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Division of Experimental Immunology and Immunopathology, Department of Pathology, and
Department of Microbiology and Immunology, University of Louisville, Louisville, KY 40292
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Mß2 integrin) functions as
both a receptor for the opsonic iC3b fragment of C3 triggering
phagocytosis or cytotoxicity and an adhesion molecule mediating
leukocyte diapedesis. Recent reports have suggested that a CR3 lectin
site may be required for both cytotoxic responses and adhesion.
Cytotoxic responses require dual recognition of iC3b via the I domain
of CD11b and specific microbial surface polysaccharides (e.g.,
ß-glucan) via a separate lectin site. Likewise, adhesion requires a
lectin-dependent membrane complex between CR3 and CD87. To characterize
the lectin site further, a recombinant baculovirus (rBv) system was
developed that allowed high level expression of rCD11b on membranes and
in the cytoplasm of Sf21 insect cells. Six rBv were generated that
contained truncated cDNA encoding various CD11b domains. Immunoblotting
of rBv-infected Sf21 cells showed that some native epitopes were
expressed by five of six rCD11b fragments. Lectin activity of rCD11b
proteins was evaluated by both flow cytometry with ß-glucan-FITC and
radioactive binding assays with [125I]ß-glucan. Sf21
cells expressing rCD11b that included the C-terminal region, with or
without the I-domain, exhibited lectin activity that was inhibited by
unlabeled ß-glucan or anti-CR3 mAbs. The smallest rCD11b fragment
exhibiting lectin activity included the C-terminus and part of the
divalent cation binding region. The ß-glucan binding affinities of
the three C-terminal region-containing rCD11bs expressed on Sf21 cell
membranes were not significantly different from each other and were
similar to that of neutrophil CR3. These data suggest that the lectin
site may be located entirely within CD11b, although lectin
site-dependent signaling through CD18 probably occurs with the
heterodimer. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M integrin, 165
kDa) and CD18 (ß2 integrin, 95 kDa) membrane
glycoproteins, is expressed by mature myeloid cells, NK cells, and
minor subsets of B and T cells. First identified as the macrophage
surface marker Mac-1 (1), it was later shown to function both as an
iC3b3 receptor (2) and an
adhesion molecule (3). As an adhesion molecule, it mediates the
diapedesis of leukocytes via generation of a high affinity binding site
for endothelial cell ICAM-1 (CD54) (4, 5, 6). Alternatively, when it
functions as an iC3b receptor, it mediates phagocytosis and
degranulation in response to micro-organisms or immune complexes
opsonized with iC3b (7, 8, 9). Regulation of these seemly disparate
functions is accomplished through a series of "inside-out" and/or
"outside-in" signaling steps that result in exposure of high
affinity binding sites and/or an altered linkage to the actin
cytoskeleton (10, 11). The presence of a lectin site in CR3 was first appreciated through the fortuitous observation that neutrophils from patients with leukocyte adhesion deficiency (a deficiency of CD18 synthesis) failed to ingest unopsonized zymosan, the insoluble polysaccharide cell wall fraction from baker’s yeast (12, 13). Later studies showed that CR3-dependent phagocytosis of iC3b-opsonized yeast required the simultaneous recognition on the yeast cell wall of opsonic iC3b and ß-glucan (14, 15). The selective blockade of these two recognition functions of CR3 by mAbs to two nonoverlapping epitopes of CD11b led to the proposal that CR3 contained separate binding sites for iC3b and ß-glucan (12, 14). These data also indicated that the ß-glucan-binding lectin site was required for phagocytosis of iC3b-opsonized yeast, as particles of pure yeast ß-glucan were phagocytosed avidly without need of opsonic iC3b, whereas iC3b-opsonized erythrocytes (EC3bi), whose membranes did not bear CR3-binding polysaccharides, were bound by CR3 without stimulating phagocytosis (12, 15).
The lack of CR3-binding polysaccharides on most mammalian cells
explains the inability of CR3 to mediate phagocytosis or
cytotoxicity of erythrocytes or tumor cells opsonized with
iC3b (16, 17, 18, 19). CR3 apparently evolved as a host defense system that
recognizes C3-opsonized bacteria and yeast, but ignores C3-opsonized
host cells by using its lectin site to recognize certain
microbe-specific polysaccharides such as ß-glucan. Nevertheless, this
tumor cell "deficit" in CR3-binding polysaccharides can be
circumvented therapeutically with small soluble
(1
3)-ß-D-glucan polysaccharides that bind with high
affinity to the lectin site of CR3 and prime the receptor on
circulating leukocytes for subsequent cytotoxic activation by
iC3b-opsonized tumor cells that are otherwise inert in stimulating
CR3-dependent cytotoxicity (20, 21). This was shown to be the mechanism
of action for a large family of soluble ß-glucan biological response
modifiers (BRM) that have been explored as potential therapeutic agents
against cancer (22, 23).
