Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression levels of adhesion molecules on neutrophils are affected
under various conditions, including ischemia, possibly because
of associated increases in cell volume. We examined the effects of cell
swelling in hypotonic media on the level of L-selectin (CD62L) and
2-integrin (CD18) on human neutrophils. In hypotonic media, neutrophils shed L-selectin. The shedding was greatly reduced by
30 µM RO31-9790, the metalloprotease (sheddase) inhibitor. Hypotonicity-induced L-selectin shedding was also time and tonicity dependent. Decreasing tonicity caused increased shedding. In 0.6× medium (0.6× the normal tonicity of 300 mosmol/kgH2O),
shedding increased over a 2-h period, after which >70% of the
neutrophils had lost L-selectin. In contrast to L-selectin, the level
of
2-integrin on the neutrophil surface was not
significantly affected. Thus L-selectin shedding, which occurs on
neutrophil activation and is usually accompanied by
2-integrin upregulation, was selectively induced by
hypotonicity without a corresponding effect on
2-integrin.
hypertonic; tumor necrosis factor-; CD62l; metalloprotease; cell
swelling
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE SELECTIN AND
INTEGRIN classes of adhesion molecules play essential roles,
respectively, in the rolling of neutrophils and then firm adhesion to
the endothelium (2, 25). Modulation of surface adhesion
molecules, such as L-selectin and 2-integrin, could
alter the host defense response and the degree of inflammation, as well
as the degree of injury caused by reperfusion after ischemia (9, 23). Neutrophils rapidly shed their L-selectin on
stimulation with chemotactic agents (1, 25) and upregulate
their
2-integrin level (4). L-selectin
shedding is mediated by proteolytic cleavage by a membrane-bound
metalloprotease, "sheddase" (14, 15, 25). Rizoli et
al. (15) have shown that cell shrinkage in hypertonic media causes L-selectin shedding in neutrophils by a mechanism that
involves the sheddase as well as the p38 mitogen-activated protein
kinase (MAPK). In contrast, very little is known about swelling-induced
L-selectin shedding.
The role played by neutrophil swelling in ischemia-reperfusion is also not well understood. During ischemia, the fall in intracellular pH would be expected to activate Na+/H+ exchange (13), causing a net uptake of NaCl and water and thereby causing cell swelling. Chemotactic factors such as N-formyl-methionyl-leucyl-phenylalanine also stimulate the Na+/H+ exchanger in neutrophils (16, 21), leading to cell swelling (16). Swelling has been shown to occur in endothelial cells during hemorrhagic shock (12), and this swelling is inhibited by the Na+/H+ exchange inhibitor amiloride (13). Activation of Na+/H+ exchange during ischemia-reperfusion is also likely to take place in neutrophils, and should lead to swelling.
When the intracellular pH of neutrophils was lowered in vitro by
exposing cells to media that were acidified by addition of HCl
(24) or by increasing CO2 (20),
there was an increase in surface 2-integrin (CD18),
indicating neutrophil activation. If a similar increase occurs during
ischemia-reperfusion, this could enhance neutrophil binding to
endothelia and increase the inflammatory response. The same experiments
showed an increase in forward light scatter as measured by flow
cytometry, which was prevented by amiloride (24),
indicating that activation of Na+/H+ exchange
causes an increase in cell volume.
Because neutrophils play a significant role in
ischemia-reperfusion injury (3, 7, 10), and
because neutrophil swelling is likely to occur under these conditions,
the aim of this study was to determine what effects, if any, cell
swelling by itself, without the accompanying complications introduced
by substantial changes in cell pH, has on the levels of L-selectin and
2-integrin and how this might affect neutrophil behavior
in the microcirculation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
Neutrophil isolation medium (NIM) was obtained from Cardinal Associates
(Santa Fe, NM). FITC-conjugated monoclonal anti-L-selectin (CD62L),
anti-2-integrin (CD18), and isotype (mouse IgG1)
antibodies were purchased from Beckman/Coulter (Miami, FL). RO31-9790
was a kind gift from Roche Products, Hertfordshire, UK. All other chemicals, including salts, were obtained from Sigma (St. Louis, MO)
and were of the highest purity available.
