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Mß2 (CD11b/CD18)1
*
The La Jolla Institute for Experimental Medicine, La Jolla, CA 92037;
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. PMN adhesion to factor H in the presence or absence
of C5a was blocked specifically by mAbs against CD11b or CD18. Affinity
purification using factor H Sepharose followed by immunoprecipitation
using mAbs to various integrin chains identified Mac-1 (CD11b/CD18) as
a factor H binding receptor. The presence of surface bound factor H
enhanced neutrophil activation resulting in a two- to fivefold increase
in the generation of hydrogen peroxide by PMNs stimulated by C5a or
TNF-. When factor H was mixed with PMNs, 1.4 to 3.8-fold more cells
adhered to immobilized heparin or chondroitin A. In addition, augmented
adhesion of PMNs was measured when factor H, but not HSA or C9, was
absorbed to wells that were first coated with heparin or chondroitin A.
The adhesion of PMNs to glycosaminoglycan-factor H was blocked by mAbs
to CD11b and CD18. These studies demonstrate that factor H is an
adhesion molecule for human neutrophils and suggest that the
interaction of factor H with glycosaminoglycans may facilitate the
tethering of this protein in tissues allowing factor H to serve as a
neutrophil adhesion ligand in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Factor H exerts a regulatory role for the alternative pathway of complement in several ways. It can impede the formation of the alternative pathway C3 convertase by competing with factor B for binding C3b, and it can also inactivate C3 convertase by serving as a cofactor for the serine protease, factor I, which converts C3b to iC3b (7, 8, 9).
In addition to its role as a control protein of the complement alterative pathway, factor H is reported to mediate several cellular responses through interaction with specific receptors. These include an ability to induce secretion of IL-1ß from monocytes, to evoke the release of factor I from lymphocytes, and to mobilize PGE2 and thromboxane B2 from macrophages (10, 11, 12).
It has been described recently that neutrophils have a specific receptor for factor H (13). However, neither the molecular identity of this receptor nor its functional role were defined. The existence of an interaction between factor H and PMNs3 suggested the possibility that, in certain circumstances, factor H could serve as an adhesion ligand for neutrophils, and thereby affect granulocyte function.
The implications of factor H acting as an adhesion ligand for PMNs are important because migration, phagocytosis, degranulation, and oxidant generation of leukocytes are all influenced by adhesion of these cells to surface molecules on other cells or on the extracellular matrix (14, 15). Indeed, many extracellular ligands have been identified that influence granulocyte adhesion and function. Examples include fibronectin, fibrinogen, thrombospondin, and laminin (15).
The findings presented in this report demonstrate that complement factor H is also a member this group of molecules that serve to promote adhesion of granulocytes. The interaction between these cells and factor H is shown to be mediated by the integrin CD11b/CD18 (Mac-1). Moreover, we show that the adherence of PMNs to factor H augments hydrogen peroxide and lactoferrin secretion. Finally, it is shown that increased PMN adhesion can be generated as a consequence of interaction of factor H with glycosaminoglycans. Because of its ability to interact with glycosaminoglycans, factor H could conceivably function similarly to fibronectin, which is also present in plasma and can deposit at sites of damaged tissue to mediate adhesive interactions with leukocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BSA (low endotoxin), human serum albumin (HSA),
anti-lactoferrin antisera, chondroitin sulfate A, crystal violet,
HEPES, lactoferrin, scopoletin, human transferrin, sodium periodate,
cytochrome c, diaminobenzidine tetrahydrochloride, PMA, and
polymyxin were purchased from Sigma (St. Louis, MO). Human fibrinogen,
octyl-ß-D-glucopyranoside, and biotin hydrazide were
obtained from Calbiochem (San Diego, CA). Peroxidase-conjugated avidin
was supplied by Bio-Rad (Hercules, CA). Factor B, factor H,
antithrombin III, plasminogen, C1q, C4b-BP, C3, C5, C6, C7, and C9 were
purified from outdated human plasma (16, 17). C3b was generated from C3
using a fluid phase C3 convertase and was purified by DEAE-Sephadex
column chromatography (17). C3bi was made by reacting C3b (1 mg/ml)
with a 1–100 weight ratio of factor H and factor I followed by
incubation at 37EC (18). C5a was generated after specific cleavage of
C5 using the fluid phase C5 convertase (19). Recombinant human IL-8 was
produced in Escherichia coli as a fusion protein with
glutathione transferase. After affinity purification, IL-8 was released
from the molecular chimera using thrombin, and the recovered
recombinant IL-8 was demonstrated to be fully functionally active (20).
