of February 21, 2013.This information is current as for Mac-1 in Taxol-Induced Gene Expression

as a Major Taxol-Binding Protein and a Role Macrophages: Identification of Murine CD18Identify Unique Cellular Targets in Murine Use of a Photoactivatable Taxol Analogue to

Stefanie N. VogelBlanco, Douglas T. Golenbock, Tanya N. Mayadas and Nayantara Bhat, Pin-Yu Perera, Joan M. Carboni, Jorge

http://www.jimmunol.org/content/162/12/73351999; 162:7335-7342; ;J Immunol 

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Use of a Photoactivatable Taxol Analogue to Identify UniqueCellular Targets in Murine Macrophages: Identification ofMurine CD18 as a Major Taxol-Binding Protein and a Rolefor Mac-1 in Taxol-Induced Gene Expression1

Nayantara Bhat,2* Pin-Yu Perera,2* Joan M. Carboni,† Jorge Blanco,* Douglas T. Golenbock,‡

Tanya N. Mayadas,§ and Stefanie N. Vogel3*

Taxol, a potent antitumor agent that binds b-tubulin and promotes microtubule assembly, results in mitotic arrest at the G2/Mphase of the cell cycle. More recently, Taxol was shown to be a potent LPS mimetic in murine, but not in human macrophages,stimulating signaling pathways and gene expression indistinguishably from LPS. Although structurally unrelated to LPS, Taxol’sLPS-mimetic activities are blocked by inactive structural analogues of LPS, indicating that despite the species-restricted effects ofTaxol, LPS and Taxol share a common receptor/signaling complex that might be important in LPS-induced human diseases. Toidentify components of the putatively shared Taxol/LPS receptor, a novel, photoactivatable Taxol analogue was employed toidentify unique Taxol-binding proteins in murine macrophage membranes. Seven major Taxol-binding proteins, ranging from;50 to 200 kDa, were detected. Although photoactivatable Taxol analogue failed to bind to CD14, the prominent Taxol-bindingprotein was identified as CD18, the;96-kDa common component of theb2 integrin family. This finding was supported by theconcomitant failure of macrophage membranes from Mac-1 knockout mice to express immunoreactive CD18 and the majorTaxol-binding protein. In addition, Taxol-induced IL-12 p40 mRNA was markedly reduced in Mac-1 knockout macrophages andanti-Mac-1 Ab blocked secretion of IL-12 p70 in Taxol- and LPS-stimulated macrophages. Since CD18 has been described as aparticipant in LPS-induced binding and signal transduction, these data support the hypothesis that the interaction of murine CD18with Taxol is involved in its proinflammatory activity. The Journal of Immunology,1999, 162: 7335–7342.

T he antitumor drug, Taxol, binds with high affinity tob-tu-bulin in microtubules, which, in turn, stabilizes polymer-ized microtubules, thus preventing mitosis (reviewed in

Refs. 1 and 2). In 1990, Ding et al. (3) found that Taxol alsoelicited in murine macrophages two responses that are alsostrongly induced by Gram-negative LPS: TNF secretion and rapidinvolution of TNF receptors. These LPS-mimetic effects were ob-served only in mouse macrophages that expressed a normalLpsgene (Lpsn), and not in macrophages from LPS-hyporesponsive(Lpsd) mice (4). Over the next few years, Taxol was shown tomimic LPS effects on mouse macrophages with respect to activa-tion of Lyn kinase activity (5), tyrosine phosphorylation of Shc andits association with Grb 2 (6), tyrosine phosphorylation of mito-gen-activated protein kinases (7–10), translocation of NF-kB (11,12), and induction of gene expression (7, 8). Like LPS, Taxol can

serve as a second signal for synergistic induction of inducible ni-tric oxide synthase mRNA and nitric oxide release, and tumori-cidal and microbicidal activities in IFN-g-primed macrophages(13, 14). A clear dissociation has been shown between Taxol’sLPS-mimetic effects on macrophage signaling versus its microtu-bule-stabilizing effects in vitro and in cells. Taxol analogues, suchas Taxotere and Epothilone, possess higher affinities forb-tubulinthan Taxol, compete with Taxol for the same binding site on mi-crotubules, yet fail to elicit LPS-mimetic activity in murine mac-rophages (7, 15–19). Conversely, Taxol induces normal microtu-bule bundling in C3H/HeJ macrophages in the absence ofdetectable 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 LPSand Taxol might engage elements within a shared receptor/signal-ing complex (15). Conversely, these same lipid A structural an-tagonists failed to inhibit Taxol-induced microtubule polymeriza-tion in macrophages (15), again suggesting that the sharedsignaling element is notb-tubulin. Macrophages express a numberof surface molecules (e.g., CD14, CD11/CD18, and others) thatcan interact with LPS (reviewed in Ref. 21). Studies using mac-rophages from CD14 knockout mice have shown that Taxol andLPS elicit gene expression by both CD14-dependent and indepen-dent 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, butnot in human macrophages; notably, the lipid A precursor, lipidIVA, shows a similar pattern of species specificity (reviewed inRef. 16). Although the proinflammatory effects of Taxol are

*Department of Microbiology and Immunology, Uniformed Services University ofthe Health Sciences, Bethesda, MD 20814;†Oncology Drug Discovery, Bristol-MyersSquibb, Princeton, NJ 08543;‡The Maxwell Finland Laboratory for Infectious Dis-eases, Boston University School of Medicine, Boston, MA 02118; and§Departmentof Pathology, Brigham and Women’s Hospital, Boston, MA 02115

Received for publication July 17, 1998. Accepted for publication March 24, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants AI-18797 (S.N.V.),NS-33296 (T.N.M.), GM-54060, and AI-38515 (D.T.G.).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, Departmentof Microbiology and Immunology, Uniformed Services University of the Health Sci-ences, 4301 Jones Bridge Road, Bethesda, MD 20814. E-mail address:vogel@bob.usuf2.usuhs.mil

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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largely limited to murine cells, identification of components of themurine Taxol-signaling apparatus is likely to lead to the identifi-cation of homologous human LPS-signaling proteins.

