Non-Neoplastic Histiocytic and Dendritic Cell Disorders in Lymph Nodes

Abstract

Benign and malignant proliferations of histiocytes and dendritic cells may be encountered in lymph nodes. Reactive histiocytic and dendritic cell infiltrates occur in response to diverse stimuli and in addition to causing lymphadenopathy, may be present unexpectedly in lymph nodes excised for other indications. This review summarizes the pathogenesis and histopathological features of the various non-neoplastic histiocytic and dendritic cell infiltrates that can occur in lymph nodes.

Introduction

Histiocytes and dendritic cells have a specialized role in antigen presentation and in the phagocytosis and removal of cellular debris and pathogens. Accumulations of these cells in lymph nodes, therefore, are often seen as part of a reactive immune response to foreign material, infection or other antigens. Benign histiocytic proliferations in lymph nodes are much more commonly encountered than their malignant counterparts. Whilst an underlying cause for a reactive histiocytic infiltrate is not always apparent on histopathological analysis, in certain situations the histological features are characteristic and may indicate a possible etiology. This review discusses the histological features, pathogenesis and differential diagnosis of the reactive, non-neoplastic histiocytic and dendritic cell proliferations that may be encountered in lymph nodes in practice.

Origin and Functions of Histiocytes and Dendritic Cells

The modern concept of the mononuclear phagocyte system commenced in the late 1960s and was based on the principle that macrophages were derived from peripheral blood monocytes, which in turn were bone-marrow derived [1, 2]. The dendritic cell was subsequently discovered in 1973 [3]. These individual cell types have been characterized based on morphology, function and phenotype. Macrophages are large cells with abundant cytoplasm and a primarily phagocytic function. Dendritic cells have a stellate appearance and present antigen to naïve T-cells on MHC molecules [4–6]. The term ‘histiocyte’ has been variously used to describe tissue macrophages [2, 4] or both macrophages and dendritic cells [7].

Macrophages are tissue resident cells, whereas subsets of dendritic cells migrate to the lymph nodes from the peripheral tissues [8]. The Langerhans cell, a specialized dendritic cell, migrates from the epidermis and mucosal surfaces to the lymph nodes upon encountering antigen [9]. The lymph nodes also have a resident population of classical or myeloid dendritic cells, in addition to plasmacytoid dendritic cells [8]. Plasmacytoid dendritic cells secrete large amounts of type I interferons in response to the recognition of certain nucleic acid sequences [10, 11]. They also have the capacity to present antigen and can exert a tolerogenic or immunogenic effect on the immune response [11].

In recent years and mainly in mouse models, significant progress has been made in our understanding of the developmental pathways of macrophages, monocytes and dendritic cells. It is now known that adult macrophages and Langerhans cells have an embryonic origin and self-renew in the tissue independently from monocytes [9, 12, 13]. Dendritic cells (classical and plasmacytoid) and monocytes derive from bone marrow hematopoietic stem cells by way of distinct precursor pathways [6, 8]. Monocyte-derived cells may replenish populations of macrophages and dendritic cells at specific sites or under certain inflammatory conditions [12, 13]. Further studies will precisely delineate the relationship between these cells and the similarities and applicability of the findings in the mouse to human dendritic and monocyte cell subsets.

In addition to dendritic cells of hematopoietic origin, lymph nodes also contain mesenchymally-derived cells including follicular dendritic cells and fibroblastic reticulum cells [14, 15]. Follicular dendritic cells are associated with the B-cell follicles, where they maintain the follicular structure and organization of the germinal center and present antigen to B-cells [16, 17]. Fibroblastic reticular cells form an interconnected network that provides structure and organization to the lymph node. They are heterogeneous and comprise a number of subsets which support the growth of and regulate the migration of different cells within the lymph node through cellular interactions and chemokine secretion [15, 18, 19].

Some of the immunohistochemical stains that are used in routine practice to identify and differentiate between the major histiocytic and dendritic cell subsets in lymph nodes are listed in Table 1.

Table 1.

Immunophenotype of major histiocytic and Dendritic cell subsets [7, 16, 185, 197, 200]

Antibody	Macrophage	Langerhans cell	Interdigitating dendritic cell	Follicular dendritic cell	Plasmacytoid dendritic cell
CD68	+	+/− Golgi	+/−	−	+
CD163	+	−	−	−	−
S100	−/+	+	+	−/+	−
Langerin	−	+	−	−	−
CD1a	−	+	−	−	−
CD21	−	−	−	+	−
CD35	−	−	−	+	−
CD123	−	−	−	−	+
TCL1	−	−	−	−	+
CD4	+	+	+	+	+

Histiocytic (Macrophage) Infiltrates

Sinus Histiocytosis

Sinus histiocytosis is a common feature in lymph node biopsies and is characterized by dilated lymph node sinuses containing variable numbers of histiocytes, with bland, indented nuclei and eosinophilic cytoplasm (Figure 1A). Many descriptions of sinus histiocytosis in the literature pertain to its prognostic significance in lymph nodes draining sites of tumor. The significance of this association varies, although a more favorable prognosis has been reported [20–22]. On occasion, the histiocytes may have a signet ring appearance, which may be mistaken for metastatic signet ring carcinoma or melanoma; however, the distinction is easily made by immunohistochemistry and special stains, as the histiocytes are positive with CD68 and negative for cytokeratin, S100 and mucin stains, although they may contain Periodic Acid-Schiff (PAS) positive globules [23–25].

Figure 1. (A) Sinus histiocytosis.

Lymph node sinuses are dilated and filled with benign-appearing histiocytes. (B–C) Hemophagocytic lymphohistiocytosis: (B) Histiocytes in the lymph node sinuses show prominent hemophagocytosis, (C) which can be highlighted using histiocytic markers (CD4 in image).

Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is an immune disorder characterized by systemic inflammation and uncontrolled hypercytokinemia. It is an uncommon [26–28] and life-threatening condition [29–31]. HLH presents with sudden onset fever, hepatosplenomegaly, generalized lymphadenopathy, malaise and cytopenias. The diagnostic criteria for HLH were updated in 2004 and require either a molecular diagnosis or 5 of 8 of the following clinical or laboratory parameters: (1) Fever, (2) Splenomegaly, (3) Cytopenias affecting 2 or more lineages, (4) Hypertriglyceridemia and/or hypofibrinogenemia, (5) Hemophagocytosis in bone marrow, spleen or lymph nodes, (6) Low or absent NK-cell activity, (7) Ferritin ≥500μg/L and (8) Soluble CD25 ≥2400U/mL [32].

HLH may be primary (familial) or secondary (acquired). Familial HLH (FHL) usually presents in the first year of life and results from mutations that interfere with the cytotoxic function of lymphocytes and NK-cells by affecting the formation of perforin or the trafficking, docking or membrane fusion of cytotoxic granules [33, 34]. In an effective cytotoxic response, lysis of the target cell removes the antigenic stimulus and terminates the inflammatory response. In HLH, it is thought that the defect in the cytotoxic pathway results in persistence of the stimulus and an inability to terminate the inflammatory response, leading to hypercytokinemia and tissue infiltration by macrophages, NK-cells and T-cells [28, 33, 35]. Mutations have been found in multiple genes in the cytotoxic pathway including: PRF1 (perforin) [36], UNC13D (Munc13-4) [37], STX11 (Syntaxin 11) [38] and STXBP2 (Munc18-2) [39, 40]. Certain mutations are associated with immunodeficiency or other manifestations such as hypopigmentation, including mutations in RAB27A, resulting in Griscelli syndrome type 2 [41], CHS1/LYST causing Chediak-Higashi syndrome [42, 43] and AP3B1 causing Hermansky-Pudlak syndrome type 2 [34, 44, 45]. X-linked lymphoproliferative disease and several primary immunodeficiencies also predispose to HLH or HLH-like manifestations usually mediated by Epstein-Barr virus (EBV) infection [4, 28, 46, 47] (Table 2). Acquired HLH is due to secondary causes including infection (EBV, Herpes viruses), malignancy and rheumatological conditions, where it is also referred to as macrophage activation syndrome (MAS) [4, 28, 48, 49]. The mechanisms of acquired HLH are still unclear, but hypercytokinemia is a common thread [33, 35].

Table 2.

Genetic Alterations associated with hemophagocytic lymphohistiocytosis [28, 34, 35]

Disease	Chromosome	Gene	Protein	Function	Associated Features
FHL1 [201]	9q21.3-22	Unknown	Unknown	Unknown
FHL2 [36]	10q21-22	PRF1	Perforin	Creates pores in target cell
FHL3 [37]	17q25	UNC13D	Munc13-4	Vesicle priming preceding vesicle membrane fusion
FHL4 [38]	6q24	STX11	Syntaxin11	Vesicle membrane fusion
FHL5 [39, 40]	19p13	STXBP2	Munc18-2	Vesicle membrane fusion	Severe diarrhea
Griscelli syndrome, type 2 [41]	15q21	RAB27A	Rab27a	Small GTPase family of proteins – vesicular docking	Partial albinism
Chediak-Higashi syndrome [42, 43]	1q42.1-42.2	LYST	LYST (lysosomal trafficking regulator)	BEACH family of proteins. Vesicle trafficking regulatory function.	Partial albinism, immunodeficiency, leukocyte giant granules, neurological disorder
Hermansky-Pudlak syndrome, type 2 [44, 45]	5q14.1	AP3B1	Subunit β3A of AP-3 complex	Lysosomal trafficking	Oculocutaneous albinism, bleeding diathesis, neutropenia, recurrent infections
X-linked lymphoproliferative disease, type 1 [46]	Xq25	SH2D1A	SAP (SLAM- associated protein)	Cell signaling	Fulminant infectious mononucleosis, EBV-related HLH, lymphoid proliferations, hypogammaglobulinemia, risk of lymphoma
X-linked lymphoproliferative disease, type 2 [47]	Xq25	BIRC4	XIAP (X-linked inhibitor of apoptosis)	NFκB and MAPK pathways, inhibitor of apoptosis	HLH (EBV, CMV or HHV-6 infection), hypogammaglobulinemia, inflammatory bowel disease
Other primary immunodeficiencies [4, 28]	CD27 deficiency - CD27 EBV-associated autosomal lymphoproliferative syndrome (ITK deficiency) - ITK X-linked immunodeficiency with magnesium defect, EBV infection and neoplasia (XMEN) - MAGT1				EBV-associated HLH/HLH-like manifestations; lymphoproliferation, immunodeficiency

The characteristic histopathological feature is hemophagocytosis, the phagocytosis of other hematopoietic cells by the activated macrophages that infiltrate the tissue (Figure 1B–C). The preferred diagnostic material is usually a bone marrow aspirate; however, hemophagocytosis may be observed in other tissues including lymph nodes, spleen and liver [35, 49]. Lymph node histology in HLH can be variable. The macrophages are cytologically benign and may diffusely infiltrate the nodal parenchyma or involve the nodal sinuses [50, 51]. In cases of HLH due to underlying EBV infection, the predominant feature may be an immunoblastic reaction with demonstrable EBV [51]. Immunohistochemistry with histiocytic markers (CD68, CD163 or others) is helpful in identifying the phagocytic macrophages (Figure 1C). The macrophages may show variably positive staining for S100, in contrast to the emperipoletic histiocytes of Rosai-Dorfman disease which are strongly positive [50, 52]. In patients with a perforin deficiency, perforin can be absent in the cytotoxic lymphocytes by flow cytometry or immunohistochemistry [53, 54]. HLH may also occur in the context of an underlying malignancy, particularly lymphoma or leukemia; therefore, such processes should be sought when examining the lymph node biopsy. The diagnosis of HLH is a clinical one, as hemophagocytosis is not entirely sensitive or specific: it is not always present on biopsy in the setting of HLH, and furthermore, it can be seen in the tissue in the absence of HLH, such as in the setting of sepsis or surgery [30, 35, 55, 56].

