Nasal-associated lymphoid tissue (NALT): frequency and localization in young children

Abstract

In mucosal immunology nasal-associated lymphoid tissue (NALT) is taken as a constitutive structure of the nasal immune system and as a target tissue in strategies of local defence and an induction site for vaccination. These concepts are based on findings in rodents, but it has not been investigated systematically whether NALT also is present in humans and if so in which amount and localization. In a postmortem study the presence of NALT in humans is documented as a morphologically distinct structure additional to the lymphoid structures of the Waldeyer's ring. Human nasal tissue blocks of 150 children who had died in the first two years of life either of sudden infant death (n = 109) without signs of respiratory tract infections or of different traumatic (n = 22) and natural causes of death (n = 19) were obtained using a specific autopsy-technique and were investigated systematically using histology. Clearly in contrast to rodents human NALT was found disseminated in the nasal mucosa with typical morphological features in 38% of all children, mainly in the middle concha, with similar morphology and frequency in the examined groups. No correlation was found between the presence of NALT and the cause of death and especially the grade of inflammation in general. Therefore, NALT might be the morphological basis for inhalative vaccination strategies in young children and play a role in mucosal host defence.

INTRODUCTION

The mucous membranes are continuously exposed to enormous amounts of antigenic material. The mucosae are not only barriers for these potentially harmful materials but are also induction sites for protective immune reactions, in which IgA plays a central role. Many concepts of the mucosa-associated lymphoid tissue (MALT) have been developed in experiments using Peyer's patches (PP). In recent years nasal vaccination protocols have demonstrated very effective immune reactions without clarifying the exact mechanism [1–4]. So far only in rodents has nasal-associated lymphoid tissue (NALT) been described as a tiny (tonsil like) aggregation of organized lymphoid tissue found on both sides of the nasopharyngeal duct dorsal to the cartilaginous soft palate and is considered analogous to the Waldeyer's ring in humans (Fig. 3b) [5–8]. It can be argued that in humans with their palatine, lingual and pharyngeal tonsils NALT would be superfluous. That might be the reason why no systematic study on organized lymphoid tissue in the nose was reported in the literature until now.

Fig. 3.

(a) Localization and distribution of NALT in the different regions of the nasal mucosa including the percentage of cases with NALT (a; H&E stained frontal section). (b) Cross-section of a rat nose at the level of the ecto- and endoturbinates showing the specific localization of NALT in rodents bilateral at the entrance of the nasopharyngeal duct (modified after Kuper et al. [7]).

NALT as defined in the present study means the presence of organized lymphoid tissue in the nasal mucosa with aggregates of lymphocyte like follicles and infiltration of the overlying epithelium of all the different parts of the nasal cavity. This is in contrast to some authors who include the nasopharyngeal tonsils in the definition of NALT [9]. Major age effects on the presence and extent of MALT in the respiratory tract were described in the larynx (LALT) in approximately 80% of children and adults [10,11], whereas in the lung bronchus-associated lymphoid tissue (BALT) was present in only 40% of children and adolescents up to 20 years of age. In contrast BALT was absent in lungs of healthy adults [12,13]. Thus, results obtained in adults should not be extrapolated to the situation in children.

In experimental animals, in particular rats and mice, local immune reactions in NALT have been studied following intranasal application of antigens. Nasal immunization with antigens and toxins induces antigen-specific IgA-producing cells in nasal passages and at other distal mucosal sites indicating the importance of NALT in host defence [1–3]. The sequence of tissue organogenesis is chronologically different for NALT, PP and peripheral lymph nodes [14]. NALT is found earlier than BALT, probably due to its strategic position [7,15]. NALT development may depend on triggering signals provided by inhaled environmental antigens after birth [14]. It has been suggested that NALT and PP are the main components in the defence of the respiratory tract and the gut, respectively, probably with different functions due to differences in distribution patterns and subset composition [8]. Thus, in rodents NALT has been shown to be an important induction site for mucosal immune responses.

Nasal mucosa can be biopsied in humans, but it remains to be shown whether single specimens are representative of the nose in general. A systematic study on the localization of lymphoid tissue in the different parts of the nasal cavity is impossible in humans. Therefore, no data exist for any age groups. The first aim of this study was to establish a technique to obtain the nasal structures without destroying the outer aspect of the face. The tissue blocks had to be decalcified to enable sections of all parts of the nose. Therefore, it was important to obtain sufficient material.

The second aim was to determine the frequency, morphology and topographical distribution of lymphoid tissue in the nasal mucosa in the first two years of life. This early phase of life is of special interest in respect to viral infections and potential vaccination strategies, e.g. to develop an effective RSV vaccine [4]. The children had either died from sudden infant death (SID) or various traumatic and natural causes.

MATERIALS AND METHODS

Thorough postmortem examinations were performed at the Department of Legal Medicine of the Hannover Medical School on 150 children (97 females, 53 males) who had died in the first two years of life (age range 6–701 days). Of these children 93·3% had died during the first year of life. The average age of the children was 155 days.

The autopsies included histological, microbiological, immunological and sometimes toxicological and neuropathological tests. Samples were obtained within a study protocol which had been approved by the local ethic committee and informed consent of the relatives was given. A total of 109 children had died from SID and 41 children from defined causes of death, so-called controls. The SID group was defined according to Beckwith [16] as ‘sudden infant death in any infant or young child which is unexpected by history and in which thorough postmortem examination fails to demonstrate an adequate cause of death’. Slight illnesses such as mild infection of the upper respiratory tract, isolated middle ear infections, and positive bacteriological or virological results unaccompanied by morphological findings were not considered to have been the cause of death. The control group consisted of 22 infants with non-natural causes of death (trauma, accident, homicide) and 19 infants with natural causes of death including congenital malformation and other severe diseases.