Neutrophil isolation.
Blood samples were obtained, with informed consent, from healthy donors
by venipuncture with heparin as anticoagulant. Whole blood and NIM were
brought to room temperature before use. Three milliliters of NIM
reagent were placed in an 8-ml Falcon polystyrene round-bottom tube
(Becton Dickinson, Franklin Lakes, NJ), and 3.5 ml of whole blood were
carefully layered on them with a serological pipette. Tubes containing
NIM and blood were centrifuged at room temperature for 40-50 min
at 1,500 rpm (470 g) in a Sorvall RC5C Plus centrifuge
(Kendro Laboratory Products, Newtown, CT). After centrifugation, there
were two visible leukocyte bands in the NIM, the upper one being
mononuclear cells and the lower one neutrophils. The upper NIM fraction
and the mononuclear cells were aspirated and discarded. The neutrophil
band was collected with a Pasteur pipette and suspended in a total
volume of 5.0 ml in a calcium-free neutrophil isolation buffer (NIB; in
mM: 135 NaCl, 4.2 KCl, 5.0 glucose, and 10.0 HEPES) containing 0.1%
BSA (fraction V, -globulin free and low endotoxin). Osmolality of
the buffer was adjusted to 290-310 mosmol/kgH2O
(measured with a Wescor vapor pressure osmometer) by using NaCl, and
the pH was adjusted to 7.4 with NaOH. Neutrophils were washed three
times in 5.0 ml NIB by centrifuging at room temperature for 10 min at
1,000 rpm (210 g). Before the last wash, 0.5 ml of the
neutrophil suspension was removed and put in 4.5 ml of a 1:6 dilution
of PBS with distilled water to hypotonically lyse erythrocytes. After
30 s, 1.5 ml of 4× PBS were added to the lysing suspension to
restore isotonicity, and neutrophils were then counted with a
hemocytometer. The rest of the neutrophil suspension, which was used
for the experiments, did not undergo hypotonic lysis to remove
erythrocytes, since they can be clearly differentiated with the flow cytometer.
L-selectin (CD62L) and 2-integrin (CD18)
measurement.
Surface L-selectin was determined by flow cytometer. Cells (5 × 105) were incubated in calcium (1.1 mM CaCl2)
and magnesium (0.9 mM MgCl2) containing NIB with decreasing
tonicity (1.0×, 0.9×, 0.7×, 0.6×, 0.5×, 0.3×) for 2 h or in
0.6× medium for various time intervals. Control cells were incubated
in 1.0× buffer for the same time intervals. Standard isotonic medium
(1.0×) had an osmolality of 300 mosmol/kgH2O. The
hypotonic buffers were made by diluting the isotonic (1.0×) buffer
with distilled, deionized and autoclaved water. The RO31-9790 cells
were preincubated at 37°C for 30 min with 30 µM inhibitor (made
from a 30 mM stock solution in DMSO) before being subjected to the
0.6× solution containing 30 µM RO31-9790 for 2 h. Tumor
necrosis factor-
(TNF-
; 3 ng/ml)-treated cells were incubated at
37°C for 2 h. All samples were placed on ice after the
incubation. For surface
2-integrin measurements,
neutrophils were incubated in 0.6× buffer or hypertonic (500 mosmol/kgH2O) buffer (by adding 100 mM NaCl to the isotonic buffer) at 37°C for 2 h.
Statistical analysis. Data are presented as means ± SE for n experiments as indicated. Significance was assessed using Student's t-test. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of hypotonicity on L-selectin shedding.