Whipple’s Disease

Whipple’s disease is a rare multisystem illness caused by the rod-shaped bacillus Tropheryma whipplei. Although initially described by George Hoyt Whipple in 1907 [57], the first case of the disease was probably reported earlier by Allchin and Hebb in 1895 [58, 59]. The pathogenic organism remained unknown until the 1990s [60]. T. whipplei is genetically diverse, but the strain of the organism has not been found to correlate with the clinical presentation of the infection [61, 62]. The classic form of the disease typically affects middle-aged white men and involves the intestinal tract, with symptoms of fever, abdominal pain, weight loss and diarrhea [58, 63–65]. Abdominal lymphadenopathy is often present. Arthralgia is also common, in particular it manifests as a prodromal stage preceding the development of gastrointestinal symptoms. The disease may affect the central nervous system and heart [64, 65]. Peripheral lymphadenopathy is present in approximately 50% [63, 66, 67]. In addition to the classic form, T. whipplei may cause an acute infection [65, 68] or infection localized to extra-intestinal sites [65]. Asymptomatic carriage of the organism also occurs and it may be detected in saliva and stool [64, 69].

Classic Whipple’s disease is rare, with an estimated incidence of between 1 and 6 new cases per 10,000,000 persons per year worldwide [65]; however, the higher asymptomatic carrier rates suggest that genetic predisposition or immune dysfunction are important factors in the pathogenesis of the disease. Studies have found an increased frequency of HLA DRB1*13 or DQB1*06 in patients with Whipple’s disease [70], and have also demonstrated abnormalities in macrophage function and T-cell responses. Macrophages in patients with Whipple’s disease have been shown to be shifted towards an alternatively activated phenotype with high expression of IL-10 and reduced IL-12 expression [64, 71–73]. There is skewing towards Th2 and T-regulatory cell responses, and a reduced Tropheryma whipplei-specific Th1 response has been found in patients with Whipple’s disease [74–78]. IL-16 has been implicated in macrophage phagosome dysfunction and bacterial replication [79, 80].

Histopathological diagnosis is usually made on duodenal or proximal small bowel biopsies which show aggregates of foamy macrophages present in the lamina propria. Involved lymph nodes may show capsular fibrosis with dilated cystic spaces and nodal sinuses [66]. The characteristic histiocytes can have a sinusoidal or paracortical distribution. Foreign body giant cells and epithelioid granulomas may be present; however, necrosis is typically absent (Figure 2A–B) [81–83]. The macrophages are positive with CD68 and the intracellular bacteria are PAS-positive [84], diastase resistant (Figure 2C). Ziehl-Neelson (ZN) stain is negative, allowing distinction from mycobacterial infection. The diagnosis can be confirmed by T. whipplei specific PCR or by specific immunohistochemistry [64, 81]. Asymptomatic carrier states of T. whipplei may occur in the gastrointestinal and respiratory tracts, so a positive PCR result from these sites necessitates correlation with additional parameters [64, 81]. The presence of epithelioid granulomas in nodal sites may mimic sarcoidosis, a pitfall documented in several reports [82, 83, 85]. Of note in these cases, the PAS stain may be negative and the intracellular bacteria present in low numbers by electron microscopy.

Figure 2. (A–C) Whipple’s disease.

(A) Small aggregates of macrophages are present in the lymph node sinuses and in the parenchyma forming loose epithelioid granulomas. (B) The macrophages have eosinophilic granular cytoplasm and occasional multinucleated giant cells are present. (C) The bacilli are positive with DPAS stain. (D) Histiocytosis following joint replacement: Sheets of polygonal histiocytes with abundant eosinophilic cytoplasm containing small black particles consistent with metal wear debris. (E–F) Silicone lymphadenopathy due to breast implant: (E) Aggregates of macrophages with abundant foamy, vacuolar cytoplasm are present within the lymph node. (F) The vacuoles are colorless and the silicone, where present, has a refractile quality and is non-polarizable.

Treatment is with antibiotic therapy and the disease is ultimately fatal if not treated [58, 63, 64]. PAS positive macrophages may persist for some time after therapy and improvement in clinical symptoms [86, 87]. Patients with Whipple’s disease may initially be misdiagnosed with a seronegative arthritis and treated with immunosuppression. Such patients are at an increased risk of developing immune reconstitution inflammatory syndrome (IRIS) during antimicrobial therapy [88]. The disease may relapse after many years and life-long follow up of patients is warranted [78].

Reactive Histiocytosis following Prosthetic Arthroplasty

Arthroplasty or joint replacement is a common surgical procedure for treatment of damaged or diseased joints and articular surfaces due to degenerative joint disease, rheumatoid arthritis, malignancy or trauma. The components of an artificial joint may be manufactured from metals such as stainless steels and cobalt-chrome, ultrahigh molecular weight polyethylene or ceramic. The prosthesis may be fixed in place using a polymethylmethacrylate cement. Usually, both articular surfaces of a joint are replaced, and the replacement surfaces may be composed of similar or different materials [89]. Histiocytic infiltrates can occur in lymph nodes draining the sites of arthroplasty prostheses in response to wear particles that are generated from the interface between the individual artificial joint components, as well as the adjacent bone [90–92]. Over time, interaction or wear at the sites of the replaced articular surfaces leads to the production and deposition of these particles in the periarticular tissue initiating a local inflammatory response with cytokine release [90, 92–95]. Wear particles may drain from the soft tissue to regional lymph nodes as free particles which are phagocytosed by macrophages forming nodal aggregates. It is also possible that some phagocytosis of the particles may occur in the periarticular tissue by macrophages which subsequently enter the lymphatics [90, 91, 95].