To identify novel Taxol-binding proteins within a shared LPSsignaling complex, we developed a novel detection system basedon a photoactivatable Taxol analogue (PA Taxol) used originallyto define Taxol’s interaction with tubulin (23). PA Taxol binds toat least seven murine macrophage membrane proteins, and themost prominent of these is the common component of theb2 fam-ily of integrins, CD18, based on the coordinate failure of macro-phages 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 wasmarkedly reduced in macrophages derived from Mac-1 knockoutmice, and anti-Mac-1 Ab blocks Taxol-induced IL-12 p70 secre-tion, suggesting that the interaction of Taxol with CD18 is criticalfor its ability to act as a full LPS mimetic.

Materials and MethodsMice

Five- to seven-week-old C3H/OuJ or C3H/HeJ mice were obtained fromThe Jackson Laboratory (Bar Harbor, ME). CD11b (Mac-1) knockout(2/2) mice and wild-type (1/1) controls (24) were bred at LongwoodMedical Research Center (Boston, MA). Mac-1 knockout and background-matched, wild-type colonies are of a mixed C57BL/129Sv background thatis maintained by ongoing heterozygous breedings. Mice were housed in avirus Ab-free facility.

Preparation of cell membrane fractions

Peritoneal exudate cells (;85% macrophages) were collected by lavagewith sterile saline 4 days after i.p. injection of mice with 3 ml thioglycollate(Difco, Detroit, MI). Cells were centrifuged (7503 g for 5 min) andresuspended in PBS (13, pH 7.4). Cell viability was.95% by trypan blueexclusion. Peritoneal exudate cells were recentrifuged (7503 g, 5 min),and the pellet was resuspended at 13 107 cells/ml in homogenizationbuffer containing 25 mM HEPES, pH 7.3, 0.5 mM EGTA, 0.5 mM sodiumorthovanadate, 0.1 mM sodium molybdate, 1 mM sodium fluoride, andprotease inhibitors (1 tablet Complete (Boehringer Mannheim, Mannheim,Germany) per 50 ml buffer). A total of 7 ml of cell homogenate was in-cubated on ice, and cells were lysed using a tight-fitting dounce homoge-nizer (100 strokes). Unlysed cells, nuclei, and cell fragments (25) wereremoved by centrifugation (10003 g, 10 min) at 4°C. The resulting nuclei-free supernatant was centrifuged again (10,0003 g, 7 min) at 4°C toremove aggregates of cytoskeletal elements (26). The membrane pellet wascollected by a final centrifugation of the above supernatant at 417,0003 g(rav) for 2 h at 4°C andresuspended and solubilized in homogenizationbuffer containing 10 mM CHAPS (Sigma, St. Louis, MO). This membranepreparation method is a modification of that reported by Liu et al. (27).Protein determinations were performed by Bio-Rad protein assay (Rich-mond, CA). In some experiments, after UV cross-linking (below), 50mlsamples were centrifuged (20,8003 g, 15 min at 4°C) before Westernanalysis. 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-humanCD18 cell lines were derived by stable transfection of Chinese hamsterovary (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 (PATaxol; m.w. 5 1043) has been described (23). Multiwell plates (Falcon96-well microtiter plates; Becton Dickinson, Lincoln Park, NJ) wereprewetted with 50ml of 50 mM Tris-HCl buffer, pH 7.4, and drained.Typically, a mixture containing 150mg membrane protein and 6ml PATaxol (835mM) was prepared in a final volume of 50ml in 50 mM Tris-HCl buffer, pH 7.4, such that the final concentration of PA Taxol was 100mM. Aliquots (50 ml) of this mixture were added to each well of theprewetted 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 ata distance of 7 cm, as described elsewhere (32). After irradiation, sampleswere diluted in 43 Laemmli buffer (32) (200 mM Tris-HCl, pH 6.8; 8%SDS; 400 mM DTT; 40% glycerol; 0.4% bromophenol blue; final concen-tration of Laemmli buffer is 13) and boiled for 5 min. Membrane proteins(30–50mg) 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 wellas to identify other proteins in membranes. For detection of CD18 byWestern blotting, nonreducing conditions were required: samples were pre-pared for SDS-PAGE as described above in the absence of DTT. FollowingSDS-PAGE, resolved proteins were transferred to Immobilon-P mem-branes (Millipore, Bedford, MA) using a Minitrans Blot ElectrophoreticTransfer Cell (Bio-Rad) for 1 h at 100 V at 4°C intransfer buffer (25 mMTris, 192 mM glycine, pH 8.3, and 20% methanol). Blots were stained for10 min in Ponceau S (Sigma) and destained in water. The positions ofstained bands of authentic standards were used to calculate m.w. of sepa-rated proteins. Membranes were blocked for 1 h at room temperature inPBS containing 1% gelatin and 5% nonfat milk. After washing (3 times, 5min each) with PBS, membranes were incubated with primary Ab dilutedin PBS plus 5% nonfat milk for 1 h atroom temperature. Membranes werewashed (3 times, 10 min each) with PBS1 Tween (0.05%) (PBST). Fi-nally, membranes were incubated in an appropriate HRP-conjugated sec-ondary Ab (i.e., goat anti-rabbit IgG (Bio-Rad), 1/2500; goat anti-mouseIgG (Bio-Rad), 1/5000; or goat anti-rat IgG, 1/2000 dilution (Santa CruzBiotechnology, Santa Cruz, CA)). Secondary Abs were diluted in PBScontaining 5% nonfat milk and incubated with membranes for 1 h at roomtemperature. Membranes were washed (five times, 5 min each) with PBST.Binding of secondary Ab was detected with the enhanced chemolumines-cence (ECL) detection method (Amersham Life Science, Little Chalfont,U.K.). In some experiments, immunoblots were stripped: membranes werewashed for 5 min at room temperature in 53 SSC (0.75 M sodium chlo-ride, 0.075 M sodium citrate, pH 7). Membranes were placed in 0.13SSCcontaining 0.1% SDS and incubated for 1 h at 65°C in a water bath withgentle rotation, followed by an additional wash for 5 min at room temper-ature in 53 SSC. Stripped membranes were blocked and reprobed using analternate primary Ab, as described above. In blocking experiments, blotswere first cut into individual lanes after the initial blocking step and indi-vidual strips were incubated overnight at 4°C (or 2 h atroom temperature)with one of the following: no Ab, rabbit anti-Taxol (1mg/ml), rat anti-CD18 (10mg/ml), or isotype-matched, affinity-purified control Abs (rabbitIgG or rat anti-Mac-3). Blots were washed with PBST and incubated witheither rat anti-CD18 (10mg/ml) or rabbit anti-Taxol (1mg/ml) primary Abto detect CD18 or Taxol-cross-linked proteins, respectively.

