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*
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;
Oncology Drug Discovery, Bristol-Myers Squibb, Princeton, NJ 08543;
The Maxwell Finland Laboratory for Infectious Diseases, Boston University School of Medicine, Boston, MA 02118; and
§
Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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50 to
200 kDa, were detected. Although photoactivatable Taxol analogue failed
to bind to CD14, the prominent Taxol-binding protein was identified as
CD18, the 96-kDa common component of the ß2 integrin
family. This finding was supported by the concomitant failure of
macrophage membranes from Mac-1 knockout mice to express immunoreactive
CD18 and the major Taxol-binding protein. In addition, Taxol-induced
IL-12 p40 mRNA was markedly reduced in Mac-1 knockout macrophages and
anti-Mac-1 Ab blocked secretion of IL-12 p70 in Taxol- and
LPS-stimulated macrophages. Since CD18 has been described as a
participant in LPS-induced binding and signal transduction, these data
support the hypothesis that the interaction of murine CD18 with Taxol
is involved in its proinflammatory activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (11, 12), and induction of gene expression (7, 8). Like LPS, Taxol can serve as a second signal for synergistic
induction of inducible nitric oxide synthase mRNA and nitric oxide
release, and tumoricidal and microbicidal activities in IFN-
-primed
macrophages (13, 14). A clear dissociation has been shown between
Taxol’s LPS-mimetic effects on macrophage signaling versus its
microtubule-stabilizing effects in vitro and in cells. Taxol analogues,
such as Taxotere and Epothilone, possess higher affinities for
ß-tubulin than Taxol, compete with Taxol for the same binding site on
microtubules, yet fail to elicit LPS-mimetic activity in murine
macrophages (7, 15, 16, 17, 18, 19). Conversely, Taxol induces normal microtubule
bundling in C3H/HeJ macrophages in the absence of detectable LPS-like
signaling (7). Taxol’s LPS-mimetic activity was not blocked by polymyxin B (15), an antibiotic that binds and inactivates LPS (20); however, lipid A analogue antagonists were inhibitory, suggesting that LPS and Taxol might engage elements within a shared receptor/signaling complex (15). Conversely, these same lipid A structural antagonists failed to inhibit Taxol-induced microtubule polymerization in macrophages (15), again suggesting that the shared signaling element is not ß-tubulin. Macrophages express a number of surface molecules (e.g., CD14, CD11/CD18, and others) that can interact with LPS (reviewed in 21). Studies using macrophages from CD14 knockout mice have shown that Taxol and LPS elicit gene expression by both CD14-dependent and independent pathways (22), suggesting the involvement of other receptors. Finally, the ability of Taxol to stimulate macrophages in an LPS-like fashion is species dependent (2), demonstrable in murine, but not in human macrophages; notably, the lipid A precursor, lipid IVA, shows a similar pattern of species specificity (reviewed in 16). Although the proinflammatory effects of Taxol are largely limited to murine cells, identification of components of the murine Taxol-signaling apparatus is likely to lead to the identification of homologous human LPS-signaling proteins.
To identify novel Taxol-binding proteins within a shared LPS signaling
complex, we developed a novel detection system based on a
photoactivatable Taxol analogue (PA Taxol) used originally to define
Taxol’s interaction with tubulin (23). PA Taxol binds to at least
seven murine macrophage membrane proteins, and the most prominent of
these is the common component of the ß2 family of
integrins, CD18, based on the coordinate failure of macrophages from
Mac-1 knockout mice to express CD18 or the major 96-kDa
Taxol-binding protein. In contrast, CD14 is not a Taxol-binding
protein. Finally, Taxol-induced IL-12 p40 mRNA was markedly reduced in
macrophages derived from Mac-1 knockout mice, and anti-Mac-1 Ab
blocks Taxol-induced IL-12 p70 secretion, suggesting that the
interaction of Taxol with CD18 is critical for its ability to act as a
full LPS mimetic.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five- to seven-week-old C3H/OuJ or C3H/HeJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). CD11b (Mac-1) knockout (-/-) mice and wild-type (+/+) controls (24) were bred at Longwood Medical Research Center (Boston, MA). Mac-1 knockout and background-matched, wild-type colonies are of a mixed C57BL/129Sv background that is maintained by ongoing heterozygous breedings. Mice were housed in a virus Ab-free facility.
