Intercellular and lymphatic pathways of the canine palatine

J. Anat. (1995), 187, pp. 93-105, with 19 figures Printed in Great Britain

Intercellular and lymphatic pathways of the canine palatinetonsils

GABRIELLE T. BELZ AND TREVOR J. HEATH

Department of Anatomical Sciences, University of Queensland, Australia

(Accepted 7 February 1995)

ABSTRACT

The palatine tonsils play a key role in initiating immune responses against antigenic material entering themouth and their lymphatic pathways are important in disseminating immunological information to thelymph nodes and other mucosal surfaces. Scanning and transmission electron microscopy and Mercox castswere used to examine the intercellular and lymphatic pathways of the palatine tonsil in dogs. Intercellularfluid within the intraepithelial passageways of the reticular epithelium flows through pores in the basementmembrane and intermingles with that of the subepithelial intercellular spaces. From there, tissue fluid entersinitial lymphatics which form a plexus surrounding each follicle. No lymphatic vessels are seen entering orleaving the follicles but intercellular pathways of the follicles are continuous with those of the adjacentinitial lymphatics. These pathways appear to provide the only route for lymphocytes leaving the follicles anddirectly entering the lymphatic pathway. Lymph then flows into sinuses adjacent to, and incompletelysurrounding, the base of follicles; or it may enter a network of sinuses between and beneath the lymphoidfollicles which convey lymph throughout the parafollicular tissue. Lymphoid cells enter the parafollicularsinuses which may be a major entry site of lymphocytes emigrating from the lymphoid parenchyma. Somelymph may bypass these sinuses by entering septal lymphatic vessels oriented perpendicular to the centralconnective tissue lamina of the palatine tonsil. All lymph is collected into basal lymphatic vessels wherevalves prevent retrograde flow. Basal vessels course within the central lamina and converge to form efferentlymphatic vessels which emerge at the caudal region of the palatine tonsil and convey lymph to the medialretropharyngeal lymph node.

Key words: Dog; tonsil; lymphoid tissue; lymphatic vessels.

INTRODUCTION

Palatine tonsils occupy a significant position at thebeginning of the gastrointestinal tract allowing in-timate contact with bacteria and other antigens thatare ingested. They play a key role in initiating immuneresponses against these antigens (Brantzaeg, 1984).The lymphatic pathways which drain these mucosalsites play an important part in conveying lymphoidcells and cell products to the draining lymph nodesand in disseminating immunological information toother mucosal surfaces (Bienenstock & Befus, 1984;Brandtzaeg, 1988).Most studies of the palatine tonsil have been made

on the histopathological features, ontogeny, cells and

cell products (Olah & Everett, 1975; Brantzaeg et al.1978; Surjan, 1987; Narita et al. 1989). Although theimportance of the tonsil in lymphopoiesis and thehigh rate of emigration of lymphocytes into and fromthe tonsil have been established (Korburg, 1967;Fichtelius, 1969; Pabst & Nowara, 1984), no detailedinformation is available on the pathways by whichlymphocytes and mediators of cellular and humoralimmunity are transported from the tonsillar epi-thelium, past the lymphoid follicles to the efferentlymphatics (Williams & Rowland, 1972; Anderson,1974; Fawcett & Raviola, 1994).The aim of this study was to describe the inter-

cellular and lymphatic pathways of the palatinetonsils in dogs. These pathways were studied using

Correspondence to Dr Gabrielle Belz, Department of Anatomical Sciences, University of Queensland, Australia 4072.

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94 Gabrielle T. Belz and T. J. Heath

Microfil and Mercox casts, by light and electronmicroscopy.

MATERIALS AND METHODS

Twenty-eight greyhound dogs, aged 9-24 months,were used. They were sedated with intravenousacepromazine (Promex 2 or Promex 10, ApexLaboratories, St Marys, New South Wales, Australia)and anaesthetised with intravenous pentobarbitonesodium (Nembutal, Boehringer Ingelheim, Artarmon,New South Wales, Australia). All animals were killedwithout regaining consciousness, either by ex-

sanguination or with an overdose of pentobarbitonesodium (Lethabarb, Arnolds of Reading, Peakhurst,New South Wales, Australia).