Histologically, involved lymph nodes show sinusoidal and interfollicular expansion by sheets of polygonal histiocytes with abundant granular or foamy cytoplasm and round nuclei, without cytologic atypia. Foreign-body type giant cells may also be present (Figure 2D) [90, 91, 93–97]. The microscopic characteristics of the wear particles depend on the material used in the prosthesis. Metal wear debris may be identified as small, irregular black particles within the cytoplasm of the histiocytes and sometimes extracellularly [90, 95, 96, 98]. Occasionally, metal particles may be numerous and produce tattooing; however, the particles can also be subtle and may be overlooked in the setting of a florid reactive histiocytic proliferation [90, 96]. The presence of metals including cobalt-chromium, titanium, molybdenum or iron in affected lymph nodes has been confirmed using energy dispersive x-ray microanalysis [90, 96] and inductively coupled plasma mass spectrometry [98].

Polyethylene wear fragments are colorless and vary in size. The particles are present as needle-like shards within macrophages and multinucleated giant cells. Polyethylene is intensely birefringent under polarized light [90, 91, 93–95]. Polymethylmethacrylate (PMMA) dissolves during tissue processing; however, is usually admixed with particles of a radiodense metal (barium or zirconium oxide) and is therefore characterized histologically in the implant bed by a large clear space with surrounding histiocytes and giant cells containing scattered granules of the admixed metal [89, 93, 99]. It is not commonly described in lymph nodes as it is not visible on paraffin sections; however, it has been suggested that phagocytosed PMMA may contribute significantly to the cytoplasmic appearance of the histiocytes in some cases [97, 100].

Resulting lymphadenopathy may be of clinical concern, particularly when the context raises the possibility of metastatic disease, such as in patients with a prosthesis resulting from treatment of a bone malignancy or the onset of pelvic lymphadenopathy in a patient with gynecologic or genitourinary tract malignancy. The identification of the metal or polyethylene fragments should prompt clinical correlation in cases where the history of a prosthesis is unknown.

Silicone Lymphadenopathy

Silicone lymphadenopathy usually occurs in regional lymph nodes draining sites of silicone-containing medical implants, typically silicone gel-filled breast implants [101] or silicone elastomer joint prostheses [102]. As local lymph nodes are involved, the axilla is the usual site of adenopathy in cases associated with breast implants and prostheses used in the replacement of the small joints of the hand; however, silicone may also travel to more distant sites in the body and has been described in supraclavicular and cervical lymph nodes [103], and in lymph nodes in the contralateral axilla to the breast implant [104].

Silicone gel from breast implants may enter the lymphovascular channels due to implant rupture or from miniscule leakages or “bleeding” from an intact implant bag [101, 105–107]. In the case of an elastomer-containing joint prosthesis, small fragments of the elastomer may become detached from the surface of the prosthesis due to wear and enter the tissue [105]. The silicone used in medical implants is composed of dimethylsiloxane polymers and the nature of the silicone (liquid, gel or elastomer) depends on the length and cross-linking of the polymer chains [106, 108].

The extent of the nodal effacement ranges from focal involvement to the presence of substantial parenchymal infiltrates. The histological features of the process differ depending on the consistency of the silicone present in the lymph nodes [105, 106]. Silicone elastomer causes a foreign body giant cell reaction which is sometimes associated with the formation of non-necrotizing granulomas composed of epithelioid histiocytes [102, 109]. The silicone fragments are present within the cytoplasm of the giant cells or within the granulomatous inflammatory reaction, which on occasion, may be marked [110]. Silicone in a liquid or gel form causes vacuoles of different sizes within the nodal parenchyma. These silicone particles are taken up by histiocytes, giving them a characteristic foamy, heavily vacuolated cytoplasm [106, 107] (Figure 2E–F). The number of giant cells present is usually much fewer than is seen with silicone elastomer [105]. The silicone may not survive tissue processing, and therefore the vacuoles may be devoid of material. If present, silicone appears as strands of a refractile, non-polarizable, clear substance within the vacuoles [101, 106, 107]. Various methods have been used in different studies to confirm the presence of silicone in the lymph nodes including energy dispersive x-ray analysis [105, 106], confocal laser-Raman microprobe (CLRM) spectroscopy [107] and Fourier transform infrared spectroscopy (FTIR) [107, 111].

Silicone lymphadenopathy may clinically mimic a malignant process, such as lymphoma [112]. An important consideration is metastatic carcinoma, particularly in patients with a breast implant for reconstructive purposes following resection of a breast malignancy. Some reports have shown that the involved nodes may be PET scan positive, which may heighten the clinical concern for a malignant process [113, 114]. In such situations, the histological features may raise the consideration of a metastatic lobular carcinoma; however, the vacuolated cells will be positive for histiocytic markers such as CD68, and negative for cytokeratin and histochemical stains for mucin, confirming the benign reactive nature of the process.

Crystal Storing Histiocytosis

Crystal storing histiocytosis is a rare disorder characterized by aggregates of histiocytes with abnormal intracytoplasmic crystals. In most cases (90% in one review [115]), the histiocytosis occurs in association with a B-cell lymphoproliferative disorder, usually lymphoplasmacytic lymphoma [116, 117], multiple myeloma [118] or monoclonal gammopathy of uncertain significance (MGUS) [119]; however, it has also been described in inflammatory and autoimmune conditions including rheumatoid arthritis [120], H. pylori infection [121] and Crohn’s disease [122]. Crystal storing histiocytosis can be classified according to the disease association or the composition of the crystals [115]. In most cases, the crystals are composed of immunoglobulin, but crystal storing histiocytes have also been described with the use of the drug clofazimine [123] to treat leprosy, in hereditary cystinosis [124] and with Charcot-Leyden crystals in the setting of eosinophilic inflammation [125].