Factor H Sepharose was prepared by coupling 5 mg of factor H per ml of
Sepharose 6B, using the cyanogen bromide reaction (21). Polyclonal Abs
to factor H were made in a rabbit, and isolated form a column of factor
H Sepharose. Anti-factor H F(ab')2 were made by digesting
anti-factor H IgG with pepsin using a 30 to 1 weight ratio of
substate to enzyme in 50 mM sodium citrate, pH 4, for 1 h at
37°C. After neutralization the uncut IgG fragments were removed by
passage through Protein A-Sepharose (Pierce Chemical, Rockford, IL).
Functional blocking mAbs with specificities to CD11a (Clone
TS1/22.1.1.13) and CD11b (Clone 44aacb) were isolated from hybridoma
cells obtained from American Type Culture Collection (Rockville,
Maryland), and CD11c (Clone CBR-p150/4G1) was from Biosource
International (Camarillo, CA) (22, 23). Monoclonal Abs to the
ß1-chain of human integrin (Clone P4C10)(24) and those
directed against the Vß3 (Clone LM609) and
Vß5 (Clone P1F6) were from Life
Technologies (Gaithersburg, MD) and Chemicon (Temecula, CA)(24, 25). A
preparation of a mAb against the ß2 integrin chain (Clone
IB4) was also used (26).
Preparation of neutrophils and eosinophils
PMNs were purified from human ACD blood from healthy donors by Percoll (Pharmacia, LKB) gradient centrifugation (27). The entire preparation was conducted at 23°C. The PMNs were greater than 98% pure, and hypotonic lysis of remaining trace amounts of erythrocytes was not performed. After the last centrifugation, the cells were suspended in buffer A: 10 mM HEPES (pH 7.4)/0.15 M NaCl/5 mM KCl/2.5 mM CaCl2/1 mM MgCl2/10 mg/ml BSA/1 mg/ml glucose. In some experiments buffer B (buffer A without BSA) was used. In other experiments buffer C was employed, which was was buffer B containing 1 mg/ml HSA.
Eosinophils were prepared by centrifugation through Ficoll-Hypaque followed by negative selection using anti-CD16 with magnetic bead separation (28, 29). The eosinophil preparations were greater than 98% pure and were suspended in buffer A for adhesion assays.
Adhesion assays
Various plasma proteins (100 µl of 0.6 mg/ml) were added to flat-bottom wells of polystyrene microtiter plates (Costar, Cambridge, MA) and were allowed to absorb for 15 h at 4°C. The wells were washed 4 times in buffer B and blocked with buffer A for 1 h at 23°C. Then the wells were washed 8 times more. Finally PMNs (100 µl of 2 x 106 cells/ml) in buffer A were added to the wells and were allowed to adhere for 1 h at 37°C. The nonadherent cells were removed, and the wells were washed 10 times in buffer B followed by fixation in 0.2% crystal violet or 0.1% Eosin Y in 10% ethanol (30). After removing the excess stain and washing, the cells contained within a central 1.2 mm square area were counted using an Olympus CK2 inverted microscope (Lake Success, NY).
The effect of the mediators C5a, TNF-, and IL-8 on the adhesion of
PMNs to several plasma proteins was performed similarly. The mediators
(within the concentration range of 10-14 to
10-6 M) were added 5 min after the PMNs had been layered
into the wells. The plates were subsequently incubated for 1 h at
37°C, and cells were counted as stated above.
Inhibition of PMN adhesion using anti-integrin mAbs
Various mAbs with specificities to human integrins were incubated with PMNs at concentration of 3.75, 7.5, and 15 µg/ml for 10 min at 23°C before performing adhesion assays as described above.