The putative lectin site differs from most animal lectins because CR3 lacks C-type lectin consensus sequences (24), and because lectin site binding of soluble polysaccharides does not require divalent cations (25). The lectin site appears to elicit bidirectional signaling. High affinity binding of a small (10–20 kDa) soluble zymosan-derived polysaccharide (SZP) to CR3 resulted both in exposure of an I domain neoepitope (inside-out signaling) and a tyrosine kinase-dependent priming of CR3 for cytotoxic degranulation responses (outside-in signaling) (20). A similar conformational change of the I domain induced by the lectin site was also proposed in another recent study that investigated the regions of CR3 involved in recognition of Candida albicans (26).
In addition to these functions with exogenous polysaccharides, a large
family of endogenous glycosylphosphatidylinositol (GPI)-anchored
membrane surface glycoproteins has been shown to form lectin-dependent
membrane complexes with CR3 as a mechanism for transmembrane signaling
(27, 28). Dependent upon the type of GPI-anchored signaling partner,
these complexes provide the critical regulation of CR3 that determines
whether it will function as an adhesion molecule or as a cytotoxic
trigger molecule. CR3 membrane complexes with CD16 (Fc
RIIIB) allow
triggering of neutrophil phagocytosis and degranulation in response to
IgG-opsonized particles that bind to the Fc receptor portion of the
complex (29, 30, 31). Conversely, CR3 is converted into an adhesion
molecule expressing its high affinity binding site for ICAM-1 by
lectin-dependent complex formation with CD87 (32, 33, 34, 35) or CD59 (36). The
requirement for complex formation between CD87 and CR3 for adhesion was
confirmed by demonstration that neutrophils from CD87 knockout mice
could not be stimulated to become adherent to ICAM-1 (37), and that
anti-CD87 mAbs either inhibited (34) or enhanced (37) the
CR3-dependent adherence of normal neutrophils. Additional reports have
implicated a similar lectin site regulation of LFA-1- and CR4-dependent
adhesion functions through formation of membrane complexes with the
same GPI-anchored membrane glycoproteins (28, 38, 39). Notably,
adhesion via LFA-1 was also deficient in neutrophils from CD87 knockout
mice (37).
Characterization of the lectin site of CR3 is critical both to understanding the molecular mechanisms that regulate CR3-dependent functions and to developing polysaccharide drugs that either prime CR3 for cytotoxic activation in response to iC3b-opsonized tumor cells or, conversely, block formation of CR3/CD87 complexes to prevent neutrophil diapedesis and tissue damage during autoimmune or inflammatory disease processes. Previous studies mapped the lectin site of CD11b by analysis of ß-glucan-FITC staining of CHO cells expressing recombinant chimeras between CD11b and CD11c in association with CD18. The prior attachment of the small SZP molecule to CR3 was also shown to block or disrupt mAb-defined epitopes of CD11b located within the C-terminal region, while having no effect on epitopes within the I domain (25). Although these data localized the lectin site to a region in the C-terminus of CD11b, it was unclear whether the lectin site included portions of CD18, or whether lectin site function required a conformation of CD11b that was induced by its association with CD18.
In the current investigation a baculovirus system was used to express various truncated rCD11b proteins at high levels in insect cells without associated CD18. Analysis of the lectin activity of these rCD11b proteins using flow cytometry and radioactive binding assays suggested that the lectin site was contained entirely within the C-terminal region of CD11b and did not require the I domain or CD18 to express its high affinity binding site for soluble ß-glucans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Spodoptera frugiperda (Sf21) cells (Invitrogen, San Diego, CA) were propagated at 27°C in Grace’s medium (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS and 10 µg/ml gentamicin. The cells were grown either as adherent cell cultures or as suspension cultures in Spinner flasks (Fisher, Fairlawn, NJ) with constant stirring at 80–90 rpm.