In 9 of the anaesthetised dogs, lymphatic pathwaysof the head were distended by ligating the trachealtrunk, and by partly occluding the jugular vein for9-20 min, to increase the movement of fluid from thecapillaries to the interstitium and so promote lymphformation.The tissue for light and scanning electron mi-

croscopy was fixed in 4% paraformaldehyde and, fortransmission electron microscopy, in a solutionof 4% paraformaldehyde and 2.5% glutaraldehyde.Heparinised saline, followed by fixative, was perfusedthrough a cannula placed in the common carotidartery. The tonsils were removed and placed in thesame fixative for a further 24 h, then washed andstored in 0.1 M sodium cacodylate buffer (pH 7.2).

In some cases, fixative (2 % paraformaldehyde and1.25% glutaraldehyde in 0.067M sodium cacodylatebuffer, pH 7.2) was dripped onto the tonsil to initiatefixation prior to removal. The tonsils were thenexcised and immersed in a second fixative (4%paraformaldehyde and 2.5% glutaraldehyde in0.067 M sodium cacodylate buffer, pH 7.2) for 24 h,then washed and stored in 0.1 M sodium cacodylatebuffer (pH 7.2).

Sections cut from each specimen were dehydratedin ethanol, cleared with xylol and mounted in wax.

Histological sections (7 jm), stained with haematoxy-lin and eosin, were photographed using an OlympusBH-2 microscope with an Olympus PM-1OAD/OM-2photographic system.

Tissue blocks for scanning electron microscopywere dewaxed, washed and dehydrated with ethanol,dried by the critical point method, sputter coated withgold and examined using a JEOL 6400F scanningelectron microscope.

Tissue for transmission electron microscopy was

postfixed in 1 % osmium tetroxide, and embedded inEpon/Araldite or Spurr's resin. Semithin sections(1 gm), were stained with toluidine blue; ultrathinsections were stained with uranyl acetate followed bylead citrate and examined with a Zeiss lOC micro-scope.

In some experiments, the epithelium of the palatinetonsil was removed to expose the underlying basementmembrane. Unfixed tonsillar tissue was immersed in a2 M solution of sodium bromide for 18 h then theloosened sheet of epithelium was separated from theunderlying tissues using fine forceps. Preparationswere placed in 4% paraformaldehyde fixative for24 h, washed in distilled water and stored in 0.1 Msodium cacodylate buffer (pH 7.2). Sections were cutfrom each specimen, dehydrated in a graded series ofethanol and dried by the critical point method.Specimens were sputter coated with gold andexamined using a JEOL 6400F scanning electronmicroscope.

Casts oflymphatic pathways were made by injectingMicrofil (Flow Tek, Boulder, Colorado, USA) orundiluted Mercox (CL-2B, Japan Vilene Hospital,Tokyo) into the subepithelial region of the tonsil. A30 G needle attached to a 2 ml syringe was used toinject by hand. The injection was stopped when thecasting medium appeared in the efferent lymph vesselsof the tonsil (usually 30-40 s).

Microfil casts were allowed to set, and the tonsil,medial retropharyngeal lymph node and interveningconnective tissue and lymphatic vessels were then dis-sected from the carcass and placed in fixative for8-12 h. They were dehydrated, then cleared in methylsalicylate and photographed with an Olympus/OM-2bellows system. Histological sections were preparedfrom some tonsils to confirm the identity of clearedtissues and the distribution of casting medium.

Tissues containing Mercox were allowed to set for1-2 h before being placed into warmed water (60 °C)for 8-12 h, and then digested in several changes of15% sodium hydroxide. The casts were washed indistilled water ethanol and air dried. They were thensputter coated with chromium using an ion-beamcoater (Gatan 681 Penning Ion-Beam Coater, GatanInc., USA) and examined using a JEOL 6400Fscanning electron microscope at 3-3.5 kV.

