Allergy and Immunology Division, Department
of Medicine, University of Pennsylvania Medical Center,
Philadelphia, Pennsylvania
Received 21 September 1998/Returned for modification 5 November
1998/Accepted 26 January 1999
Neutrophil adherence to matrix proteins likely plays an important
role in inflammatory responses. Antineutrophil cytoplasm antibodies may
activate neutrophils in certain diseases. Using an in vitro method that
allows simultaneous quantitation of neutrophil adherence and superoxide
secretion, we compared the effects of antibodies against
neutrophil granule proteins and tumor necrosis factor alpha (TNF-
),
a known neutrophil agonist. Antilactoferrin antibodies but not
antielastase or antimyeloperoxidase antibodies stimulated increased
adherence to fibronectin and laminin similar in degree to that induced
by TNF-. This, but not the simultaneous superoxide secretion, was
inhibited in the presence of anti-CD18 antibodies. Humoral immune
responses to lactoferrin, likely expressed on the
neutrophil surface, can activate neutrophils in proinflammatory responses that may be pathogenic.
 |
INTRODUCTION |
The accumulation of neutrophils
(polymorphonuclear leukocytes [PMNs]) in inflammatory sites may be
due in part to interaction of PMNs with extracellular matrix proteins
such as fibronectin, laminin, and collagen (4, 11). Several
in vitro models for the assessment of such adherence have been
described (3, 4). We have used a relatively simple method to
reliably quantitate PMN adherence to plastic wells coated with
individual proteins (20). With this approach, we have
reported that stimuli such as tumor necrosis factor alpha (TNF-) and
the phorbol ester phorbol myristate acetate will stimulate impressively
increased PMN adherence to fibronectin, less adherence to laminin, but
no increased adherence to collagen. Adherent PMNs appear to be more
activated, with the release of some intracellular contents.
Such intracellular contents may be transported to the surface membranes
of PMNs, particularly when these cells are activated by other stimuli
(17). In such cases, these PMNs may be activated further
following incubation with autoantibodies directed against certain
intracellular components. In recent years, there has been considerable investigation of the possible activation of PMNs when they
are exposed in the blood to autoantibodies against
neutrophil cytoplasmic components (ANCAs) in certain diseases. In
Wegener's granulomatosis, the ANCAs are directed mainly against
a tryptic proteinase called PR3 (12). In several types
of vasculitis, the ANCAs more commonly react with neutrophil
myeloperoxidase (MPO) (9, 12). In some individuals with
inflammatory bowel diseases, the ANCAs may be autoantibodies against
chymotrypsin and against lactoferrin, an iron-binding protein present
in the secondary granules of PMNs (6, 10). In contrast, the
ANCAs seen in the sera of some patients with rheumatoid arthritis
appear to be predominantly antielastase antibodies (11).
Bartunkova et al. (2) have shown that
zymosan-induced PMN chemiluminescence is enhanced by antibodies
against the PR3 proteinase but is inhibited by antibodies against
surface adhesion proteins CD16 and CD18. Elastase may be expressed on
the surface of activated but not resting PMNs (7). However, it is not
known whether there is similar transport of lactoferrin (Lf) or MPO to
the PMN surface or whether antibodies directed against these components
lead to increased adherence to matrix proteins. Such adherence could
play an important role in the inflammatory reactions seen in the
diseases in which serum ANCAs are found.
In the present study, we have investigated the effects of
antibodies against (i) the neutrophil granule proteins Lf elastase and
MPO and (ii) the surface determinant CD18 on PMN adherence to matrix
proteins. We have also assessed production of superoxide during this
interaction of PMNs with matrix proteins as a marker of PMN activation.
| |
MATERIALS AND METHODS |
Cells.
A granulocyte-rich fraction (over 95% PMNs) was
obtained by density gradient centrifugation from the blood of a panel
of healthy nonatopic donors receiving no medication, as described
previously by us (17). PMNs were placed in replicate wells
(105 cells per well) of polystyrene, flat-bottom microtiter
plates (Immulon-4; Fisher Scientific Co., Malvern, Pa.) coated with
either human fibronectin (25 µg/ml; NY Blood Center, New York, N.Y.) or human laminin (25 µg/ml; Biomedical Technology, Inc., Stoughton, Mass.).
Incubation.