Monoclonal Abs
Hybridomas secreting anti-CD11b mAbs were obtained as follows: MN-41 (40) from Drs. Allison Eddy and Alfred Michael, University of Minnesota (Minneapolis, MN); and OKM1 and LM2/1 from American Type Culture Collection (Manassas, VA). Other IgG anti-CD11b mAbs were provided by colleagues: VIM12, was a gift from Dr. Walter Knapp (Institute for Immunology, Vienna, Austria); CBRM1/16, CBRM1/17, CBRM1/18, CBRM1/20, CBRM1/21, CBRM1/23, CBRM1/25, CBRM1/26, CBRM1/30, and CBRM1/32 (41) were provided by Dr. Timothy Springer (Center for Blood Research and Harvard Medical School, Boston, MA). 6xHis mAb was purchased from Clontech (Palo Alto, CA). The hybridoma secreting DX17, a mouse IgG mAb to a human MHC class I framework epitope shared by HLA-A, -B, and -C (42), was a gift from Dr. Lewis L. Lanier (DNAX, Palo Alto, CA). Hybridomas were used to generate acites fluid from which the IgG fractions were isolated (21, 43). Goat F(ab')2 anti-mouse IgG-FITC was purchased from Southern Biotechnology Associates (Birmingham, AL). For flow cytometry, MN-41, OKM1, and LM2/1 were coupled to FITC (44), and for radioactive binding assays they were labeled with 125I (45).
Soluble polysaccharides
A soluble zymosan-derived polysaccharide made up primarily of
(13)-ß-D-glucan (SZP ß-glucan, 
5–10 kDa) was
isolated as previously described (21, 25). Soluble ß-glucan from
seaweed (laminarin, 8 kDa) was purchased from Sigma (St. Louis, MO).
Water-soluble baker’s yeast-derived (13)-ß-D-glucan
phosphate (127 kDa) (46) was provided by Dr. David L. Williams (East
Tennessee State University, Johnson City, TN). For use in radioactive
binding assays, the free reducing ends of SZP ß-glucan and
(13)-ß-D-glucan phosphate were first coupled to
tyramine by a modification of the reductive amination method of Cosio
et al. (47) and then radiolabeled with 125I using Iodogen
(45). Briefly, 25 mg of SZP ß-glucan or
(13)-ß-D-glucan phosphate was dissolved in 0.9 ml of
DMSO at 45°C overnight and then mixed with 0.1 ml of tyramine (200
µmol) in DMSO. After incubation (without stirring) at 45°C for
15 h, 6 mg of sodium borohydride in 1 ml of DMSO was added, and
the mixture was incubated at room temperature for 12 h with
occasional gentle shaking. An additional 6 mg of sodium borohydride in
100 µl DMSO was added to the reaction, and the mixture was incubated
at room temperature for a further 5 h with occasional gentle
shaking. The reaction was terminated by adding 5 ml of water and
lowering the pH to 4 with acetic acid. The glucan-tyramine solution was
concentrated in a rotary evaporator to 3–4 ml, and free tyramine was
removed by chromatography at 1 ml/min on a 1.6- x 50-cm column of
Sephacryl S-200HR equilibrated with PBS. Fractions containing
tyramine-coupled polysaccharides detected by OD280
absorption were pooled and concentrated using a Centricon 30
concentrator (Amicon, Beverly, MA). The tyramine-coupled ß-glucans
were labeled with Na125I, and the 125I-labeled
ß-glucans were separated from nonincorporated 125I using
Sephadex G-25 M PD-10 columns (Amersham Pharmacia Biotech, Arlington
Heights, IL). The same method was also used to couple
2-(4-aminophenyl)-ethylamine (Acros Organics, Fisher Scientific,
Pittsburgh, PA) to laminarin to provide a target amino group on
laminarin for labeling with FITC. Amino-laminarin was labeled more
efficiently with FITC than was native laminarin when using the same
FITC-labeling method described previously (25, 48).
Construction and cloning of truncated (t) CD11b donor plasmids (pFastBacHtb-tCD11bs)
PCR was employed to amplify the tCD11b fragments from wild-type
CD11b cDNA present in the plasmid pCDM1 (41) (provided by Dr. Timothy
Springer). The pCDM1, derived from pCDM8 vector, was replicated in
MC1061/P3 Escherichia coli (Invitrogen), purified using the
Qiagen Spin Miniprep kit (Qiagen, Valencia, CA), and used as the PCR
template. PCR amplification was conducted for 30 cycles of 94°C for
45 s, 60°C for 45 s, and 72°C for 1.5 or 3.5 min using a
Perkin-Elmer/Cetus DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT). Six
primers with HindIII restriction sites were synthesized for
amplifying six tCD11b fragments (Table I
). The pFastBacHtb expression vector
(Life Technologies), a donor plasmid containing sequences encoding six
histidine residues (6xHis) at its 5' terminus that was required for
the site-specific transposition of the truncated cDNA into a bacmid
construct, was manipulated to cut off nonessential multiple cloning
site sequences. Briefly, pFastBacHtb was digested with BamHI
and KpnI, the 5' and 3' overhanging ends were repaired using
native T7 DNA polymerase to generate blunt ends, and the blunt ends
were ligated using T4 DNA ligase. The amplified tCD11b cDNA was cleaved
with HindIII, subcloned into the HindIII-digested
and alkaline phosphatase-treated pFastBacHtb, and transformed into
E. coli strain DH5 (Life Technologies). Colonies were
screened by restriction enzyme digestion, and the DNA inserts were
analyzed to verify sequence and orientation. This cloning procedure
resulted in the generation of the six recombinant donor plasmids
pFastBacHtb-tCD11bs.