Preparation of cell membrane fractions
Peritoneal exudate cells (85% macrophages) were collected by
lavage with sterile saline 4 days after i.p. injection of mice with 3
ml thioglycollate (Difco, Detroit, MI). Cells were centrifuged
(750 x g for 5 min) and resuspended in PBS (1x, pH
7.4). Cell viability was >95% by trypan blue exclusion. Peritoneal
exudate cells were recentrifuged (750 x g, 5 min), and
the pellet was resuspended at 1 x 107 cells/ml in
homogenization buffer containing 25 mM HEPES, pH 7.3, 0.5 mM EGTA, 0.5
mM sodium orthovanadate, 0.1 mM sodium molybdate, 1 mM sodium fluoride,
and protease inhibitors (1 tablet Complete (Boehringer Mannheim,
Mannheim, Germany) per 50 ml buffer). A total of 7 ml of cell
homogenate was incubated on ice, and cells were lysed using a
tight-fitting dounce homogenizer (100 strokes). Unlysed cells, nuclei,
and cell fragments (25) were removed by centrifugation (1000 x
g, 10 min) at 4°C. The resulting nuclei-free supernatant
was centrifuged again (10,000 x g, 7 min) at 4°C to
remove aggregates of cytoskeletal elements (26). The membrane pellet
was collected by a final centrifugation of the above supernatant at
417,000 x g (rav) for 2 h
at 4°C and resuspended and solubilized in homogenization buffer
containing 10 mM CHAPS (Sigma, St. Louis, MO). This membrane
preparation method is a modification of that reported by Liu et al.
(27). Protein determinations were performed by Bio-Rad protein assay
(Richmond, CA). In some experiments, after UV cross-linking (below), 50
µl samples were centrifuged (20,800 x g, 15 min at
4°C) before Western analysis. Membranes were also prepared from the
following cell lines: ST2 (a CD14-negative murine bone marrow-derived
stromal cell line) (28, 29); CHO/Neo, CHO/murine CD14 (SAM8), and
CHO/human CD11b-human CD18 cell lines were derived by stable
transfection of Chinese hamster ovary
(CHO)4 cell fibroblasts (30, 31).
UV-induced cross-linking of PA Taxol to membrane proteins
A 5-azido-2-nitrobenzoic acid C-7 photoaffinity analogue of Taxol (PA Taxol; m.w. = 1043) has been described (23). Multiwell plates (Falcon 96-well microtiter plates; Becton Dickinson, Lincoln Park, NJ) were prewetted with 50 µl of 50 mM Tris-HCl buffer, pH 7.4, and drained. Typically, a mixture containing 150 µg membrane protein and 6 µl PA Taxol (835 µM) was prepared in a final volume of 50 µl in 50 mM Tris-HCl buffer, pH 7.4, such that the final concentration of PA Taxol was 100 µM. Aliquots (50 µl) of this mixture were added to each well of the prewetted plate and incubated at 37°C (6% CO2) for 20 min. The plate was set on ice and UV irradiated at 4°C for 30 min with a mineral light lamp (model R52G; Ultraviolet Products, San Gabriel, CA; 0.16 A) at 254 nm at a distance of 7 cm, as described elsewhere (32). After irradiation, samples were diluted in 4x Laemmli buffer (32) (200 mM Tris-HCl, pH 6.8; 8% SDS; 400 mM DTT; 40% glycerol; 0.4% bromophenol blue; final concentration of Laemmli buffer is 1x) and boiled for 5 min. Membrane proteins (30–50 µg) were resolved by SDS-PAGE (typically, 9 or 10% acrylamide) under reducing conditions (33) (Mini Protean II; Bio-Rad, Hercules, CA). Prestained m.w. markers (low range; Bio-Rad) and unstained m.w. markers (wide range, Mark 12; Novex, San Diego, CA) were included in each gel.