RESULTS

Microscopic appearance

The oropharyngeal mucosa of the palatine tonsil ofdogs was formed by a continuous layer of non-

Lymph pathways ofpalatine tonsil

Fig. 1. Photomicrograph of a transverse section through the palatine tonsil in the dog. The epithelium (E) invaginates (large black arrow)dividing the lymphoid tissue into lobes. The lymphoid tissue consists of the subepithelium (S), follicles (F) with prominent germinal centres,and parafollicular tissue (asterisk) which fold around a central connective tissue lamina (L). Septa (arrowheads) may arise from this laminawhich is continuous with the capsule (small black arrows). G, salivary glands; white arrows, basement membrane. Bar, 500 gtm.

keratinised stratified squamous epithelium. Much ofthe epithelium, particularly that overlying the apicalregion of follicles, was infiltrated by a great number oflymphoid and other mononuclear cells, therebyforming a reticular epithelium (Fig. 1) (Belz & Heath,1995). The basement membrane supporting the epi-thelium separated the mucosa from the subepitheliallymphoid tissues (Fig. 1). The mucosa did notinvaginate to form crypts but was folded, dividing thelymphoid tissue into several lobes (Fig. 1) (Belz &Heath, 1995).The subepithelial lymphoid tissue was composed of

a moderate infiltrate of lymphoid cells between theepithelium and the lymphoid follicles, lymphoidfollicles containing prominent germinal centres, andparafollicular tissue between and beneath the follicles(Figs 1, 2). This lymphoid tissue folded around acentrally placed connective tissue lamina which sentthin partitions (or septa) into, and merging with, theparafollicular tissue (Fig. 1). The central lamina wascontinuous with a connective tissue capsule whichseparated the lymphoid and glandular tissues of thetonsil from the surrounding buccal fascia (Fig. 1). Thegross morphology and the epithelium of the palatinetonsil have been described (Belz & Heath, 1995); this

paper is concerned primarily with the prelymphaticintercellular pathways and the lymphatic pathways ofthe tonsil (Fig. 2).

Intercellular pathways of the mucosa andsubepithelium

When Mercox was injected into the subepithelialparenchyma it entered either the intercellular spacesor lymph sinuses, or superficial lymphatic vessels (Figs3, 4). Although most of the Mercox which entered thelymphatic vessels was constrained by valves to movein the normal direction of lymph flow, that whichentered sinuses or intercellular spaces flowed in bothdirections. This resulted in the filling of initial lymphvessels (Fig. 3).

Within the reticular epithelium overlying the apexof a follicle, Mercox formed a densely packed networkof interconnected casts (Fig. 4). Discrete roundedholes, 180-450 nm across, were present on the castsurface (Fig. 4). The oropharyngeal aspect of the castswas irregular and roughened and terminated at auniform plane (Fig. 4). No Mercox was seen to flowfrom the epithelial stroma into the pharyngeal lumen.

Tubular casts, 5-12 pm across, connected the casts

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Fig. 2. Scanning electron micrograph of a section of palatine tonsil from the epithelial surface (E) to the central connective tissue lamina(C) in a preparation in which the lymphatic sinuses have been slightly distended. The intercellular spaces of the reticular epithelium (RE)are continuous with those of the subepithelium (S). Initial lymphatic vessels (L) arise at the periphery of the apex of the follicle (F) and coursearound the margin of the follicle. Lymph from the initial lymph vessels may flow (black arrows) to (1) parafollicular sinuses (asterisk) situatedin the parafollicular lymphoid tissue (P), or (2) to sinuses bordering the perifollicular connective tissue at the base of the follicle (arrowhead)which drain to other parafollicular sinuses, or (3) pass directly to septal lymphatic vessels (not shown). Lymph then drains to basal vessels(B) which pass along the central lamina and coalesce to form efferent vessels. Valves (outlined arrows) are prominent in the basal vessels.Bar, 100 hm.

of intercellular spaces of the reticular epithelium withthose of the subepithelial tissue (Fig. 4). Removal ofthe epithelium using sodium bromide revealed a veryporous basement membrane overlying the lymphoidfollicles (Figs 5, 6). Numerous rounded or oval holesranging from 2.6 to 6.8 gm in diameter were seen inthis membrane and they were smaller and fewer in

number over the apical regions of the follicles (Fig. 5).Towards the periphery of the follicles, these holesmeasured up to 12 gim across. Many lymphoid cellswere seen lying within these holes (Fig. 6).Mercox casts of the subepithelial spaces were

smaller and less densely arranged than those withinthe reticular epithelium (Fig. 4). In some areas