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Recombinant Bv were constructed using the Bac-to-Bac Expression
System (Life Technologies). DH10Bac competent E. coli
containing a bacmid and helper plasmid was used to generate tCD11b
recombinant bacmids. After transformation of DH10Bac with
pFastBacHtb-tCD11b as described in the manufacturer’s instructions,
white colonies that signified insertion of tCD11b cDNA within the
lacZ gene were identified. High m.w. recombinant bacmid
DNA was isolated using the method described in the manufacturer’s
instructions and analyzed by PCR with PUC/M13 amplification primers to
confirm the presence of the tCD11b cDNA in the recombinant bacmid. The
recombinant bacmid DNA was used to transfect Sf21 cells to generate rBv
stocks. After harvesting the virus-containing medium, the viral titer
was determined by a plaque assay using the method of O’Reilly et al.
(49). The rBv stock was amplified by infecting 50 ml of Sf21 cell
suspension culture (2 x 106/ml) at a multiplicity of
infection (MOI) of 0.1. The rCD11b was expressed as a fusion protein
containing 6xHis at its amino terminus by infecting Sf21 cells with
the amplified viral stock.
SDS-PAGE and immunoblot assay
Baculovirus-infected or uninfected Sf21 cells were harvested at different times postinfection (p.i.) and with various MOI by centrifugation at 150 x g for 5 min. Cell lysates were prepared by solubilizing the cells (5 x 104 cells/20 µl) in sample buffer (70 mM Tris-HCl (pH 6.8), 6% glycerol, 2% SDS, 100 mM DTT, and 0.002% bromophenol blue) in the presence of 2-ME, followed by boiling for 5 min. After electrophoresis on 7.5 or 15% SDS-polyacrylamide gels, the proteins bands were either visualized by Coomassie brilliant blue staining or electrotransferred to PolyScreen polyvinylidene difluoride membranes (New England Nuclear Research Products, Boston, MA) using a Tris/glycine/SDS buffer for immunoblot analysis. Western hybridization was performed using the enhanced chemiluminescence kit (Amersham Pharmacia Biotech) according to the manufacturer’s protocol. Briefly, the nonspecific binding to polyvinylidene difluoride membranes was blocked with 5% nonfat dry milk in PBS/0.5% Tween-20 buffer, probed with mAbs (5 µg/ml) specific for either 6xHis-tagged proteins or CD11b, and then incubated with sheep anti-mouse IgG-horseradish peroxidase-conjugated Ab at a 1/2500 dilution. Ab binding to the blots was visualized using the enhanced chemiluminescence detection reagents, and blots were exposed to Fuji RX x-ray film (Fuji, Tokyo, Japan).
Immunofluorescence staining and flow cytometry assay
Immunofluorescence staining was performed on either live or fixed Sf21 cells for cell surface or intracellular staining, respectively. Uninfected or wild-type virus (Htb)-infected Sf21 cells were used as negative controls. Infected or uninfected Sf21 cells were collected by centrifugation at 150 x g for 2 min. For surface staining, the cells were washed twice with ice-cold HBSS/2% FBS, and 1 x 106 cells in a 12- x 75-mm tube were incubated on ice with anti-CD11b-FITC mAbs or laminarin-FITC for 30 min, in either the presence or the absence of an excess of unlabeled anti-CD11b or laminarin in 100 µl. Alternatively, the cells were incubated with a primary mAb, washed twice with ice-cold HBSS/2% FBS, and stained with goat F(ab')2 anti-mouse IgG-FITC. As an additional negative control, cells were stained by the same procedure with DX17 anti-MHC class I. After two washes with ice-cold HBSS/2% FBS, the stained cells were resuspended in 1 ml of HBSS/2% FBS containing 10 µg/ml propidium iodide (Sigma) to stain the nuclei of dead cells. For intracellular protein staining, the CytoFix/CytoPerm kit (PharMingen, San Diego, CA) was used. After washing Sf21 cells twice with staining buffer (PBS/1% FBS/0.09% sodium azide), 1 x 106 cells were pelleted in a 12- x 75-mm tube, fixed, and permeabilized by incubation in 250 µl of Cytofix/Cytoperm solution for 20 min on ice, and then washed twice in 1 ml of 1x Perm/Wash solution. The cells were resuspended in 100 µl of 1x Perm/Wash solution containing anti-CD11b-FITC mAbs in the presence or the absence of an excess of unlabeled anti-CD11b mAbs and incubated for 30 min on ice. After two washes with 1x Perm/Wash solution, the cells were resuspended in 1 ml of staining buffer and maintained on ice until analyzed. Flow cytometry was performed as described previously (25, 48) with analysis of listmode data files using WinList 3.0 from Verity Software House (Topsham, ME).