Primary Abs

Anti-Taxol Ab was generated by hyperimmunizing rabbits with Taxolcross-linked at C7 to keyhole limpet hemocyanin. Polyclonal Abs wereammonium sulfate precipitated, affinity purified by passage over a proteinA column, and dialyzed in PBS. For Western analysis, rabbit anti-Taxol Abwas used at a final concentration of 1mg/ml. The following primary Abswere also used: rabbit anti-BSA antiserum (kindly provided by Dr. BobRoberson, University of Maryland, College Park, MD; 1:1500), rabbit anti-murine CD14 antiserum (kindly provided by Dr. Richard Ulevitch, ScrippsResearch, 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; 5mg/ml).

Preparation of Taxol-cross-linked proteins for liquidchromatography quadropole (LCQ) analysis

PA Taxol-cross-linked membrane proteins (600mg) were resolved on a 9%preparative SDS-PAGE, and a single lane from each edge of the gel wasexcised and processed for Western analysis to detect Taxol-bound proteins(see above). The remaining gel was stained with 0.1% Coomassie blue in10% acetic acid, 50% methanol for 30 min. The gel was destained for$2h in 10% acetic acid, 50% methanol until the background was clear. Thepolyvinylidene difluoride membrane strips that were developed with anti-Taxol Ab were positioned next to the Coomassie-stained gel. The region onthe stained gel that coincided positionally with the;96-kDa species wasexcised. A blank gel piece was excised as a control. Gel slices were placedin Eppendorf tubes, frozen, and sent to the W. M. Keck Foundation (New

4 Abbreviations used in this paper: CHO, Chinese hamster ovary; GaR, goat anti-rabbit IgG Ab; HPRT, hypoxanthine-guanine phosphoribosyltransferase; LCQ, liquidchromatography quadropole; PA, photoactivatable; PBST, PBS1 Tween (0.05%);sCD14, soluble CD14; TLR, Toll-like receptor.

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Haven, CT) for LCQ analysis. Briefly, 32 pmol of the;96-kDa gel slicewas digested with trypsin, along with the blank gel slice. Approximately10% of the digest was subjected to LC-MS/MS using an LCQ ion trap massspectrometer (35). A Sequest search of the OWL database indicated thatone of the reconstructed MS/MS spectra had significant similarity (i.e., thedifference in the normalized cross-correlation functions as defined by Enget al. (36) of the first and second ranked searches was 0.32) to the observedspectra. This reconstructed spectra corresponded to an 18-residue peptide,SAVGELSDDSSNVVQLIK, found in mouse cell surface adhesion glyco-proteins (LFA-1, CR3, and P150, 95,b subunit precursor), preceded by alysine.

Other reagents

Recombinant human soluble CD14 (sCD14) and recombinant murine sol-uble CD14 (mu sCD14) were kindly provided by Dr. Henri Lichenstein(Amgen Boulder, Boulder, CO) and Dr. Christine Schutt (Arndt-Univer-sitat Greifswald, Greifswald, Germany), respectively. Tubulin, devoid ofmicrotubule-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 affinitypurified from the M3/84 myeloma cell line (American Type Culture Col-lection (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 secretionexperiments. Taxol was provided by the Drug Synthesis and ChemistryBranch (National Institutes of Health), andEscherichia coliK235 LPS wasprepared 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 hypox-anthine-guanine phosphoribosyltransferase (HPRT) as a housekeepinggene (39). Immunoreactive IL-12 p70 was measured by ELISA (PharM-ingen), following the manufacturer’s instructions.