To groups of four replicate wells each was added
either (i) human TNF- (2 × 106 units/mg; R&D,
Minneapolis, Minn.) at various concentrations; (ii) sheep anti-human Lf
(anti-Lf) antibody (Dako, Carpenteria, Calif.) at various dilutions;
(iii) sheep antielastase (anti-El) antibody (Binding Site, Inc., San
Diego, Calif.) at various dilutions; (iv) sheep anti-MPO antibody
(Binding Site, Inc.) at various dilutions; (v) sheep
anti-immunoglobulin G (anti-IgG) antibody (Binding Site, Inc.) diluted
1:100 (final concentration); and (vi) additional media instead of an
agonist to assess spontaneously occurring PMN events (referred to as
"cells alone" hereafter).
In some experiments, the effects of possibly modulation were
investigated by incubating PMNs with the following combinations: (i)
anti-Lf antibody plus TNF-, (ii) anti-El antibody + TNF-, (iii) anti-Lf plus anti-CD18 antibodies, (iv) anti-Lf plus anti-OX8 (isotope control) antibodies, (v) TNF- and anti-CD18 antibody, (vi)
anti-Lf antibody plus soluble Lf (10 µg/ml; Sigma, St. Louis, Mo.),
and (vii) anti-Lf antibody plus soluble human IgG (10 µg/ml; Dako).
Cells and potential agonists were incubated in a medium of Hanks'
balanced salt solution containing cytochrome c and gelatin to permit assessment of superoxide secretion during the incubation (total reaction volume/well, 100 µl), as described previously by us
(20).
Superoxide secretion.
After incubation at 37°C in 5%
CO2-air for 30 min, the reduction of the cytochrome
c in the medium was assessed with a plate-reading spectrophotometer set at a wavelength of 550 nm as described previously by us (20). The superoxide levels in such wells were
determined as the difference in cytochrome c reduction in
the absence and presence of superoxide dismutase.
Cell adherence.
The wells were then drained thoroughly and
washed with warmed (37°C) buffered saline. Absolute methanol was
added to each well to fix the cells adherent to the well surfaces. A
solution of amido black was added to each well, and the plates were
incubated at room temperature for 30 min and then the wells were
drained. After thorough washing of the well 100 µl of a 10% solution
of sodium dodecyl sulfate (SDS) was added to lyse the cells. The amount
of released dye was quantitated by measuring the absorption by this dye
at 595 nm on a plate-reading spectrophotometer.
In previous quality control studies, we had found that amido black is
taken up only by adherent fixed cells and not by the matrix proteins or
plastic. Quantitation of adherent cells by determination of the amount
of amido black released after SDS lysis of the cells had been
previously validated by comparing amido black levels in the well fluid
with quantitation of amido black-containing adherent cells in replicate
wells (not subjected to SDS lysis after 30 min of incubation with or
without agonists at various doses) by computer-based image analysis
(r = 0.95).
| |
RESULTS |
Studies in fibronectin-coated wells. (i) Adherence
studies.
As expected, TNF- induced a dose-dependent
increase in PMN adherence, as reflected in the increased amount of
amido black released from such adherent cells after subsequent cell
lysis (Fig. 1).
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FIG. 1.
Dose-response effects of TNF- on neutrophil (PMN)
adherence to fibronectin-coated wells (e.g., TNF/1 = TNF- at 1 U/ml).
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|
Incubation of other aliquots of these PMNs with anti-Lf antibody also
led to a dose-dependent, significantly increased adherence compared to
the level of spontaneous cell adherence (Fig.
2). In contrast, there was no increased
adherence of PMNs incubated with antibodies against two other
neutrophil cytoplasmic proteins (anti-El, anti-MPO) or anti-IgG
antibodies; the last one was used as an antibody that might bind to IgG
on the surfaces of the PMNs (Fig. 2).
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FIG. 2.
Effects of (i) various dilutions of anti-Lf antibody
(A-Lf), (ii) anti-El antibody (A-El), (iii) anti-MPO antibody (A-MPO),
and (iv) anti-IgG antibody (A-IgG) on PMN adherence to
fibronectin-coated wells.
|
|
(ii) Modulation of adherence.
We had previously found that the
increased PMN adherence to fibronectin induced by TNF- was
significantly inhibited in the presence of anti-CD18 antibody
(5). Therefore, we compared the effects of adding anti-CD18
antibody to incubations of PMNs with TNF- or anti-Lf antibody. As
expected, TNF--induced adherence was almost completely inhibited in
the presence of anti-CD18 antibody but was unaltered by similar
concentrations of anti-OX8 (isotype control monoclonal antibody) (Fig.
