Histological and Histochemical Observations on Mast Cell and Glands in Chronic Tonsillitis

Abstract

Mast cell take active part in immune system of body, clinical manifestations are closely related to hypersensitivity. Reactions and potent biological mediators released by these cells are responsible for different clinical manifestation like allergic reaction. Frequent and important sites of these allergic reactions occur in—upper aero digestive tract, nasal, pharyngeal and respiratory allergies. The study aims to demonstrate different types of heparin in mast cells having role in tonsillitis. Present work is to study distribution and histochemical nature of mast cells in human palatine tonsil in patients suffering from chronic tonsillitis. 20 cases were studied during the study. Diagnosis was done on clinical criteria—Anterior pillar flushing. Expulsion of cheesy material from the tonsillar crypts on pressing with tongue depressor. Palpable jugulodigastric group of lymph nodes. Dissected Tonsillar material fixed into fixative—staining done. For qualitative histological observation Harris Haemotoxylene with eosin used as counter stain. Normal Histological study of mast cell like size, shape, granules were done. Distribution of mast cells in different parts of Tonsillar Tissue was observed. Histochemical study of mast cells to determine nature of reactive substance was done. Tonsil contains mucous glands, no granular tissue in present study of chronic inflammatory reactions feasible in form of fibrosis, lymphatic infiltration in epithelium. Morphology of mast cells with toluidine blue at pH 2 and 4.4 gave better picture. Mast cells had varied shape and size in different places and points of same field of focus, oval, elongated, fusiform, pleomorphic in shape. Nucleus is central ill-defined masked by granules. Cytoplasm contains mast cell granules that stained blue with Alcian blue stain, purple to bluish purple with toluidine blue. Distribution of mast cells in present study: Mast cells were found at all places mostly concentrated at capsules. Lamina propria-1–2/hpf, capsule-4–5/hpf, connective tissue septa-1–2/hpf. Histochemical Feature- In present study, histochemical study remained confined to connective tissue septa of mast cells and biochemical nature of Heparin contained in it. In present work, the identification of mast cells is based on metachromatic reactions of its granules with acidified toluidine blue at pH 2 and buffered toluidine blue at pH 4.4 to identify mast cell granules containing higher and lower sulphates of heparin.

Introduction

It is well known fact today that the mast cells take active part in the immune reaction of body, like the immediate hypersensitivity reactions and the different potent biological mediators which are released are responsible for the clinical manifestations of different types of allergic reactions. One of the frequent and important sites of these allergic reactions to occur is the upper aerodigestive tract. Nasal, pharyngeal and respiratory allergies are frequently encountered in day to day otolaryngologic practice. It has been found that the nasal mucosa is rich in mast cells and their role in the development of allergic rhinitis is very well substantiated [1]. Similarly in bronchial asthma the mast cell play the same role and are responsible for its clinical manifestations. There are also other diseases in the field of Otorhinolaryngology whose aetio-pathology are ill understood and as it is not unusual practice that an allergic basis is put forward to account for them. One of its classical examples is secretory otitis media.

Though the aetio-pathology of chronic tonsillitis is well understood and its infective origin has been proved beyond doubt, the high mast cell content of the normal and chronically inflamed tonsils as demonstrated by previous workers suggest that an allergic reaction might also play a part in the development of chronic tonsillitis, moreover due to their position in the oropharynx the tonsils are constantly exposed to the external antigenic substances which enter the body as inhalants and ingestants.

Not many works have been done before in this particular subject and a few workers have opined that the tonsils together with the adenoids may contribute to the allergic reactions of the nasopharynx and possibly otitis media [2].

Keeping these facts in mind the present work has been taken up to find out the mast cell population in the tonsils in the cases of chronic tonsillitis and to compare the observations with those obtained by previous workers.

A histochemical study of the mast cells also have been undertaken in an attempt to demonstrate different types of heparin in mast cells which may have some role in tonsillitis and as of late chondroitin sulphate has also been demonstrated in mucosal mast cells [3].

Materials and Method

The aim of the present work is to study the distribution and histochemical nature of mast cells in the human palatine tonsil obtained after tonsillectomies in patients suffering from chronic tonsillitis. Cases of chronic tonsillitis were diagnosed the basis of clinical criteria. These were (1) Flushing of the anterior pillars of tonsil. (2) Expulsion of cheesy material from the tonsillar crypts on pressing upon the anterior pillars with tongue depressor. (3) Palpable jugulodigastric group of lymph nodes in the absence of any other pharyngeal pathology. The dissected tonsillar specimens were cut into several pieces and were put into the fixative fluid immediately. All together 20 cases were taken for observation.