ResultsUse of a photoaffinity-labeled Taxol analogue (PA Taxol) toidentify novel Taxol-binding proteins in murine macrophages

To determine whether novel Taxol-binding proteins could be de-tected in murine macrophage membranes, macrophage membranes

were subjected to UV cross-linking in the presence or absence ofPA Taxol, an analogue developed to identify the Taxol-microtu-bule binding domain (23). Fig. 1A shows that Taxol-cross-linkedC3H/OuJ membranes (XL C3H/OuJ), but not uncross-linkedmembranes (C3H/OuJ), show a distinct pattern of immunoreactivebands when detected by Western analysis with a primary rabbitanti-Taxol Ab (RaTaxol); bands were not detected in the presenceof secondary Ab only (GaR), or if an irrelevant primary Ab, rabbitanti-BSA, were used. Inclusion of 50mM Taxol at the time of theincubation of the blot with anti-Taxol Ab resulted in nearly com-plete competition of all bands (data not shown). The seven majorspecies detected in 9–10% polyacrylamide gels migrate with av-erage molecular mass of 197 kDa, 149 kDa (sometimes resolvableas a doublet), 114 kDa, 96 kDa (always the major species thatappears as a broad band), 80 kDa, 56 kDa, and 53 kDa (also some-times resolvable as a doublet). Bands detected by Western analysisdid not coincide with major bands seen after Coomassie staining(data not shown). In certain experiments, an additional centrifu-gation step was included after UV cross-linking. Ninety percent ofthe total membrane protein subjected to cross-linking remained inthe supernatant, with an enrichment of the 114-, 96-, and 80-kDaspecies. These species are presumably associated with membranemicrodomains that remain soluble following UV cross-linking, incontrast to those recovered in the pellet fraction. All major specieswere detected in the pellet fraction (data not shown).

Earlier studies have shown that [3H]Taxol could be UV cross-linked tob-tubulin without PA derivitization (32) and that bindingof a PA Taxol analogue similar to that used in these studies couldbe competed by underivatized Taxol (40). Therefore, macrophagemembranes were UV cross-linked in the presence of underivatizedTaxol or PA Taxol (Fig. 1B). Under our standard conditions inwhich PA Taxol was used at 100mM (i.e., similar to those used instudies withb-tubulin) (40), the seven major immunoreactive spe-cies were readily detected. A total of 10mM PA Taxol resulted in

FIGURE 1. Detection of PA Taxol-cross-linked proteins in murine macrophage membranes.A, C3H/OuJ macrophage membranes were UV cross-linked(XL) in the presence of 100mM PA Taxol, and subjected to SDS-PAGE and then Western analysis using rabbit anti-Taxol (RaTx) or rabbit anti-BSA(RaBSA) as primary Abs and GaR as the secondary Ab.B, C3H/OuJ macrophage membranes were UV cross-linked (XL) in the presence of underivatizedTaxol (100mM) or PA Taxol (10 or 100mM), and subjected to SDS-PAGE and Western analysis using rabbit anti-Taxol (RaTx) as the primary Ab andGaR as the secondary Ab.C, C3H/OuJ and C3H/HeJ macrophage membranes were UV cross-linked (XL) in the presence of 100mM PA Taxol, andsubjected to SDS-PAGE and then Western blot analysis using rabbit anti-Taxol (RaTaxol) as the primary Ab and GaR as the secondary Ab. A total of 30mg protein/lane was analyzed using 9% SDS-PAGE gels in all panels. Dots indicate the position of the major PA Taxol-cross-linked species. Molecularmasses of marker proteins are indicated in kilodaltons (KD). Results are representative of two to five separate experiments.

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less intense bands, although positionally indistinguishable fromthose detected after cross-linking with 100mM PA Taxol. Underi-vatized Taxol, used at a 100mM, also resulted in detection of thesame immunoreactive species, although the efficiency of cross-linking was much more like that seen with the suboptimal (10mM)concentration of PA Taxol. These data support the hypothesis thatthe PA Taxol interacts with the same major membrane proteins asunderivatized Taxol.

C3H/HeJ mice express a gene defect (Lpsd) that renders themrefractory to LPS in vivo and in vitro (reviewed in Ref. 41). Apoint mutation in the intracytoplasmic domain of the TLR4 trans-membrane receptor underlies this defect (42). Since Taxol hasbeen shown to act only on macrophages that possess normal LPSresponsiveness (Lpsn), and not in C3H/HeJ macrophages (4),membranes from C3H/OuJ (Lpsn) and C3H/HeJ (Lpsd) mice weresubjected to UV cross-linking with PA Taxol. Fig. 1Cshows noobvious differences in the pattern of immunoreactive species inWestern blots derived from the two mouse strains. Collectively,the data in Fig. 1, A–C, illustrate the specificity and reproducibilityof the detection system.

