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CORRESPONDENCE
Neutrophil Serine Proteases Are Most Probably Involved in the Release of CD43 (leukosialin, sialophorin)
From the Neutrophil Membrane During Cell Activation
To the Editor:
Inthe September 15, 1995 issue of Blood, Remold-O'Donnel et
al' report on the cleavage of neutrophil CD43 by neutrophil elastase.
This confirms previous works, including
showing that neutrophi1 CD43 is resistant to serine proteases such as trypsin or chymotrypsin but exquisitely sensitive to leukocyte elastase and, to a lesser
extent, to cathepsin G by a mechanism that does not require a previous desialylation of the CD43 molecule. When analyzing the similarities between the cleavage of CD43 by exogeneous elastase and
the proteolytic release of the molecule observed during neutrophil
activation, we observed the following points:
(1) The activation-induced release of CD43 shows different susceptibilities to elastase inhibitors according to the cell-activation
CORRESPONDENCE
1201
Unstimul.
Eglin
PMSF
Stimul.
DFP
C
B
-200
-
-116-
- 92- 66-
Elastase
FMLP
+
+
-
Fig l. (A) Neutrophils were activated for 30 minutes at 37°C with 10
ng/mL PMA or with 10 pg/mL cytochalasin B for 5 minutes at 37°Cthen
10.‘ mollL FMLP for 10 minutes,
with or without 1 mmol/L PMSF, 1
mmolll. DFP, or 200 pg/mL eglin C.
Cells were analyzed by flow cytometry using Leu 22 anti;CD43 MoAb;(B)
’251-labeledPMN were treated either
with 30 pg/mL elastase for 20 minutes at 37°C or with cytochalasin B
and FMLP. Cell supernatants were
immunoprecipitated with Leu 22
anti-CD43 MoAb and analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 7%
acrylamide gradient gel.
agonist. During PMN activation by FMLP in the presence of cytochalasin B, the cleavage of CD43 is blocked by all serine-protease
inhibitors tested, including DFP, phenylmethylsulfonyl fluoride
(PMSF). eglin C (Fig IA), pefabloc SC, or SBTI (data not shown).
This appears also to be the case of the release of CD43 triggered
by tumor necrosis factor-a (TNF-CY) andadherence’ or the “spontaneous” shedding of CD43 described by Remold-O’Donnell and Parent4 By contrast, confirming previous report^,'.^ we observed that
the release of CD43 induced by phorbol myristate acetate (PMA)
activation is not inhibited by DFP, SBTI. or eglin C, while efficiently
blocked by PMSF or pefabloc SC (Fig IA and data not shown).
(2) Similar 55,000 and47.000Mr CD43 soluble fragments. or
155,OOO and 95,000 Mr asialofragments, are immunoprecipitated
from supernatants of PMA- or FMLP and cytochalasin B-activated
cell and of elastase-treated cells (Fig IB). The 120.000 Mr fragment
released during cell activation’.’” is not observed with exogeneous
elastase. However, this does not exclude that this large fragment is
released by an endogeneous elastase-like enzyme, either membranebound or released at the membrane level, which, unlike the exogen-
eous enzyme, would have access to a CD43 proteolytic cleavage
site close to the membrane.
Remold-O‘Donnell and Parent proposed two different pathways
of CD43 cleavage“: a “spontaneous” cleavage mediated by an elastase-like enzyme resulting in the release of 55.000 and 47,000 fragments and another pathway involving an unknown non serine-protease “CD43’ase” activated by PMA.Althoughno
soluble CD43
fragment specifically released by PMA could be identified, this hypothesis was basedonthe
observation that PMA decreases cell
labeling by the T30Smonoclonal antibody (MoAb) which recognizes
the residual cell-associated CD43 fragment after cell treatment with
elastase. However, we would like to point out that thePMA-induced
CD43 cleavage was inhibited by PMSF and pefabloc SC, which are
serine protease inhibitors. Furthermore, we always immunoprecipitated similar proportions ofthe various CD43 fragments, whether
cells were activated by PMA or by cytochalasin B and FMLP. suggesting thatthe same elastase-like and/or putativeCD43’ase are
involved in all situations. In our hands, if cells are isolated by “nonactivating” cell isolation procedures and incubated at physiological
concentrations, no “spontaneous” release of CD43 is observed in
the absence of activators.’
Alternative hypotheses to explain the different susceptibilities of
CD43 release to protease inhibitors may be proposed: ( I ) PMA and
FMLP or TNFmay release and/or activate different serine-proteases,
more or less accessible to inhibitors such as DFP or the macromolecular eglin C. (2) PMA activation, butnot FMLP or TNF, would
modify CD43, possibly through an hyperphosphorylation of the molecule. and alter its T305 epitope as well as its susceptibility to
proteases. allowing it to be cleaved byvery low concentrations of
an elastase-like enzyme at the membrane level, even in the presence
o f some inhibitors.
Furthermore, with regard to possible functional relevance of CD43
shedding. we observed the following: ( I ) that elastase treatment does
in
notbyitselfinduce
homotypicneutrophilaggregation.measured
stirred conditions by flow cytometry. This suggests that elastase not
onlyremoves CD43 butpossibly also degradesadhesionmolecules
andor that homotypicadhesionrequirestheactivation
ofadhesion
receptors such as integrins as well as the shedding of repulsive molecules: (2) that elastase inhibitors PMSFand eglin C prevent TNFe or
PMA-induced cell adhesion to proteincoated plates, further suggesting
that the release of CD43 is required for cell adhesion.
L. Halbwachs-Mecarelli
G . Bessou
P. Lesavre
INSERM U 90
H6pital Necker
Pnris. France
P. Renesto
M. Chignard
IP/INSERM U285
Insriflct Pasfew
Paris. Fronce
REFERENCES
1. Remold-O’Donnell E, Parent D: Specific sensitivity of CD43
to neutrophil elastase. Blood 86:2395, 1995
2. Nathan C, Wie Q, Halbwachs-Mecarelli L,Jin W: Albumin
inhibits neutrophil spreading andhydrogen
peroxide release by
blocking the shedding of CD43 (sialophorin. leukosialin). J Cell Biol
122:243. 1993
3. Halbwachs-Mecarelli L,Bessou G . Renesto P, Chignard M,
Lesavre P Neutrophil elastase and cathepsin G release CD43 (sialophorin, leukosialin) fromtheneutrophil membrane: A possible
1202
mechanism for CD43 shedding during neutrophil activation. J Leukoc Biol 37, 1994 (abstr, suppl)
4. Remold-O’Donnell E, Parent D: Two proteolytic pathways for
down-regulation of the barrier molecule CD43 of humanneutrophils.
J Immunol 152:3595, 1994
5.Rieu P, Porteu F, Bessou G , Lesavre P, Halbwachs-Mecarelli
L: Human neutrophils release their major membrane
sialoprotein,leukosialin (CD43) during cell activation. Eur J Immunol 22:3021, 1992
CORRESPONDENCE
6. Kuijpers W ,Hoogenverf M, Kuijpers KC, Schwartz BR. Harlan JM: Cross-linking of sialophorin (CD43) induces neutrophil aggregation in a CDl8-dependent and CDI8-independent way. J I m munol 149:998, 1992
7. Bazil V, Strominger JL: CD43, the major sialoglycoprotein of
human leukocytes, is proteolytically cleaved from the surface of
stimulated lymphocytes and granulocytes. Proc Natl Acad Sci USA
90:3792, 1993