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From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
1991 77: 2553-2555
Lectin-like cell adhesion molecule 1 mediates leukocyte rolling in
mesenteric venules in vivo
K Ley, P Gaehtgens, C Fennie, MS Singer, LA Lasky and SD Rosen
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RAPID COMMUNICATION
Lectin-Like Cell Adhesion Molecule 1 Mediates Leukocyte Rolling in
Mesenteric Venules In Vivo
By Klaus Ley, Peter Gaehtgens, Christopher Fennie, Mark S. Singer, Laurence A. Lasky, and Steven D. Rosen
*
During the inflammatory response, granulocytes and other
leukocytes adhere to and emigrate from small venules.
Before firm attachment, leukocytes are observed rolling
slowly along the endothelium in venules of most tissues
accessible to intravital microscopy. The molecular mechanism underlying this early type of leukocyte-endothelial
interaction is unknown. Leukocyte rolling was investigated
in venules (diameter, 40 pm) of the,exposed rat mesentery.
Micro-infusion of a recombinant soluble chimera (LEC-lgG) of
the murine homing receptor lectin-like cell adhesion molecule 1 (LEC-CAM 1; gp90MEL)
into individual venules reduced
the number of rolling leukocytesby 89% f 2% (mean SEM,
n = 20 venules), while a similar CD4 chimera (CD4-lgG) had
no effect (inhibition 14% f 7%. n = 25). Rolling was also
greatly reduced by a polyclonal serum against LEC-CAM 1
(inhibition 84% f 3%, n = 35); preimmune serum was ineffective (11% -c 13% inhibition, n = 28). These findings indicate
that LEC-CAM 1 mediates the adhesive interaction underlying leukocyte rolling and thus may play an important role in
inflammation and in pathologic conditions involving leukocytes.
o 1991 by The American Society of Hematology.
R
with warmed (37°C) bicarbonate-buffered physiologic saline solution equilibrated with 5% CO, in N,. Glass micropipettes (tip
diameter 7 to 10 pm) were filled through 0.45-pm filters (Millipore
HV, Eschborn, Germany) and were introduced into side branches
(approximate diameter 20 pm) of the investigated venules using a
piezo'-driven micromanipulator (PM 10; Marzhauser, Wetzlar,
Germany).
The following reagents were miro-infused for 1-minute periods
by pressurizing an air-filled chamber connected with the micropipette (applied volume approximately 100 to 200 nL): (1) a
recombinant chimera consisting of the extracellular domain of
murine LEC-CAM 1linked to human IgG Fc regions (LEC-IgG)23
at 100 pgimL in phosphate-buffered saline (PBS); (2) a similar
CD4 chimera (CD4-IgG)24at 100 &mL in PBS; (3) a polyclonal
serum against murine LECCAM 1, which cross-reacts with rat
homing receptor (data not shown), diluted 1:lO in PBS; and (4) a
matching preimmune serum, also diluted 1:10. The polyclonal
serum was produced by injecting 10 pg of purified mouse LECCAM 1'' in Freund's complete adjuvant into multiple subcutaneous sites of a rabbit, and obtaining serum after 1 month.
Observations were made on a modified Letiz intravital microscope with transillumination, using a 25~10.60N.A. salt water
immersion objective, and recorded on video tape (final magnification on monitor approximately 700x). With transillumination
microscopy, rolling leukocytes are identified as difiactive, slowly
moving objects. The number of rolling leukocytes passing per time
(rdling leukocyte flux) was measured from the video recordirrgs for
each 2-second interval. Flow velocity was measured on line by
temporal cross-correlation (auto tracking correlator 102B; Instrumentation for Physiology and Medicine, San Diego, CA) of signals
ECRUITMENT OF granulocytes, monocytes, and
lymphocytes is essential for the inflammatory process.'.* Their emigration is preceded by firm attachment to
the venular endothelium, which is dependent on p2integrins (leukocyte adhesion molecules [LEUCAMS]).24Leukocyte rolling, on the other hand, is unaffected by-anti+,
antibodies that block firm adhesion? Rolling is induced
within minutes upon exteriorization of tissues for intravital
microscopy5" and persists for hours? Rolling cells are
predominantly granulocyte^,^‘^ but mononuclear cells also
participate.6 The low rdling velocity relative to blood flow
v e l o c i p is caused by an adhesive interaction between
leukocytes and the endothelium. Leukocyte rolling is effectively inhibited by dextran sulfate and sulfated glycosaminoglycan~'~'~
as well as by fucoidin, a sulfated fucose polymer
(own unpublished observation, 1990). Dextran sulfate also
reduces adhesion of isolated polymorphonuclear granulocytes (PMNs) to cultured human umbilical vein endothelial
cells in the presence of shear stress." Earlier in vitro
~ t u d i e s ' ~have
~ ' ~ shown that fucoidin and other sulfated
sugars also inhibit the lectin-like cell adhesion molecule 1
(LEC-CAM 1)-mediated adhesion of lymphocytes to high
endothelial venules.
