Effect of divalent cations on adhesion of polymorphonuclear leukocytes to matrix molecules in vitro Evy Lundgren-Akerlund, La Jolla
Institute
for
Experimental
Elaine Medicine,
Berger,
and
La Jolla,
Abstract: Adhesion of N-formyl-methionyl-leucylphenylalanine-stimulated human polymorphonuclear leukocytes (PMNs) to dishes coated with laminin, fibronectin, or collagen types I and IV was dependent on the presence of magnesium (Mg) but not calcium (Ca2). Addition of manganese (Mn ) in combination with Ca2 and Mg further increased the number of PMNs adhering to the matrix proteins. Monoclonal antibody 60.3 (mAb 60.3) was equally effective at inhibiting adhesion of PMNs to all the matrix proteins. The presence of Mn2 (50 tM), in addition to 1 mM Ca2 and Mg required higher concentrations of mAb 60.3 to inhibit adhesion of PMNs to collagens type I or IV, suggesting increased affinity of PMNs for these substrates. These findings suggest that the PMNs may regulate the affinity of CD11/CD18 lor multiple ligands by binding different divalent cations to the receptor. J. Leukoc. Biol. 51:
,
603-608;
Key 60. 3
1992.
Words: .
Mn2”
neutrophil
#{149} CDJJ/CDJ8
#{149} monoclonal
antibody
collagen
Karl-E
that is common for all the different ligands or if other mechanisms are involved. The present investigation was undertaken to increase the understanding of the molecular mechanisms involved in PMN adhesion in vitro. Adhesion of PMNs to various matrix proteins was studied, and requirements for different divalent cations were investigated.
MATERIALS
During the acute inflammatory response polymorphonuclean leukocytes (PMNs) adhere to the endothelial cells ofthe postcapillary venules and migrate through the extnacellular matrix toward the focus ofinflammation [14]. The molecular mechanisms supporting adhesion and migration are not yet elucidated, although several proteins that participate in these interactions have been identified [23, 26, 28]. In vitro adhesion of stimulated PMNs has been studied on cultured endothelial cells [19, 20, 27, 32, 35, 38] and protein-coated plastic dishes [4, 19, 21, 37]. This adhesion is mediated primarily by the heterodimeric leukocyte adhesion receptors CD11/CD18 (LFA-1, Mac-i, gpiSO/95) [1, 22, 24, 27, 35, 38]. These neceptons are members of the integnin superfamily of matrix receptors [16-18, 25] and have the same 13 subunit (CD18) but different a subunits (CDiia, CD11b, CDlic). Binding of integnins to their ligands has been shown to require the presence ofdivalent cations such as Ca2 and Mg2 [26]. Furthermore, the presence of Mn2 in addition to Ca2 and Mg2 has been shown to increase the affinity of integnins for their ligands [9]. Leukocyte adhesion also appears to depend on the presence ofdivalent cations [4, 11, 16, 33], and the amino acid sequence of CD11b from cDNA cloning predicts three divalent cation binding sites on this molecule [2, 6]. Monoclonal antibodies such as mAb 60.3 [3, 35] and IB4 [36], recognizing epitopes on the /3 chain (CD18), effectively inhibit the adhesion of activated PMNs to cultured endothelial matrix if these
cells [19, proteins antibodies
20] and to immobilized [4, 21] and albumin [11, block a specific binding
proteins including 19]. It is not known site on CD11/CD18
AND METHODS
Materials Fibronectin (FN), collagen type I (CI), bovine serum albu(BSA), N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), and phorbol myristate acetate (PMA) were obtamed from Sigma Chemical Co., St. Louis, MO; laminin (LM) from Bethesda Research Laboratories, Bethesda, MD; and collagen type IV (CIV) from Collaborative Research, Bedford, MA. Monoclonal antibody 60.3 was prepared as described earlier [3].
mm
PMN
INTRODUCTION
Arfors
Calfomia
Isolation
Human PMNs were isolated from freshly drawn hepaninized (10 U/mi) venous blood collected from healthy adult donors. Erythnocyte sedimentation was accelerated using Macrodex (Pharmacia, Piscataway, NJ) followed by Percoll (Pharmacia) gradient separation of leukocyte-nich plasma as descnibed [15]. The PMN suspension was washed twice in isotonic saline and brought to a final concentration of 5 x 106/ml by adding adhesion buffer [Dulbecco’s phosphate-buffered saline (PBS) with 0.1% BSA].