Western blot analysis
Western blot analysis was utilized to detect Taxol-bound proteins, as well as to identify other proteins in membranes. For detection of CD18 by Western blotting, nonreducing conditions were required: samples were prepared for SDS-PAGE as described above in the absence of DTT. Following SDS-PAGE, resolved proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) using a Minitrans Blot Electrophoretic Transfer Cell (Bio-Rad) for 1 h at 100 V at 4°C in transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, and 20% methanol). Blots were stained for 10 min in Ponceau S (Sigma) and destained in water. The positions of stained bands of authentic standards were used to calculate m.w. of separated proteins. Membranes were blocked for 1 h at room temperature in PBS containing 1% gelatin and 5% nonfat milk. After washing (3 times, 5 min each) with PBS, membranes were incubated with primary Ab diluted in PBS plus 5% nonfat milk for 1 h at room temperature. Membranes were washed (3 times, 10 min each) with PBS + Tween (0.05%) (PBST). Finally, membranes were incubated in an appropriate HRP-conjugated secondary Ab (i.e., goat anti-rabbit IgG (Bio-Rad), 1/2500; goat anti-mouse IgG (Bio-Rad), 1/5000; or goat anti-rat IgG, 1/2000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA)). Secondary Abs were diluted in PBS containing 5% nonfat milk and incubated with membranes for 1 h at room temperature. Membranes were washed (five times, 5 min each) with PBST. Binding of secondary Ab was detected with the enhanced chemoluminescence (ECL) detection method (Amersham Life Science, Little Chalfont, U.K.). In some experiments, immunoblots were stripped: membranes were washed for 5 min at room temperature in 5x SSC (0.75 M sodium chloride, 0.075 M sodium citrate, pH 7). Membranes were placed in 0.1x SSC containing 0.1% SDS and incubated for 1 h at 65°C in a water bath with gentle rotation, followed by an additional wash for 5 min at room temperature in 5x SSC. Stripped membranes were blocked and reprobed using an alternate primary Ab, as described above. In blocking experiments, blots were first cut into individual lanes after the initial blocking step and individual strips were incubated overnight at 4°C (or 2 h at room temperature) with one of the following: no Ab, rabbit anti-Taxol (1 µg/ml), rat anti-CD18 (10 µg/ml), or isotype-matched, affinity-purified control Abs (rabbit IgG or rat anti-Mac-3). Blots were washed with PBST and incubated with either rat anti-CD18 (10 µg/ml) or rabbit anti-Taxol (1 µg/ml) primary Ab to detect CD18 or Taxol-cross-linked proteins, respectively.
Primary Abs
Anti-Taxol Ab was generated by hyperimmunizing rabbits with Taxol cross-linked at C7 to keyhole limpet hemocyanin. Polyclonal Abs were ammonium sulfate precipitated, affinity purified by passage over a protein A column, and dialyzed in PBS. For Western analysis, rabbit anti-Taxol Ab was used at a final concentration of 1 µg/ml. The following primary Abs were also used: rabbit anti-BSA antiserum (kindly provided by Dr. Bob Roberson, University of Maryland, College Park, MD; 1:1500), rabbit anti-murine CD14 antiserum (kindly provided by Dr. Richard Ulevitch, Scripps Research, La Jolla, CA; 1:400), and rat anti-mouse CD18 Ab, GAME 46 (34) (kindly provided by Dr. Ed Roos, The Netherlands Cancer Institute, Amsterdam; 5 µg/ml).
Preparation of Taxol-cross-linked proteins for liquid chromatography quadropole (LCQ) analysis
PA Taxol-cross-linked membrane proteins (600 µg) were resolved
on a 9% preparative SDS-PAGE, and a single lane from each edge of the
gel was excised and processed for Western analysis to detect
Taxol-bound proteins (see above). The remaining gel was stained with
0.1% Coomassie blue in 10% acetic acid, 50% methanol for 30 min. The
gel was destained for 
2 h in 10% acetic acid, 50% methanol until
the background was clear. The polyvinylidene difluoride membrane strips
that were developed with anti-Taxol Ab were positioned next to the
Coomassie-stained gel. The region on the stained gel that coincided
positionally with the 96-kDa species was excised. A blank gel piece
was excised as a control. Gel slices were placed in Eppendorf tubes,
frozen, and sent to the W. M. Keck Foundation (New Haven, CT) for
LCQ analysis. Briefly, 32 pmol of the 96-kDa gel slice was digested
with trypsin, along with the blank gel slice. Approximately 10% of the
digest was subjected to LC-MS/MS using an LCQ ion trap mass
spectrometer (35). A Sequest search of the OWL database indicated that
one of the reconstructed MS/MS spectra had significant similarity
(i.e., the difference in the normalized cross-correlation functions as
defined by Eng et al. (36) of the first and second ranked searches was
0.32) to the observed spectra. This reconstructed spectra corresponded
to an 18-residue peptide, SAVGELSDDSSNVVQLIK, found in mouse cell
surface adhesion glycoproteins (LFA-1, CR3, and P150, 95, ß subunit
precursor), preceded by a lysine.