The objective of the present study was to investigate
whether the calcium-dependent lectin-like leukocyte homing receptor LEC-CAM 1'4,'5may be involved in leukocyte
rolling. This molecule is known to bind f ~ c o i d i n . ' ~LEC,'~
CAM 1was originally shown to mediate lymphocyte homing
to high endothelial venules of lymph n ~ d e s , ' ~ ~
but
' ~ is
* 'also
~
expressed on PMNs, where it is rapidly downregulated
upon chemotactic ti mu la ti on.'^^^^ LEC-CAM 1 has previously been shown to be critical for recruitment of PMNs to
inflammatory sites~1*22
but it is unknown at what stage of
PMN-endothelial interaction LEC-CAM 1 is involved.
MATERIALS AND METHODS
Female Sprague Dawley rats (approximately 300 g body weight)
anesthetized with ketamine and pentobarbital were catheterized
(carotid artery and iugular vein) and surgically prepared for
microscopic observation of mesenteric microvessels. Blood pressure and heart rate remained constant, systemic leukocyte counts
increased slightly (from 7,000 to 8,200lpL) during the experimental
period of approximately 90 minutes. Animal core temperature was
thermostated to 37°C. The mesenteric preparation was superfused
Blood, Vol77, No 12 (June 15),1991:pp 2553-2555
From the Department of Physiology, Freie Universitat Berlin,
Germany; the Department of Immunobiology, Genentech, South San
Francisco, CA; and the Department of Anatomy and Program in
Immunology, University of Calijomia at San Francisco, San Francisco,,CA.
Submitted January 24,1991; accepted March 25,1991.
Supported by a grant from the German Research Council (DFG: Le
57313-1) to K L., and by grants from the National Institutes of Health
(GM23547and P6OAR20684) and from Genentech to S.D.R.
Address reprint requests to Klaus Ley, MD, Dept. of Physiology,
Freie UniversitatBerlin, Amimallee 22, 0-IO00 Berlin 33, Germany.
The publication costs of this article were defrayed in part by charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. section 1734 so1ely:toindicatethis fact.
0 I991 by The American Society of Hematology.
0006-4971/91/7712-0034$3.00l0
2553
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
2554
LEY ET AL
from a photo-transistor pair on which the microscope image was
projected.
160
c
E
x’
3
RESULTS
Leukocyte rolling was investigated in 108venules (diameter 40 f 1 km, mean t SEM) of the exposed rat mesentery, several junctions downstream from the site of microinfusion. Rolling leukocyte flux was reduced significantly
from 83 2 11 to 7 f 1 cells/min (mean 5 SEM, n = 20
venules) by micro-infusion of LEC-IgG (100 kg/mL), but
was virtually unaffected (74 t 10 during micro-infusion v
64 5 11 cells/min during control, n = 25) by CD4-IgG.
Micro-infusion of anti-LEC-CAM 1 antiserum (diluted
1 : l O in PBS) decreased leukocyte rolling from 90 f 9
to 13 f 3 cells/min (n = 35), while infusion of preimmune
serum had no effect (69 2 10 and 60 t 12 cells/min, respectively; n = 28) (Fig 1). Micro-infusion of neither of the
reagents had any effect on blood pressure, heart rate, or
systemic leukocyte counts of the animals.
Because the increase of blood flow velocity induced
locally by micro-infusion may affect rolling leukocyte
velocities were measured in each group and found to be
increased during micro-infusion to a similar extent: by
131% f 26% above control velocity in the LEC-IgG group,
by 200% f 24% in the CD4-IgG group, by 159% 2 19% in
the anti-LEC-CAM 1 group, and by 187% 2 30% in the
preimmune serum group. On termination of micro-infusion
of both LEC-IgG and anti-LEC-CAM 1, leukocyte rolling
reached its original value within about 15 seconds (Fig 2).
