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The Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Integrins are a large family of 
/ß heterodimeric adhesion
receptors and can be divided into subfamilies according to their common
ß subunits. CD11b/CD18 (Mß2, Mac-1, CR3)
is a member of the CD18 subfamily of integrins, which also contains
CD11a/CD18 (Lß2, LFA-1), CD11c/CD18
(Xß2), and a recently discovered fourth
member, D/CD18(Dß2) (10).
CD11b/CD18 is found on all major classes of leukocytes, including
virtually all neutrophils and macrophages as well as subsets of NK
cells, T cells, and B cells (3, 10). CD11b/CD18 has been shown to play
key and often the principal role in leukocyte adhesion, extravasation,
migration, phagocytosis, and degranulation-cytotoxicity (3). Two
domains have been implicated in the binding functions of CD11b/CD18
(3). The integrin recognizes protein ligands via a segment of about 200
amino acids in the CD11b subunit, termed the I domain (10). CD11b/CD18
recognizes mannose and ß-glucan carbohydrate structures by a
lectin-like ligand binding domain also in the CD11b () subunit (11).
I domain ligands include ICAM-1, C3bi, fibrinogen
(Fg),3 and the helminth
ligand neutrophil inhibitory factor (NIF) (12, 13, 14, 15). The lectin domain
ligands include zymosan, Saccharomyces cereviseae, and
nonspecific activators of cell-mediated immunity such as lentinan (11).
For Leishmania sp., B. dermatitidis, and B.
pertussis the protein ligands that bind to the I domain and the
carbohydrate ligands that bind to the lectin domain have been
identified (1, 2). Previous studies have suggested that recognition of
C. albicans (8), as well as other fungi (4), by CD11b/CD18
depends upon both the I domain and the lectin domains within its CD11b
subunit.
The structural basis for the ligand binding to these two domains of CD11b/CD18 has begun to emerge. The crystal structure of the CD11b I domain was solved and revealed a novel divalent cation coordination site, the MIDAS motif, which is essential for the ligand binding functions of CD11b/CD18 (16). Certain ligands of CD11b/CD18 bind to the expressed I domain in a cation-dependent manner (17, 18, 19, 20), and many CD11b/CD18 blocking Abs map to the I domain (21). The lectin-like adhesion site of CD11b/CD18 was also mapped using carbohydrate ligands (11). This domain resides in an area proximal to the cell membrane and exhibits a broad specificity for ß-glucan (glucose) and mannan (mannose) polysaccharides. Subsequent studies showed that binding of such carbohydrates to the lectin domain of CD11b resulted in priming of the receptor for phagocytosis and cytotoxicity, which involved a tyrosine kinase, a Mg-dependent conformational change in the I domain, and exposure of the mAb CBRM1/5 activation epitope (22). Consistent with this observation, certain mAbs can activate CD11b/CD18 adhesion by binding to the lectin domain (23). These studies support data showing that the I and the lectin domains are both involved in CD11b/CD18-mediated adhesion to intact microorganisms, including C. albicans (5, 8).
Previously, we have used the homologue-scanning mutagenesis strategy
(24) to analyze the binding site for the I domain ligands of CD11b/CD18
(25). This strategy takes advantage of the known crystal structures of
the CD11a and CD11b I domains and entails swapping homologous
structural motifs, either -helical segments, ß-sheets, or their
connecting loops from CD11a into CD11b to identify requisite sites for
ligand binding function while conserving the structure of CD11b/CD18.
Mutated CD11b/CD18 receptors containing CD11b I domain segments of
6–10 amino acids "swapped" with their corresponding segments of
CD11a I domain have been stably expressed in 293 cells. We have
demonstrated previously that these mutant CD11b/CD18 receptors exhibit
the conformation of the wild-type receptor by a variety of criteria
(25). Using these mutant cell lines, we have shown that the I domain
binding pockets for NIF, C3bi, and Fg are overlapping but not identical
(26). Recently, these mutants were used to map the CD11b/CD18 binding
pocket for NIF (25). The experiments in the present study describe the
utilization of these mutants to map the segments of the CD11b/CD18 I
domain that mediate adhesion to C. albicans and identify
C. albicans as a unique CD11b/CD18 ligand. Additional mutant
receptors have been used to examine the roles of the lectin-like domain
and the CD18 subunit in the recognition of this fungal ligand.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. albicans (58716, American Type Culture Collection, Manassas, VA) was provided by Dr. Herbert L. Mathews, Loyola University of Chicago (Maywood, IL), and used throughout this investigation. Cultures were stored at 25°C on Sabouraud’s dextrose agar (Difco, Detroit, MI). Cells used for experimentation were cultured overnight at 37°C on Sabouraud’s dextrose agar, collected as isolated colonies, washed once in complete HBSS, and counted with a hemocytometer.