Section

All specimens were cut at a thickness of 6–8 microns with a rotatory microtome.

Staining

1. For qualitative histological observation Harri’s haematoxylene, with eosin as counter stain was used.

2. For histochemical test the following staining procedure were applied.

   1. For highly sulphated acid mucopolysaccharides

      • i.
        Alcian blue pH 1.0
      • ii.
        Alcian blue pH 1.0 after active methylation.
      • iii.
        Aldehyde fuschin.
   2. For weakly sulphated acid mucopolysaccharide

      • i.
        Alcian blue pH 2.5 [4].
      • ii.
        Alcian blue pH 2.5 after active methylation.
   3. For simultaneous demonstration of highly and weakly sulphated acid mucopolysaccharides.

      • i.
        Alcian blue- Safranin procedures.
   4. Toluidine blue for metachromatic properties at pH 2.0 and 4.4.

Method of Study

• I.
  Normal histological study of mast cells:

  1. The size, shape and granule content of mast cells have been observed mainly in toluidine blue solution at pH 4.4 and pH2.0.
• II.Distribution of mast cell has been studied in different parts of the tonsillar tissue.
• III.Histochemical study of mast cell:

Histochemical reactions of mast cells granules have been studied in order to determine the nature of reactive substance with respect to:

1. Heparin trisulphate.

2. Heparin monosulphate.

Results

Normal Histological and Histopathological Features of the Tonsil

The tonsil lies in the lateral wall of oropharynx between palatoglossal and palatopharyngeal arches. In routine haematoxylene and eosin stain the tonsil shows a condensation of dense connective tissue on its deep surface laterally called capsule which separates it from the superior constrictor muscle and the styloglossus muscle. It’s medial surface is covered by mucous membrane which shows 12–15 crypts. The surface and the crypts are lined by stratified squamous epithelium of non keratinizing variety. Underneath the epithelium lies the lamina propria, a layer of connective tissue. It sometimes extends above into the soft palate, below into the base of the tongue, and anteriorly and posteriorly it may go beyond the confines of the palatoglossal and palatopharyngeal folds. The tonsillar substance consists of lymphoid tissue which is present both as diffuse mass and as follicles which is probably due to reactive changes it undergoes in inflammatory process. Tonsil contains mucous glands but in none of the 20 specimens under observation any glandular tissue was observed.

The specimens of tonsil under observation in this work were all obtained from cases of chronic tonsillitis. Chronic inflammatory reactions were visible in most of the specimens showing fibrosis, lymphocytic infiltration in the epithelium and also prominent blood vessels.

Morphology and Distribution of Mast Cells

Morphology

The structure of mast cell has been studied after staining with toluidine blue at pH 2 and 4.4 at pH 2 the background does not stain and this gives a better picture for counting mast cells. Alcian blue at pH 2.5 has also been used to study the histochemical properties of mast cells as well as to doubly verify their occurrence. The mast cells have varied shape and size at different places and at different points of the same field of focus. They have been found to be oval, elongated, fusiform and of pleomorphic shape. The nucleus is usually central but not well defined and remains masked by the granules. The cytoplasm contains mast cell granules which are blue with alcian blue and take purple to bluish purple stain with toluidine blue.

Distribution of Mast Cells

Since mast cell is a connective tissue cell, its presence in the tonsil is expected to be confined wherever there is connective tissue in the organ. The connective tissue is confined to the capsule at the lateral surface of the tonsil and also in the lamina propria on the medial surface as well as in relation to the crypts. At other places like connective tissue septa which are seen here and there, are also expected to contain the mast cells (Table 1; Fig. 1a, b, c).

Table 1.

