Divergent Response to LPS and Bacteria in CD14-Deficient Murine Kathryn J. Moore, Lorna P. Andersson, Robin R. Ingalls, Brian G. Monks, Rui Li, M. Amin Arnaout, Douglas T. This information is current as Golenbock and Mason W. Freeman of October 1, 2021. J Immunol 2000; 165:4272-4280; ; doi: 10.4049/jimmunol.165.8.4272 http://www.jimmunol.org/content/165/8/4272 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Divergent Response to LPS and Bacteria in CD14-Deficient Murine Macrophages1

Kathryn J. Moore,2* Lorna P. Andersson,2* Robin R. Ingalls,† Brian G. Monks,† Rui Li,‡ M. Amin Arnaout,‡ Douglas T. Golenbock,† and Mason W. Freeman3*

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of . The GPI-linked membrane , CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-␣ and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-␬B and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, Downloaded from in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-␣ release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14- deficient macrophages produced significantly less TNF-␣ than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by blockade using inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-␣ in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate http://www.jimmunol.org/ for the loss of CD14 in response to whole bacteria. The Journal of Immunology, 2000, 165: 4272–4280.

ipopolysaccharide, the major constituent of the outer acute phase reactant LPS binding protein (LBP)4 and soluble membrane of Gram-negative bacteria (1, 2), plays a cru- CD14 (sCD14). When expressed on the surface of cells, CD14 has L cial role in mediating host responses to Gram-negative no intrinsic signaling capabilities, but is postulated to present LPS infections by stimulating the release of inflammatory mediators, to its high affinity signal transducer, Toll-like receptor (TLR) 4 including , from various target cells. LPS activation of (5–8). CD14 also enhances the responses of phagocytes to bacte- these mediators is thought to be responsible for the clinical man- rial products that activate cells via TLR2 (e.g., peptidoglycan and