NIF protein and mAbs
The NIF protein was provided by Corvas International (La Jolla, CA). The mAbs used were as follows: OKM1 (anti-CD11b, IgG2b), 44a (anti-CD11b, IgG1), LM2/1 (anti-CD11b, IgG1), IB4 (anti-CD18, IgG2a), and W6/32 (anti-MHC class I, IgG1; all from American Type Culture Collection). These Abs were purified from conditioned tissue culture medium using a column of recombinant protein G as described by the manufacturer (Zymed, San Francisco, CA). All anti-human mAbs were of mouse origin, and the secondary Ab used for immunofluorescence analysis was FITC goat anti-mouse IgG (Zymed). Protein concentrations were determined spectrophotometrically.
Cell lines and culture
The human kidney 293 fibroblastoid cell line was maintained as described previously (27) in DMEM-F12 plus 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (all from BioWhittaker, Walkersville, MD). Adherent cells were removed for passage and experiments using enzyme-free cell dissociation buffer (Life Technologies, Grand Island, NY). Construction of 293 cells stably expressing wild-type (WT) or mutant forms of CD11b/CD18 has been previously described (26). Before use in adhesion assays, expression levels of CD11b/CD18 were verified to be similar for all mutant and WT cells using FACS analysis. CD11b/CD18 mutants with low expression levels were enriched for clones with expression levels similar to WT using limiting dilution followed by FACS analysis to select for higher expressing clones. For cases in which this step was required, data were pooled from at least two separate clones. CD11b/CD18 expression also was verified at the time of the experiments to insure similar levels of CD11b/CD18 expression between WT and mutant cells. The mean fluorescent intensities for the WT and mutants used in this study are approximately 300 when stained with an anti-CD11b mAb (OKM1), compared with 5 for mock-transfected cells. All FACS analysis was performed with FACStar (Becton Dickinson, San Jose, CA) in the core flow cytometry facility at The Cleveland Clinic supported by the Keck Foundation. The expression levels of the cell lines used in this study differed by <50%. There was no correlation between the adhesive activity and the CD11b/CD18 expression levels within this range. For example, two subclones of WT-CD11b/CD18 with mean fluorescence intensities of 358 and 697 had 2.5 x 104 and 1.5 x 104 adherent cells, respectively, when a total of 5.0 x 104 cells were added to the assay. The average was 1.99 ± 0.39 x 104 adherent cells over 20 independent experiments.
IL-2-activated human lymphocytes
Human PBMC were obtained by venipuncture from normal donors with informed consent and were isolated using Ficoll-Histopaque (Pharmacia, Piscataway, NJ) by centrifugation at 2000 x g for 25 min. Lymphocytes at the interface were removed with a sterile Pasteur pipette, washed once in HBSS, and cultured in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µM nonessential amino acids, and 2 mM L-glutamine (all from BioWhittaker, Walkersville, MD) as well as 50 µM 2-ME. The cells were distributed at 2.5 x 106 cells/ml into Costar 24-well plates (Becton Dickinson, Lincoln Park, NJ) with 1500 U/ml IL-2 (Sigma, St. Louis, MO), and they were harvested after 7 days at 37°C in 5% CO2. These cells were >99% lymphocytes as judged by Wright staining.
Calcein labeling of 293 cells and lymphocytes
Human 293 kidney cells and IL-2-activated lymphocytes were labeled with the fluorochrome calcein-AM (Molecular Probes, Eugene, OR) for use in adhesion assays. The calcein-AM solution was prepared fresh for each experiment using 50-µg aliquots provided by the manufacturer by initial solubilization in 10 µl of DMSO followed by 950 µl of HBSS without Ca2+ or Mg 2+. Cells were suspended at a concentration of 107/ml in DMEM F-12 with 0.5% BSA (Sigma) and 5 µg/ml calcein-AM (100 µl) for 30 min at 37°C in 5% CO2. Labeling was determined to have no effect on cell viability as assessed by trypan blue staining.