With increasing incubation time in 1.0× (isotonic) medium at 37°C,
the percentage of neutrophils positive for L-selectin
decreased slightly (Fig. 1),
falling from 95 ± 1% to 83 ± 3% after 2 h (Fig. 1),
probably because of nonspecific activation due to the artificial incubation conditions. In 0.6× medium (Fig. 1), the number of cells
positive for L-selectin dropped from 90 ± 1% to 19 ± 9% within 2 h. The shedding was clearly detectable in 10-30 min
and statistically significant after the first hour, during which over half of the neutrophils lost their L-selectin (Fig. 1).
|
|
|
Effect of hypotonicity and hypertonicity on
2-integrin (CD18) level in neutrophils.
Incubation of neutrophils for 2 h in 0.6× medium or in a
hypertonic (by adding 100 mM NaCl to the isotonic solution) medium had
no significant effect on the
2-integrin level, but 3 ng/ml of TNF-
showed a significant increase in
2-integrin (Fig. 4).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypotonicity, which swells neutrophils (22), caused
L-selectin shedding in a time- and osmolarity-dependent manner. The inhibition of the shedding by the metalloprotease (sheddase)
inhibitor RO31-9790 shows that the sheddase is involved in the process. In addition, the fact that supernatant from hypotonically treated cells
failed to shed L-selectin indicates that the effect does not
involve any factors secreted from the cells, and is consistent with
activation of the specific membrane-bound enzyme that mediates activator-induced shedding. Although most chemotactic agents
downregulate L-selectin levels (25) and concomitantly
upregulate 2-integrin levels (4) on
neutrophil activation, hypotonicity (Fig. 3), like hypertonicity
(15), had a selective effect on L-selectin, and did not
influence the
2-integrin level (Fig. 4). This indicates that L-selectin shedding and
2-integrin upregulation are
separately controlled and that volume changes activate some but not all
of the pathways stimulated by cytokines. Considered in light of
evidence that low pH increases the
2-integrin level
(20, 24), the lack of an effect of cell swelling on
2-integrin suggests that this
2-integrin
upregulation is primarily caused by the decrease in pH, with little or
no contribution from the resulting increase in cell volume.
As previously shown by Rizoli et al. (15), cell shrinkage decreases L-selectin in neutrophils by activating the sheddase via a pathway involving the p38 MAPK. In some cells, cell shrinkage also activates protein kinase C (8) and triggers several proteins involved in the MAPK cascade that leads to Jun NH2-terminal kinase (JNK) activation (11). Cell swelling has been shown to stimulate myeloperoxidase exocytosis in neutrophils (18) and, in other cells, to activate protein kinase C, encourage tyrosine phosphorylation, and stimulate the MAPK cascade as well as activate JNK (8, 17). Considering the common pathways involved in both swelling and shrinkage, it is conceivable that the shedding observed with hypertonicity (15) and hypotonicity could involve a common downstream cascade, which then leads to the activation of the sheddase. The possible involvement of tyrosine phosphorylation in this process requires further investigation.
Ischemia, which is a consequence of hypoperfusion caused either by hemorrhagic shock or interruption of blood flow, leads to intracellular acidification that causes Na+ influx through the Na+/H+ exchanger (12, 13). This event leads to endothelial cell swelling (12, 13), a process that is exacerbated during reperfusion (5). Under these conditions, a similar process could also cause neutrophil swelling and therefore a decrease in the L-selectin level. This would be expected to impair the ability of neutrophils to roll on venular endothelium, which should make it more difficult for activated neutrophils to adhere firmly to vessel walls under conditions of flow. The slow time course of the shedding induced by cell swelling alone (Fig. 1), however, indicates that, even for neutrophils within the ischemic region, the loss of L-selectin by this mechanism would require 1-2 h of ischemia. Perhaps more significantly, the swelling-induced shedding, because of its slow time course, could not decrease L-selectin sufficiently to have much effect on rolling of neutrophils entering a local ischemic region on reperfusion. Other events that take place simultaneously during ischemia and reperfusion, such as changes in pH and intracellular calcium, however, may speed up the process.