3). A marked inhibition of the
adherence-inducing effects of anti-Lf antibody was seen when anti-CD18
antibody was placed in the incubation medium (Fig.
4). Addition of the OX8 isotype control
monoclonal antibody had no effect on the adherence induced by anti-Lf
antibody.
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FIG. 3.
Effects of (i) TNF- (5 U/ml), (ii) TNF- (5 U/ml)
plus anti-CD18 antibody (1:100 dilution) (a-CD18), and (iii) TNF- (5 U/ml) plus OX8 antibody (1:100 dilution) on PMN adherence to
fibronectin-coated wells.
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FIG. 4.
Effects of (i) anti-Lf antibody (1:100 dilution),
(A-Lf), (ii) anti-Lf antibody plus anti-CD18 antibody (1:100 dilution)
(A-Lf + A-CD18), (iii) anti-Lf antibody plus OX8 control antibody
(A-Lf + OX8), and (iv) anti-Lf antibody (1:100 dilution) plus
soluble lactoferrin (A-Lf + Lf).
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|
The likelihood that the anti-Lf antibody was interacting with Lf on the
PMN surface to induce increased PMN adherence is suggested by the
observation that addition of soluble Lf (10 µg/ml) along with the
anti-Lf antibody to the PMNs at the beginning of the incubation period
almost completely blocked the increase in PMN adherence seen when
anti-Lf antibody alone was added (Fig. 4). Addition of similar
concentrations of human serum albumin and IgG to the anti-Lf antibody
did not inhibit the adherence-promoting effects of anti-Lf antibody.
The soluble Lf, albumin, and IgG did not stimulate adherence by themselves.
Previous studies suggested that prior stimulation of PMNs with agonists
like TNF- might induce translocation of cytoplasmic proteins such as
Lf to the PMN surface, where they might be a target for interaction
with anti-Lf antibody. Therefore, we compared the adherence of PMNs
preincubated with TNF- at 5 U/ml or buffered saline in siliconized
tubes (to inhibit cell adherence) when these two cell populations were
subsequently incubated in fibronectin-coated wells with anti-Lf and
anti-El antibodies. As shown in Fig. 5, the adherence of the PMN upon first exposure to TNF- when anti-Lf antibody was added was somewhat (but not significantly) greater than
the adherence of PMNs preincubated in buffered saline in response to
added anti-Lf antibody. It turned out that these levels of adherence
were similar to those in other PMN aliquots incubated with TNF-
alone. PMNs preincubated with TNF- at 5 U/ml in siliconized tubes
and then added along with anti-El antibody (diluted 1:100) to
fibronectin-coated wells adhered to these wells to a degree similar to
that seen when PMNs were incubated with TNF- at 5 U/ml alone in
fibronectin-coated wells (Fig. 5).
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FIG. 5.
Adherence of PMNs to fibronectin-coated wells (i) alone,
(ii) with TNF- at 5 U/ml (TNF), (iii) with preincubation in
TNF- at 5 U/ml and then incubation in wells with anti-Lf
antibody (1:100 dilution) (TNF + Anti-Lf), (iv) with anti-Lf
antibody (1:100 dilution) (Anti-Lf), (v) with preincubation in
TNF- at 5 U/ml and then incubation in wells with anti-El
antibody (1:100 dilution) (TNF + anti-Elast), and (vii) with
anti-El antibody (1:100 dilution) (Anti-Elast).
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|
(iii) Superoxide secretion.
As expected, TNF- stimulated a
dose-dependent secretion of superoxide (O2
)
during the 30-min incubation period in the fibronectin-coated wells
described above (Fig. 6). However,
O2 secretion was not inhibited in the
presence of a concentration of the anti-CD18 antibody which had almost
completely blocked PMN adherence (Fig. 6). There was a modestly
increased secretion of O2 by PMNs incubated
with anti-Lf antibodies for 30 min (Fig. 6). Again, this increased
O2 secretion was not inhibited in the
presence of anti-CD18 antibody. Anti-MPO and anti-El antibodies
modestly stimulated O2 secretion by suspended
PMNs in siliconized tubes (2.1 ± 0.3 nM). However, such
O2 secretion was not significantly different
when the incubation of these antibodies with PMNs was carried out in
fibronectin-coated wells (2.3 ± 0.4 nM).
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FIG. 6.
