Actividad S 1.3: Evaluar prácticas de seguridad organizacional

Scanning electron microscopy studies demonstrate that the HEC form a continuous

lining of the HEV wall, with cytoplasmic extensions of individual HEC overlapping

adjacent HEC (Anderson and Anderson, 1976). These overlaps, which may prevent

non-specific vascular leakage, form deep crevices in the luminal surface of the HEC.

Approximately 80 % of lymphocytes interacting with the HEC bind at, or near these

crevices (Anderson and Anderson, 1976). Basal foot processes extending from one

HEC under adjacent HEC, viewed under EM may also form part of a barrier to vascular

leakage (Anderson et al., 1976).

Intercellular junctions between HEC are discontinuous ‘spot-welded’ junctions, located

at luminal and basilar regions, and occasionally at mid-portions of adjacent HEC

(Anderson and Anderson, 1976). These are similar to non-occluding junctions found

in other postcapillary venules (Anderson and Shaw, 1993). Scanning and transmission

electron microscopy studies indicate that the junctions between adjacent HEC facilitate

the transendothehal migration of lymphocytes (Anderson and Anderson, 1976),

although it was previously suggested that lymphocytes penetrate the HEC cytoplasm in

order to extravasate (Marchesi and Gowans, 1964). Simultaneous transcellular and

inter-cellular migration of T lymphocytes across cytokine-stimulated human brain

microvessel endothehal cells has recently been demonstrated in vitro (Wong et al.,

1999). The migration of lymphocytes from the vessel lumen, across HEV wall is

%

&

Figure 1.3. View of the structure of HEV in rat lymph nodes. HEV are lined by cuboidal endothelial cells (High Endothelial Cells, HEC) that are joined to each other by discontinuous ‘spot-welded’ junctions. Basal foot processes (FP) extend beneath adjacent HEC. The endothelium rests upon a basement membrane, which is surrounded by a network of reticular cells (RC). The sequence of lymphocyte migration across the venular wall is illustrated. Lymphocytes are shown; adhering to the luminal surface of HEC (1); transmigrating HEC (2); crossing the basement membrane (3) and traversing the reticular cell sheath (4). (Magnification x 3200). (Figure reproduced from Anderson et.al. (1976)).

Currently, the mechanism(s) used by lymphocytes to penetrate the HEC wall is not

fully understood, although several molecules which may modulate endothelial cell

adhesive interactions have been identified (Girard et ah, 1999). An ECM protein

termed Kevin, approximately 60 % homologous to an anti-adhesive protein called

SPARC (secreted protein acidic and rich in cysteine) has been identified in human

tonsilar HEV, and is associated with the basolateral, luminal and apical HEC surface in

mouse HEV (Girard and Springer, 1995a; Girard and Springer, 1996). In vitro, hevin

fails to support adhesion of endothehal cells, or the spreading of endothelial ceUs on

fibronectin substrates. Hevin-treated endothehal cehs have a rounded morphology and

fail to form focal adhesions structures between adjacent cells (Girard and Springer,

1995a; Girard and Springer, 1996). Since hevin is located both on luminal and

basolateral HEC surfaces, it is possible that the anti-adhesive activity of Hevin

modulates the junctions between adjacent HEC, thereby allowing lymphocyte entry

through the HEC and basement membrane into LN (Girard and Springer, 1995a; Girard

and Springer, 1996). Hevin may also weaken the interactions between HEC and ECM

proteins of the underlying basement membrane, fachitating lymphocyte migration

through this barrier.

A member of the Ig superfamily. Vascular Endothehal Junction-Associated Molecule

(VE-JAM), was recently identified during screening of a subtracted human tonsilar

HEV cDNA library (Palmeii et al., 2000). This protein is homologous to junction

adhesion molecule (JAM), which is found at intercehular junctions of epithehal and

endothehal cehs and is imphcated in the transendothehal migration of monocytes cehs

(Martin-Padura et al., 1998). VE-JAM is locahsed to the boundaries between adjacent

HEC, however it is also found on the endothehal lining in other organs and is not

specific to HEC.

Underlying the HEC is a thickened basement membrane, composed of pericytes and

Shaw, 1993; Girard and Springer, 1995b). It is not clear how lymphocytes cross this

prominent basement membrane although it must be a tightly regulated process, since the

basement membrane is a barrier to soluble molecules administered intra-arterially

(Anderson and Anderson, 1976). It is possible that matrix metaUoproteinases (MMPs),

which control the metastatic spread of tumour cells by regulating the degradation of

ECM components (Nelson et al., 2000), could allow lymphocytes to enter LN tissue by

degrading the structure of the HEC and underlying basement membrane. Recently, it

was demonstrated that expression of MMP-2 is induced upon Thl lymphocyte binding

to specific cell adhesion molecules on endothehum in vitro, and that this was a

prerequisite for transendothehal migration of the lymphocytes (Madri et al., 1996).

MMP-2 or other MMPs may regulate migration of naïve lymphocytes across HEC in a

similar manner.

In addition to hevin, several other matricellular molecules preferentially expressed by

HEC have recently been characterised. These may also modulate HEC-HEC

interactions or HEC-extracellular matrix interactions, and could thus be involved in

facilitating lymphocyte extravasation.

Thrombospondin (TSP)-l, like hevin, inhibits ceh spreading and formation of focal

adhesions, however TSP-1 is detected at the basement membrane level and may

therefore play a greater role in weakening HEC binding to ECM proteins, allowing

lymphocytes to cross the HEV basal lamina (Girard et al., 1999; Murphy-Ullrich and

Hook, 1989; Sage and Bomstein, 1991). The mouse homologue of mac25, an ECM

polypeptide that is 20-25 % homologous to human insulin-like growth factor binding

protein, has been detected in association with luminal and basolateral regions of HEC

(Girard et al., 1999; Izawa et al., 1999). Mac25 has been implicated in the organisation

of blood vessels in tumours by modulating the interactions of endothelial cells with

collagen (Akaogi et al., 1996). In vitro, TSP-1 and mac25 bind to heparan sulfate

microvillus processes of HEC (Girard et al., 1999; Murphy-Ullrich and Hook, 1989;

Sage and Bomstein, 1991). This is significant since microvilli harbour several

adhesion molecules that mediate the initial contact between HEC and blood-bome

lymphocytes (Berlin et al., 1995; Stein et al., 1999), and proteoglycan molecules are

also the sites of chemokine presentation to lymphocytes (Tanaka et al., 1993a; Tanaka

et al., 1993b). Therefore, in addition to facilitating lymphocyte entry through the HEC

wall or basement membrane, mac25 or TSP-1 may play a role in the earlier stages of

lymphocyte extravasation, e.g. presentation of chemoattractant molecules to the

lymphocytes.

Extensive collagen fibres and reticular cell processes extend the basement membrane

into the tissue regions of the paracortex (Anderson and Anderson, 1976). In contrast to

the barrier afforded to soluble particles in the bloodstream, the flow of soluble material

from the lymphatics to the HEV lumen is not blocked by this ECM and reticular cell

network, or at the level of the basement membrane (Anderson and Anderson, 1976).

Although the significance of this ‘one-way’ traffic remains to be resolved, it is possible

that it may be a mechanism which allows soluble factors derived from the LN stroma

e.g. chemoattractants, to enter the HEV lumen and mediate lymphocyte adhesion and

transendothehal migration. Recent identification of chemokines expressed within

secondary lymphoid tissues makes this more plausible (Cyster, 1999).