Other reagents
Recombinant human soluble CD14 (sCD14) and recombinant murine soluble CD14 (mu sCD14) were kindly provided by Dr. Henri Lichenstein (Amgen Boulder, Boulder, CO) and Dr. Christine Schütt (Arndt-Universität Greifswald, Greifswald, Germany), respectively. Tubulin, devoid of microtubule-associated proteins, was isolated from calf brain, as described (37). Affinity-purified rabbit IgG was kindly provided by Dr. Mark Lynch (Bristol-Myers Squibb, Princeton, NJ). Rat anti-Mac-3 (IgG1) was affinity purified from the M3/84 myeloma cell line (American Type Culture Collection (ATCC), Manassas, VA; TIB168). Affinity-purified rat anti-Mac-1 (IgG2b; M1/70; ATCC; TIB 128) and isotype-matched control (anti-CD122; PharMingen, San Diego, CA) Abs were used in IL-12 secretion experiments. Taxol was provided by the Drug Synthesis and Chemistry Branch (National Institutes of Health), and Escherichia coli K235 LPS was prepared by the method of McIntire et al. (38).
Analysis of IL-12 p40 mRNA and IL-12 p70 secretion
IL-12 p40 mRNA was measured by quantitative RT-PCR, using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a housekeeping gene (39). Immunoreactive IL-12 p70 was measured by ELISA (PharMingen), following the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether novel Taxol-binding proteins could be
detected in murine macrophage membranes, macrophage membranes were
subjected to UV cross-linking in the presence or absence of PA Taxol,
an analogue developed to identify the Taxol-microtubule binding domain
(23). Fig. 1
A shows that
Taxol-cross-linked C3H/OuJ membranes (XL C3H/OuJ), but not
uncross-linked membranes (C3H/OuJ), show a distinct pattern of
immunoreactive bands when detected by Western analysis with a primary
rabbit anti-Taxol Ab (RaTaxol); bands were not detected in the
presence of secondary Ab only (GaR), or if an irrelevant primary Ab,
rabbit anti-BSA, were used. Inclusion of 50 µM Taxol at the time
of the incubation of the blot with anti-Taxol Ab resulted in nearly
complete competition of all bands (data not shown). The seven major
species detected in 9–10% polyacrylamide gels migrate with average
molecular mass of 197 kDa, 149 kDa (sometimes resolvable as a doublet),
114 kDa, 96 kDa (always the major species that appears as a broad
band), 80 kDa, 56 kDa, and 53 kDa (also sometimes resolvable as a
doublet). Bands detected by Western analysis did not coincide with
major bands seen after Coomassie staining (data not shown). In certain
experiments, an additional centrifugation step was included after UV
cross-linking. Ninety percent of the total membrane protein subjected
to cross-linking remained in the supernatant, with an enrichment of the
114-, 96-, and 80-kDa species. These species are presumably associated
with membrane microdomains that remain soluble following UV
cross-linking, in contrast to those recovered in the pellet fraction.
All major species were detected in the pellet fraction (data not
shown).
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B). Under our standard conditions in
which PA Taxol was used at 100 µM (i.e., similar to those used in
studies with ß-tubulin) (40), the seven major immunoreactive species
were readily detected. A total of 10 µM PA Taxol resulted in less
intense bands, although positionally indistinguishable from those
detected after cross-linking with 100 µM PA Taxol. Underivatized
Taxol, used at a 100 µM, also resulted in detection of the same
immunoreactive species, although the efficiency of cross-linking was
much more like that seen with the suboptimal (10 µM) concentration of
PA Taxol. These data support the hypothesis that the PA Taxol interacts
with the same major membrane proteins as underivatized Taxol.
C3H/HeJ mice express a gene defect (Lpsd) that
renders them refractory to LPS in vivo and in vitro (reviewed in 41). A point mutation in the intracytoplasmic domain of the TLR4
transmembrane receptor underlies this defect (42). Since Taxol has been
shown to act only on macrophages that possess normal LPS responsiveness
(Lpsn), and not in C3H/HeJ macrophages (4),
membranes from C3H/OuJ (Lpsn) and C3H/HeJ
(Lpsd) mice were subjected to UV cross-linking with
PA Taxol. Fig. 1C shows no obvious differences in the
pattern of immunoreactive species in Western blots derived from the two
mouse strains. Collectively, the data in Fig. 1, A–C,
illustrate the specificity and reproducibility of the detection system.