Radioactive ligand binding assay and Scatchard analysis
Human blood neutrophils were isolated as described previously
(50), washed twice, and resuspended in HBSS/10% FBS. Infected or
uninfected Sf21 cells (viability, 
90%) were harvested gently, washed
twice, resuspended in HBSS/10% FBS, and analyzed for Ag- or
receptor-specific binding of 125I-labeled anti-CD11b
mAbs, SZP ß-glucan, or (13)-ß-D-glucan phosphate as
described previously for leukocytes (21). Evaluation of the binding
affinity of the 125I-labeled
(13)-ß-D-glucan phosphate to rBv-infected Sf21 cells
or human neutrophils was conducted by Scatchard plot analysis as
previously described (21, 25) with concentrations of
125I-labeled (13)-ß-D-glucan phosphate
ranging from 10-9–10-7 M and 1 x
106 cells tested in triplicate. Prism 2.01 (GraphPad
Software, San Diego, CA) was used for generation of graphs and
statistical analysis of data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six tCD11b-rBv were used to produce six rCD11b proteins in Sf21
cells (Fig. 1 and Table II). SDS-PAGE of detergent-solubilized
Sf21 cell lysates confirmed the synthesis of six rCD11b proteins of the
size expected for each tCD11b-rBv (Fig. 2). An MOI of 0.1 was determined to be
optimal for expression of all six rCD11b proteins and therefore was
used in all subsequent experiments. Fig. 2 shows rCD11b expression in
Sf21 cells infected with tCD11b-rBv at an MOI of 0.1 and harvested
72 h p.i. With the exception of the small and N terminus of
divalent cation-binding region (DCB) proteins, the four larger rCD11bs
appeared to be the major proteins in cell lysates. No protein bands of
the same predicted size as the rCD11b proteins were detected in either
Htb-infected or uninfected cells.
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Western blot analysis was conducted with mAbs specific for various
CD11b epitopes or a fusion protein-specific mAb for the 6xHis tag
(Table III and Fig. 3). The rCD11b proteins were detected
only in cell lysates (Fig. 3) and not in spent cell-free medium (data
not shown), indicating that rCD11b expression was nonsecretory (as
expected since the tCD11b cDNAs lacked a signal peptide sequence). A
strong reaction of all six rCD11b proteins with the 6xHis mAb
confirmed high expression levels of the appropriate sized rCD11b
proteins (Fig. 3). The anti-CD11b mAbs LM2/1, MN-41, OKM1,
CBRM1/21, CBRM1/23, and CBRM1/25 each reacted with the rCD11b proteins,
whereas no reaction was detected with mAbs CBRM1/16, CBRM1/17,
CBRM1/18, CBRM1/20, CBRM1/30, CBRM1/32, and VIM12. Only fragments
containing the I domain (ID, ID/DCB, and ID to C terminus (ID/TM)
reacted with mAbs to I domain epitopes, and likewise, only fragments
containing the C-terminal region (TM1, TM2, and ID/TM) reacted with
mAbs to C-terminal region epitopes (Table III). Fig. 3 shows the
reactivity of rCD11bs with 6xHis mAb, LM2/1 (specific for the I
domain), and OKM1 (specific for the C-terminal region). The proteins
from uninfected (data not shown) or Htb-infected Sf21 cells showed no
reactivity with either the 6xHis mAb or any of the anti-CD11b
mAbs.
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Flow cytometry analysis for cell surface vs cytoplasmic staining
was conducted with Sf21 cells harvested at 5 days p.i. Cell
surface-positive fluorescence staining with the 6xHis mAb, defined by
comparison to the nonspecific staining obtained with the DX17 mAb to
human MHC class I, showed that all six of the rCD11b fusion proteins
were expressed on the cell surface (data not shown). This result was
confirmed by the positive surface staining obtained with MN-41-FITC
(Fig. 4A). Analysis of cells
following intracellular staining with MN-41-FITC indicated that the
rCD11b proteins also accumulated in the cytoplasm (Fig. 4B).