Our results demonstrate that L-selectin levels can be regulated
separately from 2-integrin expression, in agreement with studies of the effects of hypertonicity (15), metabolic
inhibitors (6), and 5-nitro-2-(3-phenylpropylamino)benzoic
acid (19). These findings provide an experimental means
for selectively altering the surface density of L-selectin to test
predictions about the effects this may have on the rolling behavior of
neutrophils, without any additional complications caused by changing
the level of
2-integrins.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Vivian Leung and Yvette Law for technical assistance, as well as Dr. Peter Keng and Tara Calcagni of the flow cytometry facility for their assistance.
![]() |
FOOTNOTES |
---|
This research was supported by National Heart, Lung, and Blood Institute Grant P01 HL-18208.
Address for reprint requests and other correspondence: P. A. Knauf, Dept. of Biochemistry and Biophysics, Box 712, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: Philip_Knauf{at}urmc.rochester.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 January 2001; accepted in final form 19 June 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahmed, NA,
Yee J,
Giannias B,
Kapadia B,
and
Christou NV.
Expression of human neutrophil L-selectin during the systemic inflammatory response syndrome is partly mediated by tumor necrosis factor .
Arch Surg
131:
31-36,
1996[Abstract].
2.
Aplin, AE,
Howe SK,
Alahari K,
and
Juliano RL.
Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins.
Pharmacol Rev
50:
197-263,
1998
3.
Engler, FL,
Dahlgren MD,
Morris D,
Peterson MA,
and
Schmid-Schoenbein G.
Role of leukocytes in the response to acute myocardial ischemia and reflow in dogs.
Am J Physiol Heart Circ Physiol
251:
H314-H323,
1986
4.
Fernandez-Segura, E,
Garcia JM,
and
Campos A.
Topographic distribution of CD18 integrin on human neutrophils as related to shape changes and movement induced by chemotactic peptide and phorbol esters.
Cell Immunol
171:
120-125,
1996[ISI][Medline].
5.
Gidlöf, A,
Lewis DH,
and
Hammersen F.
The effect of prolonged total ischemia on the ultrastructure of human skeletal muscle capillaries. A morphometric analysis.
Int J Microcirc Clin Exp
7:
67-86,
1987[ISI].
6.
Gómez-Gaviro, MV,
Dominguez-Jiménez C,
Carretero JM,
Sabando P,
González-Alvaro I,
Sánchez-Madrid F,
and
Díaz-González F.
Down-regulation of L-selectin expression in neutrophils by nonsteroidal anti-inflammatory drugs: role of intracellular ATP concentration.
Blood
96:
3592-3600,
2000
7.
Ito, Y,
Imai S,
Ui G,
Nakano M,
Imai K,
Kamiyama H,
Naganuma F,
Matsui K,
Ohashi N,
and
Nagai R.
A Na+-H+ exchange inhibitor (SM-20550) protects from microvascular deterioration and myocardial injury after reperfusion.
Eur J Pharmacol
374:
355-366,
1999[ISI][Medline].
8.
Larsen, AK,
Jensen BS,
and
Hoffmann EK.
Activation of protein kinase C during cell volume regulation in Ehrlich mouse ascites tumor cells.
Biochim Biophys Acta
1222:
477-482,
1994[ISI][Medline].
9.
Lozano, DD,
Kahl EA,
Wong HP,
Stephenson LL,
and
Zamboni WA.
L-selectin and leukocyte function in skeletal muscle reperfusion injury.
Arch Surg
134:
1079-1081,
1999
10.
Lucchesi, BR,
Werns SW,
and
Fantone JC.
The role of the neutrophil and free radicals in ischemic myocardial injury.
J Mol Cell Cardiol
21:
1241-1251,
1989[ISI][Medline].
11.