Biotinylation of PMN surface proteins and immunoprecipitation of a factor H receptor
Freshly isolated PMNs were biotinylated as described earlier (31). Briefly, PMNs (107 cells/ml) were first oxidized with 1mM sodium periodate for 30 min on ice. The cells were then washed three times with PBS containing 0.1% glucose and 10 mM biotin hydrazide for 30 min at room temperature. The cells were washed sequentially to remove unbound biotin hydrazide and kept frozen until use. The biotin-labeled proteins were extracted from PMNs obtained from 12 donors in 10 mM Tris-HCl (pH 7.4)/0.15 M NaCl/50 mM octyl-ß-D-glucopyranoside/2.5 mM CaCl2/1 mM MgCl2/1 mM PMSF. The biotinylated cell lysate was centrifuged and subjected to affinity chromatography using a 10-ml column of factor H Sepharose, which was equilibrated with extraction buffer. The unbound material was removed by washing, and the bound proteins were recovered using the same buffer without divalent metal ions but containing 20 mM EDTA and 300 mM NaCl. As a control, a similar procedure was applied using unconjugated Sepharose 6B. Protein in eluted fractions was detected by absorbance at 280 nm, and this material was electrophoresed through 5 to 10% polyacrylamide gels under nonreducing conditions. After SDS-PAGE, protein was detected by silver staining (31).
Integrins contained within the eluted material were identified using immunoprecipitation followed by Western blotting. To the protein eluted from factor H Sepharose, CaCl2 and MgCl2 were added to a final concentration of 20 mM. After immunoprecipitation using mAbs against several different integrin chains, the samples were subjected to SDS-PAGE. The electrophoresed immunoprecipitated protein was transferred to a nitrocellulose paper, which was blocked with PBS containing 1% BSA and 0.1% Tween-20. The filters were incubated with 1:2000 dilution peroxidase-conjugated avidin for 15 min at 37°C, and the biotinylated proteins were then detected using 3,3'-diaminobenzidine tetrahydrochloride as a substrate, as described earlier (32). Furthermore, all the mAbs were tested for the ability to immunoprecipitate their cognate integrins following a similar procedure using unfractionated membrane protein.
Hydrogen peroxide release assay
The release of hydrogen peroxide was conducted by measuring the
quenching of the fluorescent dye scopoletin (33). Microtiter plates
were coated as described above with factor H, BSA, and C9. Then,
neutrophils (104 cells per well) in buffer B were
pipetted into the wells. The neutrophils were allowed to absorb to the
wells for 15 min at 37°C, and this was followed by the addition of
IL-8 (10-9 to 10-6 M), C5a (10-9
to 10-6 M), PMA (0.1–100 ng/ml), or TNF- (0.1–100
ng/ml). Scopoletin was added rapidly to a final concentration of 25 nM,
and the plates were assayed immediately for fluorescence using the
excitation and emission wavelengths of 360 and 460 nm, respectively, in
a CytoFluor 2300 Fluorescence Measurement System by Millipore
(Philadelphia, PA). The plates were incubated at 37°C, and subsequent
readings were taken at 15-min intervals. Data were plotted as the
amount of H2O2 generated by 104
PMNs in a 2-h time period.
Superoxide anion assay
Superoxide generation by adherent PMNs was determined by measuring the superoxide dismutase (SOD) inhibitable reduction of cytochrome c. PMNs (2 x 106), adherent on HSA, fibrinogen, C9 or factor H, were incubated for various times in buffer C containing 1 mg/ml cytochrome c, 10-9 M C5a with or without 500 µg/ml factor H. In control wells 300 U/ml of superoxide dismutase was also added. The molar extinction coefficient difference (21,000 OD x M-1 x cm-1) between reduced and oxidized cytochrome c at 550 nm was used to calculate the molar amounts of superoxide generated (34).
Lactoferrin release assay
Microtiter dishes were coated as described with HSA, C9,
fibrinogen, or factor H, and PMNs (4 x 104
cells) in buffer B were layered into the wells. After 20 min at 23°C,
mediators C5a (10-8 M) or TNF- (1 ng/ml) were added to
the wells. For experiments involving TNF-, polymyxin (76.4 U/ml) was
added to BSA to block the wells, and polymyxin (76.4 U/ml) was also
incorporated into the PMN buffer B. Ten microliters of the supernatants
and 90 µl of 50 mM sodium bicarbonate, pH 9.6, were added to wells of
a second microtiter plate. The protein was allowed to absorb for
15 h at 4°C. Subsequently, an ELISA was conducted similar to
that described previously, with the modification that
2,2'-azino-di-(3-ethylbenzathiazolinsulfonate) (Boehringer-Mannheim,
Indianapolis, IN) was used as the substrate (35).