Adhesion of lymphocytes to C. albicans
This assay was performed as described previously (28) with some modifications. Briefly, C. albicans hyphae were prepared by growth of 105 yeast in 50 µl of RPMI 1640/well (without serum) for 3 h at 37°C in flat-bottom 96-well plastic plates (Corning, Corning, NY). After 3 h, 90–100% confluence of hyphae was obtained, and 100 µl of DMEM/Ham’s F-12/0.5% BSA was added per well to reduce nonspecific binding. After 30 min, calcein-AM-labeled cells were added to wells of the assay plates, the final volume was adjusted to 250 µl/well using DMEM/Ham’s F-12/0.5% BSA, and the plate was incubated in a 5% CO2 incubator at 37°C for 1 h. The assay was terminated by removal of unbound cells from each well by gently washing three times with 200 µl of DMEM/Ham’s F-12/0.5% BSA using a multichannel pipette. For each cell concentration tested and in each experiment, triplicate wells were washed, and another triplicate set remained unwashed for calculation purposes. Fluorescence was then measured using a fluorescence plate reader (Cytofluor, San Diego, CA), and the values obtained were converted to cells bound per well using the Excel software program (Microsoft, Seattle, WA) to construct a standard curve from the fluorescence of the unwashed wells at the various cell concentrations added and to compute mean fluorescence per cell. Adhesion of WT CD11b/CD18 293 cells was assessed in each experiment and was assigned a value of 100% adhesion. Background (nonspecific) adhesion was determined for each cell type for every experiment as the cells remaining bound after washing when the cells were added to wells containing no hyphae. Data for all cell types (experimental) were then expressed as the percentage of WT adhesion after subtraction of background from each value using the formula: % WT adhesion = [(mutant cells bound - background) ÷ (WT cells bound - background) x 100]. Background adhesion averaged 7–10% for all cells tested. Data represent the mean percent WT adhesion for triplicate values of three or more experiments ± SD.
Competition for binding of lymphocytes to C. albicans
This procedure was performed as described for the adhesion of cells to C. albicans, except that 5 x 104 calcein-AM-labeled cells were preincubated for 1 h with the indicated proteins or mAbs at 37°C in 200 µl of DMEM/Ham’s F-12 in polypropylene test tubes. The entire 200-µl preincubation mixture was transferred to wells containing C. albicans. Background was determined and subtracted from total counts as described above. For all inhibition experiments, fluorescence per cell was determined as described above and expressed as the percent inhibition of cell adhesion relative to binding of untreated cells using the formula: % inhibition = [1 - [(cells bound treated experimental - background) ÷ (cells bound untreated - background)] x 100]. Data are calculated as the mean percent inhibition for triplicate values of three or more experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous studies (10, 12, 29), including those from our laboratory
(26), have demonstrated that CD11b/CD18 can be expressed at high levels
on cell surfaces as functional heterodimers. We initially assessed the
ability of the WT-CD11b/CD18 receptor expressed on 293 cell lines to
mediate cell adhesion to C. albicans. The hyphal form of
C. albicans was used in these experiments and was obtained
by growing the yeast for 3 h at 37°C in 96-well plates. The
formation of an extensive hyphal network was verified by microscopic
examination in each experiment. Transfected 293 cells, labeled with
calcein, were added over a range of cell concentrations to the C.
albicans hyphae, and the extent of adhesion was quantitated at
1 h. As shown in Fig. 1
, adhesion
increased as a function of the number of cells added. The optimal
number of cells added was determined to range from 104 to
5 x 104 cells/well, and individual concentrations of
104, 2 x 104, 3 x 104,
and 5 x 104 cells/well were examined in subsequent
experiments. As a negative control, the adhesion of mock-transfected
cells also was evaluated at these same cell concentrations in adjacent
wells. To account for variability among individual experiments and to
allow comparison between experiments, data were expressed as binding of
cells, as a percentage of WT transfectants. Binding of WT-CD11b/CD18
cells was 5- to 10-fold greater than that of mock-transfected cells at
all cell concentrations tested. As shown in Fig. 1, the recombinant
WT-CD11b/CD18 behaved in a similar manner as the IL-2-activated human
lymphocytes. Previous studies have shown that adhesion of such
activated lymphocyte preparations to C. albicans is
CD11b/CD18 mediated (30). Fig. 1 shows that the extent of adhesion of
both cell types to the hyphae was similar. In contrast, 293
transfectants expressing CD11a/CD18 at a similar level as the
WT-CD11b/CD18 transfectants adhered minimally to the hyphae; the extent
of adhesion of the CD11a/CD18 transfectants was comparable to that of
the mock-transfected cells.
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). These inhibitors included several
mAbs (OKM1, 44a, and IB4) to different regions of CD11b/CD18. The
anti-MHC class I mAb W6/32, which does bind to the 293
transfectants, was used as a negative control. mAb 44a to the I domain
of CD11b strongly inhibited the adhesion of WT-CD11b/CD18 transfectants
and IL-2-activated lymphocytes to the hyphae; adhesion of both cell
types was inhibited by >80%. Treatment with the control mAb (W6/32)
had no effect on the adhesion of either cell type to the hyphae.
Consistent with a role for the I domain of CD11b in hyphal recognition,
NIF, a CD11b/CD18 I domain-specific ligand (25), eliminated adhesion of
both the WT-CD11b/CD18 transfectants and IL-2-activated lymphocytes to
the hyphae. In addition, an anti-CD18-blocking mAb, IB4, also
strongly inhibited (
80% inhibition) WT-CD11b/CD18 and
IL-2-activated lymphocyte adhesion to the hyphae. OKM1, a mAb that
blocks the function of the lectin domain of CD11b (11), inhibited
adhesion of WT-CD11b/CD18 cells and IL-2-activated lymphocytes by 50%,
and a further increase in the OKM1 concentration did not substantially
increase the extent of inhibition. Taken together, these data
demonstrate the role of CD11b/CD18 in mediating cell adhesion to
C. albicans hyphae and are consistent with a contribution of
multiple domains in recognition of the pathogen by the receptor (8).
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To dissect the roles of individual CD11b and CD18 chains in
adhesion, we conducted binding experiments with the transfected cells
expressing only the CD11b subunit or the heterodimeric WT-CD11b/CD18
(Fig. 3). Cell surface expression of
CD11b in the absence of CD18 on 293 cells and lack of association with
other endogenous ß subunits have been verified previously using
several independent approaches, including FACS analysis with a
CD11b-specific mAb (44a) and a CD18-specific mAb (IB4) and surface
labeling and immunoprecipitation (25). The cells expressing only CD11b
bound hyphae well, although at a reduced level compared with
WT-CD11b/CD18 (54–76% of WT). To insure that the decreased adhesion
was not a result of small differences in receptor expression, several
subclones of the CD11b-only cells were developed and tested. These
subclones expressed from 50–100% of WT levels of CD11b by FACS
analyses, and no correlation was found between expression level and the
extent of adhesion among these cell lines (not shown). The specificity
of CD11b-C. albicans interaction was verified using the
specific blocking mAbs noted above. Both OKM1 and 44a inhibited
adhesion of CD11b cells to hyphae, while the CD18-specific mAb IB4,
which inhibited binding of the cells expressing the heterodimer (see
above), had no effect on adhesion of CD11b-only cells. Thus, the CD11b
subunit alone is sufficient to mediate hyphal binding and suggests that
the CD18 subunit does not provide a major contact site for
C. albicans.
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, mutation of Ser138
to Ala abolished CD11b/CD18 adhesion to C. albicans. Cell
surface expression of the WT and mutant CD11b/CD18 was similar as
judged by FACS (26). Therefore, although CD18 is not required for
adhesion to C. albicans, i.e., CD11b in the absence of CD18
supports cell adhesion, mutation of Ser138 abrogates the
adhesive activity of the CD11b/CD18 complex. Roles of the lectin-like domain in CD11b/CD18-mediated adhesion to C. albicans
To investigate the role of the CD11b lectin-like domain in
adhesion to C. albicans, cells were developed expressing a
heterodimer in which the I domain of CD11a was substituted with that of
CD11b (termed L/M). CD11a/CD18 has been shown to lack the lectin
binding activity found in CD11b/CD18 (11). In the absence of the CD11b
lectin-like domain, the chimeric CD11a/CD18-expressing cells (L/M),
which contains only the I domain of CD11b, bound C. albicans
well, ranging from 60–82% of the WT-CD11b/CD18 adhesion (Fig. 4). Verifying the specificity, the
wild-type CD11a/CD18-expressing cells failed to adhere to the fungi.
These data demonstrate that the CD11b/CD18 lectin-like domain is not
required for CD11b/CD18-mediated adhesion to hyphae.
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). Each carbohydrate was used at a
concentration of 150 mM, previously shown to maximally inhibit the
function of the lectin-like domain (11). ß-D-glucose was
used as a negative control. As shown in Fig. 5, N-acetyl-D-glucosamine and
methyl-ß-glucopyranoside inhibited adhesion of the WT-CD11b/CD18
cells by 60 and 70%, respectively, demonstrating a contribution of the
lectin-like domain in CD11b/CD18 to adhesion. In contrast, these
carbohydrates had no effect on L/M adhesion (Fig. 5). In separate
experiments, we evaluated the effects of these sugars on CD11b/CD18-NIF
interaction. At 150 mM, none of the three carbohydrates had any effect
on NIF binding to the transfected cells as judged by FACS analysis
using biotinylated NIF and avidin-FITC conjugate (25) (data not shown).
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, the
inhibitory carbohydrates, N-acetyl-D-glucosamine
and methyl-ß-glucopyranoside, had no effect on adhesion of this
constitutively active CD11b/CD18 receptor, in contrast to their potent
inhibition of the WT-CD11b/CD18 receptor. Mapping the C. albicans binding region within the CD11b I domain
Since mAbs to the CD11b I domain and NIF blocked CD11b/CD18
adhesion to C. albicans (Fig. 2), and since the chimeric
CD11a/CD18 receptor containing the CD11b I domain adhered well to the
fungus (Fig. 4), we sought to localize the specific segments within the
CD11b/CD18 I domain that mediate this adhesion. The adhesion to
C. albicans of 10 homologue-scanning mutants within the I
domain was assessed. These previously described mutants contain swaps
of homologous small structural elements, -helixes, ß-sheets, and
connecting loops, of the CD11a I domain into the CD11b I domain (25).
The adhesion of these mutants to C. albicans hyphae was
assessed over the range of cell concentrations shown in Fig. 1, but
only data from a representative concentration of 5 x
104 cells are shown (Fig. 6).
The 10 mutants assessed were CD11b (P147-R152), CD11b (M153-T159),
CD11b (Q190-S197), CD11b (E178-T185), CD11b (R208-K217), CD11b
(K245FG), CD11b (D248-Y252), CD11b (E253-R261), CD11b (D273-K279), and
CD11b (R281-I287). Adhesion of these mutants to the hyphae fell into
two general categories; those that are similar to WT-CD11b/CD18,
including CD11b (M153-T159), CD11b (Q190-S197), CD11b (E178-T185),
CD11b (K245FG), and CD11b (D273-K279) (Fig. 6a), and those
that demonstrate virtually no adhesion to C. albicans,
including CD11b (P147-R152), CD11b (R208-K217), CD11b (D248-Y252),
CD11b (E253-R261), and CD11b (R281-I287) (Fig. 6b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In undertaking the present analyses, we were greatly concerned that differences in expression levels of the various mutants might influence CD11b/CD18-mediated adhesion to the fungal hyphae. Accordingly, we only compared cells that expressed equivalent levels of WT and mutated forms of CD11b/CD18 as assessed by FACS analyses. These analyses were performed with each cell type before and after adhesion experiments, and low expressing mutants were subcloned to select for high expression equivalent to WT. Thus, the expression level of the transfectants used in our study differed by <50%. In addition, WT-CD11b/CD18 and mock-transfected cells as well as background binding for each cell type to wells containing no fungus were determined as internal controls for each experiment. The physiologic relevance of the CD11b/CD18-expressing 293 cells used in our study was verified by the demonstration that these cells behaved qualitatively and quantitatively similarly to IL-2-activated lymphocytes, which are known to use CD11b/CD18 to bind C. albicans (30). Furthermore, CD11a/CD18-expressing 293 cells did not bind to hyphae specifically, and this integrin does not play a role in C. albicans binding to leukocytes (8, 30). The failure of CD11a/CD18 to recognize the hyphae allowed implementation of the homologue-scanning strategy in which segments of CD11a were exchanged for the corresponding segments of CD11b to map the I domain contact segments for C. albicans.
The role of the CD11b subunit in CD11b/CD18 ligand binding is well
documented (17, 20, 21, 32) and is confirmed in this study. Transfected
cells expressing only CD11b bound specifically to the hyphae. An mAb to
the CD11b subunit that blocked binding of the heterodimeric receptor to
the fungus remained inhibitory, whereas a mAb to the CD18 subunit lost
inhibitory function. The CD11b cells did not bind to C.
albicans as efficiently as the WT cells (65% WT; Fig. 3), but the
modest reduction in binding precludes the existence of a substantial
number of direct contacts between the hyphae and CD18. However, the
function of the CD18 subunit in C. albicans binding to the
heterodimeric receptor or, for that matter, of other ligands to this or
other integrins is significant but remains difficult to define. The
D134XSXS sequence of CD18 has been previously demonstrated to be
critical for the recognition of several CD11b/CD18 ligands. Mutation of
D134, S136, or S138 eliminated adhesion of CD11b/CD18-expressing cells
to dOva, C3bi, ICAM-1, and fibrinogen (26, 29), and C.
albicans can be added to this list. The homologous region within
ß3 also is critical to the ligand binding function of
this integrin subfamily (33), and a naturally occurring point mutation
at one of these residues has been reported to fully inactivate these
receptors (34). Recently, it has been proposed that the ß subunits of
integrins, including CD18, contain an I domain-like structure, and the
DXSXS sequence is part of a MIDAS motif providing cation coordination
sites (35, 36, 37, 38) and also may contain direct ligand contact sites as well
(39, 40, 41). However, as demonstrated in this study, cells expressing
CD11b alone adhered well to C. albicans, and removal of the
entire CD18 subunit had limited effects on the recognition of this
ligand, suggesting that, at least for this ligand, the CD18 subunit is
not required for ligand engagement. Thus, the consequence of mutation
of Ser138 is the creation of a dominant negative effect,
which masks the intrinsic activity of the CD11b subunit. The
mechanism of this effect is unclear. One possibility is that the
absence of this residue changes the cation coordination complex to one
that is incompatible with ligand binding. Clearly, the relationship
between ligand and cation binding is vital to integrin function (42).
Previous studies with C. albicans (8) and with other ligands
(22, 43) have implicated the lectin-like domain in CD11b/CD18
functions. In this study, the lectin domain blocking mAb OKM1 and two
carbohydrate ligands of the lectin-like domain significantly reduced
the adhesion of both WT transfectants and IL-2-activated lymphocytes.
Although a large proportion of the surface of C. albicans is
composed of ß-glucan polymers, this domain is not sufficient for
binding of C. albicans to the receptor. This conclusion is
supported by the observations that 1) certain receptors with mutations
only in the I domain and without alterations in the lectin domain
failed to adhere to C. albicans; and 2) the chimeric
CD11a/CD18 receptor that contained only the I domain of CD11b and
lacked the lectin-like domain (the carbohydrates that inhibited WT
adhesion had no effect on the function of the chimeric receptor) was
able to bind the fungus to an extent similar (80%) to that of the
WT-CD11b/CD18 cells. The mechanism by which the lectin-like domain
modulates CD11b/CD18 function appears to reside in its modulation of I
domain conformation. The mutant CD11b/CD18 receptor,
CD11b(Q190-S197)/CD18, contains an activating mutation within the I
domain (31), and the inhibitory carbohydrates did not block C.
albicans binding by this activated receptor. These data and
interpretation are consistent with the capacity of the lectin
domain-specific mAb VIM12 to exert an activating effect on I
domain-mediated adhesion of CD11b/CD18 as observed in association with
homotypic aggregation (23). Recently, several studies have suggested
the existence of a promiscuous lectin binding site with somewhat
distinct sugar specificity within CD11a/CD18 (44). In the same study,
N-acetyl-D-glucosamine was shown to inhibit the
association between CD11a/CD18 and the Fc
R333 receptor (44).
Although this polysaccharide had no effect on L/M chimera adhesion to
hyphae (Fig. 5), our data cannot rule out the possibility that a lectin
site with different carbohydrate specificity may exist in CD11a/CD18
and could participate directly in L/M chimera interactions. Hyphae
binding to a low affinity I domain may require the assistance of the
lectin domain (in the cases of WT and L/M chimera), whereas a high
affinity I domain can overcome the requirement for an additional
contact site in the lectin domain (in the case of the CD11b(Q190-S197)
mutant). Nevertheless, since cells expressing WT CD11a/CD18 and mutant
CD11b/CD18 with intact lectin domain did not bind hyphae, the direct
contribution from the lectin domain to hyphae binding, if any, cannot
be substantial. The lectin domain also could be involved in directly
priming CD11b/CD18 for antifungal cytotoxicity, a possibility that was
not addressed in this study (22, 30).
That the I domain provides the major contact interface for C.
albicans is supported by the following experiments. First, CD11b I
domain-specific mAbs and the high affinity CD11b/CD18 ligand (NIF) that
has been shown to interact exclusively with the I domain (25) blocked
CD11b/CD18 adhesion to C. albicans. Second, the chimeric
CD11a/CD18 receptor containing the CD11b I domain adhered well to the
fungi. Accordingly, we sought to localize the functional segments
within the I domain using our existing homologue-scanning mutants. Of
the 10 representative mutants we tested, five (CD11b (M153-T159), CD11b
(Q190-S197), CD11b (E178-T185), CD11b (K245FG), and CD11b (D273-K279))
had similar C. albicans adhesion to WT-CD11b/CD18 (Fig. 6a). Of note are mutants CD11b (K245FG) and CD11b
(D273-K279). CD11b (K245FG) has approximately 60% the adhesive
activity of WT-CD11b/CD18. This mutant binds significantly less well to
C3bi (<20% of WT; our manuscript in preparation), thus
distinguishing C. albicans from C3bi as a CD11b/CD18 ligand.
The CD11b (D273-K279) mutant has been shown previously to exhibit
virtually no adhesion to Fg (26), although it bound well to hyphae.
This result distinguishes C. albicans from Fg as a
CD11b/CD18 ligand. Five other mutants, including CD11b (P147-R152),
CD11b (R208-K217), CD11b (D248-Y252), CD11b (E253-R261), and CD11b
(R281-I287), demonstrate virtually no adhesion to C.
albicans (Fig. 6b). The first four of these segments
have been shown to constitute the NIF binding site within the I domain
(Fig. 7) (25). Thus, C.
albicans shares an overlapping ligand binding site with NIF,
providing the molecular basis for the potent inhibitory activity of NIF
toward C. albicans binding. However, the CD11b (R281-I287)
and CD18 (S138A) mutations have no effect on NIF recognition (25) while
eliminating adhesion to C. albicans, thus setting these
hyphae apart from NIF as a microbial ligand for CD11b/CD18. Taken
together, our data show that the NIF binding site (P147-R152,
R208-K217, D248-R261) represents the core structure of a broad surface
that is involved in establishing the oligospecificity of the CD11b/CD18
integrin toward its multiple and unrelated ligands. The C.
albicans recognition site encompasses the entire NIF binding site
plus an additional segment (R281-I287; Fig. 7).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Li Zhang, The Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.
3 Abbreviations used in this paper: Fg, fibrinogen; NIF, neutrophil inhibitory factor; WT, wild-type.
Received for publication April 30, 1998. Accepted for publication July 29, 1998.
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subunit of integrin CR3 (CD11b/CD18). Cell 80:631.[Medline]
-chain (C11b) potentially involved in intramembrane complex formation with glycosylphosphatidylinositol-anchored Fc-RIIIB (CD16) molecules. J. Immunol. 154:5452.[Abstract]
Mß2 for a specific and high affinity ligand, NIF. J. Biol. Chem. 272:17558.Mß2 integrins. J. Biol. Chem. 271:18211.-carboxyglutamic acid () variant (6D, 7D) of human activated protein C displays greatly reduced activity as an anticoagulant. Biochemistry 29:10828.[Medline]
Lß2, CD11a/CD18) and MAC-1 (Mß2, CD11b/CD18). J. Biol. Chem. 270:94.Mß2. J. Biol. Chem. 271:29953.IIbß3 is dependent on a MIDAS-like domain in the ß3 subunit. J. Biol. Chem. 271:21978.Lß2 and Mß2 ligand interactions. J. Biol. Chem. 271:23729.vß1 and vß3 integrins by swapping a short diverse sequence of the ß subunit. J. Biol. Chem. 272:19794.receptor III on human neutrophils. J. Immunol. 150:3030.[Abstract]
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