Distribution of mast cells in human tonsil

Sl No. of specimen	Toluidine blue pH 4.4			Toluidine blue pH 2.0			Alcian blue pH 2.5
	Capsule	Septa	SECT	Capsule	Septa	SECT	Capsule	Septa	SECT
1	3–4	1–2	1–2	2–3	o	o	3–4	1–2	1–2
2	3–4	o	o	3–4	1	1	4–5	1	o
3	4–5	1–2	1	4–5	1–2	1–2	5–5	1–2	O
4	3–4	o	o	4–5	o	o	3–4	1–2	O
5	2–3	o	o	3–4	o	O	2–3	1–2	O
6	4–5	1–2	1–2	4–5	o	o	4–5	1–2	1
7	6–8	2–3	1–2	8–10	2–3	o	6–8	2–3	1–2
8	5–6	2–3	1–2	5–6	2–3	1–2	4–5	O	O
9	5–6	1–2	O	5–6	1–2	o	4–5	O	O
10	4–5	1–2	O	6–8	1–2	1–2	5–6	1–2	1–2
11	5–6	1–2	1–2	8–10	3–4	2–3	5–6	1–2	O
12	3–4	1–2	1–2	3–4	1–2	o	3–4	1	O
13	3–4	1–2	1	4–5	1–2	O	4–5	1–2	O
14	3–4	o	o	4–5	1–2	o	4–5	1–2	O
15	3–4	o	o	3–4	1	o	3–4	o	O
16	4–5	1	O	4–5	1–2	1	4–5	1–2	o
17	6–8	2–3	1–2	8–10	3–4	1–2	5–6	2–3	1–2
18	3–4	1–2	o	3–4	1	o	3–4	1	O
19	3–4	1–2	1	3–4	1–2	o	3–4	1	O
20	6–8	1–2	1–2	8–10	2–3	1–2	6–8	2–3	1–2

O/O occasional, SECT sub epithelial connective tissue

Fig. 1.

Distribution of mast cells in human tonsil (Total specimen 20). a Toluidine blue pH 4.4 stain, b Toluidine blue pH 2.0, c Alcian blue pH 2.5

In this present work the mast cells have been found at all the places though they are mostly concentrated in the capsule.

1. With toluidine blue at pH 4.4 mast cells are present occasionally 1–2 in number per high power field in lamina propria. In the capsule the mast cells varied from 4 to 5 per high power field in most of the specimens. In some there were 6–8 per high power field. Mast cells were 1–2 per high power field in connective tissue septa.

2. With toluidine blue staining at pH 2, the mast cells at all sites were far more numerous than those with toluidine blue at pH 4.4. In most of the specimens with toluidine blue at pH 2.0 the mast cells were on an average 5–6 per high power field and in some 8–10 per high power field, in the capsule. Comparatively fewer mast cells were seen in lamina propria of the mucosal surface as well as the crypts. Mast cells were comparatively fewer in connective tissue septa but more than with toluidine blue at pH 4.4.

3. With alcian blue at pH 2.5 the distribution of mast cell were almost at par with toluidine blue at pH 2 and fewer mast cells stained with aldehyde fuchsin staining.

Histochemical Features of Mast Cells

The mast cells contain many biologically active substances including the proteoglycans, amines and certain enzymes. The proteoglycan in mast cell is contained in its granule as heparin. It is due to this glycosaminoglycan that the metachromatic property has been imparted to the mast cell granules. Other constituents of mast cell granules are histamine, 5 H–T, neutral proteases and eosinophil chemotactic factor of anaphylaxis(ECF-A). Mast cells also release leukotriene (Formerly known as SRS-A) but these substances are not stored in mast cell granules but in phospholipids of cell membrane and is immediately released upon appropriate stimulation.

At least two population of mast cells have been found in connective tissue. One type is called connective tissue mast cell whose granules contain heparin. In the second type, termed mucosal mast cells, the granules contain chondroitin sulphate instead of heparin. These two types react differently to pharmacologic agents.

In the present work the histochemical study on mast cell has remained confined to the connective tissue mast cell and the biochemical nature of heparin contained in it.

Heparin exist in mast cell in two forms the mono and trisulphates of heparin. The monosulphate form contains one sulphate radical while the trisulphate form contains two and half sulphate radicals per disaccharide unit. It consists of N-acetyl-d-glucosamine and sulphated-d-glucosamine and d-glucronic acid and a sulpho amino group. The sulphate radicals are found at the C-6 position of glucosamine and C-2 position of the glucronic acid. Both these forms of heparin in mast cells have been described by various workers especially Riley and West [5], Combs et al. [6] and Spicer et al. [4].

Heparin monosulphate, accordingly to Spicer et al. [4] and Bancroft and Stevens [7], falls in weakly sulphated group and heparin trisulphate in strongly sulphated group.