Effects of glycosaminoglycans on PMN adhesion
Adhesion assays of PMNs to wells coated with chondroitin A, heparin, as well as HSA, C9, and factor H were performed with PMNs initially suspended in buffer C, or buffer C containing 500 µg/ml factor H. In additional experiments, chondroitin A and heparin (10 mg/ml) were absorbed to microtiter plates for 15 h at 4°C. These were drained, and test proteins HSA, C9, or factor H were added. The secondary absorption was allowed to continue for an additional 3 h at 23°C. PMN adhesion assays were then conducted as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purity of the factor H preparation, as well as of other
proteins used in this study, was demonstrated by SDS-PAGE and Western
blotting. Figure 1
shows samples of
factor H, C3b, and C3bi stained after SDS-PAGE. Also, factor H was
tested for contamination by plasma proteins known to serve as adhesion
ligands, namely, C3, fibronectin, and vitronectin (Fig. 1). Western
blots failed to detect C3, fibronectin, or vitronectin in our factor H.
Since the sensitivity of a Western blot is at least 4 ng, and 4 µg of
factor H was added to each track, the maximal possible contamination,
if any, of factor H by C3, fibronectin, or vitronectin is 0.1%.
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The observation that PMNs have a specific receptor for complement
factor H (13) motivated us to examine the possibility that factor H
could serve as an adhesion ligand for granulocytes. To obtain
comprehensive results, a comparison with 18 other plasma proteins was
undertaken (Table I). The results of this
experiment demonstrate that, relative to other plasma proteins, factor
H is an effective adhesion ligand for neutrophils. The data for this
experiment were derived from 12 different donors. Compared with several
control proteins including HSA (110 ± 30 cells) and BSA (280
± 50 cells), greater numbers of PMNs (640 ± 100 cells) were
found to adhere to factor H. The SE of the means observed were fairly
large, but such variations were seen with all other ligands as well.
For instance, PMN adhesion to wells coated with C3b gave 940 ±
190 cells, and that to wells coated with IgG gave 850 ± 160
cells. The adhesion to factor H was specific for neutrophils because
eosinophils did not bind appreciably to factor H (70 ± 30 cells)
but did so to IgG (1270 ± 380 cells) and C3b (840 ± 240
cells) (Table II).
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).
This indicates that, like binding of soluble factor H to PMNs, adhesion
of PMNs to factor H-coated wells is divalent metal ion dependent.
PMNs from half of the donors evinced some degree of polarization when
adherent to factor H. An example of the morphology of neutrophils
adherent to factor H and BSA from one donor is shown in Figure 2. Cell polarization was not seen when
the PMNs were resident on any of the other plasma proteins tested
except plasminogen.
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Since many adhesive responses of neutrophils are mediated by
integrins (14, 15), we examined the possibility that binding of PMNs to
factor H was integrin dependent. Monoclonal Abs with specificities to
different integrin chains were tested for inhibition of factor
H-mediated PMN adhesion. Similar studies were performed using C3bi as a
positive control. The results shown in Figure 3 demonstrate a substantial
dose-dependent inhibition of PMN adhesion to factor H or C3bi-coated
wells by mAbs against CD11b (M) and CD18
(ß2), whereas mAbs to CD11a (L), CD11c
(X), ß1, Vß3, and
Vß5 had no significant effect. These
results suggested that Mac-1 (CD11b/CD18) is a major neutrophil
adhesion receptor for factor H.