Fig. 3. Scanning electron micrograph of a Mercox cast of intercellular and lymphatic pathways of a palatine tonsil. Epithelial casts (IE) arecontinuous with those of the subepithelium (IS). Initial lymphatic vessels (arrowheads) arise at the periphery of follicles (F), some of whichcontain Mercox. Lymph from initial lymphatic vessels flows into sinuses (P) located in the parafollicular tissue. These sinuses may then joinseptal lymphatics (S) coursing with connective tissue septa between follicles. *Space representing the perifollicular connective tissue. Bar,100 gm.Fig. 4. Scanning electron micrograph of a Mercox cast of the intercellular spaces of the epithelial and subepithelial region of a palatine tonsil.Epithelial casts (IE) are continuous with subepithelial casts (IS) by tubular casts (arrows) which extend through the space occupied by thebasement membrane (asterisk). Subepithelial casts are continuous with, and coexist with, casts of initial lymphatic vessels (L). Both theepithelial and subepithelial casts are punctuated by round impressions (arrowheads), 180-450 nm across. Bar, 20 jm.

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Fig. 5. Scanning electron micrograph of the basement membrane overlying the apical portion (A) of a follicle after removal of the epitheliumusing 2 M sodium bromide. The holes are larger and more abundant towards the periphery (arrows) of the follicle. Bar, 20 gm.Fig. 6. Scanning electron micrograph showing holes (H) in the basement membrane (BM) overlying the peripheral region of the follicle. Anumber of lymphoid cells can be seen passing through the holes (arrows). Bar, 10 gim.

overlying the apex of the follicles, casts of theintercellular spaces were absent (Fig. 3).

Lymphatic pathways of the subepithelium

Lymphatic pathways associated with follicles. Theinitial lymphatic vessels were single or branched blind-ending vessels, 7-11 gm across, which arose towardsthe periphery of the apical region of the lymphoidfollicles and formed plexuses which almost completelysurrounded the follicles (Figs 7, 8). No lymphaticvessels were seen entering the follicles themselves.

Initial lymphatic vessels coexisted with, and werecontinuous with casts of the subepithelial intercellularspaces (Fig. 4). Scanning electron microscopy revealedthat these vessels had a smooth endothelial lining withsome microvillous protrusions (Fig. 9). Adjacentendothelial cells were usually overlapping; however,in distended preparations these were drawn apartexposing intercellular holes through the wall of theinitial lymphatic vessel (Fig. 9). Abluminally, a finemeshwork of reticular fibres adjoined the endothelialwall and was supported by collagen bundles andfibroblasts.Each follicle was separated from the parafollicular

tissue by connective tissue. This perifollicular tissueconsisted of an external lamina of collagen fibres andsmall collagen bundles interspersed among severallayers of fibroblasts extending concentrically aroundthe follicle (Fig. 10). It appeared more denselyarranged towards the basal boundaries of the follicles.

Fluid spaces, packed with macrophages and lympho-cytes, separated adjacent layers of fibroblasts (Fig.10). At some places, lymphoid cells, which appearedto be moving through discontinuities in this peri-follicular tissue, were interposed between the con-tiguous processes of fibroblasts (Fig. 10).The confluence of intercellular spaces in para-

follicular tissue and lymphoid follicles wasdemonstrated with Mercox casts (Fig. 3). In castsof the 22 tonsils examined, Mercox enteredapproximately 60% of follicles, flowing between thelymphoid cells to form a network of casts (Fig. 3). Aspace, representing the connective tissue layers, sepa-rated the stroma of the follicle from the parafolliculartissue (Fig. 3). Mercox casts connected intercellularspaces of the lymphoid follicles and the initiallymphatics in the parafollicular tissues (Fig. 8).The initial lymphatic vessels coursed around the

margins of the follicles adjacent to the surroundingconnective tissue, then flowed into flattened sinuses,measuring up to 100 gim across, which lay adjacent tothe base of the follicle (Figs 2, 11). These sinuses werelined by a convoluted endothelium, possessing en-dothelial flaps and, more prominently, cytoplasmicextensions and nuclear protrusions. Some smoothmuscle cells, fibroblasts and connective tissue adjoinedthe abluminal aspect. Lymph flowed from thesesinuses into parafollicular sinuses immediately be-neath the follicles through rounded or oval holes,10 gim across (Fig. 12). When Mercox was injectedinto the tonsillar parenchyma and filled the para-