1. Identification of heparin trisulphate

It stains purple with toluidine blue at pH 2, purple with aldehyde fuchsin, blue with alcian blue at pH 1 and gives a reddish tinge with safranine. The alcian blue at pH 1 is abolished by active methylation.

• 2.Identification of heparin monosulphate

It stains blue to bluish purple with toluidine blue at pH 4.4 and blue with alcian blue at pH 2.5. This alcianophillie is abolished by mild methylation.

In the present work, the mast cells in tonsils have been subjected to toluidine blue staining at pH 2 and 4.4, alcian blue staining at pH 2.5 and 1.0 and alcian blue pH 2.5 safranine procedure for simultaneous identification of highly and weakly sulphated forms of heparin.

The tonsillar mast cells stained blue to bluish purple with toluidine blue at pH 2 and 4.4. They stained moderate to deep blue with alcian blue at pH 2.5 and 1. This alcianophilia is greately reduced after mild methylation at pH 2.5 but is completely abolished after active methylation at pH 1. Aldehyde fuchsin stains mast cells light purple in some specimen while in others no staining is seen. With alcian blue pH 2.5 safranine procedure, only a few mast cells show bluish red staining while most of the mast cells take only bluish stain of alcian blue.

The above staining reactions of mast cells have been found at all the sites, that is, in the capsule, in lamina propria as well as in thin septa seen here and there.

This toluidine blue staining procedure has also shown deep purple staining of basophils in the region of the capsule which indicates the similar histochemical properties in basophils as in mast cells so far as heparin is concerned.

Discussion

In the present work the identification of mast cell is based on metachromatic reaction of its granules with acidified toluidine blue at pH2 and buffered toluidine blue at pH 4.4. The two pH levels have been used to identify both types of mast cell granules containing higher and lower sulphates of heparin. Heparin trisulphate has greater affinity for toluidine blue at lower pH level, while at higher pH even the monosulphate form of heparin reacts with the dye.

Combs et al. [7] suggest that the purple to red purple colour of rat mast cell granules is due to higher sulphates of heparin maturation of mast cell granules begins with sulphation of heparin in the immature mast cells. Initially the heparin has only one sulphate group, but as the mast cell granules mature the number of sulphate group increases in its mucopolysaccharides and in fully mature mast cells heparin is found in its trisulphate form.

Apart from mastocytosis and mastocytomas in which the mast cell count is greatly increased, considerable alterations (increase or decrease) in the mast cell density undoubtedly occur as a consequence of inflammation and sclerosis in the surroundings of diverse specific morbid processes and the direction of the change appears to depend mainly upon the intensity and chronicity of the lesion [8]. In most of the chronic inflammatory disorders mast cell count is raised as in ulcerative colitis, chronic gastritis and peptic ulcer.

The paucity of mast cells in the subepithelial connective tissue and in the mucosa of tonsil appears baffling. This exposed surface shows many features of chronic inflammatory processes yet the mast cell count in this region is comparatively sparse. No description of mast cell has been found in the lymphoid mass of tonsil anywhere in the literature and in none of the 20 specimens under observation in this work.

Conclusion

1. Observations have been made on the histological and histochemical studies on mast cells in chronic tonsillitis. Altogether 20 specimens of tonsil have been studied in the present work.

2. The tissues have been fixed in Lillie’s buffered neutral formalin(10 %) and the sectioned tissue have been subjected to a battery of histochemical tests for identification of mast cells and characterization of the biochemical nature of heparin.

3. Apart from routine haematoxylene and eosin stain, the histochemical staining procedures used are—Toluidine blue at pH 2.0 and 4.4, Alcian blue at pH 1.0 and 2.5, with or without blocking procedures for heparin, Aldehyde fuchsin, and Alcian blue safranine staining procedures.

4. The number of mast cells have been found to be 4 to 5 peer high power field in the capsule of the tonsil; they are fewer in the septa and less so in the lamina propria.

5. Most of the mast cells have been found to contain heparin trisulphate with a smaller amount of heparin monosulphate, at almost all the sites which indicates that the mast cells in the tonsils are all mature mast cells.

6. Though allergy has little role in chronic tonsillitis, yet the occurrence of mast cells in the tonsil in insufficient number and has got no significant role in chronic tonsillitis.