Membrane CD14,;53–56 kDa molecular mass, is the majorLPS-binding protein on macrophages (reviewed in Ref. 43). Stud-ies using macrophages from CD14 knockout mice showed a CD14dependency for Taxol stimulation, albeit more limited than ob-served for LPS (22). Therefore, we tested the hypothesis that eitherthe 53- or 56-kDa Taxol-binding proteins were CD14. MurinesCD14 failed to be cross-linked by PA Taxol, under conditions inwhich the 53- and 56-kDa Taxol-cross-linked species were de-tected in the C3H/OuJ membrane preparation (Fig. 2). Similarly,no anti-Taxol immunoreactive bands were detected in cross-linkedhuman sCD14, under conditions in which tubulin was extensivelycross-linked and readily detected by Western analysis (i.e., itformed high m.w. complexes). Thus, CD14 is not a target forTaxol binding.

Taxol cross-linking to various membrane preparations

An analysis of membrane preparations derived from several LPS-responsive cell lines was undertaken to evaluate the potential forcommon Taxol-binding proteins. Fig. 3 compares PA Taxol-cross-linked membranes derived from CHO cells that express humanCD11b/CD18, SAM8 (murine CD14 transfectant), and the control

CHO-NEO cell line, the murine ST2 cell line, and C3H/OuJ andC3H/HeJ macrophages. Both the CD11b/CD18 and SAM8 trans-fectants respond to rough LPS (29, 30), but not to Taxol (unpub-lished observations); the control CHO-NEO cell line responds toneither LPS nor Taxol. The murine ST2 cell line, derived frombone 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 in-cluded as controls. Western analysis of PA Taxol-cross-linkedmembranes from the three CHO cell lines was indistinguishable,but exhibited a pattern of anti-Taxol immunoreactive bands dis-tinct from that exhibited by the ST2 cell line, and from the patterncommon to C3H/OuJ and C3H/HeJ macrophage membranes. Anumber of bands comigrate in all of the membranes, includingthose with apparent molecular mass of;149 kDa,;114 kDa (al-though considerably fainter in the macrophage membranes),;80kDa, and;53 kDa (the latter was somewhat more prominentlydetected in the macrophage membranes), and the;56-kDa speciesis more pronounced in the ST2 cell line. Most significantly, the;96-kDa species appears to be predominant and specific to themurine macrophage membrane preparations.

Identification of the major Taxol-binding species in murinemacrophages as CD18

LCQ analysis of peptides derived from the preparative;96-kDagel slice indicated that the mass/charge ratio of one tryptic frag-ment was derived from murine CD18, the commonb-chain of theb2 family of integrins (reviewed in Ref. 44). CD18 is expressed asan obligate ab heterodimer in noncovalent association withCD11a, CD11b, CD11c, or CD11d (reviewed in Ref. 44). SinceCD11b/CD18 (also referred to as Mac-1) is the predominantb2

integrin on murine macrophages (reviewed in Refs. 44 and 45),and since CD11b/CD18 has been found to mediate LPS signalingin 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 tookadvantage of mice with a targeted mutation in CD11b, thea sub-unit of Mac-1 (24). Previous FACS analysis revealed that neutro-phils and macrophages derived from CD11b knockout mice lack

FIGURE 2. PA Taxol cross-linking of murine soluble CD14 (musCD14). Mu sCD14 (10mg) or C3H/OuJ macrophage membranes wereUV cross-linked (XL) in the presence of PA Taxol or not (no modifier), andwere subjected to 10% SDS-PAGE and Western analysis with anti-Taxolor anti-CD14 Ab, as described for Fig. 1. Results are representative of threeseparate experiments.

FIGURE 3. Detection of PA Taxol-cross-linked proteins in membranesprepared from CHO cell transfectants (human CD11b/CD18 (CHO-huCD11b/CD18), murine CD14 (CHO-SAM8), and control (CHO-NEO)),murine ST2 stromal cells, and murine C3H/OuJ and C3H/HeJ macro-phages. SDS-PAGE and Western analysis were conducted as described forFig. 1. Results are representative of five separate experiments.

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surface CD11b (24) and that surface expression of CD18 on thio-glycollate-elicited macrophages was reduced by.85% (Coxonand Mayadas, unpublished). This observation is consistent withfinding that CD11b/CD18 is the most abundantb2 integrin onmacrophages (45), and that in the absence ofa subunits, CD18 isnot translocated to the cell surface (reviewed in Ref. 44). Thus, wereasoned that if CD18 were the predominant Taxol-binding proteinin murine macrophages, there should be a significant reduction inthe expression of the;96-kDa Taxol-binding protein in macro-phage membranes derived from CD11b knockout mice.