Matsuda, SH,
Kawasaki H,
Moriguchi T,
Gotoh Y,
and
Nishida E.
Activation of protein kinase cascades by osmotic shock.
J Biol Chem
270:
12781-12786,
1995
12.
Mazzoni, MC,
Borgström P,
Intaglietta M,
and
Arfors K-E.
Lumenal narrowing and endothelial cell swelling in skeletal muscle capillaries during hemorrhagic shock.
Circ Shock
29:
27-39,
1989[ISI][Medline].
13.
Mazzoni, MC,
Intaglietta M,
Cragoe EJ, Jr,
and
Arfors K-E.
Amiloride-sensitive Na+ pathways in capillary endothelial cell swelling during hemorrhagic shock.
J Appl Physiol
73:
1467-1473,
1992
14.
Preece, G,
Murphy G,
and
Ager A.
Metalloproteinase-mediated regulation of L-selectin levels on leucocytes.
J Biol Chem
271:
11634-11640,
1996
15.
Rizoli, SB,
Rotstein OD,
and
Kapus A.
Cell volume-dependent regulation of L-selectin shedding in neutrophils: a role for p38 mitogen-activated protein kinase.
J Biol Chem
274:
22072-22080,
1999
16.
Rosengren, S,
Henson PM,
and
Worthen GS.
Migration-associated volume changes in neutrophils facilitate the migratory process in vitro.
Am J Physiol Cell Physiol
267:
C1623-C1632,
1994
17.
Sadoshima, J,
Qui ZH,
Morgan JP,
and
Izumo S.
Tyrosine kinase activation is an immediate and essential step in hypotonic cell swelling induced ERK activation and c-Fos gene expression in cardiac myocytes.
EMBO J
15:
5535-5546,
1996[Abstract].
18.
Sato, N,
Wang X,
and
Greer MA.
Hyposmolarity stimulates myeloperoxidase exocytosis from human polymorphonuclear leukocytes.
Am J Med Sci
299:
309-312,
1990[ISI][Medline].
19.
Schultz JB and Knauf PA. Short exposure to NPPB causes human
neutrophils to shed L-selectin but has little effect on surface
expression of 2-integrins (Abstract). Proc Int
Congr Physiol Sci 34th Christchurch New Zealand 2001. In
press.
20.
Serrano, CV, Jr,
Fraticelli A,
Paniccia R,
Teti A,
Noble B,
Corda S,
Faraggiana T,
Ziegelstein RC,
Zweier JL,
and
Capogrossi MC.
pH dependence of neutrophil-endothelial cell adhesion and adhesion molecule expression.
Am J Physiol Cell Physiol
271:
C962-C970,
1996
21.
Simchowitz, L.
Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils.
J Biol Chem
260:
13237-13247,
1985
22.
Simchowitz, L,
Textor JA,
and
Cragoe EJ, Jr.
Cell volume regulation in human neutrophils: 2-(aminomethyl)phenols as Cl channel inhibitors.
Am J Physiol Cell Physiol
265:
C143-C155,
1993
23.
Strausbaugh, HJ,
Green PG,
Lo E,
Tangemann K,
Reichling DB,
Rosen SD,
and
Levine JD.
Painful stimulation suppresses joint inflammation by inducing shedding of L-selectin from neutrophils.
Nat Med
5:
1057-1061,
1999[ISI][Medline].
24.
Trevani, AS,
Andonegui G,
Giordano M,
López DH,
Gamberale R,
Minucci F,
and
Geffner GR.
Extracellular acidification induces human neutrophil activation.
J Immunol
162:
4849-4857,
1999
25.
Walcheck, B,
Kahn J,
Fischer JM,
Wang BB,
Fisk RS,
Payan DG,
Feehan C,
Betageri R,
Darlak K,
Spatola AF,
and
Kishimoto TK.
Neutrophil rolling altered by inhibition of L-selectin shedding in vitro.
Nature
380:
720-723,
1996[ISI][Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |