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EL4 studies were performed in the first instance, with the stable expression of human wildtype and (non-shedding) AM-N L-selectin molecules. The AM-N mutation in human L-selectin has

already been shown to abolish phorbol ester (PMA) stimulated shedding, when expressed in the mouse pre-B cell line 300.19 and in COS cells (Chen et al., 1995). The abolition of PMA induced shedding has now been demonstrated for the first time in a T cell line, suggesting a common mechanism of shedding is operative in both B and T cells.

Before transfectants could be tested in binding and migration assays, two criteria had to be satisfied. Firstly, human L-selectin expressed by the transfectants was shown to sustain binding to L-selectin ligand(s) purified from rat HECs Sgpl50/>200 (Derry et al., 1999). Secondly, attempts were made to reproduce the phenomenon of HEC induced shedding already reported with primary lymphocytes. Shedding of (wildtype) human L-selectin from transfectants was unimpressive, which was a matter of concern. The biggest concern was that

the rat L-selectin expressing EL4 transfectant known as RMl (the positive control) also showed very poor levels of L-selectin shedding, which in itself pointed to a peculiarity in the behaviour of the EL4 line.

HEC-induced shedding seen with primary cells, had originally been put forward as a potential indication of the role of shedding in vivo, namely as a prerequisite to transendothelial migration (Ager and Wood, 1994). Therefore it was important to scrutinise the undramatic levels of HEC

induced shedding seen with EL4 transfectants, since divergence from the behaviour of normal lymphocytes in this key regard undermined the value of the EL4 transfection system. Low levels of L-selectin shedding (as measured by antibody staining) were investigated further using time course experiments along with ELISA analysis, to measure shed L-selectin levels.

These experiments refuted the hypothesis that HECs induced substantial shedding of (wildtype human) L-selectin which had been masked by reexpression within 60 minutes (the period of initial experiments). Instead it appeared that shed L-selectin was rapidly replenished at the cell surface and that interaction with HEC shifted a dynamic balance between shedding and reexpression rates towards shedding. Nonetheless, it appears that wildtype human L-selectin is indeed shed by the EL4 line, in response to HEC stimulation. In addition, a clear abrogation of shedding is demonstrated with the AM-N mutation with clone D2 with measurable quantities of wildtype but not AM-N L-selectin being shed into the supernatant. Taken together, these data

lend to the tentative conclusion that HEC and PMA induced shedding are not distinct shedding events.

There are several other factors which may contribute to the paucity of L-selectin shedding in EL4 cells - other than merely the metabolic idiosyncrasies of this particular cell line. EL4 cells

are substantially larger than primary lymphocytes (3-4 times in diameter) and hence a much smaller proportion of a cell’s surface area is likely to contact the HEC surface. Hence if some contact mediated signal is required, a much greater proportionate signal can be delivered to the smaller primary cells. An alternative explanation for low shedding is that the expression of

human or indeed rat (in the case of RM l) L-selectin in the murine EL4 line, may have revealed some species effects. This was addressed subsequently with mouse L-selectin transfectants.

An unexpected observation was the downregulation seen with high expressing AM-N clone

2D36, after incubation with HECs. Concerns that this mutation did not, after all, abolish shedding, were mitigated in part by the demonstration that there was at least substantial inhibition of shedding with AM-N clone D2 as compared with (expression matched) wildtype

transfectant 2L10. That such deletions may not necessarily abolish shedding was subsequently suggested by Stoddart et al where antibody crosslinking (under static conditions)

downregulated L-selectin on a similar deletion mutant form of L-selectin known as 321del.9 (Stoddart et al., 1996).

Binding and migration behaviour of human L-selectin transfectants was investigated in vitro

using cultured HEC monolayers. Firm adhesion and migration is principally integrin mediated, so these assays were essentially a readout of the effects of L-selectin expression on integrin binding. Early results indicated that high expression of AM-N L-selectin (clone 2D36) affected

binding but not migration - suggesting that these two events are differentially regulated and that L-selectin shedding may be involved in the former. Poor binding might be explained in terms of steric hindrance of integrins by L-selectin. Alternatively, the high expression of (non­ shedding) L-selectin may have perturbed any L-selectin mediated integrin activation signals.

Adhesion of transfectants to HECs was inhibited by ROD peptides, confirming integrin mediated binding. A subsequent paper published recently implicated such ROD peptides in the induction of apoptosis (Buckley et al., 1999). The relevance of this unexpected finding is that

apoptotic cells, in addition to displaying well established patterns associated with apoptosis, for example, DNA fragmentation or membrane blebbing, also tend to detach from any substrate. The role of apoptosis was not investigated in this experiment, although an element of binding

and de-adhesion was recorded in the experimental observations. Therefore, the experiment does not exclude apoptotic mechanisms for reduced binding.

Nonetheless, antibody characterisation of EL4 integrins heavily implicated LFA-1 as the

operative (leukocyte) integrin involved in adhesion to HECs. This is similar to naive lymphocyte:HEC interactions in vivo (with the exception of Peyer’s patches, where this is dependent) and in this respect further qualifies EL4 transfectants for modelling these interactions. The anomalies in binding seen with the 2D36 clone remain unresolved. In characterising the integrins mediating firm adhesion, the low binding phenotype seen with

2D36 was no longer reproducible. Hence a preliminary conclusion at this point, was that high expression of L-selectin may be an obstacle to integrin mediated binding, but no involvement of either shedding or indeed L-selectin expression per se is seen to affect transmigration across HECs.