Endothelial
Cell Culture
Endothelial cells were harvested and cultured from human umbilical veins as described previously [31, 34]. Briefly, endothelial cells were isolated by collagenase digestion and suspended in culture medium 199 with Earle’s salt (MA. Bioproducts, Walkersville, MD) supplemented with HEPES buffer (25 mM; Gibco, Grand Island, NY), fetal bovine serum (17%; M.A. Bioproducts), endothelial growth factor (25 jg/ml; Sigma), heparmn (20 U/mI), and antibiotics. The
Abbreviations:
I and
IV;
BSA,
FACS,
bovine
serum
fluorescence-activated
albumin;
CI
cell
and
sorter;
CIV,
collagen
Flit,
types
fluorescein
isothiocyanate; IMLP, N-formyl-L-methionyl-L-leucyl-L-phenylalanine; FN, fibronectin; HUVEC, human umbilical vein endothelial cell; LM, laminin; PBS, phosphate-buffered saline; PMA, phorbol myristate acetate; PMN, polyrnorphonuclear leukocyte. Reprint requests: Dr. Evy LundgrenAkerlund, Department of Medical and Physiological Chemistry, Lund University, P.O. Box 94, 5-221 00 Lund, Sweden. Received June 18, 1991; accepted January 2, 1992.
Journal
of Leukocyte
Biology
Volume
51, June
1992
603
harvested cells were seeded onto 75-mm2 culture flasks (Corning, Corning, NY) coated with 2% gelatin (Sigma) in PBS. Once confluent, the cells were mechanically scraped off, diluted three times with culture medium, and plated onto tissue culture treated 48-well plastic dishes (Costar, Cambridge, MA) coated with gelatin. Confluent monolayers were formed 5-7 days after plating.
PMN Adhesion Tissue culture treated 48-well plastic dishes (Costar) were coated overnight at room temperature with 20 g/ml of either LM, FN, CI, CIV, on BSA diluted in PBS. Before the adhesion assay the dishes were washed three times with PBS. Endothelial cell cultures were washed twice with PBS prior to the adhesion experiment. The PMNs, suspended in adhesion buffer, were added to the wells (2.5 x 10 cells/well) together with the different reagents and allowed to adhere for 30 mm at 37#{176}C.After this incubation period the wells were gently washed twice with PBS to remove nonadherent cells and adherence was quantitated as myeloperoxidase extracted from the adherent PMNs.
Myeloperoxidase
Assay
Mycloperoxidase was determined as described by Suzuki et al. [30]. Briefly, PMNs were solubilized by addition ofO.5 ml of 0.5% hexadecyltnimethylammonium bromide in 50 mM potassium phosphate buffer (pH 6.0) to each well. Samples of the extracts were added to 96-well microtiter plates (Dynatech) containing 80 mM potassium phosphate buffer (pH 5.4), 0.5 mM hydrogen peroxide, 0.16 mM tetramethylbenzidine, and 8% N,N-dimethylformamide. The samples were incubated for 15-20 mm at 37#{176}Con an orbital shaker and the reaction was stopped by addition of 0.2 M sodium acetate (pH 3.0). The absorbance of the developed color was measured at 650 nm in a microtiten plate reader (Flow Laboratonies, McLean, VA). A known number of PMNs in suspension were serially diluted, extracted, and analyzed for mycloperoxidase content on the micnotiter plates and used for calculation of a standard curve. Adherence is expressed as a percentage of the total number of cells present in the adherence assay.
Statistics Results are presented as means ben of individual experiments. was used to determine statistical
± SEM and The Wilcoxon significance.
n = rank
the numsum test
RESULTS Adhesion of PMNs stimulated with fMLP (106 M) to the immobilized matrix proteins LM, FN, CI, and CIV in the presence of 1 mM CaC12 and MgCl2 is shown in Table 1. fMLP-stimulated cells also adhered to dishes coated with BSA or to uncoated plastic dishes as shown in Table 1. Nonstimulated cells did not adhere to any of the protein-coated dishes but adhered to uncoated plastic dishes as well as did fMLP-activated PMNs (data not shown). Furthermore, adhesion to the different substrates was studied in the absence of Ca2 or Mg2 or when either of the divalent cations alone was added. In the absence of divalent cations, there was only slight adhesion of PMNs to the various matrix proteins (Fig. 1). When Ca2 (1 mM) was added to the adhesion buffer as the only divalent cation, a minor increase in adhesion to the matrix proteins was seen compared to adhesion in the ab-
604
Journal
of Leukocyte
Biology
Volume
51, June
1992
TABLE
1.
Adhesion
of fM LP-stimulated unc oated plastic
Substrate
and
strates and allowed a percentage of the numbers represent
sence added
25.0 39.7 37.5 27.4 50.2 82.2
I IV albumin
fMLP
to protein-coate
(%)
Adhesion
Laminin Fibronectin Collagen type Collagen type Bovine serum Plastic apMNs
PMNs dishesa
(106
M)
were
added
d and
4.0 3.8 2.6 6.2 1 .4 4.5
simultaneously
to the various
to adhere for 30 mm at 37#{176}C.Adhesion is expressed total number of cells added in the adherence assay. the average of n individual experiments ± SEM.
of divalent cations. as the only divalent
However, cation,
n
SEM
when Mg2 the adhesion
(1 mM) increased
6 6 6 6 4 3 subas The
was to
levels seen in experiments in which both Ca2 and Mg2 were present (Fig. 1). Adhesion in the presence of both Ca2 and Mg2 is expressed as 100% in Fig. 1. We also studied adhesion to the different matrix proteins by PMNs stimulated with PMA (108 M) instead of fMLP (data not shown). Although more cells adhered to the immobilized substrates when activated with PMA, the results showed that the adhesion to the different matrix proteins was dependent on the presence of Mg2 but not Ca2. Furthermore, we studied the effect of Ca2 and Mg2 on the adhesion of stimulated PMNs to confluent cultures of human umbilical vein endothelial cells (HUVECs). In these experiments the PMNs were stimulated with PMA (108 M). It was found that PMN adhesion to HUVECs was also dependent on the presence of Mg2. When Ca2 was added to the adhesion buffer as the only divalent cation, 4.5 ± 0.8% of the PMNs adhered to the HUVECs. In contrast, 24.1 ± 0.2% of the cells adhered when Mg2 was added as the only divalent cation. We also found that adhesion to HUVECs, in the presence of both Ca2 and Mg2, was slightly lower (13.7 ± 0.9%) than adhesion in the presence of Mg2 only. The numbers represent averages of five individual experiments ± SEM. The nequirement for Mg2 in adhesion of PMNs to HUVECs and to immobilized matrix proteins might indicate that similar mechanisms are involved in supporting adhesion of PMNs to these substrates. In contrast to these experiments, it was found that BSA-coated dishes and uncoated plastic dishes were able to mediate adhesion of PMNs both in the absence of divalent cations and divalent cation, although strates when both Ca2
when Ca2 was added more cells adhered and Mg2 were present
as
to
the only these sub(Fig. 1).
Figure 2 shows the results of experiments on the effect of 50 M Mn2 on adhesion offMLP-stimulated PMNs to LM, FN, CI, and CIV in addition to Ca2 and Mg2. Because others have shown that Mn2 can increase the affinity between some matrix receptors (integnins) and their ligands ( see Introduction) we tested whether Mn2 had any effect on PMN adhesion to the various matrix proteins. When 50 M Mn2 was added to the adhesion buffer, the attachment increased on all substrates. This dose of Mn2 had no detectable effect on superoxide production by the PMNs (data not shown), suggesting that it does not induce damage to the PMNs. To study the affinity of PMNs for the different substrates, mAb 60.3, which recognizes the /3 chain of the adhesion complex CD11/CD18, was used to inhibit the adhesion. The monoclonal was added at different concentrations together with PMNs and fMLP. It was found that mAb 60.3 inhibited PMN adhesion in a dose-dependent manner and that the
0
120
C 0 0
100
0
80 C 0 C,)
60
0
40
0 (8
z
20
ci-
0
LM
FN
CI
CIV
BSA
plastic
Fig.
1. Effect of 1 mM Ca2 and Mg2 on adhesion of fML}tstimulated (106 M) PMNs to plastic dishes coated with laminin (LM), fibronectin (FN), collagen type I (CI), collagen type IV (CIV), or bovine serum albumin (BSA) and to uncoated dishes (plastic). The PMNs were allowed to adhere for 30 mm at 37#{176}Cin the absence of divalent cations (0) or in the presence of either 1 mM Ca2 () or 1 mM Mg2 (0). Adherence of PMNs in the presence of both divalent cations is set to 100% and is represented by the dashed line. The numbers represent the average of at least three individual experiments ± SEM.
efficiency
was
similar
on the
different
substrates
(Fig.
3a and
b). At an mAb 60.3 concentration of 15 tg/ml the inhibition of adhesion was almost maximal on all the substrates. In parallel experiments, 50 cM Mn2 was added together with PMNs, mAb 60.3, and #{163}MLPin the Ca2, Mg-containing buffer.
It was for the
PMNs
found that immobilized
Mn2 could substrates
alter because
the
only at 5 g/ml of mAb 60.3. Mn2 had no significant effect on the other two substrates, LM or FN, at any concentration of mAb 60.3. The possibility that the altered affinity of PMNs for collagen was a result of increased expression of CD11/CD18 on the PMN surface was tested by fluorescence activated cell sorter (FACS) analyses. It was found that 50 M Mn did not increase the expression of the receptor CD11/CD18 as studied by binding of fluorescein isothiocyanate (FITh)-labeled mAb 60.3 to fMLP-stimulated PMNs (data not shown). Taken together, these results showed that CD11/CD18 is the major adhesion receptor on fMLP-stimulated PMNs and that binding of the receptor to the immobilized matrix proteins LM, FN, CI, and CIV requires Mg2 but not Ca2. In contrast, adhesion of PMNs to immobilized BSA and to uncoated plastic dishes occurs in the absence of divalent cations. Furthermore, Mn2 can synergize with Mg2 to increase the affinity of CD11/CD18 for immobilized collagen, especially for CI.
DISCUSSION The present study of the effect of divalent cations on PMN adhesion found that adhesion to matrix proteins immobilized on plastic dishes required the presence of Mg. We found that the absence of any divalent cations or the presence of Ca2 alone supported only minimal PMN adhesion to matrix proteins. These results are somewhat contradictory to those previously described by Bohnsack et al. [4], who reported
affinity of higher doses
ofmAb 60.3 were required to inhibit attachment (Fig. 4a-d). However, only the affinity for the collagen substrates increased to a significant degree and especially the affinity of PMNs for CI appeared to be changed. As shown in Fig. 4c, mAb 60.3 inhibition of PMN attachment to CI was significantly lower when Mn2 was present, indicating an increased affinity of PMNs for this substrate. This was seen at concentrations of mAb 60.3 up to 15 g/ml. At higher concentrations of mAb 60.3 (40 tg/ml), Mn2 had no significant effect on the PMN affinity for CI. Addition of Mn2 also appeaned to increase the affinity of PMNs for CIV; however, the change in inhibition was highly significant (P < .01)
40
30
20
0
a a
10
-C
z 0
ci0
b
a .0
40
E
80
0
C
30
0 -C 0
60
Co
20
z ci-
40
10
0 0
:::T
20
.0
E
,
.
.
0
C
0
10
20
30
40
0
LM
Fig.
2.
plastic
(CI),
Effect dishes
or
of Mn2 coated
collagen
type
FN on
adhesion
with
laminin
IV
Cl
CIV
of fMLP-stimulated (LM),
(CIV).
The
(106
fibronectin PMNs
were
mAb
M)
PMNs
(FN),
collagen
allowed
to
adhere
Fig. 3. Adhesion
to
type
I for
ious
collagen adhere
numbers
the
the
average
of
six
individual
experiments
±
SEM.
Lundgren-Akerlund
of mAb
simultaneously
30 mm at 37#{176}C in the presence of 1 mM Ca2 and Mg2 () and in the presence of 50 iM Mn2 in addition to Ca2 and Mg2 (0). Adherence is cxpressed as a percentage of the total of cells added in the adhesion assay. The represent
ofIMLP-stimulated
concentrations
total
to
plastic
(106
60.3. dishes
coated
number
et al.
of of
cells six
Divalent
added
individual
cations
in
the
PMNs
in the
antibody, with
adhesion
and
laminin
IV (A) and is expressed
experiments
and
(pg/mI)
M)
PMNs,
type I (L:), or collagen type for 30 mm at 37#{176}C.Adherence
average
60.3
(0),
the
assay.
presence
fMLP
of var-
were
added
(#{149}),
fibronectin
cells were allowed to as a percentage of the
The
numbers
represent
± SEM.
PMN
adhesion
in vitro
605
100 80
60 40
20 0
C 0 .0 -C C
100 80 60
40 20
0
10
20
30
40
0
mAb
.,
Fig. 4. Inhibition PMNs, antibody,
of fMLIstimulated
fMLP, + Mn2); (c) collagen at 37#{176}C. Inhibition was
of antibody. determined experiments
Statistical
significance
with the Wilcoxon ±
(106
and Mn’ type I (L calculated
M)
adhesion
by
various
added simultaneously to plastic , - Mn2; A, + Mn2), or (d) collagen for each concentration of mAb 60.3 of differences
rank
PMN
were
sum
in inhibition,
test for each
ofmAb
lated PMN adhesion to BSA-coated or uncoated plastic dishes was seen both in the absence of divalent cations and in the presence of either Ca2 or Mg2, different mechanisms are probably involved in adhesion to these substrates as compared to matrix proteins or endothelial cells. It was reported by Dransfield and Hogg [7] that Mg can regulate the binding activity of a specific CD11/CD18 epitope present on all Journal
60.3.
of
with , -
in the absence or in the presence of Mn2 (50 sM). (0, - Mn2; + Mn); (b) fibronectin ((), - Mn2; V , + Mn2) and the cells were allowed to adhere for 30 mm in the presence of antibody with adherence in the absence
Mn2; the
.01;
40
#{149},
mAb 60.3,
(a)laminin
adherence in
P