Antibody blocking studies in the mouse suggest that the
MEL-14 antigen is involved in neutrophil-endothelial cell interactions and may be important in neutrophil extravasation to sites of
inflammation in vivo. We recently showed that
chemotactic factor activation causes a rapid (within minutes) shedding of a large fragment of the
MEL-14 antigen from the surface of neutrophils. We report here that
chymotrypsin, at low doses (0.1 units/1 x 10(6) cells), but not
trypsin,
elastase, or
collagenase, causes an activation-independent rapid loss (greater than 90%) of the
MEL-14 antigen from the surface of murine neutrophils. Under the same treatment conditions
chymotrypsin has no effect on the expression of four other neutrophil
surface antigens, including the Mac-1 adhesion
protein.
Chymotrypsin treatment has no effect on neutrophil adhesion to
plastic, migration to C5a, regulation of the
Mac-1 antigen, but causes a greater than 95% reduction in neutrophil binding to high endothelial venules (HEV) in peripheral lymph nodes measured in the ex vivo frozen section HEV binding assay. The level of inhibition of neutrophil adhesion to HEV was comparable to that seen with the MEL-14 antibody. This experimental system allows us for the first time to specifically examine the consequences of removing the
MEL-14 antigen from the surface of neutrophils on function in vivo. We show that treatment with
chymotrypsin blocks greater than 85% of the ability of neutrophils injected back into the animal to home to the inflamed peritoneum. In similar in vivo experiments the MEL-14 antibody blocks neutrophil homing by 60-70%. These results further support the importance of the
MEL-14 antigen in neutrophil extravasation in vivo and indicate that
chymotrypsin could be useful in examining the molecular mechanisms involved in extravasation of leukocytes into a variety of diverse tissue sites of
inflammation.