Similar results were obtained when using mAbs (e.g., LM2/1 and OKM1) to
other specific epitopes of CD11b (data not shown). These data indicated
that the rCD11b proteins were distributed both on the cell membrane and
in the cytoplasm of the infected cells.
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Flow cytometry analysis of ß-glucan binding to rCD11b fragments
The polysaccharide binding (lectin) activity of the rCD11b
proteins was evaluated by flow cytometry analysis of cells staining
with laminarin-FITC. Receptor-specific (saturable/reversible) staining
by laminarin-FITC was observed with infected Sf21 cells expressing
rCD11b proteins containing the C-terminal region of CD11b (TM1, TM2,
and ID/TM), because this staining was blocked by excess unlabeled
laminarin. No receptor-specific laminarin-FITC staining occurred with
cells expressing rCD11b proteins lacking the C-terminal region or with
Htb-infected or uninfected cells. Fig. 5
shows positive laminarin-FITC staining of cells expressing TM2 or ID/TM
vs negative laminarin-FITC staining of cells expressing either ID/DCB
or wild-type virus. Staining with MN-41-FITC (Fig. 5) and 6xHis mAb
(data not shown) confirmed equivalent expression of each rCD11b
protein.
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The ß-glucan binding activity of the rCD11b proteins was next
examined by a radioactive binding assay with 125I-labeled
SZP ß-glucan. Fig. 6 shows the binding
of 125I-labeled MN-41 or OKM1 to rBv-infected Sf21 cells,
where specific binding was defined as net radioactivity after
subtraction of the nonspecific binding obtained in the presence of a
100-fold excess of the homologous unlabeled mAb. Only cells expressing
rCD11b proteins containing the I domain (ID, ID/DCB, and ID/TM)
exhibited specific binding of [125I]MN-41; likewise, only
cells expressing rCD11b proteins containing the C-terminal region (TM1,
TM2, and ID/TM) showed specific binding of [125I]OKM1. No
specific binding of either mAb occurred with uninfected or Htb-infected
cells. This assay demonstrated the feasibility of using the radioactive
binding assay to detect the surface ligand binding activity of
rBv-infected Sf21 cells expressing rCD11b proteins. The specific uptake
of these 125I-labeled anti-CD11b mAbs with this assay
became detectable at 2 days p.i. and reached its highest level at 3
days p.i. Maximum specific binding activity occurred before the cells
were maximally expressing rCD11b on day 5 (as determined by flow
cytometry), because after 3 days the nonspecific uptake of radiolabeled
mAb by the increasing proportion of dead cells exceeded the specific
uptake of radiolabeled mAbs by the remaining viable cells (data not
shown).
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The three rCD11b proteins containing the C-terminal region (TM1, TM2,
and ID/TM) exhibited ß-glucan-specific binding activity, whereas no
lectin activity was detectable with the three rCD11b proteins that
lacked the C-terminal region (ID, DCB, and ID/DCB). Also, no ß-glucan
receptor-specific binding activity was detectable with the uninfected
or Htb-infected cells. The presence of a 50-fold excess of unlabeled
SZP ß-glucan produced 40–60% inhibition of receptor-specific
binding activity of the TM1, TM2, and ID/TM proteins compared with the
100% inhibition (i.e., zero specific binding by definition) obtained
with a 100-fold excess of unlabeled SZP ß-glucan. The lectin activity
of these rCD11b proteins was inhibited by OKM1, and with ID/TM that
contained the I domain in addition to the C-terminal region, lectin
activity was also inhibited by MN-41.
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Scatchard plot analysis showed that 125I-labeled
(13)-ß-D-glucan phosphate bound to Sf21 cells
expressing TM1, TM2, or ID/TM with affinities of 68.2–125.3 nM, which
were not significantly different (p > 0.05)
from each other (Fig. 8). Parallel
Scatchard analysis of 125I-labeled
(13)-ß-D-glucan phosphate binding to human neutrophils
calculated an affinity of 34.6 nM (Fig. 8), which was not significantly
different (p > 0.05) from those of the three
rCD11b proteins. Specific uptake of 125I-labeled
(13)-ß-D-glucan phosphate was virtually abolished when
neutrophils were incubated with a mixture of unlabeled anti-CD11b
mAbs (MN-41 and OKM1) before Scatchard plot analysis, indicating that
CR3 was responsible for most of the (13)-ß-D-glucan
phosphate-receptor activity (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The use of rBv to express cell surface fragments of rCD11b provided the opportunity to examine the lectin site function of CD11b in the absence of CD18 and any conformational changes in CD11b induced by the native heterodimeric complex with CD18. Based on SDS-PAGE, optimal expression levels of the rCD11b proteins occurred at an MOI of 0.1 and were diminished when cells were infected at higher MOI. Flow cytometry analysis with the 6xHis mAb revealed that maximum expression of rCD11b proteins occurred at 4 or 5 days p.i. This time was different from the 3-day optimal p.i. period determined with the radioactive binding assay because of the increasing numbers of dead cells that take up labeled mAbs nonspecifically. Only with the flow cytometry assay was it possible to exclude the high level of dead cell uptake of labeled probes through propidium iodide staining, so that only the specific uptake of label by viable cells was measured.
