The rabbit is often taken as the most typical animal for the bronchus-associated lymphoid tissue (BALT). We studied the lungs of adult rabbits of four groups: A. hobby breeder, B. medical school central animals facilities from experiments on joints. C. Animals kept on the central animal facilities from experiments on muscle cells and D. from rabbits which were directly delivered to the laboratory of biochemistry. The presence of BALT was definitely dependent on the environment: larger and active in group A (as known from the literature) In group B and C the BALT was smaller in some sections was no BALT. In group D BALT was often absent or very small. In conclusion, BALT is also in rabbits not a contribution lymphoid organ like lymph nodes or spleen.

• Lymphoid Tissue

• Muscle Cells

• Spleen

Pabst R, Peter M, Höpfel C, Tschernig T (2024) The Bronchus-Associated-Lymphoid-Tissue (BALT) in Rabbits is Also Dependent on Environmental Stimulation. J Vet Med Res 11(1): 1264.

In many organs with a mucosa, lymphoid cells are found aggregated in mucosa-associated-lymphoid tissue (MALT) [1]. The best characterized MALTs are the Peyer’s patches in the small intestine which are part of the gut-associated-lymphoid tissue (GALT). Bienenstock et al. [2,3], described in 1973 comparable lymphoid aggregates in the lung and introduced the term bronchus-associated-lymphoid tissue (BALT). In these initial studies the basic histology was described and 3 compartments defined in adult rabbits. In the following decades BALT was mentioned in textbooks of anatomy, immunology and pulmonology and reviews on MALT as a typical structure found in many species. However, BALT - as a normal structure - had not been discovered in various species, including adult humans [4]. The schematic drawings showed a central lymphoid follicle consisting mainly of B lymphocytes and some follicular dendritic cells. In the periphery T lymphocytes and interdigitating dendritic cells were localized and high-endothelial-venules (HEV) were observed in the parafollicular or interfollicular area. The third compartment is the dome area which bulges into the lumen of the bronchus. This compartment consists of T- and B lymphocytes and a specialized epithelium of membranous cells (M cells), which are known from Peyer’s patches to be specialists in antigen uptake. This important function was studied by electron microscopy [5] and the uptake of horseradish peroxidase and bacteria was documented [6]. In the dome epithelium there are few goblets cell and the secretory compartment of IgA is absent. This compartment is essential for the transepithelial transport of IgA.

The structure and function of BALT in farm animals had been reviewed [7]. Randell et al. [8], reviewed BALT in detail and also supported the use of the term “induced BALT” as being typical for a tertiary lymphoid organ. The aim of this study was to quantify BALT in rabbits, bred under very different environmental conditions

This study was carried out on the lungs of four different groups of adult rabbits: A. This group consisted of adult rabbits from a hobby breeder. They were sacrificed using a captive bolt pistol, followed by exsanguination. B. Six rabbits used for myoglobin studies by Prof Brenner molecular physiology, Medical School Hannover. C. Eighteen large white female rabbits from studies on the regeneration of cartilage performed of the orthopedic surgery (average age: 1 year, body weight: 3.8 to 4.6 kg). D. Adult rabbits which were directly delivered from a breeder (Charles River Sulzfeld, Germany) to the laboratory of biophysical chemistry (Dr. G. Tsiavaliaris), without contact to the central facility. The B and C group rabbits were kept in the central animal facilities of the medical school. These rabbits were killed by concussion followed by exsanguination. Two samples were excised from the right middle and lower lobe. These were fixed for 3 days by immersion in 10% and later in 3% formaldehyde. The tissues were processed for routine paraffin histology, a section of 5µm thickness was stained with haematoxylin-eosin (H&E).

Evaluation

The area of the whole section was measured with an ocular micrometre, the presence of BALT was first established with a high-magnification scan and its size then measured. The mean and standard deviation was calculated for each of the four samples.

All sections of the lungs from the rabbits provided by the hobby breeder (group A) displayed accumulations of lymphoid cells associated with a bronchus (Figure 1).

Figure 1: Histological section of a rabbit lung from the hobby breeder with a large accumulation of lymphoid cells in the bronchial wall (haematoxylin-eosin staining, 5 µm section magnification).

Many of these BALT structures had obvious germinal centres (Figure 2).

Figure 2: Histological section of an adult rabbit lung (from the hobby breed group) with a large BALT containing a germinal center) (hematoxylin-eosin staining, 5 µm section, and magnification).

The number of BALT structures per section (> more than 1.5 cm2) were calculated and shown in Figure 3.

Figure 3: Graphical documentation of the mean number of BALT per lung sample in the four groups of adult rabbits.

There were no obvious differences between male and female animals. Three rabbits of group B displayed no BALT at all. The lung sections of the rabbits used for experiments of cartilage defect replacement performed by the orthopedic surgery (group C) also showed BALT but these were smaller. The quantitative data was also documented in Figure 3. The BALT in the group of 8 rabbits delivered directly to the Institute of biophysical biochemistry (group D) was not only low in numbers but also in size (Figure 4).

Figure 4: Histological section of a lung from group 4 (Biophysical chemistry) displaying a bronchus without any BALT (hematoxylineosin staining, 5 µm section, magnification).

Bienenstock et al. [2,3], described accumulations of lymphoid cells in the walls of bronchi and introduced the term bronchusassociated lymphoid tissue (BALT). This structure was quickly integrated in the system of mucosa-associated lymphoid tissue (MALT) (for review and details see Pabst [9]). A great variability in the frequency and size of BALT in different species was described, e.g. the absence of BALT in adult humans [10]. Our group later documented BALT in sudden infant death children and in control children: 36.4% and 44.1% respectively [11]. In some species, such as the mouse, BALT was completely lacking [12].

Thus, the main aim of this study was to demonstrate the presence of BALT and to investigate a possible environmental influence on its development. Previously vaccinated and later boostered pigs did not display BALT after microbial stimulation with a pig-specific lung pathogen (Actinobacillus Pleuropneumoniae) [13]. Repeated intratracheal application of MALP-2 (a macrophage product which stimulates the Toll-like receptors 2 and 6 of the immune system) to adult LEWIS rats resulted in larger-sized BALT [14]. Our aim was to establish whether this is a typical environmental microbial stimulation. Several of the rabbits delivered directly to the laboratory in a sterile container had no BALT. Some time ago Pabst and Tschernig [15], proposed a two-step vaccination protocol for the lung. The concept was to increase M-cell activity in BALT, for example through stimulation by bacterial products. The second step foresaw subsequent aerosol vaccination in order to induce a specific BALT antibody pronation.

Our data show that BALT in rabbits (an excellent species for BALT) also depends on environmental factors and is not a typical secondary lymphoid organ like lymph nodes or spleen. Nevertheless, it could be clinically relevant to stimulate this structure in a first step and to later use the antigen uptake for potential specific protective immune reaction.

This study was supported by the “Gesellschaft der Freunde der Medizinischen Hochschule Hannover” (the Society of Friends of the Hannover Medical School). We thank Susanne Faßbender who helped us to contact the „Kleintierzuchtverein” (“small animal breeding club”) and Mrs. Comado-, who contributed the lungs of the conventionally bred adult rabbits and A. Söther for correcting the English. We also thank the colleagues of the Medical School who gave us the opportunity to take samples of the lungs of their rabbits: 1. The late Prof. Erich Brenner, Institute of Molecular physiology. 2. Dr. Frank Witte, Clinical orthopedic surgery and 3. Dr. Georgios Tsiavaliaris, Institute of Biophysical chemistry

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