ifestations of septic shock (3) and it may also play a role in the lipopeptide), although the exact contribution of TLR2 to LPS re- by guest on October 1, 2021 pathophysiology of chronic inflammatory disorders such as sponses remains to be clarified. Although all of the above receptors , pelvic inflammatory disease, and inflammatory bowel dis- are components of the host response to endotoxin, the extraordi- ease. Although the targets of LPS activation include a broad array nary sensitivity of the macrophage response to LPS appears to of cells, including polymorphonuclear cells and epithelial and en- depend upon the presence of either membrane-bound or dothelial cells, macrophages are especially responsive to LPS. Expo- soluble CD14. sure of macrophages to nanogram quantities of LPS results in the The presence of sCD14 in serum and the expression of mem- rapid synthesis and release of myriad cytokines, such as IL-1, -6, -8, brane-bound CD14 on myeloid cells have complicated the analysis and TNF-␣, as well as proinflammatory eicosanoids and NO (1). of alternative receptors involved in activation by bacterial prod- ␤ Three cloned families of molecules on the surface of leukocytes ucts. This is particularly true of the 2 integrins (CD11/CD18), are known to bind the toxic moiety of LPS. These include which bind LPS, especially when LPS is presented to phagocytes CD14, the macrophage scavenger receptors (SR-A family), and the as a component of a membrane (e.g., whole bacteria; Ref. 9). Al- ␤ 2 or CD11/CD18 leukocyte integrins (4). LPS binding to CD14 is though phagocytes bind the LPS moiety of Gram-negative bacteria enhanced by serum factors; these serum components include the via CD11/CD18, this interaction was felt initially to be biologi- cally insignificant because children with congenital CD11/CD18 deficiency responded normally to LPS (10). In retrospect, this ob- *Lipid Metabolism Unit, Massachusetts General Hospital and Harvard Medical School, servation might have been predicted because the cells that were Boston, MA 02114; †Maxwell Finland Laboratory for Infectious Diseases, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, harvested from these children expressed CD14. When expressed in ‡ ␤ MA 02118; and Leukocyte Biology and Inflammation Program, Renal Unit, Massachu- a cell line that does not coexpress CD14, the 2 integrins function setts General Hospital, Harvard Medical School, Boston, MA 02114 as low affinity LPS receptors (11–13). Although the ED50 for Received for publication March 22, 2000. Accepted for publication July 24, 2000. CD11/CD18 engagement is higher than that for CD14, few qual- The costs of publication of this article were defrayed in part by the payment of page itative differences in LPS activation by these two distinct receptor charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. systems have been described (14). Like CD14, CD11/CD18 can ac- tivate cells in the absence of cytoplasmic signaling residues, as dem- 1 This work was supported by R01GM54060 (to D.T.G.), U19AI3815 (to D.T.G.), DK50305 (to D.T.G./M.A.A./M.W.F.), and HL56985 (to M.W.F.). onstrated by experiments that showed equivalent LPS responses to a 2 K.J.M. and L.P.A. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Mason W. Freeman, Massachu- 4 Abbreviations used in this paper: LBP, LPS binding protein; TLR, Toll-like recep- setts General Hospital, 55 Fruit Street, GRJ1328, Boston, MA 02114. E-mail address: tor; NIF, neutrophil inhibitory factor; ES, embryonic stem; JNK, c-Jun N-terminal [email protected] kinase; sCD14, soluble CD14.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 4273 full length and “tailless” integrin (12). It seems likely that other, less well-characterized, cell surface that bind bacteria may also activate phagocytes, but these will be challenging to identify as long as the highly sensitive CD14 activation pathway can be engaged. To explore the contributions of both CD14-dependent and -in- dependent signaling mechanisms to LPS and bacterial stimulation, we chose to generate mice lacking this receptor. Macrophages from our CD14-null mice proved to be unresponsive to LPS stim- ulation over an LPS dosage range of two log orders (1–100 ng/ml), confirming similar observations Haziot et al. previously made in CD14-null mice generated with a different gene-targeting strategy (15). In this work, we further confirm that the lack of responsiveness in LPS-stimulated CD14-null macrophages is as- sociated with impaired activation of both the NF-␬B and the c-Jun N-terminal mitogen-activated protein kinase (MAPK) pathways (16, 17). In contrast with their unresponsiveness to LPS, CD14- null macrophages retained cytokine responses to whole bacterial particle stimulation. On exposure to 10 heat-killed Escherichia coli, per cell or greater, CD14-null and wild-type macrophages produced similar amounts of TNF-␣.At1E. coli per cell, CD14- Downloaded from null macrophage cytokine responses were definitely impaired but not lost. This CD14-independent cytokine production was substan- tially reduced when neutrophil inhibitory factor (NIF), a compet- ␤ itive inhibitor of the 2 integrin CD11B, was included in the ex- periments. Inhibition of phagocytosis by cytochalasins completely abolished CD14-independent responses. These data indicate that http://www.jimmunol.org/ CD14-null mice will be of considerable value in dissecting the contributions of the multiple response pathways macrophages use to combat bacterial infections. FIGURE 1. Targeting of the mouse CD14 gene. A, Schematic diagram Materials and Methods of the targeting vector KO3CD14, the endogenous CD14 locus, and the Reagents targeted CD14 locus. KO3CD14 contains 4 kb of 5Ј sequence immediately Protein-free LPS derived from E. coli K235 was provided by Dr. S. Vogel upstream of the initiator methionine codon in the CD14 gene, followed by a neor cassette derived from PGKneopA, and 2.5 kb of sequence immedi-

(Department of Microbiology and Immunology, Uniformed Services Uni- by guest on October 1, 2021 versity of the Health Sciences, Bethesda, MD). Recombinant CD14 was ately 3Ј of the coding region. Homologous recombination results in the purified from the conditioned medium of Chinese hamster ovary/CD14 replacement of the entire coding region of CD14 with the neo r cassette. cells as described elsewhere (18). The Ancyclostoma caninium (hook- The targeting vector also contains a thymidine kinase gene that acts as a worm) protein, NIF, was provided by Dr. Matthew Moyle (Corvas Inter- negative selectable marker to eliminate nonhomologous recombinants. national, San Diego, CA) (19). Endotoxin-free cell culture media and ad- Screening of selected ES cells was performed by Southern blot analysis of ditives were purchased from Life Technologies (Grand Island, NY). SacI-digested DNA using the indicated NheI-NcoI fragment located out- Generation of CD14-deficient mice side of the targeting vector as a probe. This probe recognizes a 6-kb frag- ment in the endogenous locus and a 17-kb fragment in the targeted locus A P1 clone with ