**4. Bronchus-associated lymphoid tissue (BALT)**

BALT, an important part of MALT, is classically used to refer to intrapulmonary lymphoid tissue in connection with the pulmonary vessels and adventitia of the bronchi [11, 28]. Macklin [29] named this lymphoid tissue in 1955 as 'sumps' or 'pulmonary tonsils' in which dust and organisms are retained. Subsequently, Bienenstock et al. [28, 30] identified these formations as subepithelial follicular lymphoid aggregates, primarily composed of lymphocytes, organized in the bronchial mucosa in contact with the surface epithelium, and coined the term BALT to describe them.

Although BALT, a secondary lymphoid tissue that plays an important role in the maintenance and regulation of lung mucosal immune homeostasis [8], was initially claimed to resemble Peyer's patches in the small intestine [11]; it was later revealed

that it was quite different from these formations [31]. Compared to GALT where in the founder Peyer's patches are located, it is accepted that BALT is not regularly present during fetal life due to embryonic preprogramming; however, it occurs with antigenic stimulation during the postnatal period [32, 33]. In other words, it is claimed that there is a relatively special lymphoid tissue in the development of BALT. However, studies have shown that BALT exhibits great differences between species [34, 35].

BALT, which was first identified in the bronchial wall of rabbits by Bienenstock et al. [28], is frequently detected in these animals and has the highest number of regions [28, 34]. In terms of the presence and distribution of BALT, rats and guinea pigs [34] follow rabbits, whereas germ-free pigs [28, 34], cats, dogs, and Syrian hamsters [34, 36] do not have this lymphoid tissue. BALT is frequently present in poultry, particularly hens [37]. In mice and humans, the situation with BALT is a little more contradictory [19]. Some scientists suggest that BALT is present in germ-free mice when antigenic stimulation is absent [12], whereas others report that it is not [38, 39]. Besides the differing viewpoints on the presence of BALT in mice, it is assumed that it is only observed infrequently after the neonatal period.

Further, it is claimed that BALT is not present in structurally healthy humans [31] because the features similar to BALT in mice are also found in humans [8]. BALT, in particular, is detectable if it is induced in adults; however, it is only observed in 40% healthy children and adolescents. Factors inducing the presence and distribution of BALT in these adults include infection, pathogen exposure, chronic pulmonary inflammation or autoimmune disease, etc. [32, 33, 40]. Moreover, it is suggested that the formation, size, and amount of BALT depend on the type and duration of exposure [41]. Therefore, it is concluded that BALT varies in different species as well as indifferent physiological states of the same species [8].

#### **4.1 Inducible BALT (**İ**BALT)**

Most of the secondary lymphoid organs found in mice and humans develop embryonically in the absence of microbial stimulation or environmental antigens [42]. Furthermore, the structure and function of several secondary lymphoid organs, particularly those on the mucosal surfaces, are dramatically altered upon exposure to foreign antigens and commensal organisms [43]. Peyer's patches of MALT demonstrate a striking increase in size and complexity following the colonization of commensals [44, 45]. Similarly, in rodents, NALT is not completely developed until the postnatal period; however, microbial exposure accelerates this process [46]. On the other hand, the appendix tissue of rabbits has the characteristics of the primary and secondary lymphoid tissues in terms of being functionally dependent on microbial colonization [47]. However, some lymphoid tissues, known as tertiary lymphoid tissues, develop only after environmental exposure to microbes, pathogens, or inflammatory stimulations. Interestingly, although the lungs of mice and humans normally lack organized lymphoid tissue, tertiary lymphoid structures are frequently observed in lung tissue [38, 48].

BALT is recognized as an inducible tertiary or ectopic lymphoid tissue, unlike the related secondary lymphoid organs. BALT develops during the postnatal period and at anatomically non-lymphoid sites. In terms of disease states characterized by chronic inflammation, infection, or autoimmunity, BALT formation can be induced, and these areas are then known as iBALT [32, 38]. iBALT is a classic example of tertiary lymphoid tissue because it does not develop on a preprogrammed basis; its creation, size, and number in the lungs depend on the type and duration of antigenic *Bronchus-Associated Lymphoid Tissue (BALT) Histology and Its Role in Various Pathologies DOI: http://dx.doi.org/10.5772/intechopen.99366*

exposure [31, 49]. iBALT regions are best characterized in the lungs of rodents and humans. They are observed in the lungs of mammals and birds as well as in possibly all air-breathing vertebrates [41]. The emerging arguments confirm the role of infectious agents, such as isolated lymphoid follicles in the gut, indicating that iBALT may develop in response to microbial exposure [32]. In contrast, BALT is said to have been discovered in germ-free rats [28] and mice [50] as well.

Unlike the classical BALT structure, iBALT does not always have an overlying lymphoepithelium, is not associated with a continuous airway, and can be located adjacent to small pulmonary arteries in the lung parenchyma [32]. However, as both BALT and iBALT have the same function, both tissue types are called BALT [48].

#### **4.2 Microscopic structure of BALT**

Microscopically, BALT is defined as a densely packed cluster of lymphocytes with follicular structures enveloped in a network of reticular stromal cells beneath a specialized airway epithelium devoid of cilium. These structures are claimed to be located along the main bronchial airways embedded in the airway wall with extensive lymphocytic infiltration of the epithelial layer forming a classical dome epithelium (**Figures 3** and **4**) [11].

Further, it is stated that BALT is present in bronchial tree bifurcations to capture respiratory antigens. In species, BALT develops in response to various stimulations rather than being constitutively present in the lung, whereas iBALT does not always have such a defined structure or precise localization in the lung [51].

As a part of the integrated mucosal system including GALT, NALT, and other secondary lymphoid tissue representatives, BALT is known to contain cell types that are responsible for eliciting an appropriate immune response. BALT is mainly defined as an organized structure comprising T- and B-cell domains, dendritic cells (DCs), stromal cells, and high endothelial venules (HEVs) in the T-cell region [38, 52–55].

**Figure 4.** *A higher power light microscopic view of the BALT structure, rat lung (H-E). Red star: BALT formation.*

Furthermore, it is stated that most of its cellular component consists of B cells expressing IgMlo IgDhi; however, depending on the nature of the microbe and/or antigen to which the cells respond, IgG-, IgA-, and even IgE-positive plasma cells may also be present [50, 56–58].

Moreover, in BALT, the most prominent structure is follicular-like lymphocyte accumulation, which is the common microscopic appearance of secondary lymphoid tissues, forming a classical germinal center (active site) [59, 60]. In this structure, surrounded by more mature, small lymphocytes, most of the germinal center comprises antigen-presenting macrophages [58, 61]. Lymphocytes leave the blood and migrate to BALT in the walls of HEVs, which are present at the periphery of the tissue. As there are no afferent lymphatics, these HEVs are thought to be the only entry site where lymphocytes migrate to BALT [59, 60]. In addition, the expression of chemokines in HEVs ensures accurate targeting of lymphocytes to lymphoid tissues [62].

However, in the direction of the bronchial epithelium, a dome-like protrusion similar to Peyer's patches toward the bronchial lumen is sometimes clearly observed [31]. The B-cell follicle, which is the most noticeable characteristic in classic BALT tissues with dome epithelium, is positioned below the epithelium [11]. CD4+ T cells are abundant in B-cell follicles, especially in reactive follicles with germinal centers [63], and CD8+ T cells are uncommon. Moreover, BALT is covered by a lymphoepithelium, which contains M cells that are similar to the M cells present in the dome epithelium of Peyer's patches in some species [31]. M cells are thought to transport antigens from the mucosal lumen to DCs that are in close contact with the dome epithelium [48]. Rabbits, the first and important representative of BALT, have fewer ciliated cells, few goblet cells, and many lymphocytes between epithelial and M cells. Although this basic structure appears to be valid for all species, there are some differences in details [31].

