Obtención y evaluación de los microencapsulados

It has been clearly established that L-selectin plays a critical role in the homing of lymphocytes to secondary lymphoid tissue and of neutrophils to sites of inflammation (discussed above). In

this respect, L-selectin shedding may be a means of regulating the number of L-selectin molecules on the cell surface and thus cellular migration patterns. A recent paper by Tang et al

discussed the contribution of differing levels of L-selectin on the migration pathways of lymphocyte subsets using adoptively transferred lymphocytes from normal mice, hemizygous or homozygous L-selectin knockout mice (Tang et al., 1998). Here, a correspondence is demonstrated between higher levels of L-selectin on T cells and their preferential migration into peripheral lymphoid tissues. The reasons for such subset specific “intrinsic” differences in L- selectin levels are not known. However, whether L-selectin levels are transcriptionally regulated or not, L-selectin shedding is an obvious candidate mechanism for short term regulation.

Similar homing functions have been ascribed to the amount of L-selectin on neutrophils. Stimulation of systemic L-selectin shedding from neutrophils through a painful (electrical) stimulus was suggested to inhibit neutrophil - dependent plasma extravasation in induced knee joint inflammation (Strausbaugh et al., 1999). This study showed a correspondence between reduced levels of neutrophil L-selectin and inhibition of neutrophil accumulation at inflammatory sites. The importance of this work is in the demonstration of an association between in vivo L-selectin shedding and a physiological function. The level of L-selectin downregulation reported was not extensive, with a noxious stimulus accounting for a 38% reduction in “L-selectin positive” neutrophils. However, the authors suggest that this reduction results from complete L-selectin shedding from a sub population of cells, with L-selectin levels

on the rest being unaffected.

Proposed roles for L-selectin shedding in leukocyte homing can be divided firstly, into events leading to sequestration from flow and secondly, into events associated with transendothelial

migration. In terms of the multistep adhesion cascade paradigm, sequestration of leukocytes from blood flow involves initial rolling on and firm adhesion to endothelium.

1.4.4.3.1 Rolling velocity and firm adhesion

Intuitively, the rate at which a leukocyte rolls over endothelium should impact on the likelihood

that local chemokines bind cognate receptor, or the chance that sufficient integrindigand pairings can be made to arrest a leukocyte. In addition, L-selectin has the remarkable property of only mediating leukocyte rolling above a threshold hydrodynamic shear (Finger et al.,

1996). Finger et al demonstrated in vitro a requirement of wall shear stress of above 0.6 dyn cm'^ for both neutrophils and T lymphocytes to roll on PNAd. The extent to which L- selectin levels influence this parameter is not known.

With neutrophils, L-selectin shedding has been (indirectly) imphcated in determining rolling velocity both in vitro and in vivo (Hafezi-Moghadam and Ley, 1999; Walcheck et al., 1996). Both these studies made use of a biochemical shedding inhibitor known as KD-IX-73-4, one of a class of zinc chelating hydroxamic acid based metalloprotease inhibitors. Walcheck et al examined neutrophil rolling on purified MECA-79 antigen in in vitro hydrodynamic flow assays. With the application of the inhibitor, mean rolling velocity was reduced from 27.7|ims ’ with control cells, to 9.2jims '. Further, neutrophil attachment increased from control levels of

21.8 cells/mm^ to 94.0 cells/mm^. These results were contradicted in another in vitro study where they found no changes in velocity or accumulation (Allport et al., 1997). In this study, the authors again examined the role of shedding on rolling behaviour but extended the research to examine binding and endothelial transmigration.

There were several differences in the methods used by Allport et al. Firstly, a different shedding inhibitor was used, known as Ro31-9790. In addition, flow assays were conducted over (TNFa activated) cultured human umbilical vein endothelial cell (HUVEC) cultures. The

79 antigen), allowed transendothelial migration to be examined. This work concluded that L- selectin shedding not only had no regulatory effect on rolling and attachment, but further, it

played no role in transendothelial migration.