The mechanism by which the crystals accumulate within the histiocytes is not known, but it is thought to be due to the structural properties of the immunoglobulin in combination with high levels of the immunoglobulin in the serum [120, 126]. It has been suggested that conformational changes in the immunoglobulin due to amino acid substitutions may be a pathogenic factor, with one case report identifying unusual amino acid substitutions, including at a site important for hydrophobic interactions within the protein structure [127]. Inherited or acquired processing defects in histiocytes have also been theorized [128]. Ultrastructural studies performed indicate that the immunoglobulin is endocytosed by macrophages and the crystals are formed within lysosomes during lysosomal digestion [117, 129, 130]. In the case of clofazimine, the drug has been observed within macrophage phagosomes prior to the development of the crystals [123, 131].

Histologically, the histiocytes are ovoid to spindle shaped, with abundant eosinophilic cytoplasm and contain packed elongated structures, some of which may show a parallel arrangement. The nuclear features are benign and the nuclei may be peripheralized within the cell (Figure 3). Occasional multinucleated cells may be present [116, 117, 120, 132, 133]. The histiocytic infiltrate is variable, but may be so prominent as to obscure an associated B-cell lymphoma [115, 128]. The histiocytes are positive for histiocytic markers such as CD68 (Figure 3C) and CD163 and are negative for desmin, S100 and markers of B-cell/plasma cell lineage [116, 120, 133]. Immunoglobulin crystals within the cytoplasm of the histiocytes may show monotypic light chain or heavy chain expression (Figure 3D–E); however, cases without demonstrable staining have been described. The negativity of the crystals in these cases has been attributed to the altered immunoglobulin structure or to poor tissue fixation [116]. There is no clear association with any individual heavy or light chain class [115]. Histochemically, immunoglobulin crystals stain blue with phosphotungstic acid hematoxylin, and show variable staining with PAS [116, 120, 132]. They are needle-shaped or rhomboid-shaped on ultrastructural examination [120, 129, 133]. Clofazimine crystals are red and show bright-red birefringence in frozen sections; however, they dissolve during tissue processing and are therefore colorless in formalin-fixed paraffin-embedded tissue sections [123].

Figure 3. Crystal storing histiocytosis associated with marginal zone lymphoma with plasmacytic differentiation.

(A) The lymphoma component is diffuse in areas, containing only scattered admixed histiocytes. (B) Elsewhere, there are sheets of histiocytes with abundant eosinophilic fibrillary cytoplasm. (C) The histiocytes are positive with CD68, which aids in visualizing the intracytoplasmic rhomboid and needle-shaped crystals. Immunohistochemistry for kappa (D) and lambda (E) show that the crystals and background plasma cells in this case are positive for lambda light chain.

The differential diagnosis of crystal storing histiocytosis includes rhabdomyoma[116], from which it can be distinguished by immunohistochemistry, malakoplakia and storage disorders such as Gaucher disease.

Lysosomal Storage Disorders

Lysosomal storage disorders are rare diseases, some of which are associated with histiocytic infiltrates in bone marrow and other organs including lymph nodes.

Niemann-Pick disease comprises two different abnormalities in lipid metabolism: types A and B are due to a functional deficiency of acid sphingomyelinase (ASM), whereas type C results from defective intracellular trafficking of cholesterol [134, 135]. In types A and B, many different mutations and deletions have been reported in the SMPD1 gene on chromosome 11p15.4, which encodes ASM [135]. Most patients with Niemann-Pick type C have mutations in the NPC1 gene on chromosome 18q11-q12 (approximately 95%), other patients have mutations in the NPC2 gene on chromosome 14q24.3 [134]. The Niemann-Pick cell is lipid-laden with abundant foamy cytoplasm containing vacuoles of sphingomyelin or cholesterol – the so-called ‘mulberry appearance’ [135].

Gaucher disease is an autosomal recessive sphingolipidosis caused by mutations in the glucocerebrosidase gene (GBA) on chromosome 1q21. Mutations reduce the enzymatic activity of β-glucocerebrosidase resulting in the accumulation of its substrate, glucosylceramide (glucocerebroside), in macrophage lysosomes. The characteristic Gaucher cells have abundant blue-grey cytoplasm with a fibrillary appearance that is often compared to wrinkled tissue paper [136, 137] and are positive with PAS and Prussian blue stains [136, 138]. Pseudo-Gaucher cells may be seen in association with other conditions including chronic myeloid leukemia and hemoglobinopathies. There are 3 major clinical phenotypes, the most common, type 1, is non-neuropathic and characterized by hepatosplenomegaly, cytopenias and bone marrow involvement. Types 2 and 3 both have neurological involvement, with type 2 usually fatal within the first 1–2 years of life [136, 137].

Granulomatous Lymphadenitis

Granulomatous inflammation is a form of chronic inflammation that occurs in response to infectious, autoimmune, neoplastic and unknown causes [139]. The granuloma is composed of an admixture of cells of the mononuclear phagocyte system, including epithelioid histiocytes and multinucleated giant cells. Epithelioid histiocytes form clusters and are characterized by abundant eosinophilic, granular cytoplasm, indistinct cell borders and oval or elongate nuclei. Multinucleated giant cells in granulomas were traditionally described as either Langhans type (with multiple nuclei organized at the periphery of the cell) or foreign-body type (nuclei present throughout the cytoplasm) [139, 140]. Both types can be present. Granulomatous inflammation occurs in response to an antigen that is insoluble or otherwise difficult to eliminate [139]. Macrophages enter the tissue and secrete TNF-α and pro-inflammatory cytokines including IL-12 and IL-23, recruiting other inflammatory cells and CD4-positive T-cells.

Typically, a Th1 immune response predominates with IFN-γ production resulting in granuloma formation, although the exact components of the granuloma vary with the underlying cause [140–142]. It has been suggested that a Th2 response may develop over time in certain autoinflammatory granulomata, with shifting of the macrophage phenotype from M1 to M2 as fibrosis develops [141, 142]. Macrophage polarization and diversity are also important in the response to Mycobacterium tuberculosis infection [143].

Histologically, granulomatous inflammation can be categorized as non-necrotizing, necrotizing and suppurative [50] (Table 3).

Table 3.

Differential Diagnosis of Granulomatous Inflammation in Lymph Nodes [50]

Necrotizing/Suppurative Granulomas	Clinical	Nodal Sites	Histological Features1	Histochemical stains & Immunohistochemistry	Other Investigations
Mycobacterial lymphadenitis (Mycobacterium tuberculosis) [153, 154]	History of exposure to TB	Cervical, axillary, mediastinal	Central necrosis. Acid fast bacilli	Ziehl-Neelson: + Fite: +	Tuberculin skin test IFN-γ release assay PCR/culture
Histoplasmosis (Histoplasma capsulatum) [156, 157]	Asymptomatic, pulmonary infection or disseminated depending on immune status and exposure level	Hilar or others in disseminated	2–4μm, clustered in macrophages. Difficult to appreciate in granulomas without histochemical stains	GMS: + PAS: + Mucicarmine: −	Serology Histoplasma antigen EIA Culture
Cryptococcosis (Cryptococcus neoformans, Cryptococcus gattii) [156, 202, 203]	Pulmonary involvement. May involve CNS. May be disseminated	Cervical, hilar, axillary, inguinal and others	Well-formed granulomas. May form cystic spaces with gelatinous material. 5–10μm - within clear spaces in granulomas and macrophages	PAS: + GMS: + Mucicarmine: + Fontana-Masson: +	Cryptococcal antigen EIA Culture
Cat Scratch disease (Bartonella henslae) [158, 159, 162, 163]	History of contact with cats Skin lesion	Axillary, cervical, epitrochlear	Warthin-Starry: Pleomorphic coccoid or curved bacilli in clumps or singly in vessel walls or in foci of necrosis	Brown-Hopps: Faint (gram- negative) Warthin-Starry: + Ziehl-Neelson: − Immunohistochemistry for Bartonella henslae	Serology PCR
Lymphogranuloma venereum (Chlamydia trachomatis, L1, L2, L3) [164, 204]	Mucosal or skin lesions in genital tract or rectum	Inguinal, femoral or iliac nodes. May fistulate.	Macrophages with vacuoles. Organisms form a central clump or a peripheral rim in vacuoles	Warthin-Starry: + PAS: − Ziehl-Neelson: − Brown-Hopps: Gram- negative	Serology Nucleic acid detection
1. H&E. Features on special stain if indicated
Non-Necrotizing Granulomas	Clinical	Nodal Sites	Histological Features	Histochemical stains & Immunohistochemistry	Other Investigations
Sarcoidosis [142, 144, 148]	Multi-system disease; pulmonary symptoms; erythema nodosum	Bi-hilar adenopathy	Sharply demarcated granulomas; Asteroid bodies, Schaumann bodies; Hamazaki- Wesenberg bodies	Ziehl-Neelson and fungal stains are negative. Hamazaki-Wesenberg bodies are GMS + and PAS + (Pitfall)	Serum ACE levels Chest x-ray Microbiological cultures

The prototypical example of non-necrotizing granulomatous inflammation (Figure 4A) occurs in sarcoidosis, a multi-system disease in which the underlying cause is still unknown [142, 144]. Histologically, sarcoidal granulomas are well-demarcated, usually numerous and in close proximity to one another in involved lymph nodes. They contain both epithelioid cells and giant cells, and may have focal necrosis [145, 146]. Cytoplasmic inclusions can be identified within the granulomas, including asteroid bodies (spiculated inclusions in giant cells composed of complex lipoproteins) and Schaumann bodies (calcified structures with concentric lamellations) [145, 147, 148]. Hamazaki-Wesenberg bodies are thought to represent extracellular giant residual lysosomal bodies [149] and may be present near the subcapsular sinuses [146]. They are positive with Gomori methenamine silver (GMS) and PAS stains, and therefore present a pitfall in that they may be mistaken for fungal infection [150]. These inclusions, whilst commonly described in association with sarcoidosis, are not specific and occur in other conditions. Non-necrotizing granulomata can also be present in lymph nodes in Crohn’s disease [151] and a granulomatous reaction with or without necrosis may also occur in lymph nodes in association with malignancy, including carcinoma, Hodgkin’s lymphoma and non-Hodgkin’s lymphoma [152].

Figure 4. Granulomatous Inflammation.

(A) Non-necrotizing granuloma: The granuloma is well-demarcated and composed of epithelioid histiocytes with a peripheral lymphoid cuff. (B) Necrotizing granuloma: There is a large area of necrosis surrounded by a peripheral rim of epithelioid histiocytes. A giant cell is also present. (C–E) Disseminated histoplasmosis: (C) The lymph node is effaced by sheets of macrophages with extensive necrosis. (D) The yeast is present in clusters within the cytoplasm of the macrophages and is positive with (E) GMS stain. (F) Acid-fast bacilli (Fite): Mycobacteria, a cause of necrotizing granulomatous lymphadenitis, may be few in number and difficult to identify. (G–J): Cryptococcal lymphadenitis: (G) Granulomatous inflammation with central cystic spaces. (H) The yeast is visible within the cytoplasm of the histiocytes as faintly staining structures within clear spaces. It is positive with (I) GMS and (J) mucicarmine stains.

Necrotizing granulomas (Figure 4B) usually occur due to an infectious process and are characteristically seen with Mycobacterium tuberculosis infection, in which cervical lymphadenopathy is the most common site of peripheral lymph node involvement [153]. Tuberculous granulomas are composed of a central area of necrosis surrounded by epithelioid histiocytes, Langhans-type giant cells and lymphocytes. The central necrosis typically does not contain cellular debris. With age, fibrosis and calcification of the lymph node occurs [154]. Mycobacterial organisms are acid-fast and can be demonstrated using ZN or Fite stains (Figure 4F), although demonstration of the organism is often unsuccessful in tissue sections and bacterial culture or PCR are used to identify the organism [153, 155]. Fungal infection may also result in a necrotizing granulomatous lymphadenitis, such as occurs in Histoplasma capsulatum infection. Lymph nodes involved by H. capsulatum contain epithelioid granulomas and sheets of yeast-containing macrophages which undergo necrosis (Figure 4C). The yeast is round to oval, 2–4μm in size and visible in macrophages as intracytoplasmic clusters (Figure 4D–E). It is difficult to appreciate in the epithelioid granulomas on H&E; however, is detectable with GMS stain [156, 157]. Histoplasma capsulatum must be distinguished from Cryptococcus (Figure 4G–J), which in addition to GMS, also stains with mucicarmine and Fontana-Masson stains [156].

Suppurative granulomatous inflammation is seen in Cat Scratch disease, a usually self-limited lymphadenitis caused by the gram-negative bacterium Bartonella henslae. Histologically, the lymph node shows stellate-shaped neutrophilic abscesses, with a surrounding rim of epithelioid histiocytes and Langhans-type giant cells. In the early stages of involvement, there may be follicular hyperplasia and a monocytoid B-cell reaction [158–160]. The organism can be demonstrated using a Warthin-Starry stain as pleomorphic bacilli present singly or in clumps in the foci of necrosis, in macrophages, or in the walls of capillaries [161]. Immunohistochemical stains are also useful, as they avoid the background precipitate that may make interpretation of silver stains difficult [162] and PCR can also identify the organism in formalin-fixed tissue [163]. Other causes of suppurative granulomatous inflammation include lymphogranuloma venereum (Chlamydia trachomatis L1, L2, L3) [164] and tularemia (Francisella tularensis) [165].

Mixed Histiocytic and Dendritic Cell Infiltrates

Dermatopathic Lymphadenopathy

Dermatopathic lymphadenopathy is a reactive condition that classically occurs in lymph nodes draining sites of chronic skin disorders. Some of the earliest descriptions have been variously attributed in the literature to Jadasson in 1892 [166, 167], Wise in 1917 [168, 169] or to Pautrier and Woringer in French (réticulose lipo-mélanique/lipomelanotic reticulosis) in the 1930s [169–171]. The process was eventually termed dermatopathic lymphadenitis in 1942 by Hurwitt [167]. The lymphadenopathy may be generalized or localized. Axillary and inguinal lymph nodes [169, 171] are most frequently involved and a peripheral blood eosinophilia may be present [166, 167, 169, 172].

Dermatopathic lymphadenopathy occurs at any age, but it is more common in the 5^th and 6^th decades [169, 173] and in males [166, 167, 169, 172]. In their series of 906 consecutive lymph node biopsies, Cooper et al reported an incidence of 4.8% with 15% (6/40) of their cases of dermatopathic lymphadenopathy occurring in patients with mycosis fungoides [169]. Other skin conditions described in patients with dermatopathic lymphadenopathy include psoriasis, eczema, exfoliative dermatitis, neurodermatitis, cutaneous hypersensitivity reactions, seborrheic dermatitis and non-specific chronic dermatitis [166, 167, 169].

Whilst theories regarding the pathogenesis of dermatopathic lymphadenopathy have centered on an inflammatory reaction with excessive absorption of melanin from the skin due to the underlying skin disorder [166, 167, 170, 172], some lymph nodes may have histologic features of dermatopathic lymphadenopathy in the absence of an associated skin disease [169]. Gould et al, in a study of 1181 lymph nodes from axillary dissections from patients without skin disease, observed varying degrees of dermatopathic-like histologic features, concluding that dermatopathic lymphadenopathy “may represent one end of a normally occurring histologic spectrum that may be found in the absence of a dermatitis” [170, 174]. There is also the consideration that changes may reflect an undocumented prior dermatitis [174] - in one study, a lapse of 40 years was reported in one case between the resolution of skin changes and the lymph node biopsy [166].

Histologically, the architecture of the lymph node is intact, but there is variable expansion of the paracortex by a pale staining proliferation composed of interdigitating dendritic cells, histiocytes and Langerhans cells, which may compress and peripheralize the cortical areas of the node [166, 174, 175] and surround residual atrophic follicles [172]. Mitotic activity may be present. At the margins of the proliferation [166, 169], some of the histiocytes contain phagocytized pigment, usually melanin, although some hemosiderin may be present [167, 169]. Phagocytized cytoplasmic lipid may also be evident [169, 172]. The infiltrate contains variable numbers of lymphocytes, plasma cells and eosinophils (Figure 5A–B).

Figure 5. (A–D) Dermatopathic lymphadenitis.

(A) The lymph node paracortex is expanded by pale staining nodules. (B) The pale staining areas are composed of an admixture of dendritic cells, Langerhans cells and histiocytes, some of which contain pigment. The Langerhans cells may be numerous and are identified by immunohistochemistry with (C) CD1a and (D) langerin. (E–F) Plasmacytoid dendritic cell aggregate: (E) The plasmacytoid dendritic cells have pale cytoplasm and fine chromatin. Scattered tingible body macrophages are present. (F) CD123 stains both the plasmacytoid dendritic cells and the endothelial cells, highlighting the location of the plasmacytoid dendritic cell aggregate adjacent to the high endothelial venules.

By immunohistochemistry, the interdigitating dendritic cells are positive for S100 and negative for CD1a and langerin. Langerhans cells are positive for S100, CD1a and langerin (Figure 5C–D) [175, 176]. The paracortical pattern of the infiltrate is useful in the distinction from Langerhans cell histiocytosis, which is generally sinusoidal. The associated histiocytes are positive for CD68 and lysozyme and the presence of melanin and hemosiderin can be confirmed by histochemical stains [167, 169, 177].

As dermatopathic lymphadenopathy occurs in lymph nodes from patients with mycosis fungoides and Sézary Syndrome, a well-recognized diagnostic difficulty lies in the assessment of early nodal involvement by the T-cell malignancy in dermatopathic lymph nodes. Several studies have examined histologic, immunophenotypic and electron microscopy parameters to distinguish between subtle early nodal involvement by mycosis fungoides and dermatopathic lymphadenopathy without involvement by lymphoma; however, no characteristic feature has been found that can definitively discriminate between the two processes using these methods [170, 173, 174, 178, 179]. Molecular studies for T-cell receptor gene rearrangements have proved useful in identifying monoclonal T-cell populations in dermatopathic lymph nodes from patients with mycosis fungoides in the absence of overt histological involvement, suggesting the diagnosis of early nodal involvement by lymphoma. The presence of molecular lymph node involvement in this group of patients may predict a poorer prognosis [180, 181].

Other Dendritic Cell Proliferations

Plasmacytoid Dendritic Cell Proliferations

Plasmacytoid dendritic cells (PDCs) are a normal cellular component of reactive lymph nodes; however, increased numbers and clusters have been reported in certain conditions including hyaline-vascular Castleman disease, histiocytic necrotizing lymphadenitis, Kimura disease [182] and granulomatous lymphadenitis [183]. Histologically, the PDCs are recognizable as clusters of medium-sized cells with fine chromatin and pale cytoplasm. The aggregates have interspersed tingible body macrophages, and are situated in the paracortex, usually in proximity to the high endothelial venules (Figure 5E) [184]. PDCs are identifiable by immunohistochemistry (Figure 5F), as the cells are positive for CD123, BDCA2(CD303), granzyme B and TCL-1 and negative for specific markers of B-cell, T-cell and myeloid lineages. CD2AP and CD4 are often expressed, and granular staining for CD68 may be seen. They are negative for CD34 and TdT [184, 185].

Mature plasmacytoid dendritic cell proliferations are also associated with myeloid neoplasms, mainly chronic myelomonocytic leukemia, but also with myelodysplasia and acute leukemia with monocytic differentiation [185, 186]. The proliferations occur in the lymph nodes, skin and bone marrow and may be significant, comprising numerous, large nodules of PDCs with conspicuous apoptosis which, at first glance, may be mistaken for reactive germinal centers [185, 187, 188]. The immunophenotype of the PDCs in these proliferations is similar to that of normal reactive PDCs; however, aberrant expression of lymphoid or myeloid markers has been reported [185, 187, 189]. A clonal relationship with similar chromosomal abnormalities to the associated myeloid neoplasm has been demonstrated by fluorescent in-situ hybridization or mutational analysis in some cases [186, 187]. The prognosis generally depends on the underlying myeloid neoplasm [186, 188].

Langerhans Cell Proliferations associated with Lymphoma

Langerhans cell neoplasia (histiocytosis or sarcoma) has been observed in association with other hematologic malignancies. In such cases, the Langerhans cell proliferation may share common genetic abnormalities with the associated malignancy suggesting a clonal relationship or transdifferentiation [190–193]. Small foci of Langerhans cell histiocytosis (LCH) may also represent a reactive, incidental finding in lymphoma and such aggregates have been shown to be non-clonal by HUMARA assay [194]. A recent study did not find BRAF V600E or MAP2K1 mutations in the incidental LCH component of seven cases analyzed (associated with classical Hodgkin lymphoma, mantle cell lymphoma and angioimmunoblastic T-cell lymphoma). The authors concluded that the presence of lymphoma-associated LCH is a benign process, and suggested that activation of the ERK pathway may occur due to the lymphoma or its interplay with the associated microenvironment [195].

Histiocytic and Dendritic Cell Neoplasms

Current classifications of histiocytic and dendritic cell disorders include the WHO classification of histiocytic and dendritic cell neoplasms, which includes neoplasms of both hematopoietic and mesenchymal origin [196, 197] and the revised classification from the Histiocyte Society, which divides the histiocytoses and neoplasms of the macrophage-dendritic cell lineages into five groups based on the clinical, radiological, pathological and molecular characteristics of the diseases [4]. Histiocytic and dendritic cell neoplasms are rare tumors which show morphologic and immunophenotypic features of the various dendritic cell and macrophage subsets. Although uncertainties regarding the cell of origin remain for certain neoplasms, recent transcriptomic analysis has provided some additional insights.

Histiocytic sarcoma shares morphologic and immunophenotypic features with mature tissue histiocytes. It is a diagnosis of exclusion, as the tumor is positive for non-specific histiocytic markers, and should be negative for diagnostic markers of other entities. Langerhans cell histiocytosis has similar morphologic and immunophenotypic features to the Langerhans cell, as it is positive for CD1a and langerin, and ultrastructurally contains Birbeck granules. Transcriptomic analysis has suggested closer similarity between the neoplastic cells of LCH and immature myeloid-dendritic cells than epidermal Langerhans cells, suggesting that LCH may arise from a more immature cell than the differentiated Langerhans cell from which it has been proposed to arise [198]. Interdigitating dendritic cell sarcoma is postulated to arise from the interdigitating dendritic cell, whereas follicular dendritic cell sarcoma and fibroblastic reticulum cell tumor are tumors of the mesenchymally-derived follicular dendritic and fibroblastic reticulum cells [197].

Blastic plasmacytoid dendritic cell neoplasm is probably derived from immediate precursors of plasmacytoid dendritic cells [185, 197]. It expresses CD4, CD56, CD123 and BDCA2(CD303) [185]. In contrast to the previous neoplasms, it is grouped with the myeloid neoplasms and acute leukemias and not the histiocytic and dendritic cell neoplasms in the WHO classification [199].

Conclusion

Accumulations of benign histiocytes and dendritic cells can occur in lymph nodes in response to diverse stimuli including foreign material, infection, autoimmunity, tumor and as a result of lysosomal storage disorders. Although they may present as lymphadenopathy, such proliferations are also encountered as an unexpected finding in lymph nodes excised for other purposes, such as cancer staging. Certain histiocytic and dendritic cell infiltrates have distinctive histopathological features, correct recognition of which should prompt appropriate ancillary tests or clinical correlation to identify an underlying etiology.