MICRO-INFUSION
120
LL
U
60
80
i:
W
(7
z
40
i!LL
0‘
0
_I
0
120
0
-
K
I
LEC-1gG
CD4-IgG
chimera
60
80
100
120
20
40
60
80
100
120
TIME. s
Anti-LEC-CAM 1 and LEC-IgG markedly reduced the
number of rolling leukocytes while the respective control
reagents did not, with similar hemodynamic conditions
prevailing in the investigated venules. LEC-IgG has recently been shown to inhibit PMN recruitment to an
inflammatory site.” However, it remained unclear which of
the different steps involved in the process (leukocyte
E
40
TIME. s
0
DISCUSSION
I
I
20
PolyMel
Preimmune
serum
Fig. 1. Flux of rolling leukocytes during micro-infusion. The percent of control flux, mean ? SEM, and number of applications are
indicated. LEC-IgG, recombinant chimera of murine LEC-CAM lZ3;
CD4-lgG, similar chimera of CD4U, both 100 pg/mL; PolyMel, rabbit
antimouse LEC-CAM 1 serum (1:lO); preimmune, matching preimmune serum.
Fig. 2. Flux of rolling leukocytes during and following microinfusion. Top, anti-LEC-CAM 1 antiserum (35 applications, solid line)
and preimmune serum (28 applications, broken line). Bottom, LEC-lgG
(average of 20 applications, solid line) and CD4-lgG (25 applications,
broken line). Micro-infusion was terminated at 60 seconds.
rolling, firm adhesion, emigration) was blocked by LECIgG. The present intravital microscopic data indicate that
LEC-IgG interferes with the earliest form of leukocyteendothelial interaction, ie, leukocyte rolling. A preliminary
report suggests that leukocyte rolling is a prerequisite for
firm adhesion and emigration.” Hence, it appears likely
that LEC-IgG precludes PMN recruitment” by blocking
their rolling.
On termination of micro-infusion of anti-LEC-CAM 1,
leukocyte rolling resumed within about 15 seconds, which is
in accordance with earlier observations with application of
sulfated glycosaminoglycan^.^ This behavior was to be
expected, because leukocytes exposed to micro-infused
anti-LEC-CAM 1 were swept away into the systemic
circulation and replaced by fresh leukocytes. The similar
lag-time seen after infusion of LEC-IgG was surprising,
because LEC-IgG interacts with the endothelial ligand of
LEC-CAM 1 and, hence, should remain attached to the
endothelium of the investigated venule. LEC-IgG has
previously been shown to specifically bind to lymph node
high endothelial venules and block lymphocyte attachmet~t.’~
The transitory effect of LEC-IgG infusion on
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2555
LEC-CAM 1 MEDIATES LEUKOCYTE ROLLING
leukocyte rolling indicates that LEC-IgG binding to and
release from its ligand on extra-lymphoid venular endothelium may have a short time constant of the order of several
seconds. This concept is consistent with the nature of the
bond established between the rolling leukocyte and the
endothelium. In the process of rolling, attachment and
detachment of individual bonds probably alternate very
frequently.
The conclusion that LEC-CAM 1 plays a major role in
mediating leukocyte rolling in vivo is supported by several
other observations. Both leukocyte rolling’.’’ and LECCAM 1-mediated lymphocyte adhesion to high endothelial
venules of lymph n ~ d e ” , ’are
~ , inhibited
~~
by sulfated polysaccharides, among them the sulfated fucose polymer fucoidin.
LEC-CAM 1 has been shown to be downregulated upon
chemotactic stimulation of PMNs,”,~’which correlates with
the inability of stimulated PMNs to “home” to an inflammatory site.” Likewise, leukocyte rolling in venules of the
hamster cheek pouch has been reported to be reduced
upon chemotactic stimulation.26
It is concluded that the inhibitory effects of LEC-IgG and
anti-LEC-CAM 1 antiserum on leukocyte rolling seen in
the present study are caused by interference with the
interaction between LEC-CAM 1 and its endothelial ligand.
The rapid onset (1 to 3 minutes) of leukocyte rolling upon
surgical trauma6 suggests that a pre-existing endothelial
ligand is exposed at the luminal surface of venules, or
modified to allow leukocyte adhesion via LEC-CAM 1.
ACKNOWLEDGMENTS
We thank Dr T.A. Yednock, Department of Anatomy, University of California at San Francisco, and Dr L.M. Stoolman, Department of Pathology, University of Michigan at Ann Arbor, for help
in producing and characterizing the antiserum, Dr A.R. Pries,
Department of Physiology, Freie Universitat Berlin, Germany for
revising the manuscript, and M. Ehrlich for technical assistance.
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