The reactivity of rCD11b proteins with anti-CD11b mAbs required preservation and exposure of native epitopes. The finding that mAbs LM2/1, OKM1, CBRM1/21, CBRM1/23, and CBRM1/25 reacted with rCD11b fragments confirmed a previous report (51) indicating that exposure of the epitopes defined by these mAbs on whole rCD11b did not require the CD11b/CD18 complex. Likewise, absent reactivity with mAbs CBRM/16, CBRM1/17, CBRM1/18, CBRM1/20, CMBRM1/30, and CBRM1/32 is in agreement with the finding that the CD11b/CD18 heterodimer was required for reaction with these specific mAbs (51). In addition, a positive reaction in Western blots indicated the lack of specific conformational requirements in the epitopes defined by mAbs LM2/1, OKM1, CBRM1/21, CBRM1/23, and CBRM1/25. Some conformational requirement for the exposure of the I domain epitope of MN-41 was suggested by its weak reaction in Western blots vs the strong staining activity of MN-41-FITC detected in flow cytometry assays of cells. An affinity of 38 nM for MN-41 binding to native neutrophil CR3 had been noted previously (25). Moreover, the staining activity of MN-41-FITC with insect cells expressing only the small I domain fragment (ID) showed that this epitope does not require CD18 for proper orientation.
Lectin site functional analysis of the rCD11b proteins was greatly facilitated by their surface expression on the rBv-infected insect cells. However, for reasons that are unclear, all six of the rCD11b proteins were detected on cell surfaces, including three of the rCD11b fragments that lacked the C-terminal transmembrane domain (ID, DCB, and ID/DCB). Examination of the sequences of these three rCD11b fragments did not show any other hydrophobic region that could explain their membrane expression. However, these rCD11b proteins may not be expressed in a membrane-anchored fashion resembling a normal receptor. The membranes of these virus-infected insect cells are increasingly damaged by the virus replication process, such that they become partially permeable and allow weak staining of nuclear DNA with propidium iodide. Eventually, over a 3- to 5-day period, virus replication kills the cells and completely ruptures their membranes. In between infection and cell death, the rBv directs the cells to synthesize large amounts of recombinant protein that accumulates in the cytoplasm. It thus appears possible that some of the membrane rCD11b detected by mAb or ß-glucan-binding assays might have represented intracellular protein that was exposed by viral damage to the membrane. The best specific staining for rCD11b was observed with large cells that exhibited weak staining with propidium iodide.
The surface expression of the rCD11b proteins by viable cells allowed
development of both immunofluorescence and radioactive binding assays
to evaluate the ß-glucan binding activity of the rCD11b proteins.
With both assays, saturable and reversible binding of ß-glucan could
only be detected with cells expressing rCD11b fragments containing the
C-terminal region regardless of their content of the I domain and
without requirement for coexpression of CD18. Scatchard analysis showed
that the binding affinities of 125I-labeled
(13)-ß-D-glucan phosphate to three rCD11b proteins
were not significantly different from each other. The smallest fragment
containing the C-terminal region and lacking the I domain, TM1 (699
aa), exhibited a binding affinity for 125I-labeled
(13)-ß-D-glucan phosphate similar to that of the
largest one, ID/TM (963 aa), that included the I-domain. These data
confirm findings made with CD11b/CD18 heterodimers that the lectin site
is located outside the I domain (25) and demonstrate further that CD18
is not required for expression of a functional ß-glucan binding
lectin site.
Despite the lack of a requirement for CD18 in forming the lectin site in CD11b, several previous observations indicate that the lectin site probably does have an interaction with CD18 that is required for transmembrane signaling events. For example, the activation of tyrosine kinase for adhesion or cytotoxicity that is stimulated through the lectin site requires an intact CD18 cytoplasmic domain (52). An mAb to CD18 was also shown to inhibit neutrophil CR3-dependent phagocytosis of unopsonized yeast or Staphylococcus epidermidis bacteria without blocking lectin site-dependent attachment of the yeast or bacteria to the neutrophil surface (13). Moreover, neutrophil phagocytosis of particulate ß-glucan via the lectin site of CR3 was found to stimulate phosphorylation of CD18 (53).
