic follicles are identified by an increase in number and size of follicles and conversion to secondary follicles. Hyperplasia of the paracortex is characterized by an increase in the cell density and, depending on the degree of hyperplasia, an increase in the paracortical area.
Plasma Cell Hyperplasia
Plasma cells are usually increased in number in response to antigenic stimulation that requires antibody production. Therefore B cell hyperplasia can occur simultaneously with plasma cell hyperplasia. Marked plasma cell hyperplasia, or plasmacytosis, is a common finding in rodents, particularly in the submandibular lymph nodes. The medullary cords normally contain plasma cells and their precursors as the dominant cell types and these cords are the primary sites of plasma cell hyperplasia. In cases of marked plasma cell hyperplasia the node can be greatly enlarged, composed almost entirely of plasma cells, exhibit partial effacement of normal nodal architecture, and can be difficult to differentiate from neoplasia. Findings that support hyperplasia are a lack of cortical and capsular infiltration, atypical plasma cells and metastases (Figure 8). Depending on the degree and chronicity of antigenic stimulation, some plasma cells may contain Russell’s bodies. Plasma cell precursors (immunoblasts or plasmablasts) may also be present among the more mature plasma cells.
Macrophage Hyperplasia
Macrophage hyperplasia usually results from proliferation of resident sinusoidal macrophages (Figure 9A) but can also be seen as aggregates of macrophages within any region of the lymph node. Macrophage aggregates can be peripherally located around the paracortex (Figures 9B–D) or within the cortical, paracortical and medullary regions (Figures 9E–G). Macrophage hyperplasia can also be a feature of lymph nodes that drain a site of test article application (Figures 9H–I). Specific patterns (intrasinusoidal, cortical, paracortical, medullary) of macrophage hyperplasia in the same node within a dose group would be consistent with a treatment-related effect.
Proliferation (hyperplasia) of resident macrophages can easily be confused with increased numbers of intrasinusoidal macrophages that enter through the efferent lymphatics draining an area with high numbers of macrophages. Comparison of the lesions within the lymph node with the organs and tissues that that particular node drains helps to differentiate the two. Mesenteric lymph nodes are constantly stimulated by intra-intestinal antigens and can therefore have large numbers of intrasinusoidal macrophages as well as multifocal aggregates of macrophages within the cortex and paracortex. Also, there can be considerable individual variation among animals. Comparison of a group of treated animals with control animals would help to determine if this is a treatment-related finding. Special attention should be given to lymph nodes associated with the route of administration of a test compound. For inhalation studies, bronchial and mediastinal lymph nodes should be examined and orally administered compounds may result in lesions in the submandibular and mesenteric lymph nodes. Prior knowledge of the physical and chemical properties of the test compound may help to identify phagocytized test article material. For example, insoluble particulate matter may be seen as intracytoplasmic refractile material when polarized (Figures 9J–M).
When histiocytes occur as aggregates within the sinusoids, the common term for this finding is “sinus histiocytosis.” When aggregates of histiocytes occur within the lymph node parenchyma, the terms “granulomatous inflammation,” “granulomatous lymphadenitis,” “histiocyte aggregates/infiltrates,” and “macrophage aggregates/infiltrates” have been used interchangeably. However, the degree of macrophage accumulation should help to determine if the term “granulomatous” is used. If there are histiocyte aggregates with a minimal to mild severity grade, then histiocyte or macrophage aggregates/infiltrates would be appropriate. If the severity is moderate or marked with partial or complete effacement of nodal architecture, then the term “granulomatous” would be more appropriate.
Extramedullary Hematopoiesis
Although the spleen is the principle site of extramedullary hematopoiesis (EMH) in the rodent, it can sometimes be present in the lymph node. EMH is typically a physiological response to a dramatic loss or increased need for additional blood cells from conditions such as hemorrhage or severe inflammation. It is characterized by a mixture of myelocytic, erythrocytic and megakaryocytic cells and is primarily present in the medullary cords (Figure 10).
Lymphoma and MCL
There are a variety of subclassifications of lymphoma including small lymphocyte, lymphoblastic, plasma cell, immunoblastic, follicular center and marginal zone lymphomas. A consensus system for classification of mouse lymphoid neoplasms according to their histopathologic and genetic features has been proposed as a way to model human hematopoietic diseases in mice (Morse et al., 2002). However, discussion and description of each type is beyond the scope of this paper.
Lymphoma is the most common primary neoplasm arising in lymph nodes and, in the F344 rat, must be distinguished from mononuclear cell leukemia (MCL). In the B6C3F1 mouse, lymphomas often arise in mesenteric lymph nodes, spleen and Peyer’s patches (Ward et al., 1999). Lymphoma typically consists of monomorphic sheets of neoplastic lymphocytes (Figure 11A). Lymphocyte apoptosis is a common feature of lymphoma giving the lymph node a “starry sky” appearance at low magnification (Figure 11B). At higher magnification tingible body macrophages are visualized with intracytoplasmic apoptotic bodies, which represent nuclear debris (Figure 11C). General diagnostic features of lymphoid neoplasia include the size of the lymph node, the loss of normal architecture, the presence of a monomorphic population of lymphocytes, capsular invasion and perinodal fat invasion (Figures 11D–G). It should be noted that lymphoid tissue is a common finding within the perinodal fat and should not be considered neoplastic invasion without other features of lymphoma present in the node.
The diagnostic features of lymphoma can also be features of MCL. However, the cytoplasm of MCL cells may have a characteristic eosinophilic granular appearance with a hematoxylin and eosin stain or show almost no staining and the nuclear staining can range from pale to densely basophilic. Diffuse splenic red pulp involvement is the primary feature of MCL and will help to differentiate MCL from lymphoma. The presence of leukemia in other tissues such as lung (Figure 11H), liver and kidney is a common feature of advanced MCL. For more detailed information on MCL, refer to the paper by Suttie (2006).
Metastatic Lesions
Metastatic lesions in lymph nodes can arise from neoplastic blood-born emboli from tumors that are not in close proximity to the node. Metastatic neoplasias can also be found in the lymph nodes draining the region affected by the tumor. Therefore, if metastatic neoplasia is found within a lymph node and the primary tumor has not yet been identified, the location of the primary tumor may be found by evaluating tissues within the lymphatic draining field of that particular node. Although not an exhaustive list, Table 1 lists several lymph nodes and corresponding metastases in the F344 rat based on anatomic location and draining field (Stefanski et al., 1990). Detailed information on the location of specific lymph nodes and patterns of lymphatic drainage in the rat and mouse have been described (Tilney, 1971; Sainte-Marie et al., 1982; Van den Broeck et al., 2006). The photomicrographs and descriptions for Figures 12–17 illustrate and discuss various metastatic lesions in rodent lymph nodes.
Footnotes
Acknowledgments
This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.