Membrane CD14, 53–56 kDa molecular mass, is the major LPS-binding
protein on macrophages (reviewed in 43). Studies using macrophages
from CD14 knockout mice showed a CD14 dependency for Taxol stimulation,
albeit more limited than observed for LPS (22). Therefore, we tested
the hypothesis that either the 53- or 56-kDa Taxol-binding proteins
were CD14. Murine sCD14 failed to be cross-linked by PA Taxol, under
conditions in which the 53- and 56-kDa Taxol-cross-linked species were
detected in the C3H/OuJ membrane preparation (Fig. 2). Similarly, no anti-Taxol
immunoreactive bands were detected in cross-linked human sCD14, under
conditions in which tubulin was extensively cross-linked and readily
detected by Western analysis (i.e., it formed high m.w. complexes).
Thus, CD14 is not a target for Taxol binding.
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An analysis of membrane preparations derived from several
LPS-responsive cell lines was undertaken to evaluate the potential for
common Taxol-binding proteins. Fig. 3
compares PA Taxol-cross-linked membranes derived from CHO cells that
express human CD11b/CD18, SAM8 (murine CD14 transfectant), and the
control CHO-NEO cell line, the murine ST2 cell line, and C3H/OuJ and
C3H/HeJ macrophages. Both the CD11b/CD18 and SAM8 transfectants respond
to rough LPS (29, 30), but not to Taxol (unpublished observations); the
control CHO-NEO cell line responds to neither LPS nor Taxol. The murine
ST2 cell line, derived from bone marrow stromal cells (28), responds to
both LPS and Taxol (29). C3H/OuJ (LPS- and Taxol-responsive) and
C3H/HeJ (LPS- and Taxol-hyporesponsive) macrophage membranes were
included as controls. Western analysis of PA Taxol-cross-linked
membranes from the three CHO cell lines was indistinguishable, but
exhibited a pattern of anti-Taxol immunoreactive bands distinct
from that exhibited by the ST2 cell line, and from the pattern common
to C3H/OuJ and C3H/HeJ macrophage membranes. A number of bands
comigrate in all of the membranes, including those with apparent
molecular mass of 149 kDa, 114 kDa (although considerably fainter
in the macrophage membranes), 80 kDa, and 53 kDa (the latter was
somewhat more prominently detected in the macrophage membranes), and
the 56-kDa species is more pronounced in the ST2 cell line. Most
significantly, the 96-kDa species appears to be predominant and
specific to the murine macrophage membrane preparations.
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LCQ analysis of peptides derived from the preparative 96-kDa
gel slice indicated that the mass/charge ratio of one tryptic fragment
was derived from murine CD18, the common ß-chain of the
ß2 family of integrins (reviewed in 44). CD18 is
expressed as an obligate 
ß heterodimer in noncovalent association
with CD11a, CD11b, CD11c, or CD11d (reviewed in 44). Since
CD11b/CD18 (also referred to as Mac-1) is the predominant
ß2 integrin on murine macrophages (reviewed in Refs. 44
and 45), and since CD11b/CD18 has been found to mediate LPS signaling
in macrophages and in CHO cells transfected with human CD11b/CD18 (31),
we tested the hypothesis that the 96-kDa Taxol-binding protein was
CD18. To address this possibility, we took advantage of mice with a
targeted mutation in CD11b, the subunit of Mac-1 (24). Previous
FACS analysis revealed that neutrophils and macrophages derived from
CD11b knockout mice lack surface CD11b (24) and that surface expression
of CD18 on thioglycollate-elicited macrophages was reduced by >85%
(Coxon and Mayadas, unpublished). This observation is consistent with
finding that CD11b/CD18 is the most abundant ß2 integrin
on macrophages (45), and that in the absence of subunits, CD18 is
not translocated to the cell surface (reviewed in 44). Thus, we
reasoned that if CD18 were the predominant Taxol-binding protein in
murine macrophages, there should be a significant reduction in the
expression of the 96-kDa Taxol-binding protein in macrophage
membranes derived from CD11b knockout mice.
Fig. 4 shows the results of two separate
experiments in which membranes from CD11b (Mac-1) knockout macrophages
and background-matched, wild-type macrophages were compared for their
ability to be UV cross-linked with PA Taxol. As a control, C3H/OuJ
membranes were included. First, noncross-linked (Expt. 1) or
PA Taxol-cross-linked (Expt. 2) Mac-1 (+/+), Mac-1 (-/-), and C3H/OuJ
membranes were subjected to SDS-PAGE analysis in the absence of DTT
(-DTT), conditions that are required for Western analysis with
anti-CD18 mAb (i.e., the anti-murine CD18 is directed against a
conformational epitope, precluding its use under reducing conditions)
(34). Our experiments show that the membranes of Mac-1 knockout
macrophages are highly deficient in two immunoreactive species that are
present in both the Mac-1 (+/+) and C3H/OuJ membranes, supporting the
prediction that surface expression of CD18 is diminished in
Mac-1-deficient macrophages. A comparison of the first panel in each of
the two experiments also indicates that Taxol cross-linking does not
alter detection of these immunoreactive species by Western blot
analysis. The second panel in each experiment represents the identical
blot used to detect CD18 that was stripped and reprobed with
anti-Taxol Ab. In experiment 1, no signal is detected in the
stripped blot because the membranes were not PA Taxol cross-linked;
however, in experiment 2, the PA Taxol-cross-linked Mac-1 (-/-)
membranes (-DTT) demonstrate a markedly reduced signal that
corresponds positionally to the species detected with anti-CD18.
When these same PA Taxol-cross-linked membrane preparations were
compared using our standard reducing conditions (+DTT) and Western
analysis with the anti-Taxol Ab (the third panel in both
experiments), the signal corresponding to the 96-kDa species,
present as the major species in both the Mac-1 (+/+) and C3H/OuJ
membranes, was greatly diminished in macrophage membranes derived from
the Mac-1 (-/-) mice. These data support the hypothesis that the
predominant Taxol-binding protein in the murine macrophage membranes is
CD18. Careful analysis of the Western blots that were conducted after
size fractionation of membrane components under reducing conditions
reveals that, in addition to the major 96-kDa species being
depreciated in intensity, the Mac-1 (-/-) preparations showed a
reduced signal for several other proteins (e.g., the doublet at 53
and 56 kDa), under conditions in which the intensity of the 80-kDa
species is indistinguishable.
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96-kDa Taxol-substituted protein with
anti-CD18 Ab (Fig. 5). Western
analysis of PA Taxol-cross-linked C3H/OuJ membranes, subjected to
SDS-PAGE under nonreducing conditions, revealed that rabbit
anti-Taxol, but not rabbit IgG, blocked the capacity of rat
anti-CD18 to detect immunoreactive species. Conversely, when rat
anti-CD18 was used as the blocking agent, a significant inhibition
of detection using anti-Taxol Ab was observed.
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Sutterwala et al. (46) recently showed that induction of IL-12 p40
mRNA in murine bone marrow macrophages by LPS was reduced significantly
by cross-linking Mac-1 on the cell surface. To determine whether
Taxol-induced IL-12 p40 mRNA was also Mac-1 dependent, Mac-1 (+/+) and
Mac-1 knockout (-/-) macrophages were stimulated with various
concentrations of Taxol or with LPS. Fig. 6 shows that IL-12 p40 mRNA was strongly
induced in control macrophages at all concentrations of Taxol, and
equivalently to a dose of LPS shown previously to induce suboptimal
expression of this gene (39). In contrast, Mac-1 knockout macrophages
responded only minimally to 5 and 10 µM Taxol, and less well than +/+
macrophages at higher concentrations of Taxol or to LPS, under
conditions of comparable expression of the housekeeping gene,
HPRT. These data demonstrate a Mac-1 dependency for both LPS-
and Taxol-induced IL-12 p40 mRNA expression.
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Functional IL-12 exists as a protein heterodimer (IL-12 p70),
composed of proteins derived from both the IL-12 p40 and IL-12 p35
genes. Therefore, we analyzed the capacity of anti-Mac-1 Abs to
block Taxol-induced IL-12 secretion. Table I illustrates that both Taxol- and
LPS-induced IL-12 p70 secretion is markedly reduced in macrophages
treated with anti-Mac-1 Ab versus macrophages treated with either
medium or an isotype-matched control Ab.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A comparison of the electrophoretic mobilities of Taxol-bound proteins
with those of binding or signaling proteins previously implicated in
LPS signaling permitted us to exclude a number of obvious candidate
proteins, e.g., lyn (53 and 56 kDa), hck (56 and
59 kDa), fgr (59 kDa), c-raf (74 kDa), P13-kinase
(85 kDa), rsk (90 kDa), vav (95 kDa), Sos 1, 2
(170 kDa), and other molecules (e.g., monomeric ß-tubulin), by their
failure to comigrate with Taxol-cross-linked species (data not shown).
CD14, the best-characterized macrophage LPS receptor, was also
excluded, since 1) neither murine nor human sCD14 could be PA Taxol
cross-linked (Fig. 2); 2) CHO cells that overexpress murine CD14 failed
to exhibit an augmented signal in this molecular mass range upon
cross-linking and Western analysis with anti-Taxol Ab (Fig. 3);
and, 3) Taxol-binding patterns of macrophage membranes from C57BL/6J,
background-matched CD14 knockout, and C3H mice were indistinguishable
(data not shown). However, the key finding of a comparison of membranes
derived from several LPS-responsive cell lines is that macrophage
membranes exhibit a major Taxol-binding protein at 96 kDa, not seen
in the other five membrane preparations. Peptide analysis of the
96-kDa preparative gel slice indicated the presence of CD18, a
molecule previously implicated in LPS signaling in the context of
ß2 integrin expression in macrophages and neutrophils
(reviewed in 47). Fig. 4 confirms that the Mac-1 knockout
macrophages possess markedly decreased amounts of immunoreactive CD18,
a concordant loss of a major Taxol-binding species with similar
electrophoretic mobility under nonreducing conditions, and a massive
diminution of the major 96-kDa Taxol-binding protein under reducing
conditions. The results of Fig. 5 suggest that the epitope recognized
by the anti-CD18 Ab and substituted by Taxol on CD18 must be in
close physical proximity. Thus, the major Taxol-binding protein in
murine macrophages is CD18. The failure of the LPS-responsive CHO human
CD11b/CD18 transfectant to exhibit a prominent Taxol-cross-linked
96-kDa band (Fig. 3), coupled with its lack of Taxol sensitivity to
induce NF-B translocation (data not shown), suggests a possible
basis for the species specificity of Taxol for murine macrophages.
Although Mac-1 was shown many years ago to recognize LPS and
Gram-negative bacteria (48), it is only within the past few years that
evidence has been provided for its role in LPS-induced signal
transduction. Ikeda et al. (49) demonstrated that anti-CD18 Ab
blocked acute lethality in LPS-injected, Propionibacterium
acnes-primed rabbits, accompanied by a decrease in
plasma cytokine levels. Direct evidence came from the creation of CHO
cell transfectants that expressed either human CD11c/CD18 or CD11b/CD18
(31, 50), that were responsive to stimulation by rough LPS to induce
NF-B translocation. Stimulation of these cell lines required
significantly more LPS than CD14-CHO transfectants (30), but did not
require sCD14 or LPS-binding protein. Fig. 6 and Table I show that both
Taxol- and LPS-induced IL-12 p40 gene expression and IL-12 p70
secretion in mouse are Mac-1 dependent, particularly at lower Taxol
concentrations, and support the findings of Sutterwala et al. (46), who
showed that ligation of Mac-1 severely limits induction of IL-12 p40
mRNA by LPS. That higher concentrations of Taxol, as well as LPS,
induce suboptimal IL-12 p40 gene expression in Mac-1 knockout
macrophages suggests the possibility of additional Taxol- or
LPS-binding proteins. This notion is also supported by the observation
that the Taxol-responsive ST-2 line (29) failed to express the
96-kDa Taxol-binding protein (Fig. 3).
Ingalls et al. found that the cytoplasmic domains of CD11b and CD18
could be severely truncated, yet retain their ability to serve as LPS
receptors in CHO cell transfectants (31), supporting the notion that
Mac-1 presents LPS to a second signaling receptor for NF-B
translocation. In this regard, recent studies have shown that cells
cotransfected with CD14 and TLR2 constructs exhibited greater NF-B
translocation induced by LPS than observed in cells transfected with
TLR2 only (51, 52). This suggests that CD14, which is GPI linked and
has no signaling capability of its own, presents LPS to a TLR for
signaling. Perhaps, analogously, TLR molecules serve as
signal-transducing molecules for Taxol presented by Mac-1 following its
binding to CD18. Our finding that there is no detectable difference in
the Taxol-binding patterns exhibited by Lpsn or
Lpsd macrophages (Figs. 1 and 3) is consistent with
the widely held hypothesis that the C3H/HeJ defect, recently identified
as a single amino acid change in the intracytoplasmic domain of TLR4
(42), is distal to receptor/ligand interaction at the surface of the
membrane (reviewed in 41). Since certain normal LPS-induced
signaling events have been observed in C3H/HeJ macrophages within the
first few minutes of LPS or Taxol stimulation (5; reviewed in 41),
the failure of C3H/HeJ macrophages to respond to both LPS and Taxol is
most likely due to an interruption in the signal transduction machinery
shared by these two stimuli.