Lvmph pathways ofpalatine tonsil

Fig. 7. Scanning electron micrograph ot a Mercox cast snowinginitial lymphatic vessels (filled retrogradely) (arrowheads) arising as

single or branched vessels at the periphery of the apical portion ofeach follicle (asterisk) and draining towards larger lymphatic vessels(L) which occur between adjacent follicles. Bar, 100 gm.Fig. 8. Scanning electron micrograph of a Mercox cast of a portionof a lymphatic plexus surrounding a follicle. Casts of intercellularspaces of the follicle (asterisk) are continuous with the initiallymphatic vessels (L). Bar, 100 gm.Fig. 9. Scanning electron micrograph of an initial lymphatic vesselin a preparation in which the lymphatic spaces were slightlydistended. The endothelial cells have overlapping edges (arrow),however, in some areas these have drawn apart exposing inter-cellular pores (arrowheads). *Microvillous protrusions. Bar, 1 Am.

follicular sinuses and basal lymphatics, it did not flowretrogradely into the sinuses surrounding the follicles.

Lymphatic pathways of the parafollicular tissue.Lymph flowed to the basal lymphatics along two

Fig. 10. Transmission electron micrograph of a lymphatic vessel (L)coursing between and separating the parafollicular lymphoid tissue(P) from a follicle (F). The follicle is surrounded by a layer ofcollagen fibres (C) adjoining the lymphatic endothelium (E)abluminally. Elongated processes of fibroblasts (arrowheads),between which lie many lymphoid cells, form laminae at theperiphery of the follicle. At some points (asterisk) lymphoid cellsoccur between contiguous processes of the fibroblasts. BV, bloodvessel. Bar, 10 gim.

pathways. In the first pathway, lymph flowed from theinitial lymphatics or the sinuses at the base of follicles,into lymphatic sinuses lying within the parafollicularparenchyma (Figs 2, 13). These parafollicular sinuseswere 20-80 jim across and infrequently containedluminal bridging processes. They were lined by a layerof gently undulating endothelium (Fig. 14). Endo-thelial cells overlapped adjacent cells forming flapsand lymphoid cells, which appeared to be migratingfrom the parafollicular tissue into the sinuses, wereoccasionally seen (Fig. 15). Although these vessels didnot contain valves, the contiguous walls of the vesselsformed single valve-like flaps at their confluence withseptal and basal lymphatics (Fig. 16).

In the second pathway, lymph within initiallymphatics flowed directly into vessels which coursedwithin the connective tissue septa (Fig. 1). Thesevessels were oriented approximately perpendicular tothe basal lymphatics into which they delivered lymph.These septal vessels were lined by a slightly convolutedepithelium (Fig. 17) and contained bicuspid valves.The connective tissue support of the septa, centrallamina and capsule surrounding the lymphoid tissueprovide a protective conduit for lymphatic and bloodvessels entering and draining the tonsil (Fig. 1).

Basal lymphatic vessels, 75-115 gm in diameter,were continuous with the parafollicular sinuses (Fig.2) and septal vessels. Bicuspid valves were prominentin these vessels. They were lined by a continuous layerof flattened endothelial cells, supported by bundles ofcollagen fibres and fibroblasts, and surrounded by a

99


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Fig. 11. Scanning electron micrograph showing the entry (arrow) from a lymph sinus (S) adjacent to the base of a follicle (F) into aparafollicular sinus (P). Bar, 10 jm.Fig. 12. Scanning electron micrograph showing openings (arrows) punctuating the roof of a parafollicular sinus. These openings communicatewith a flattened lymphatic sinus which incompletely surrounds the base of the follicle. Bar, 10 im.

Fig. 13. Scanning electron micrograph of parafollicular sinuses(asterisks) passing between and beneath adjacent follicles (F). Somelymphatic vessels (white arrows) adjoin the connective tissuedemarcation surrounding each follicle. Bar, 20 km.

Fig. 14. Transmission electron micrograph of the wall ofa lymphaticsinus in the parafollicular tissue. The lymphatic endothelium (E) issupported by a lamina of collagen fibres (asterisk) which areintimately associated with fibroblasts (F). Bundles of nerve fibres(arrows) are distributed throughout the parafollicular tissue and liein close proximity to lymphoid cells. P, plasma cell; L, lumen oflymphatic sinus. Bar, 1 km.

layer of smooth muscle cells (Figs 2, 18). Bundles ofunmyelinated nerve fibres were often intimatelyassociated with the lymphatic endothelium. Around50 basal lymphatics coursed between the layers offibroblasts and collagen fibres and longitudinally

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Fig. 15. Scanning electron micrograph of the endothelial lining of aparafollicular sinus. Endothelial cells have a pitted appearance(asterisks) and overlapping endothelial edges (black arrows). Asmooth-surfaced lymphoid cell (ML) with trailing plasmamembrane-bound extensions appears to be passing betweenoverlapping endothelial cells (white arrow). L, lymphoid cells withsurface microvilli. Bar, 1 gm.