Fig. 4 shows the results of two separate experiments in whichmembranes from CD11b (Mac-1) knockout macrophages andbackground-matched, wild-type macrophages were compared fortheir ability to be UV cross-linked with PA Taxol. As a con-trol, C3H/OuJ membranes were included. First, noncross-linked(Expt. 1) or PA Taxol-cross-linked (Expt. 2) Mac-1 (1/1), Mac-1(2/2), and C3H/OuJ membranes were subjected to SDS-PAGEanalysis in the absence of DTT (2DTT), conditions that are re-quired for Western analysis with anti-CD18 mAb (i.e., the anti-murine CD18 is directed against a conformational epitope, pre-cluding its use under reducing conditions) (34). Our experimentsshow that the membranes of Mac-1 knockout macrophages arehighly deficient in two immunoreactive species that are present inboth the Mac-1 (1/1) and C3H/OuJ membranes, supporting theprediction that surface expression of CD18 is diminished in Mac-1-deficient macrophages. A comparison of the first panel in each ofthe two experiments also indicates that Taxol cross-linking doesnot alter detection of these immunoreactive species by Westernblot analysis. The second panel in each experiment represents theidentical blot used to detect CD18 that was stripped and reprobedwith anti-Taxol Ab. In experiment 1, no signal is detected in thestripped blot because the membranes were not PA Taxol cross-linked; however, in experiment 2, the PA Taxol-cross-linkedMac-1 (2/2) membranes (2DTT) demonstrate a markedly re-duced signal that corresponds positionally to the species detectedwith anti-CD18. When these same PA Taxol-cross-linked mem-brane preparations were compared using our standard reducingconditions (1DTT) and Western analysis with the anti-Taxol Ab(the third panel in both experiments), the signal corresponding tothe ;96-kDa species, present as the major species in both theMac-1 (1/1) and C3H/OuJ membranes, was greatly diminished inmacrophage membranes derived from the Mac-1 (2/2) 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 aftersize fractionation of membrane components under reducing con-ditions reveals that, in addition to the major;96-kDa species be-

ing depreciated in intensity, the Mac-1 (2/2) preparations showeda reduced signal for several other proteins (e.g., the doublet at;53and 56 kDa), under conditions in which the intensity of the;80-kDa species is indistinguishable.

To confirm that CD18 is the major Taxol-binding protein, com-petition experiments were conducted to block detection of CD18 inthe Western analysis using anti-Taxol Ab as the blocking agent,and conversely, to block detection of the;96-kDa Taxol-substi-tuted protein with anti-CD18 Ab (Fig. 5). Western analysis of PATaxol-cross-linked C3H/OuJ membranes, subjected to SDS-PAGEunder nonreducing conditions, revealed that rabbit anti-Taxol, butnot rabbit IgG, blocked the capacity of rat anti-CD18 to detectimmunoreactive species. Conversely, when rat anti-CD18 wasused as the blocking agent, a significant inhibition of detectionusing anti-Taxol Ab was observed.

Mac-1 knockout macrophages exhibit diminished IL-12 p40mRNA induction in response to Taxol and LPS

Sutterwala et al. (46) recently showed that induction of IL-12 p40mRNA in murine bone marrow macrophages by LPS was reducedsignificantly by cross-linking Mac-1 on the cell surface. To deter-mine whether Taxol-induced IL-12 p40 mRNA was also Mac-1dependent, Mac-1 (1/1) and Mac-1 knockout (2/2) macro-phages were stimulated with various concentrations of Taxol or

FIGURE 4. Detection of CD18and PA Taxol-cross-linked proteinsin membranes derived from Mac-1(1/1), Mac-1 (2/2), and C3H/OuJmacrophages. Macrophage mem-branes were UV cross-linked in thepresence of PA Taxol (XL) or not(no modifier), and subjected to SDS-PAGE under nonreducing (2DTT)or reducing (1DTT) conditions,then subjected to Western analysis.Results are representative of six sep-arate experiments.

FIGURE 5. Anti-Taxol Ab blocks detection of CD18, and anti-CD18Ab blocks detection of the Taxol-cross-linked;96-kDa species in PATaxol-cross-linked C3H/OuJ membranes. C3H/OuJ macrophage mem-branes were UV cross-linked in the presence of 100mM PA Taxol andsubjected to SDS-PAGE under nonreducing conditions (2DTT). Westernanalysis was conducted using rat anti-CD18 (rat a CD18) or rabbit anti-Taxol (RaTx) following incubation of individual lanes in the absence orpresence of blocking Abs or their isotype-matched controls (indicatedabove each lane), as described inMaterials and Methods. Results are rep-resentative of two separate experiments.

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with LPS. Fig. 6 shows that IL-12 p40 mRNA was strongly in-duced in control macrophages at all concentrations of Taxol, andequivalently to a dose of LPS shown previously to induce subop-timal expression of this gene (39). In contrast, Mac-1 knockoutmacrophages responded only minimally to 5 and 10mM Taxol,and less well than1/1 macrophages at higher concentrations ofTaxol or to LPS, under conditions of comparable expression ofthe housekeeping gene, HPRT. These data demonstrate a Mac-1dependency for both LPS- and Taxol-induced IL-12 p40 mRNAexpression.

Anti-Mac-1 Ab blocks both LPS- and Taxol-elicited IL-12 p70secretion in C3H/OuJ macrophages

Functional IL-12 exists as a protein heterodimer (IL-12 p70), com-posed of proteins derived from both the IL-12 p40 and IL-12 p35genes. Therefore, we analyzed the capacity of anti-Mac-1 Abs toblock Taxol-induced IL-12 secretion. Table I illustrates that bothTaxol- and LPS-induced IL-12 p70 secretion is markedly reducedin macrophages treated with anti-Mac-1 Ab versus macrophagestreated with either medium or an isotype-matched control Ab.