Zarewych et al. (53) demonstrated a transient association of CD14 and
Mac-1 on human neutrophils that was observed in the presence of LPS and
serum or LPS-binding protein. Additional studies (reviewed in 47)
suggest that the physical interaction of ß2 integrins
with GPI-linked proteins, including CD14, urokinase plasminogen
activator, and FcRIIIB, may enable signal transduction to occur in
response to engagement of the GPI-linked protein with its specific
ligand (i.e., LPS, in the case of CD14). In this model, the primary
interaction of LPS with CD14 would lead to a transient association with
a ß2 integrin, such as Mac-1, that in turn would initiate
signaling through the activation of the G protein, rho, and
the assemblage of an intracellular platform that includes protein
kinase C, protein tyrosine kinases, and mitogen-activated protein
kinases, components previously implicated in LPS signaling (reviewed in
54). Using gentle immunoprecipitation that resulted in recovery of
Mac-1/urokinase plasminogen activator complexes, as well as associated
src family kinases (55), Petty et al. (56) found five
proteins associated with Mac-1 with estimated molecular mass of 40, 50,
74, and 120 kDa, in addition to the Mac-1 - and ß-chains. Careful
examination of Fig. 5 revealed that not only was the 96-kDa
Taxol-binding protein largely absent in the Mac-1 knockout membranes,
but there also was a concomitant disappearance of the 53- and
56-kDa species. It is tempting to speculate that these are
components of the signaling pathway normally associated with Mac-1.
Hence, one could envision a model in which both CD14 and Mac-1 are
brought together following interaction of CD14 with LPS or of Mac-1
with Taxol or LPS to engage signaling components, such as TLR
molecules, and/or to activate additional signaling pathways through
Mac-1. This model also accounts for the observation that CD14-mediated
LPS signaling, CD11/CD18-mediated LPS signaling (57), and Taxol
signaling are blocked by LPS analogue antagonists. Previous studies
support the hypothesis that these antagonists act at sites distinct
from CD14 or Mac-1 (30, 58). Since TLR2 binds LPS (51), the LPS
analogue antagonists may also bind the same signal-transducing
molecule, and thereby inhibit intracellular signaling components
normally activated by LPS or Taxol. Thus, it is possible that LPS and
Taxol are inhibited by these compounds, despite the fact that they bind
initially to distinct pattern recognition receptors (59).
At this time, the identities of the other major Taxol-cross-linked
species are unknown. However, an 80-kDa LPS receptor was first
identified by Morrison and colleagues (reviewed in 21) in murine
and human macrophages using a photoaffinity-labeled,
125I-labeled LPS analogue. mAb raised against this molecule
induced LPS-like signaling. Dziarski (60) later suggested that this
molecule was murine albumin. In this regard, our 80-kDa
Taxol-binding protein was completely separable from bovine, murine, and
human albumins, based on clearly differing electrophoretic mobilities
after Western blot analysis with anti-Taxol versus anti-BSA Abs
(data not shown). Schletter et al. identified an 80-kDa LPS-binding
protein as the GPI-linked protein, decay-accelerating factor, in human
macrophage membranes (61, 62). It is tempting to speculate that our
80-kDa Taxol-cross-linked protein might represent one of these. We
are continuing to approach the identification of each of these novel
Taxol targets by a combination of biochemical, immunologic, and genetic
approaches, as demonstrated herein for the identification of the
96-kDa species as CD18.
|
Acknowledgments |
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Footnotes |
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2 N.B. and P.-Y.P. contributed equally to the work presented in this study.
3 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. E-mail address:
4 Abbreviations used in this paper: CHO, Chinese hamster ovary; GaR, goat anti-rabbit IgG Ab; HPRT, hypoxanthine-guanine phosphoribosyltransferase; LCQ, liquid chromatography quadropole; PA, photoactivatable; PBST, PBS + Tween (0.05%); sCD14, soluble CD14; TLR, Toll-like receptor.
Received for publication July 17, 1998. Accepted for publication March 24, 1999.
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