Dose-response stimulation of superoxide secretion by
PMNs in fibronectin-coated wells by TNF- (at 0.25, 1.0, and 5.0 U/ml) and anti-Lf antibody (at 1:1,000, 1:500, and 1:100 dilutions).
The effects of the addition of anti-CD18 to anti-Lf antibody (1:100
dilution) (a-Lf 1:100 + a-CD18) or to TNF- at 5 U/ml
(TNF/5 + a-CD18) on stimulation of superoxide secretion were
assessed.
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|
The extent of PMN adherence and O2 generation
by PMNs in the same wells was compared. There was no impressive
correlation between adherence and O2
secretion stimulated by either TNF- (r = 0.35; P was
not significant) or anti-Lf antibody (r = 0.3; P was
not significant) (P values were determined by the Spearman
rank order correlation).
Studies in laminin-coated wells. (i) Adherence.
As expected,
TNF- stimulated increased PMN adherence to laminin-coated wells
in a dose-dependent manner, although the increased adherence required
TNF- concentrations somewhat higher than those required
for PMNs in fibronectin-coated wells. TNF- at concentrations as
low as 0.25 U/ml stimulated significantly increased adherence to
fibronectin, with peak increased adherence (optical density, 0.30 ± 0.03) seen with TNF- at 10 U/ml (Fig. 1). In comparison, significantly increased PMN adherence to laminin was stimulated by TNF-, but only at concentrations of 
5 U/ml, with peak
adherence seen with TNF- at 20 U/ml (optical density, 0.20 ± 0.03).
There was considerably increased adherence to laminin of PMNs incubated
with the anti-Lf antibody diluted 1:100 (P = 0.001 versus the spontaneous adherence of these PMNs) (Fig.
7). A less prominent adherence was
stimulated by a 1:500 dilution of the anti-Lf antibody, but this
adherence was similar in degree to that induced by TNF- at 5 U/ml (P = 0.01 versus spontaneous adherence). The
adherence of PMNs incubated with anti-Lf antibody diluted 1:1,000 was
not significantly greater than spontaneous adherence. Incubation with anti-El or anti-IgG antibodies did not induce increased PMN adherence to laminin-coated wells (Fig. 7).
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FIG. 7.
Adherence of PMNs to laminin-coated wells (i) alone;
(ii) with anti-Lf antibody (Anti-Lf) at 1:100, 1:500 and 1:1,000
dilutions; (iii) with a mixture of anti-Lf and anti-CD18 antibodies
(A-Lf + A-CD18); (iv) with anti-El antibody (1:100 dilution)
(Anti-El); (v) with anti-IgG antibody (1:100 dilution) (Anti-IgG); and
(vi) with TNF- at 5 U/ml (TNF).
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(ii) Modulation of adherence.
Addition of anti-CD18 antibody
or soluble Lf to the incubation mixture significantly inhibited the
adherence to laminin stimulated by anti-Lf antibody so that such
adherence was not significantly different from the spontaneous
adherence (Fig. 7).
(iii) Superoxide generation.
Both TNF- and anti-Lf
antibody stimulated significantly increased the level of superoxide
generation by PMNs during incubation in laminin-coated wells (3.8 ± 0.3 and 2.3 ± 0.2 nmol, respectively). This superoxide
generation was not inhibited in the presence of anti-CD18
antibody. There was no significant correlation between the mean
degree of adherence and the mean amount of superoxide generation in
individual wells containing the agonists (r = 0.4 and
0.35, respectively; P was not significant).
| |
DISCUSSION |
The findings described here indicate that antibodies against
Lf, a protein present in the secondary granules of the PMN
cytoplasm, will stimulate resting neutrophils to adhere more readily to
the matrix proteins fibronectin and laminin. The effect likely
involves an immune interaction of the anti-Lf antibody with Lf on the
PMN surface since the increased adherence is inhibited in the presence of soluble Lf. This increased adherence is likely mediated by increased
expression of the beta-integrin CD11b/CD18 complex on the PMN surface
since the increase in adherence was markedly inhibited in the presence
of anti-CD18 antibodies. In contrast, antibodies against El and MPO,
which are components of primary granules in the PMN cytoplasm, did not
stimulate such increased adherence to fibronectin or laminin.
The increased adherence induced by anti-Lf antibodies was quite
prominent, in the same range as that seen in PMNs stimulated by
TNF-, a potent PMN agonist. The TNF--induced adherence to fibronectin and laminin was also inhibited by anti-CD18 antibodies, suggesting similar pathways for the adherence stimulated by both agents.
Also, the adherence induced by both anti-Lf and TNF- was
accompanied by increased superoxide generation. Using the nitroblue tetrazolium assay for detection of intracellular superoxide, we have
found that most of the cells containing formazan (the product formed by the reduction of nitroblue tetrazolium by superoxide) were
predominantly in the PMNs adhering to fibronectin and not in the
nonadherent PMNs after the 30-min incubation. Nevertheless, the
superoxide generation induced by anti-Lf antibody or TNF- was
not altered when the added anti-CD18 antibodies blocked PMN adherence.
It is unclear whether the superoxide generation occurs during
initiation of PMN adherence or later during the 30-min incubation.
Others have reported more prominent superoxide generation induced by
TNF- in PMNs previously plated on fibronectin-coated wells; this
superoxide generation was not blocked by prior treatment of the
adherent PMNs with an anti-CD18 antibody (15). No similar studies with antibodies against neutrophil cytoplasm proteins have
previously been reported. However, one report described increased chemiluminescence induced by anti-Lf antibodies in PMNs pretreated with
the formylated tripeptide (but not in untreated PMNs); no results of
adhesion studies were reported (16).
However, there is evidence that antibodies against some other
cytoplasmic components of PMNs may activate PMNs. Several reports have
described the generation of reactive oxygen species (ROS) by PMNs
incubated with ANCA-positive sera, particularly when the PMNs were
primed with substimulating concentrations of TNF- (2, 7,
13). It was felt that this ROS production was due mainly to MPO
activation (13). Previous studies have shown that anti-MPO antibodies can stimulate PMNs to damage human vascular
endothelial cells in vitro (8). More recently, antibodies
against the proteinase 3 found in PMN granules enhanced PMN
chemiluminescence (3). ANCA-positive sera can also induce
increased adherence of PMNs to the vascular endothelium and cause
endothelial injury. Most ANCA-positive sera have been found to have
antibody activities against proteinase 3 or MPO (9, 12).
However, a minority of ANCAs have anti-Lf or anti-El activities
(6, 14, 18). Therefore, our findings that anti-Lf antibodies
but not anti-El or anti-MPO antibodies stimulate PMN adherence and
superoxide generation have potential clinical relevance.
The mechanisms underlying the stimulation by antibodies against
proteins present in the PMN cytoplasmic granules have not been
completely defined. Previous studies have shown reasonable amounts of
proteinase 3 and cationic protein 57 and very small amounts of MPO on
the PMN surface (7). The surface expression of these
proteins is markedly increased on TNF--primed PMNs. Although
there have been no analogous studies of the surface expression of Lf on
PMNs, our preliminary flow cytometry studies with labeled anti-Lf
antibodies suggest that Lf is constitutively expressed on the surface.
To be sure that our process of separating the PMNs from the blood by
density gradient centrifugation did not "activate" the PMNs to
express more Lf on the surface, we repeated the studies on several
occasions using leukocyte suspensions obtained after spontaneous
gravity sedimentation in several experiments. Although we were dealing
with a more heterogeneous leukocyte suspension in such
experiments, there was evidence of increased adherence stimulated
by anti-Lf antibodies.
It is perhaps not surprising that anti-El and anti-MPO antibodies did
not stimulate increased PMN adherence to the matrix proteins. El
and MPO, like other primary granule components, are secreted more into
intracellular phagocytic vesicles than into the extracellular milieu
(19). Therefore, there may be little constitutive El on the
PMN surface. As noted above, impressive amounts of MPO were found on
the PMN surface only after stimulation of the PMN by an agent like
TNF- (7).
Therefore, we can conclude from these findings that anti-Lf antibodies
can stimulate PMN adherence and production of ROS as much as
TNF-, a potent agonist, can. Anti-Lf antibodies have been found
in the sera of some patients with rheumatoid vasculitis, systemic
lupus, ulcerative colitis, and sclerosing cholangitis (6,
14). Peen et al. (16) have described the
activation of PMNs added with anti-Lf antibodies to Lf-coated
endothelial cells. Therefore, it is possible that the
proinflammatory activities of anti-Lf antibodies described here may
play roles in the pathogenesis of these disorders.
This work was supported by NIH grant RO1-AI-14332 and the
Immunology Research and Education Fund.
The advice and collaboration of Steven Albelda and Paul Atkins in the
initial development of the adherence assay used in the current study
are greatly appreciated.
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Abstract
Introduction
