Abstract
Keywords
Dear Dr. Elmore,
Knowledge of spontaneous or incidental tissue findings is important to toxicologic pathologists, but inappropriate categorization of such findings can be a confounding variable for toxicity studies. I believe that a recent article, “Spontaneous Findings in the Eyes of Cynomolgus Monkeys (Macaca fascicularis) of Mauritian Origin” by Woicke et al. (Toxicol Pathol 46, 273–282, 2018), has inappropriately identified and reported normal acquired mucosal-associated lymphoid tissue (MALT) in the eye as inflammatory infiltrates. The readership should be aware that (1) healthy mucosal tissues frequently contain acquired lymphoid aggregates in response to antigenic exposure, (2) understanding the characteristic features of MALT will assist in identification of these acquired structures relative to inflammation, and (3) normal MALT structures do not represent an inflammatory change, but MALT can undergo non-neoplastic and neoplastic changes similar to those found in other lymphoid tissues.
The Woicke et al. (2018) retrospective review describes a number of spontaneous lesions of the eye including inflammatory changes in the conjunctiva and uvea of cynomolgus monkeys. This category lists lymphocytic and mononuclear cell infiltrates and accumulations (Figures 1–3), without typical features of inflammation, as a common finding. The authors description of the conjunctival infiltrates as, “A lymphoid nodule, protruding outward, infiltrating, and replacing the limbal epithelium with a loss of goblet cells,…,” is entirely consistent with conjunctiva-associated lymphoid tissue (CALT). CALT is acquired in the postnatal period after exposure to antigens and is a well-known normal component of the MALT system. These structures do not represent foci of inflammation or a lymphoid/mononuclear cell infiltrate of unknown origin (Knop and Knop 2000; Cain and Phillips 2008; Chodosh, Nordquist, and Kennedy 1998; Siebelmann et al. 2013; Steven and Gebert 2009).
MALT structures can be histologically identified as nonencapsulated subepithelial lymphoid follicles that generally focally elevate the epithelium. The specialized covering of follicle-associated epithelium (FAE) may appear flattened, lacks cilia, and contains few to no goblet cells. Intraepithelial lymphocytes and occasional granulocytes can transmigrate thorough loose intercellular junctions of the FAE and M (microfold) cells that selectively sample antigens, transport antigenic information into the follicles to initiate a local mucosal immune response, and contribute to systemic immune responses (Knop and Knop 2000; Cain and Phillips 2008; Cesta 2006). MALT can be found as a postnatal, age-related, and acquired lymphoid tissue of variable location, number, size, and degree of maturation. These structures are associated with many mucosal surfaces including the oral-pharyngeal cavity, conjunctiva, eustachian tube, respiratory tract (nasal cavity, trachea, larynx, and epiglottis), gastrointestinal tract (gallbladder [except Peyer’s patches]), and the urogenital tract. Similar MALT-like structures are also described in some epithelial-associated tissues including around glandular ducts (duct-associated lymphoid tissue [DALT]) and in skin (skin-associated lymphoid tissue [SALT]). Conversely, certain MALT structures are constitutive as they develop in utero and have consistent locations at birth. MALT in this category includes Peyer’s patches, nasal-associated lymphoid tissue (NALT) in rodents and small mammals that are obligate nasal breathers, nasopharyngeal tonsils in larger mammals (NALT equivalent), and other tonsils (Cesta 2006).
CALT, as with all MALT, will undergo further development and organization with postnatal antigenic stimulation, resulting in a change in number, size, and characteristics of the lymphoid tissue. Early CALT formation may consist of loose aggregates of a few lymphocytes, progressing to complex follicles with germinal centers with repeated antigenic exposure. CALT often persists throughout adulthood but may regress in aged animals (Cain and Phillips 2008; Siebelmann et al. 2013). Tissue sections of any MALT structure captured at the edge of the follicle may also consist of only loose aggregates of or small foci of lymphocytes. Serial sections may be required to clearly demonstrate typical follicles and other characteristic features of MALT. Figure 2 of Woicke et al. (2018) appears to be CALT captured at the edge of a lymphoid follicle with only a small area of overlying FAE. Figure 3 appears to be a central section of highly organized CALT overlaid by classic FAE (elevated nodule covered by flattened epithelium devoid of goblet cells). The authors also noted that paranodularly, “Blood vessels exhibit swollen endothelial nuclei.” The described paranodular vessels are typical of specialized high endothelial venules (HEV), lined by cuboidal (“high”) endothelial cells. HEV are sites of extravasation of lymphocytes into MALT and emigration of lymphocytes from MALT to regional lymph nodes and systemic lymphoid tissues (Miyasaka and Tanaka 2004; Cain and Phillips 2008; Siebelmann et al. 2013). HEV may contain interepithelial or intraluminal lymphocytes as depicted in Figure 3. The contralateral conjunctiva of Figure 3 was presented in Figure 4 as subacute inflammation. At the magnification presented, I was unable to confirm necrosis or other inflammatory changes. However, these contrasting figures demonstrate the need for pathologists to be familiar with the location and characteristics of normal MALT when inflammation may be concurrently present in mucosal tissues.
CALT is an important part of the eye-associated immune system conserved across mammals, birds, and reptiles (Knop and Knop 2000; Steven and Gebert 2009). The immune system of the eye and periocular structures also includes physical barriers, complex secretions in tears, and lymphoid tissues including CALT, lymphoid follicles in the nictitating membrane, and DALT in the lacrimal gland and lacrimal gland drainage system (Dua et al. 1994; Siebelmann et al. 2013; Cesta 2006). Uveal tract (iris, ciliary body, and choroid) lymphocyte aggregates, which the authors have described and depicted in Figure 1, and retinal (ora serrata) lymphocyte aggregates (not reported by the authors), can be a common finding in monkeys (Sinha, Cartwright, and Johnson 2006) and other mammals (McMenamin 1997). In the normal state, the highly vascularized ora serrata and uveal tract contain lymphocytes, tissue resident macrophages, and dendritic cells that are capable of antigen capture and presentation (Sinha et al. 2006; Greaves 2011). The eye is not a closed system and the so-called immune privilege of the eye does not preclude the need for an active immune response in which lymphoid structures may be acquired (Wayne 2003). In my opinion, lymphoid aggregates observed in the ciliary body and choroid and less frequently iris could also be categorized as normally acquired lymphoid structures associated with epithelium, similar to DALT or SALT. This opinion is not supported by the current literature, and by tradition intraocular lymphoid aggregates are presumed to be abnormal findings, and generally reported as nonspecific lymphoid or mononuclear cell infiltrates of the ora serrata, ciliary body, iris, or choroid.
When identified as a spontaneous finding in tissues, MALT should be appropriately categorized as a normal acquired lymphoid tissue. Except for Peyer’s patches, NALT, and sometimes palatine tonsils, MALT are not intentionally examined in toxicity studies, unless there is a study-specific need (e.g., conjunctiva and lacrimal gland outflow tract for topical ocular therapeutic testing). Therefore, pathologists are often not familiar with the potential for normal MALT throughout the body, and these normal follicles may be inappropriately categorized as inflammation or a lymphoid infiltrate of unknown origin. Typically, pathologists would not record normal lymphoid structures such as Peyer’s patches or NALT as a study finding, although my preference is to record their presence and localization in a comment, particularly for the lesser known CALT, DALT, and any MALT-like aggregate in mucosal or nonmucosal tissues. During conduct of the histopathology evaluation in a toxicity study, it may become apparent that the incidence, number, size, or characteristics of MALT are different between control and treated animals and such differences should be recorded. As with all lymphoid tissues, MALT may undergo non-neoplastic and neoplastic changes that require robust reporting of appropriate morphological diagnoses using standard or enhanced histopathology methods (Elmore 2006).
In conclusion, mucosa-associated lymphoid aggregates should be carefully examined to determine whether they fit the criteria for normal MALT structures. In postweaning animals, organized submucosal lymphoid follicles, without evidence of inflammation, are most likely MALT. These normal lymphoid structures found in healthy tissues should not be automatically and inappropriately categorized as inflammatory changes. Rather, any MALT can be simply recorded as present, unless findings in test animals relative to temporal controls provide justification for reporting specific morphological changes in MALT in toxicity studies.
Submitted by,