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Fig. 16. Scanning electron micrograph showing a partly collapsedsingle valve flap (arrow) at the convergence of adjacent para-follicular sinuses (P). Bar, 10 gm.

oriented smooth muscle cells forming the centrallylocated connective tissue lamina of the tonsil (Figs 2,19). These vessels passed between and around thelobules of the adjacent salivary glands (Fig. 1).The lobules of the salivary glands were separated

from the lymphatics by connective tissue whichcontained fibroblasts surrounding an interlacing net-work of collagen fibres (Fig. 1). Lymphoid cells wereoften. seen within the interstices of this meshwork. In

Fig. 17. Transmission electron micrograph of the wall of a septallymphatic vessel. The endothelium (E) is supported by bundles ofcollagen (C) arranged in various orientations, and fibroblasts (F).L, vessel lumen. Bar, 2 gm.Fig. 18. Transmission electron micrograph of a basal lymphaticvessel (LV) which is accompanied by a blood vessel (BV) in thecentral connective tissue lamina. The lymphatic endothelium (E) issupported by a lamina of collagen fibres (asterisk), fibroblasts (F)and a layer of smooth muscle cells (M). Bar, 2 gim.

many cases the abluminal surface of basal lymphaticsadjoined the connective tissue margin of the glands.The many basal lymphatics converged and joined to

form 3-5 efferent lymphatic vessels which emergednear the caudal hilar region of the tonsil (Fig. 19).They conveyed lymph to the ventromedial region ofthe medial retropharyngeal lymph node.

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Fig. 19. Photograph of a Microfil cast showing basal lymphaticvessels converging and forming a few efferent vessels (E) whichleave at the caudal pole of the palatine tonsil. Valves (arrowheads)are prominent in these vessels. Bar, 100 gm.

DISCUSSION

In the palatine tonsil of the dog, intercellular fluid andlymphoid cells flow within the intraepithelial passage-

ways of the reticular epithelium, through holes in thebasement membrane, to reach the subepithelium.From there, lymph constituents pass into initiallymphatic vessels which form a plexus surroundingeach follicle. Lymph then enters sinuses adjacent tothe base of the follicle; or it may enter a network ofparafollicular sinuses which drain directly into septaland basal lymphatics. The basal lymphatics coalesceto form efferent vessels which convey lymph to themedial retropharyngeal lymph node.The intercellular spaces of the reticular epithelium

are continuous with those of the subepitheliumthrough openings in the basement membrane.Lymphoid cells were often seen projecting throughthese pores. Such porosity of the basement membraneappears to be a feature of gut-associated lymphoidtissues (Low & McClugage, 1984; McClugage & Low,1984; McClugage et al. 1986; Higashikawa et al.1990; Kihara et al. 1991). Even though the process bywhich pores are formed and repaired is unknown,they appear to represent a phase of transitionaldeformation of the basement membrane (McClugage& Low, 1984; Komuro, 1985) permitting lymphoid

cells and molecular aggregates derived from immuneresponses to be transported between the epitheliumand the subepithelial lymphoid tissues (Falk & Mootz,1973; Sato & Wake, 1990).

Discontinuity of the basement membrane has beenobserved in some studies (Olah et al. 1972; Sato &Wake, 1990) but Maeda et al. (1982) could notdemonstrate direct connections between the intra-epithelial passageways and the underlying lymphoidtissues. Although Mercox initially entered the inter-cellular spaces of the reticular epithelium from thesubepithelium by passing retrogradely through thepores in the basement membrane, interconnectedportions of the intraepithelial network would havebeen filled by normograde flow. Therefore, Mercoxcould re-enter the intercellular spaces of the sub-epithelium and thus flow into initial lymphatic vessels.The pores in the basement membrane, therefore,appear to provide a significant route for the passage ofintercellular fluid, immune cells and cell products andallow their bidirectional movement between themicroenvironment of the epithelium and the sub-epithelial lymphoid tissues.