At autopsy an oscillating saw was used to remove blocks from the anterior base of the skull. The nasal cavities prepared by this procedure were decalcified using Althoff's method (for details see [17]). In short, the blocks were incubated in 8%formic acid for 24 h and 29% formic acid for 3 × 1 h. Parafrontal and in some cases serial sections were processed. Hematoxilin eosin (HE), Masson-Goldner, Gieson's elastica and PAS staining were performed. All sections were investigated for typical lymphoid follicles meeting the criteria of NALT which are the presence of (1) lymphoid follicles (2) lymphoepithelium and (3) HEVs. The localization and number of these follicles were determined by light microscopy by three independent investigators.

RESULTS

The tissue blocks enabled sections to be taken from all parts of the nose and despite the decalcification routine histological staining was possible. Thus, for the first time a systematic study of the nasal cavity of young children was possible. NALT was found in different localizations of the nasal mucosa not only dorsal at the choanae but mainly in the ventral regions of the conchae. It showed the typical morphological features such as lymphoid follicles sometimes with germinal centres, lymphocytes in the overlying epithelium and high endothelial venules (Fig. 1a–d). In some cases the lymphoid follicles were even found like pearls of a necklace, resembling the PP in the gut. NALT was present in 38% of all cases examined. Between the SID group and the children who had died of defined traumatic or nontraumatic causes no differences were detected regarding morphology, frequency, localization or size. No obvious influence of age on the presence of NALT was observed within the first two years of life (Fig. 2). The local distribution found in the described material was not homogeneous. NALT was mainly found in the middle concha (Fig. 3a). This is obviously in contrast to the findings in rodents where NALT is only situated in a single localization (not diffuse-tonsil like) bilateral at the entrance of the nasopharyngeal duct (Fig. 3b). The frequency of NALT did not differ with the cause of death of the children (Table 1). Furthermore, the presence or amount of NALT was not related to the occurrence of concomitant inflammation. Within the SID children but also within the other groups NALT occurred in about 40% of the investigated cases not indicating any relation to significant inflammation. For example, NALT was only partially found in cases suffering from purulent pneumonia, whereas some children who had died of traumatic causes and without positive microbiological or virological isolates showed impressive formation of NALT.

Fig. 1.

H&E histology of two human mucosal samples containing NALT at three different magnifications (a–f, bar = 500 µm). a,c,e, Nasal septum, SID, 99 days of age, multiple follicles resembling pearls of a necklace, note the lymphocytes infiltrating the overlaying epithelium in the dome area called lymphoepithelium. At the highest magnification high endothelial venules can be seen. b,d,f Medial nasal cavity, SID, 308 days of age, similar findings as in the previous sample but with the section beneath the dome area.

Fig. 2.

Presence of NALT in the investigated cases depending on age (each circle represents one investigated case).

Table 1.

Frequency of NALT

		NALT
Group	n	No. of cases	%
SID	109	40	37
Controls	41	17	41
Natural death	19	8	42
Traumatic death	22	9	41
Total	150	57	38

DISCUSSION

To our knowledge this is the first systematic investigation of the mucosa of the human nose presenting evidence of the presence of NALT in the sense of disseminated lymphoid, subepithelial follicles in the whole nasal mucosa (Fig. 3a). In contrast, in rodents NALT is described exclusively as paired lymphoid structures found only at the opening of the nose to the pharynx (Fig. 3b) [6,7]. So far, lymphoid structures have not been shown in the nose of humans but Waldeyer's ring is thought to be functionally equivalent lymphoid tissue. The collection of oropharyngeal and nasopharyngeal lymphoid tissues in humans is known as Waldeyer's ring. Thus, in children NALT is present in addition to Waldeyer's ring. It remains to be shown whether the size and presence of NALT correlates to the size of the tonsils in this age group, i.e. whether NALT is a compensatory lymphoid structure when the pharyngeal tonsil is small.

NALT might play a role in mucosal host defence and could be useful in vaccination strategies in young children as discussed in recent papers [1,3,14]. In rodents NALT has been shown to be an important inductive site for priming of immunocompetent cells to induce antigen-specific mucosal immune responses in the upper respiratory tract and other mucosal effector tissues [2,18,19]. The injection of antigens directly into NALT seems to be an effective immunization route eliciting high levels of antibodies [3]. Nasal vaccination induced protection without pathology and can even protect distant mucosal sites from a variety of infectious diseases in animal models [4]. In summary, the present data support the hypothesis that human nasal MALT also plays a role as a part of the integrated common mucosal immune system also in humans and are the basis for clinical trials of nasal vaccination and immunization procedures.

It was a surprising finding that the frequency of NALT did not differ between the different reasons of death and SID as has been discussed [20,21]. The preference of NALT for the middle concha (26%) and upper nasal cavity (30%) is hard to explain as this was similar in the very young children and can therefore not be caused by turbulence and continuous antigen exposure.

It remains unclear whether NALT also exists in older children, adolescents and adults like LALT or is lacking in healthy adults like BALT. One has to consider that investigation of human nasal tissues is very difficult because of the complex preparation. Probably due to the decalcification procedure, immunohistology for T lymphocytes and their subsets resulted in very faint staining (data not shown). Modifications of the staining procedure might improve this technique in future. It would further be of interest to look at other age groups to follow up the development of NALT during maturation and ageing but the increasing ossification hinders the histological procedure. Taking small tissue samples of the nasal mucosa from defined areas of the nasal cavity by endoscopy might in part overcome this problem.