Another cell type that makes up the cellular component of BALT is follicular DCs (FDCs). These cells depend on the lymphotoxin signaling pathway to differentiate

### *Bronchus-Associated Lymphoid Tissue (BALT) Histology and Its Role in Various Pathologies DOI: http://dx.doi.org/10.5772/intechopen.99366*

into conventional lymphoid tissues and BALT [38]. Located at the center of B-cell follicles, these cells present antigen to B cells [64] and provide costimulatory signals that increase B-cell activation and proliferation in germinal centers [65, 66]. FDCs in mice are characterized by their ability to bind to antibodies against CD21/CD35 [38], FDCM1, or FDCM2 [57] and to sequester their immune complexes [67]. In addition, FDCs are responsible for the organization of the follicle and expression of CXCL13, which is responsible for the recruitment of B cells and some T cells in the B-cell area [68]. DCs located at the highest concentration in the T-cell areas of BALT are reportedly capable of preserving the BALT architecture as well as their antigenpresenting ability [48].

BALT is induced to produce IgA<sup>+</sup> cells that secrete polymeric IgA, mainly due to its role in immunity. When polymeric IgA is transported into the lumen, it induces the formation of S-IgA, which has considerable immunological importance [8]. Thus, when BALT is identified as part of the integrated mucosal immune system, the term should be restricted to structures tightly associated with an epithelium infiltrated by lymphocytes. In the integrated mucosal immune system, specific antigen uptake and antigen presentation by M cells occur and immune reactions are initiated, including IgA responses [31].

Immunohistological studies in humans show a preferential central localization of B cells mixed with some CD4+ lymphocytes and macrophages. CD4+ lymphocytes are also present in the area around the HEV, at the edge resembling a crown, and in the epithelium. In addition to the few proliferative cells positive for Ki67 observed in the follicles, many cells positive for the human leukocyte antigen-DR isotype, which is associated with various autoimmune conditions, disease susceptibility, and disease resistance, are evenly distributed in the follicle [69]. This basic structural distribution of lymphoid and non-lymphoid cells has also been noted in BALT in pathological conditions such as rheumatoid arthritis [70], hypersensitivity pneumonia [71], or diffuse panbronchiolitis [72]. Therefore, it is reasonable to conclude that BALT plays an important role in many respiratory system-related pathologies.

### **4.3 Role of BALT in various pathologies**

BALT plays an important role in pulmonary immunity such as regulating microbial homeostasis [73], inducing immune tolerance [74], inhibiting inflammation [75], and supporting immune clearance [76]. Therefore, BALT frequently encounters many pathologies associated with infectious disease agents, allergens, environmental antigens, air-borne particles, autoimmune disease agents, and factors causing malignancy. As these pathological conditions have a broad spectrum, it is not possible to discuss all the roles of BALT; therefore, only a few have been addressed.

### *4.3.1 Role of BALT in resistance to infectious diseases*

The respiratory tract is a typical entry site for viruses. This makes it difficult for the immune system to effectively eliminate viruses and virus-infected cells without causing much damage and inflammation, which jeopardizes the lung's structural and functional integrity. The balance between eliciting an immune response to effectively eliminate viruses and virus-infected cells and to cause less damage and inflammation is maintained by a complex network of innate and adaptive immune mechanisms as well as immunomodulatory and anti-inflammatory mechanisms. Accordingly, BALT could be one of the mechanisms that facilitates viral clearance by eliciting immune

responses and decreasing inflammatory responses [48]. BALT reportedly initiates pulmonary immune responses that are faster and more protective than those initiated at systemic sites. It has been proposed that once generated, BALT could play a key role in combating successive rounds of the same infection as well as assisting in establishing local immunity against unrelated viruses or pathogens [51]. For example, it has been suggested that *Lta*\_/\_ mice without lymph nodes and Peyer's patches are more susceptible to the influenza virus and although they elicit immune responses, both Band T-cell responses are delayed. Based on flow cytometric identification of germinal center B cells in the lung to question where immune responses might be initiated, it was concluded that both B- and T-cell responses are probably produced in the lungs [77]. BALT is suggested to be formed in the lungs of *Lta*\_/\_ mice and locally initiates immune responses against influenza because the germinal center is present only in secondary lymphoid tissues. Another study reported that, in addition to germinal centers, plasma cells specific to influenza nucleoprotein were detected in BALT after influenza infection [58]. However, B-cell responses to influenza are accelerated in mice with pre-existing BALT, and morbidity and mortality rates are markedly reduced in response to a variety of viruses, including influenza, severe acute respiratory syndrome coronavirus, and mouse pneumovirus [78].