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Confirmation of the identity of CD11b/CD18 as a factor H binding
protein was obtained by affinity chromatography and
immunoprecipitation. Cells were labeled with biotin, and membrane
lysates were passed through a factor H Sepharose column. Since we
observed that adhesion of PMNs to factor H was divalent metal ion
dependent and inhibited by EDTA, the bound receptor was eluted
specifically using 20 mM EDTA along with 0.3 M NaCl. A control
absorption and elution was performed similarly using unconjugated
Sepharose. Visualizaiton of the entire eluted pool after SDS-PAGE
followed by silver staining revealed three major bands and a fourth
band that was less intensely stained. The two largest major bands were
determined to be of sizes Mr 
156,000 and
Mr 97,000, which are approximately the sizes
of the chains of Mac-1. A smaller well-stained band of
Mr 85,000, along with a minor band of
Mr 141,000, which may be a degraded form of
the largest protein on the gel, were also seen. Only a faint band of
Mr 220,000 was detected from material eluted
from Sepharose alone (Fig. 4A).
Immunoprecipitation of the eluted protein pool from factor H Sepharose
with various anti-integrin mAbs followed by SDS-PAGE and Western
blotting revealed that anti-M (anti-CD11b) and
anti-ß2 (anti-CD18) specifically recognized
factor H binding protein (Fig. 4B). An explanation as
to why the -chain stains less intensely than the ß-chain is that
this may be a consequence of differential labeling by biotin. Proof of
the specificity of the immunoprecipitation was made by demonstrating
that all the mAbs have the capacity to precipitate their cognate
integrins (Fig. 4C). From these studies it can be
concluded that Mac-1 (CD11b/CD18) expressed on human neutrophils
functions as a specific receptor for factor H and can mediate binding
and adhesion in vitro.
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, and IL-8 on PMN adhesion to factor H
Next we examined whether the adhesion of PMNs to wells coated with
factor H could be increased when the cells were incubated with various
concentrations of C5a, TNF-, or IL-8. Both C5a and TNF- evoked a
dose-dependent increase of PMN adhesion to factor H up to a fourfold
augmentation, but IL-8 had only a negligible effect (Fig. 5). The augmented adhesion of PMNs to
factor H in the presence of 10-8 M C5a was blocked by
mAbs to CD11b and CD18 to a more appreciable extent than other
anti-integrin Abs tested. A similar pattern of inhibition was
observed with C3bi as the adhesion ligand (Fig. 6). C5a and TNF- had only a minor
influence of adhesion of cells to C9 and BSA.
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Since TNF- and C5a augmented the adhesion of PMNs to factor
H-coated wells, we examined whether these mediators could have an
influence on the respiratory burst of PMNs in a manner dependent on the
adhesion molecule. Accordingly, the effects of surfaces coated by
factor H or other proteins on the release of hydrogen peroxide from
neutrophils activated by C5a, TNF-, IL-8, or PMA were measured. The
results shown in Figure 7 demonstrate
that, when PMNs adherent to factor H were stimulated by C5a or TNF-,
an augmentation of hydrogen peroxide secretion occurred dependent on
the concentration of C5a or TNF-. The maximal rate of hydrogen
peroxide generated from PMNs, in wells coated with factor H, induced by
C5a or TNF-, was 0.6 to 0.8 nmoles/104 cells/2 h. A
smaller effect was seen when fibrinogen was the surface protein, but
C5a and TNF- had only a minor influence on hydrogen peroxide release
when PMNs were in wells coated with C9 or BSA. In these experiments,
IL-8 had no influence on hydrogen peroxide secretion regardless of the
protein that was used to coat the wells. Since PMA is an activator of
protein kinase C (36), it served as a positive control and evoked
increased release of hydrogen peroxide from PMNs independently of the
surface coating of the wells (Fig. 7). Thus, simultaneous utilization
of PMN adhesion and chemotactic receptors appears to provide
costimulatory signals for cellular activation.
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To examine further the effects of adhesion of PMNs to factor H,
the rate of release of lactoferrin, a specific granular protein of
neutrophils, was measured. The results shown in Figure 8 demonstrate that PMNs that were
resident on factor H released 1.5- to 2-fold more lactoferrin after 15
min than cells on HSA, C9, or fibronectin. The addition of C5a
(10-8 M) had only a small effect (1.5-fold at 20 min)
on lactoferrin release when cells were adherent to factor H. However,
TNF- (1 ng/ml) caused an approximate 10-fold augmentation in the
release of lactoferrin from neutrophils after 20 min of incubation, and
the results were most appreciable when the cells were adherent to
factor H. For experiments involving TNF-, wells were blocked with
polymyxin b (76.4 U/ml), and this drug was also incorporated into the
PMN buffer to obviate the hazard of LPS activation of the neutrophils.
Since cytochalasin B was not used in these experiments, the
microfilamentary network of the cells was not artificially altered, and
only the specific granular constituents were secreted. Azurophil
granular contents would not have been expected to have been released,
and indeed no myeloperoxidase activity was detected in any
circumstances.
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The experiments presented thus far demonstrate that factor H can
support adhesion of neutrophils in vitro. In turn, factor H-mediated
adhesion can be related to augmented hydrogen peroxide production and
increased specific granule release in response to C5a and TNF-
stimulation. Since factor H is a soluble protein present in human
plasma at a concentration of about 500 µg/ml (8), we explored
possibilities as to how factor H could function as an adhesion ligand
in vivo. Since factor H is reported to have a specific
glycosaminoglycan binding region in module 7 (37), we hypothesized that
perhaps, during episodes of tissue damage, factor H could adsorb to
proteoglycans presented by activated endothelial cells and/or to
glycosaminoglycans exposed on the interstitial matrix. Once bound by
surface glycosaminoglycans, factor H could act as a bridge between the
granulocyte and the endothelial cell or extracellular matrix.
Experiments were designed to examine whether factor H could enhance PMN
adhesion to glycosaminoglycans. Adhesion of PMNs to wells coated with
heparin or chondroitin A in the presence or absence of 500 µg/ml
soluble factor H was compared. The adherence of PMNs to wells coated
with chondroitin A and heparin was 1.4 to 3.8 times greater when factor
H was present in the assay buffer than in its absence (Fig. 9A). Another important
result of this experiment was that adhesion to factor H was not
reduced, but was even somewhat higher, when soluble factor H was
present in buffer containing PMNs. Since the C3b receptor on
neutrophils (CD35, CR1) is comprised of tandem Sushi units like factor
H, a diminished adherence of PMNs to C3b was predictably observed when
soluble factor H was included in the buffer (38).
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, B and
C). The augmentation by factor H was especially
notable (about 13-fold) in the case where the initial absorbed molecule
was chondroitin A sulfate (Fig. 9 C). To examine
which integrins were involved in the augmented adhesion of PMNs to
glycosaminoglycans on factor H, we employed mAbs to block PMN
adherence. The results indicate that the adhesion of PMNs to complexes
of glycosaminoglycan and factor H was blocked by mAbs to CD11b and CD18
to a greater degree than by other anti-integrin Abs that were
tested (Fig. 9, D and E).
Since factor H is present in plasma at a concentration of 500 µg/ml,
it was important to determine whether soluble factor H could affect
oxidant radical production by adherent PMNs. The sensitive fluorescence
assay that we used to measure hydrogen peroxide (Fig. 7) could not be
used to answer this question because soluble factor H could quench the
fluorescence. Therefore, we employed a less sensitive colorimetric
assay for superoxide generation by PMNs. The assay is based on the
change in absorbance at 550 nm experienced by ferricytochrome
c when it is reduced by superoxide anion. In this experiment
PMNs were allowed to adhere to wells coated with HSA, fibrinogen, C9,
or factor H. Figure 10 shows that the
superoxide anion generation by cells adherent on factor H was
considerably larger (twofold after 2 h) than on the other proteins
tested. Moreover, the presence of soluble factor H at the levels
normally found in plasma (500 µg/ml) did not inhibit this effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The fact that this phenomenon occurs even in half the cases
suggests that the affected cells are being activated by surface-bound H
to alter their cytoskeletal networks.
Two lines of evidence indicated that integrin CD11b/CD18 (Mac-1)
mediates PMNs adhesion to factor H. Monoclonal Ab inhibition of PMN
adhesion provided the first indication that CD11b/CD18 (Mac-1) is a
factor H receptor. The pattern of inhibition is similar for factor H
and C3bi; the latter protein is known to be recognized by CD11b/CD18
(46) (Fig. 3). The identity of Mac-1 (CD11b/CD18) as a factor H binding
protein was confirmed by affinity chromatography on factor H Sepharose,
followed by immunoprecipitation and Western blotting (Fig. 4). Factor H
shares CD18-integrin binding function with other well-characterized
leukocyte adhesion ligands. Previous studies using mAbs demonstrated
that ß2-integrins (CD18) mediate PMN adhesion to
matrix proteins laminin, collagen, and fibrinogen (42, 47, 48). The
oligospecificity of CD11b/CD18 (Mac-1) is well documented because it is
reported to interact with C3bi, fibrinogen, factor X, and denatured
albumin (23, 46, 49, 50, 51, 52). We demonstrate here that factor H can be
added to this collection of Mac-1 ligands.
In addition to visualizing the chains of Mac-1 after affinity
chromatography of PMN extracted protein, we observed an unknown band of
Mr 85,000 (Fig. 4A).
Therefore, the observation that Mac-1 can interact with factor H does
does not rule out the possibility that other PMN membrane proteins may
also bind this plasma protein. However, the collection of all data
converge on the conclusion that CD11b/CD18 is a factor H receptor.
The involvement of CD11b/CD18 in PMN adhesion to factor H cannot be a
consequence of a simple passive interaction between receptor and ligand
because, although eosinophils express this integrin, this cell type
failed to adhere to factor H (Table II). These results indicate that
CD11b/CD18 interacts with factor H, but the presence of this integrin
alone is not sufficient for factor H to mediate cell adhesion. Possible
explanations for this could be derived from conformation, focal
concentrations, and/or functional dynamics of Mac-1 (CD11b/CD18)
molecules (14, 15, 53). Eosinophils are a heterogeneous cell type that
can be brought to various stages of development and activation by
cytokines such as granulocyte-macrophage (GM)-CSF, IL-3, and IL-5 (54).
While the current studies indicate that these cells, as they are
isolated from blood, do not employ factor H for adhesion, the
possibility exists that some activated state of eosinophils could
do so.
In keeping with the known requirement of divalent metal ions for
integrin-ligand interaction (43, 55), both the binding of factor H to
PMNs and adhesion of PMNs to factor H-coated surfaces was dependent on
divalent metal ions in the buffer (Table I) (13).
The results presented here indicate that many effects of surface-bound
factor H on PMN function have parallels with those of other adhesive
ligands for this cell type. C5a and TNF- augmented the adhesion of
PMNs to factor H but not to BSA or C9; however, IL-8 had little effect
on any of these (Fig. 5). Corresponding with those results C5a and
TNF-, but not IL-8, released greater amounts of hydrogen peroxide
when the cells were resident on factor H, but not when they were on BSA
or C9 (Fig. 7). The data presented here describing the influence of
factor H on PMN function are consistent with published findings for
effects of other adhesion ligands on PMNs. TNF- and FMLP, but not
IL-8, were found to augment the adhesion of PMNs for fibronectin (56),
and TNF- was reported to increase the integrin-dependent
(CD11b/CD18) adhesion to fibrinogen (35). TNF- evoked a prolonged
release of hydrogen peroxide from PMNs adherent to fibrinogen,
fibronectin, laminin, and thrombospondin but not from cells in
suspension (57, 58). These adhesion-dependent responses could be a
consequence of up-regulation of CD11b/CD18 from stored pools as a
consequence of C5a or TNF- stimulation (59, 60).
In addition to providing a support for an enhanced respiratory burst,
factor H stimulated the release of the specific granule protein,
lactoferrin (Fig. 8). The degranulation of this specific granule
protein was most appreciable when TNF- was employed as a mediator
(Fig. 8C). Lactoferrin appears to function in the
host defense as an anti-microbial protein (61). Thus, factor H
could stimulate PMNs to kill microorganisms by facilitating the
respiratory burst as well as by evoking increased specific granule
release.
Although factor H is an adhesive ligand for neutrophils, it does not support fibroblast adhesion. However, plasma contains a truncated form of factor H (FHL-1) (which consists of the first seven of the twenty CCP modules of factor H) that does have adhesive activity for fibroblasts. This interaction is mediated through integrins that recognize the canonical Arg-Asp-Gly sequence, which is found in module 4 (62). Therefore, factor H and FHL-1 serve adhesive roles for the host defense system in addition to the well-established regulatory role for the alternative pathway of complement.
It is instructive to compare factor H with other plasma proteins that can engage in adhesive interactions with leukocytes. The group of proteins that includes factor H, FHL-1, the von Willibrand factor, fibrinogen, fibronectin, derivatives of C3, and vitronectin exhibit only limited modular homology among themselves. Yet all these molecules are large proteins that are present in reasonable abundance in plasma; all interact with leukocyte integrins; all contain the tripeptide sequence (Arg-Asp-Gly) (although this is not a recognition unit in all cases); all except fibrinogen interact with heparin; and all are involved in some way with the host defense to trauma and infection (63, 64, 65, 66).
Factor H is a soluble protein present in plasma at a concentration of
500 µg/ml, and it may operate similarly to vitronectin and
fibronectin. These proteins are also present in plasma at high
concentrations and are able to deposit at sites of tissue injury or
inflammation to mediate adhesive interactions with leukocytes (64, 65).
One plausible mechanism as to how factor H could serve as an adhesion
ligand would be if it could become immobilized to proteoglycans
expressed on activated endothelium or on the extracellular matrix after
tissue injury. Credence for this hypothesis comes from the reported
observation that factor H contains a specific glycosaminoglycan binding
site at module 7 (37). Experiments presented here show that factor H
can interact with heparin or chondroitin A to provide a more suitable
surface for the adherence of PMNs (Fig. 9). Although heparin itself
supports neutrophil adhesion (67), addition of factor H to heparin
creates a better adhesive surface (Fig. 9). Moreover, soluble factor H
neither inhibited adhesion of neutrophils to wells coated with factor H
(Fig. 9A), nor did it inhibit the generation of
superoxide anion by PMNs resident on factor H (Fig. 10). The fact that
soluble factor H does not act as a competitive inhibitor with
surface-associated factor H for cell adhesion implies that plasma
factor H will not interfere with the function of immobilized H in
mediating adherence for PMNs in vivo. The lack of competition between
soluble and bound factor H for PMN adhesion can be rationalized by the
fact that cell adherence is not a simple equilibrium reaction but a
complicated cellular behavior involving, among other things,
ligand-cytoskeletal association and cytoskeletal reorganization. Tissue
edema and microvascular leakage of plasma proteins occur in regions of
trauma, inflammation, or infection. These processes expose
glycosaminoglycans that may be suitable for the fixation and
immobilization of factor H (66). Hence, the absorption of factor H to
glycosaminoglycans on basement membranes or activated endothelium may
mediate PMN adherence regardless of the high concentration of this
protein in plasma. Whether injury, infection, or inflammation in vivo
actually do result in factor H adsorption will require further study,
but the data presented here at least indicate that this idea is
plausible.
In summary, a novel function is ascribed to complement factor H;
namely, it is an adhesion ligand for neutrophils. These cells employ
Mac-1 (CD11b/CD18) to adhere to this protein. The adhesive interaction
of factor H with these cells augments the oxidant burst evoked by C5a
or TNF- and increases lactoferrin release. Proteoglycans may serve
to adsorb factor H in vivo, which would enable this protein to
facilitate selective neutrophil accumulation and activation at sites of
injury or inflammation. These new results can be integrated into the
current understanding dealing with leukocyte migration. It is now
recognized that leukocyte mobilization in vivo involves a multistep
adhesion cascade including sequential rolling, firm adhesion, and
trans-endothelial migration (68). It is possible that rolling PMNs may
utilize immobilized factor H in addition to several other adhesion
molecules exposed at sites of vascular injury or inflamed endothelium
to fulfil their function.
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Footnotes |
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2 Address correspondence and reprint requests to Richard G. DiScipio, La Jolla Institute for Experimental Medicine, 505 Coast Boulevard South, La Jolla, CA 92037.
3 Abbreviations used in this paper: PMNs, polymorphonuclear leukocytes; HSA, human serum albumin.
Received for publication May 2, 1996. Accepted for publication December 19, 1997.
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), C9 (
), or
factor H (
). Various concentrations of TNF-
), or factor H (
), C9 (
), fibrinogen (
),
C9 (