Hafezi-Moghadam and Ley recently addressed this conflicting data by applying shedding inhibitor KD-IX-73-4 in in vivo intravital microscopy studies. Injecting the inhibitor via a catheter local to the exteriorised microvasculature of the mouse cremaster muscle, the authors demonstrated reductions in leukocyte rolling velocity from 55|Lims‘' (controls) to 35pms \ In an

elegant use of knockout mouse resources, experiments were conducted on both L-selectin and E-selectin deficient mice. Experiments using L-selectin knockout mice confirmed an L-selectin specific role in the observations, with velocity of (L-selectin null leukocyte) rolling being unaffected by the inhibitor. Which leukocytes were rolling however, was not established in this study. A possible extension to this work therefore, would be to adoptively transfer fluorescently labelled homogenous cell populations to enable formal identification of the rolling cells. Interestingly, comparison of TNFa treated wildtype mice with TNFa treated E-selectin

knockouts seemed to explain the differences seen by Allport et al. The results indicated that the E-selectin mediated component of rolling (invoked by activation of endothelial cells by TN Fa),

dominated that of L-selectin, hence masking the effects of inhibiting L-selectin shedding.

The relevance of L-selectin shedding in leukocyte rolling however, is strongly disputed in a number of biophysical studies, notably from the Springer group. In a recent study, Alon et al immobilised L-selectin and examined the rolling behaviour of neutrophils (Alon et al., 1998). The authors make use of the fact that neutrophils (unlike lymphocytes) bear L-selectin ligands, to demonstrate that the kinetics of rolling and transient tethering on immobilised L-selectin remain remarkably consistent with previous studies where neutrophils roll on the immobihsed L-selectin ligand PNAd (Alon et al., 1997; Puri et al., 1998). They conclude from this that since L-selectin is clearly not being proteolytically shed from the flow chamber wall on which it is immobilised, that the “inherent properties of L-selectin tethers to glycoprotein ligands” are sufficient to explain rolling and tethering behaviour. This controversy remains unresolved.

1.4.4.3.2 Transendothelial migration

At sites of inflammation, infiltrating neutrophils have greatly reduced levels of cell surface L- selectin (Jutila et ah, 1989). This indicates that somewhere between initial capture from flow and final migration into tissue, L-selectin has been lost. In the above study, (mouse)

neutrophils were harvested 3 hours after an inflammatory stimulus (thioglycollate) was injected intraperitoneally, which does not preclude regulation of L-selectin levels at a transcriptional level. Nor, however, does it preclude a function for L-selectin shedding in the period between sequestration from flow and extra vascular localisation.

However, it is not known whether naive lymphocytes behave in this way. In vitro, extensive downregulation of L-selectin has been reported following binding of lymphocytes to primary cultures of high endothelial cells (HECs) (Ager and Wood, 1994). This observation is extremely important for two reasons. Firstly, it points to HECs as a source of the elusive physiological shedding stimulus. Secondly, it suggests that L-selectin shedding might be a prerequisite for lymphocyte migration across high endothelium ((Ager and Wood, 1994) and unpublished data). In contrast to the data from neutrophils above, HEC induced shedding was seen within 60 minutes.

Further, transmigration of lymphocytes across cultured monolayers of primary HECs, is reduced after shedding is blocked with the shedding inhibitor Ro31-9790, (Ager and Preece, unpublished data) pointing to a role in migration. Interestingly, this data could not be rephcated with neutrophils on TNFa activated HUVEC cultures (Allport et al., 1997). However, a recent

study showed that the Ro31-9790 delayed emigration of lymphocytes from lymph node HEVs

in vivo, again implicating shedding in transendothehal migration (Christelle Faveeuw, manuscript in preparation). The reasons for such disparities between lymphocyte and neutrophil data are not clear although as mentioned earlier, biochemical inhibitors such as Ro31-9790 are likely to have several undefined cellular effects.

The function of L-selectin shedding remains unresolved. Insofar that shedding results in total

loss of L-selectin, then it follows from L-selectin knockout studies, that it may act as an anti homing mechanism, both for lymphocytes and other leukocytes (Arbones et al., 1994; Tang et al., 1998; Tedder et al., 1995). However, this is not a satisfactory explanation for the high

levels of circulating L-selectin in the blood of normal healthy individuals. That L-selectin is crucial in determining rolling velocity is not resolved. In addition to biophysical arguments, hydroxamic acid based inhibitors are promiscuous and potent compounds and results should be

interpreted with caution. Evidence for a role in migration of lymphocytes across HECs, is sparse. But, the observations of Ager and Wood, if correct, are absolutely key - both in imphcating HECs as a source of the elusive physiological lymphocyte L-selectin shedding

stimulus, as well as pointing to a role for shedding in transendothelial migration (Ager and Wood, 1994).