In contrast to the relative paucity of intraepitheliallymphoid cells observed in follicle-associated epi-thelium of other gut-associated lymphoid tissues (Abe& Ito, 1978; Smith & Peacock, 1980), tonsillarepithelium is infiltrated by a great number oflymphoidcells which transform the stratified squamous epi-thelium into a reticular network of intraepithelialpassageways (Falk & Mootz, 1973; Anderson, 1974).Although reticular transformation of tonsillar epi-thelium and fragmentation of the basement membraneby migrating lymphoid cells commences in utero(Chen et al. 1990; Slipka, 1992), full development ofthe lymphoid tissue and its associated epitheliumappears dependent on exogenous stimulation (Falk &Mootz, 1973). In the dog tonsil (Belz & Heath, 1995),as in man (Howie, 1980), M cells appear morefrequent toward the periphery of the lympho-epithelium overlying the follicle, which may facilitateantigen interaction as fluid and particulate matterflow from the apex to the periphery of follicles. Theirbasolateral membrane enfolds many lymphoid cellsallowing antigen, transported within the cells(Williams & Rowland, 1972; Wolf& Bye, 1984), to bewidely distributed among lymphocytes enmeshedwithin the cytoplasmic pockets. Towards the peripheryof follicles the porosity of the overlying basementmembrane is greater and this may facilitate anincreased passage of lymphocytes into the epitheliumto the region of the intraepithelial cytoplasmic web ofthe M cells. This study suggests that a distinct


relationship exists between the distribution ofM cells,lymphocyte infiltration and the porosity of thebasement membrane: direct transepithelial transportof antigenic material by M cells augments an influxand recruitment of lymphoid cells. The porosity of thebasement membrane may reflect the current antigenicstimulation of the lymphoid tissues and this is likely tovary over time.

Tonsils differ from the lymph nodes for they lackafferent lymphatics which deliver lymph to thelymphoid parenchyma (Yoffey & Courtice, 1970;Williams & Rowland, 1972; Fawcett & Raviola,1994). The intercellular pathways of the subepitheliumappear similar to the 'lymphatic labyrinth' describedin the human palatine tonsil (Sato & Wake, 1990).These passageways which form between the con-nective tissue elements lack an endothelial lining andconstitute prelymphatic intercellular pathways(Casley-Smith, 1976; Castenholz, 1988) in tonsillartissues. They provide routes for antigenic materials,lymphoid cells and cell products to be carriedthroughout the subepithelial tissues. More import-antly, however, they may provide the major route bywhich antigens and antigenic information aretransported to the follicles where immune responsesare initiated (Curran & Jones, 1978).Towards the periphery of follicles, casts of the

subepithelial intercellular spaces coexisted with, andwere continuous with, initial lymphatic vessels. Dis-tension of the underlying interstitium, as would haveoccurred as a result of partial occlusion of the venousdrainage used to demonstrate the lymphatic pathways,would be expected to increase fluid flow into initiallymphatic vessels. The openings noted within theendothelium of initial lymphatics appeared to be openintercellular clefts in which the distension wassufficiently great as to reveal an open pore. Anabluminal collagen fibre network, produced by ad-jacent fibroblasts supporting the lymphatic endo-thelium, may regulate stretching of the endothelialwall to accommodate variations of interstitial fluidvolume. Collagen fibres are strongly attached to theabluminal filaments (Leak & Burke, 1966), and joinwith unattached borders of lymphatic endothelialcells. These fibres allow overlapping endothelial cells,acting as unidirectional flap-valves, to withdrawslightly when forming open intercellular clefts(Casley-Smith & Florey, 1961; Casley-Smith, 1972;Castenholz, 1988). In addition, specialised junctionalcomplexes (complexus adhaerentes) which connectadjacent lymphatic endothelial cells (Schmelz &Franke, 1993), together with intracytoplasmic cont-

be involved in cell contractions and local plasmamembrane retractions and thus contribute, in a

dynamic way, to the formation of these apertures.Retrograde filling of the lymphatic network with

Mercox revealed single or branched blind-endinginitial lymphatic vessels. The increase in intraluminalpressure promotes closure of the interendothelialflaps (Casley-Smith, 1977), thereby preventingreflux of Mercox (or lymph) into the subepithelialintercellular spaces. Therefore, occlusion of efferentpathways, together with interstitial distension, may benecessary to raise intraluminal pressure sufficiently torender lymphatic microvalves incompetent.From the intercellular spaces of the reticular

epithelium and subepithelium, the lymph flows into a

plexus of lymphatic sinuses surrounding each lymph-oid follicle and into an interconnecting network ofsinuses within the parafollicular tissue. Some lymphmay bypass these sinuses by entering septal vessels.This differs from reports of the lymph pathways of thepalatine tonsil in other species where the tonsilsapparently lack lymph sinuses; the first observablelymph vessels are blindly-ending lymph capillariessurrounding the capsule (Williams & Rowland, 1972;Anderson, 1974; Fawcett & Raviola, 1994).Although many lymphocytes migrate from tonsillar

lymphoid tissue (Pabst & Nowara, 1984) no reportshave documented the sites where lymphocytes may

enter the lymphatic vessels. Lymphoid cells from theparafollicular tissue appear to enter the parafollicularsinuses by passing between the overlapping endo-thelial cells. These cells exhibit the main features ofthe moving lymphocyte; they adopt a 'hand-mirror'morphology in which the cell body headed by thenucleus moves in toto, trailed by plasma membrane-bound extensions of these cells and accompanied bythe loss of surface microvilli (de Bruyn, 1946; Norberget al. 1973; van Ewijk et al. 1975). The parafollicularsinuses, therefore, may be a major entry site oflymphocytes emigrating from the lymphoidparenchyma.

Lymphatic vessels were not found over the apices offollicles but a small proportion of proliferatinglymphocytes may enter initial lymphatic vesselsleading from, and forming, a network encircling theperiphery of follicles. However, the vast majority oflymphoid cells originating from the follicles appear toreturn to the epithelium where they encounter secon-

dary signals (Korburg, 1967; Curran & Jones, 1978)before entering the efferent lymphatic pathway.

In this study, intercellular pathways were shown toexist within the follicle and join the surrounding

ractile proteins (Casley-Smith & Florey, 1961), may

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lymphatic plexus although drainage of the lymphoid


follicles through lymphatic vessels appeared absent.Discontinuities seen in the connective tissue sur-rounding the follicles were connected with channelsforming perifollicular lamellae. These intercellularpathways resemble a similar arrangement observed insecondary follicles in human tonsil (Watanabe et al.1992) and appear to provide the only route forlymphocytes leaving the follicles. In the tonsillarfollicles T helper/inducer lymphocytes, which assist inthe movement of B lymphocytes, predominate withinthe interstices of this perifollicular complex(Yamanaka et al. 1983).

Valves within the parafollicular sinuses are less welldeveloped than the bicuspid valves of the basallymphatic vessels. Although these single valve-flapsmay permit some reflux of lymph (Wenzel-Hora et al.1987 a, b) their main contribution may be in directingthe flow of lymph throughout the parafollicular sinusnetwork.

In the tonsil, like some other gut-associated lymph-oid tissue (Lowden & Heath, 1994), smooth musclecells adjoining and supporting the walls of lymphsinuses are poorly developed. The paucity of smoothmuscle cells associated with tonsillar vessels suggeststhat compression and expansion of the initiallymphatics and lymphatic sinuses may be initiated by,and depend largely on, external tissue stresses whicharise during deglutition. During swallowing, posteriorpressure of the tongue against the soft palate and thetonsils, which are drawn medially, produces com-pression of the enclosed tissues, including the palatinetonsils (Guyton, 1991). Therefore, lymph within thetonsillar sinuses could be propelled towards the basalvessels which contain valves to prevent retrogradeflow. In addition, this upward and backward com-pression exerted over the surface of the tonsil, coupledwith contraction of the smooth muscle cells within thewall of the basal lymphatic vessels, may combine totransport lymph towards efferent vessels which emergeat the caudal region of the tonsil and subsequentlyconvey lymph to the medial retropharyngeal lymphnode.

ACKNOWLEDGEMENTS

We are grateful to Gary Godbold for obtaining dogs,Rodney Williams for help with photography, and theUniversity of Queensland Centre for Microscopy andMicroanalysis and Tina Chua for assistance withelectron microscopy.

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Intercellular and lymphatic pathways of the canine palatine