Experiments that attempted to measure the binding affinities of three
rCD11b fragments expressed by insect cells using
125I-labeled (13)-ß-D-glucan phosphate
found affinities of 68.2–125.3 nM that were similar to both the 34.6
nM affinity measured with neutrophils and 125I-labeled
(13)-ß-D-glucan phosphate and the 67 nM affinity
reported previously with neutrophils and 125I-labeled SZP
(25). Moreover, with both
[125I](13)-ß-D-glucan phosphate and
[125I]SZP ß-glucan, 85–90% of the receptor-specific
uptake by neutrophils was blocked by a mixture of two mAbs to CD11b.
However, a low affinity binding site for
(13)-ß-D-glucan phosphate has been noted on
CR3-deficient U937 cells (54) that fail to bind SZP (25).
Most reports about the functional sites of CR3 have focused on the I domain and the structure of its high affinity metal ion-dependent adhesion site for ICAM-1 (41, 55, 56, 57). The functional contributions of regions outside the I domain are only beginning to be explored. The C-terminal location of the lectin site of CD11b was recently confirmed in a study of rCR3 binding to Candida albicans that suggested that ligation of Candida polysaccharides to the lectin site caused an increased affinity of a second binding site for Candida located in the I domain (26). Our previous studies suggested that the lectin site became covered or hidden when mAbs were attached to distal sites in the I domain (25). A similar finding of lectin site blockade by an mAb to the I domain was recently also made with mouse CR3 (21). The current study showed that even with rCD11b alone, the binding to 125I-labeled ß-glucan could be blocked by mAbs to the I domain as well as by mAbs to C-terminal region epitopes. It appears possible that a dynamic state exists in CD11b such that attachment of polysaccharides to the lectin site in the C-terminal region causes a rearrangement of the distal I domain evident by the expression of either the I domain neoepitope defined by mAb CBRM1/5 (25) or the high affinity metal ion-dependent adhesion site conformation (26). Conversely, attachment of a mAb to the I domain may reverse CD11b folding such that the distal lectin site in the C-terminal region is no longer exposed.
The rBv expression system developed for this investigation should be useful for the generation and functional analysis of progressively smaller truncated CD11b fragments to determine the exact site and structure of this unusual non-C-type lectin. Moreover, small truncated CD11b fragments containing the lectin site could potentially be used to screen soluble forms of the GPI-anchored membrane glycoproteins to determine how they couple to the lectin site for regulation of CR3-dependent cytotoxicity vs adhesion. Finally, these CD11b fragments could also be used to screen potential drug agonists or antagonists of the lectin site. Soluble ß-glucan BRM (agonists) appear to hold promise as antitumor agents through their ability to prime the CR3 of circulating leukocytes for cytotoxicity of iC3b-opsonized tumor cells (23). Conversely, an antagonist of the lectin site could theoretically block the formation of membrane CR3/CD87 complexes, thereby preventing neutrophil-mediated tissue damage in the inflammatory reactions associated with autoimmunity, hypersensitivity reactions, transplantation, or ischemia/reperfusion injury (8).
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Acknowledgments |
|---|
3)-ß-D-glucan phosphate. We thank Dr. Stephen C.
Peiper (University of Louisville) for critical guidance concerning
methods for working with baculovirus and insect cells as well as for
numerous helpful discussions. We further acknowledge the generous
donation of mAbs from the following scientists: Drs. Allison Eddy and
Alfred Michael (University of Minnesota, Minneapolis, MN), Dr. Walter
Knapp (Institute for Immunology, Vienna, Austria), and Dr. Lewis L.
Lanier (DNAX Research Institute, Palo Alto, CA). We particularly thank
Margareta Hanik
ová for her excellent technical
assistance. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Yu Xia, Division of Experimental Immunology and Immunopathology, Department of Pathology, University of Louisville, KY 40292. E-mail address:
3 Abbreviations used in this paper: iC3b, opsonic fragment of C3 that binds to CR3 and CR4; CR3, complement receptor type 3 (Mac-1, CD11b/CD18, Mß2 integrin); BRM, biological response modifiers; SZP, soluble zymosan polysaccharide; 6xHis, six histidine residues; tCD11b, truncated CD11b; rBv, recombinant baculovirus; MOI, multiplicity of infection; p.i., postinfection; ID, I domain; DCB, N terminus of divaalent cation-binding region; TM1, partial divalent cation-binding region of C terminus; ID/TM, ID to C terminus; TM2, divalent cation-binding region to C terminus.
Received for publication February 9, 1999. Accepted for publication April 5, 1999.
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