DiscussionTaxol is best known for its antitumor activity, which is affected byits ability to interact withb-tubulin to hyperstabilize microtubulesand block mitosis. However, Taxol is a full LPS mimetic in murinemacrophages, an activity that is mechanistically distinct from itsb-tubulin-binding activity. Based on the observation that LPS an-alogue antagonists block the LPS-mimetic effects of Taxol, wehypothesized that Taxol’s LPS-mimetic activity was due to its in-teraction with a membrane structure(s) distinct fromb-tubulinwithin a putative LPS receptor complex that engages signal trans-duction pathways common to both LPS and Taxol. To date, iden-tification of proteins within an LPS signaling complex has been

fraught with technical difficulties due to the extremely hydropho-bic nature of LPS-derived probes. To obviate this concern, we useda novel, photoactivatable Taxol analogue (PA Taxol), used origi-nally in studies of Taxol’s interaction with microtubule proteins, tocross-link components of this putative, common signaling com-plex within murine macrophage membranes. We sought to identifystructures associated with Taxol-induced proinflammatory actionsin murine macrophages that might ultimately lead to the identifi-cation of homologous human proteins involved in LPS signaling.We found that PA Taxol binds to a number of proteins in cellmembranes in a reproducible and specific fashion, in addition to itswell-characterized ability to interact withb-tubulin in the contextof microtubules.

A comparison of the electrophoretic mobilities of Taxol-boundproteins with those of binding or signaling proteins previously im-plicated in LPS signaling permitted us to exclude a number ofobvious candidate proteins, e.g.,lyn (53 and 56 kDa),hck (56 and59 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., monomericb-tubulin), by their failure to comigrate withTaxol-cross-linked species (data not shown). CD14, the best-char-acterized 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 toexhibit an augmented signal in this molecular mass range uponcross-linking and Western analysis with anti-Taxol Ab (Fig. 3);and, 3) Taxol-binding patterns of macrophage membranes fromC57BL/6J, background-matched CD14 knockout, and C3H micewere indistinguishable (data not shown). However, the key findingof a comparison of membranes derived from several LPS-respon-sive cell lines is that macrophage membranes exhibit a majorTaxol-binding protein at;96 kDa, not seen in the other five mem-brane preparations. Peptide analysis of the;96-kDa preparativegel slice indicated the presence of CD18, a molecule previouslyimplicated in LPS signaling in the context ofb2 integrin expres-sion in macrophages and neutrophils (reviewed in Ref. 47). Fig. 4confirms that the Mac-1 knockout macrophages possess markedlydecreased amounts of immunoreactive CD18, a concordant loss ofa major Taxol-binding species with similar electrophoretic mobil-ity under nonreducing conditions, and a massive diminution of themajor;96-kDa Taxol-binding protein under reducing conditions.The results of Fig. 5 suggest that the epitope recognized by theanti-CD18 Ab and substituted by Taxol on CD18 must be in closephysical proximity. Thus, the major Taxol-binding protein in mu-rine macrophages is CD18. The failure of the LPS-responsiveCHO human CD11b/CD18 transfectant to exhibit a prominentTaxol-cross-linked;96-kDa band (Fig. 3), coupled with its lack

FIGURE 6. Induction of IL-12 p40 mRNA by Taxol and LPS in macrophages derived from Mac-1 (1/1) and Mac-1 (2/2) mice. Macrophages wereplated at a final concentration of 6.53 106 cells/well in six-well culture plates and treated for 4 h with the indicated concentration of Taxol or LPS. TotalRNA was harvested and subjected to quantitative RT-PCR, as described previously (39). HPRT was included as the housekeeping gene. This Southern blotis representative of three separate experiments.

Table I. Effect of anti-Mac-1 or control Ab on LPS- and Taxol-inducedIL-12 p70 secretion in C3H/OuJ macrophagesa

Stimulus

IL-12 p70 (pg/ml)

Medium Anti-Mac 1 Anti-CD122 (control)

Medium 0 0 9 6 12b

LPS (1 ng/ml) 7826 56 3706 112 8036 118Taxol (20mM) 4466 39 1906 5 4656 47

a C3H/OuJ macrophages (2.33 105/well in a 96-well plate) were pretreated with25 mg/ml anti-Mac 1 or control anti-CD122 Ab for 15 min before treatment with LPSor Taxol for 24 h. Culture supernatants from duplicate wells were pooled and assayedfor IL-12 p70 by ELISA.

b The data represent the mean6 SD from two separate experiments.

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of Taxol sensitivity to induce NF-kB translocation (data notshown), suggests a possible basis for the species specificity ofTaxol for murine macrophages.

Although Mac-1 was shown many years ago to recognize LPSand Gram-negative bacteria (48), it is only within the past fewyears that evidence has been provided for its role in LPS-inducedsignal transduction. Ikeda et al. (49) demonstrated that anti-CD18Ab blocked acute lethality in LPS-injected,Propionibacteriumacnes-primed rabbits, accompanied by a decrease in plasma cyto-kine levels. Direct evidence came from the creation of CHO celltransfectants that expressed either human CD11c/CD18 or CD11b/CD18 (31, 50), that were responsive to stimulation by rough LPSto induce NF-kB translocation. Stimulation of these cell lines re-quired significantly more LPS than CD14-CHO transfectants (30),but did not require sCD14 or LPS-binding protein. Fig. 6 and Ta-ble I show that both Taxol- and LPS-induced IL-12 p40 gene ex-pression and IL-12 p70 secretion in mouse are Mac-1 dependent,particularly at lower Taxol concentrations, and support the findingsof Sutterwala et al. (46), who showed that ligation of Mac-1 se-verely limits induction of IL-12 p40 mRNA by LPS. That higherconcentrations of Taxol, as well as LPS, induce suboptimal IL-12p40 gene expression in Mac-1 knockout macrophages suggests thepossibility of additional Taxol- or LPS-binding proteins. This no-tion is also supported by the observation that the Taxol-responsiveST-2 line (29) failed to express the;96-kDa Taxol-bindingprotein (Fig. 3).