*Mycobacterium tuberculosis* (Mtb) infection is one of the serious health threats worldwide and is typically confined to the lungs. Although local immune mechanisms are primarily responsible for keeping Mtb infection under control, once the infection has settled in the lungs, immune mechanisms alone do not appear to be capable of eliminating these bacteria [79]. In humans, Mtb is localized to the granulomas comprising a central nucleus surrounded by macrophages, multinucleated giant cells, and lymphocytes [80]. The lymphocyte clusters surrounding these granulomas are B cells that form structures similar to BALT. These BALT areas associated with granuloma have B-cell follicles, and T-cell areas are present at the outer edge of the follicles [81]. Similar BALT domains, for example, have been discovered in murine models of Mtb infection, where B-cell clusters surrounding the granuloma were observed. Well-defined B-cell domains with FDCs are formed as early as day 42 after pulmonary infection and are protected from infection until at least day 90 [82]. Considering the link between B follicular structures surrounding the granuloma and Mtb uptake, another study showed that B-cell follicles formed around Mtb lesions in mice developed large germinal centers, and the B cells responded to the antigen [83]. Therefore, it is indicated that BALT initiates local pulmonary immune responses against Mtb infection via B cells.

### *4.3.2 Role of BALT in pulmonary responses to allergens and environmental antigens*

Endotoxin, known as lipopolysaccharide (LPS), is a component of the gramnegative bacteria [84, 85] that is commonly present in the environment [86, 87]. The development or exacerbation of asthma [86, 87], bronchitis, and chronic obstructive pulmonary disease [88, 89] is linked to considerable LPS exposure. LPS, a classical T-cell-independent B-cell antigen, and mitogen are thought to bind to TLR4 signaling pathway [84, 85], triggering B-cell activation, proliferation, and differentiation into antibody-secreting cells [90]. TLR4 signaling activates macrophages and DCs, epithelial cells, and even fibroblasts, causing them to produce inflammatory cytokines and chemokines [91, 92]. Experimentally, pulmonary exposure of rats to endotoxin has been found to cause increases in pre-existing BALT and pulmonary plasma cells,

### *Bronchus-Associated Lymphoid Tissue (BALT) Histology and Its Role in Various Pathologies DOI: http://dx.doi.org/10.5772/intechopen.99366*

ultimately leading to the formation of germinal centers [93]. Sustained dosing of LPS prior to pulmonary inflammation in BALT-deficient mice appeared to result in BALT development in the major airways with an accumulation of B cells, T cells, and macrophages in the lungs, and even in BALT-deficient areas [94]. Thus, environmental exposures to LPS, often with additional antigenic or inflammatory components, cause BALT reactivity and pulmonary physiology alterations [95].

Considering the importance of pulmonary inflammation in asthma, a correlation between BALT development and asthma is likely. However, some believe that the presence of BALT is not always associated with asthma [96], but that the reactivity of BALT in patients with asthma is elevated [97]. Further, there is evidence that specific allergens, such as *Aspergillus fumigatus*, might cause pulmonary allergies that are similar to asthma. In allergic bronchopulmonary aspergillosis, large BALT regions characterized by diffuse and IgE-stained germinal centers have been found [98]. Thus, it is suggested that BALT can potentially contribute to allergic reactions by producing IgE locally in response to *A. fumigatus*.

Hypersensitivity pneumonia is defined as an inflammatory disease of the alveoli induced by hypersensitivity to inhaled organic antigens [99]. In contrast to asthma, which affects the airways, this condition affects the alveoli [48]. An occupational exposure often is the cause of hypersensitivity pneumonia; it can occur particularly when farmers are exposed to mold and fungi in barns [100]. Considering that hypersensitivity pneumonia results from chronic pulmonary exposure to the antigen, the emergence of well-developed BALT areas with vast germinal centers and FDC networks is not surprising for researchers [61].