Ingalls et al. found that the cytoplasmic domains of CD11b andCD18 could be severely truncated, yet retain their ability to serveas LPS receptors in CHO cell transfectants (31), supporting thenotion that Mac-1 presents LPS to a second signaling receptor forNF-kB translocation. In this regard, recent studies have shown thatcells cotransfected with CD14 and TLR2 constructs exhibitedgreater NF-kB translocation induced by LPS than observed in cellstransfected 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, TLRmolecules serve as signal-transducing molecules for Taxol pre-sented by Mac-1 following its binding to CD18. Our finding thatthere is no detectable difference in the Taxol-binding patterns ex-hibited byLpsn or Lpsd macrophages (Figs. 1 and 3) is consistentwith the widely held hypothesis that the C3H/HeJ defect, recentlyidentified as a single amino acid change in the intracytoplasmicdomain of TLR4 (42), is distal to receptor/ligand interaction at thesurface of the membrane (reviewed in Ref. 41). Since certain nor-mal LPS-induced signaling events have been observed in C3H/HeJmacrophages within the first few minutes of LPS or Taxol stimu-lation (5; reviewed in Ref. 41), the failure of C3H/HeJ macro-phages to respond to both LPS and Taxol is most likely due to aninterruption in the signal transduction machinery shared by thesetwo stimuli.

Zarewych et al. (53) demonstrated a transient association ofCD14 and Mac-1 on human neutrophils that was observed in thepresence of LPS and serum or LPS-binding protein. Additionalstudies (reviewed in Ref. 47) suggest that the physical interactionof b2 integrins with GPI-linked proteins, including CD14, uroki-nase plasminogen activator, and FcgRIIIB, may enable signaltransduction to occur in response to engagement of the GPI-linkedprotein with its specific ligand (i.e., LPS, in the case of CD14). Inthis model, the primary interaction of LPS with CD14 would leadto a transient association with ab2 integrin, such as Mac-1, that inturn would initiate signaling through the activation of the G pro-tein, rho, and the assemblage of an intracellular platform that in-cludes protein kinase C, protein tyrosine kinases, and mitogen-activated protein kinases, components previously implicated in

LPS signaling (reviewed in Ref. 54). Using gentle immunoprecipi-tation that resulted in recovery of Mac-1/urokinase plasminogenactivator complexes, as well as associatedsrc family kinases (55),Petty et al. (56) found five proteins associated with Mac-1 withestimated molecular mass of 40, 50, 74, and 120 kDa, in additionto the Mac-1a- and b-chains. Careful examination of Fig. 5 re-vealed that not only was the;96-kDa Taxol-binding proteinlargely absent in the Mac-1 knockout membranes, but there alsowas a concomitant disappearance of the;53- and;56-kDa spe-cies. It is tempting to speculate that these are components of thesignaling pathway normally associated with Mac-1. Hence, onecould envision a model in which both CD14 and Mac-1 arebrought together following interaction of CD14 with LPS or ofMac-1 with Taxol or LPS to engage signaling components, such asTLR molecules, and/or to activate additional signaling pathwaysthrough Mac-1. This model also accounts for the observation thatCD14-mediated LPS signaling, CD11/CD18-mediated LPS signal-ing (57), and Taxol signaling are blocked by LPS analogue antag-onists. Previous studies support the hypothesis that these antago-nists act at sites distinct from CD14 or Mac-1 (30, 58). Since TLR2binds LPS (51), the LPS analogue antagonists may also bind thesame signal-transducing molecule, and thereby inhibit intracellularsignaling 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 recogni-tion receptors (59).

At this time, the identities of the other major Taxol-cross-linkedspecies are unknown. However, an;80-kDa LPS receptor wasfirst identified by Morrison and colleagues (reviewed in Ref. 21) inmurine and human macrophages using a photoaffinity-labeled,125I-labeled LPS analogue. mAb raised against this molecule in-duced LPS-like signaling. Dziarski (60) later suggested that thismolecule 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 electrophoreticmobilities after Western blot analysis with anti-Taxol versus anti-BSA Abs (data not shown). Schletter et al. identified an;80-kDaLPS-binding protein as the GPI-linked protein, decay-acceleratingfactor, in human macrophage membranes (61, 62). It is tempting tospeculate that our;80-kDa Taxol-cross-linked protein might rep-resent one of these. We are continuing to approach the identifica-tion of each of these novel Taxol targets by a combination ofbiochemical, immunologic, and genetic approaches, as demon-strated herein for the identification of the;96-kDa speciesas CD18.

AcknowledgmentsWe thank Sheila Hauck and Dr. Vittorio Farina for the conjugation of BSAand keyhole limpet hemocyanin to Taxol as well as the many people whogenerously provided reagents for this study: Drs. Bob S. Roberson, RichardUlevitch, Ed Roos, Henri Lichenstein, Christine Schutt, and Mark Lynch.Finally, we appreciate the advice of Drs. S. Shaw and E. Roos during thepreparation of this manuscript.

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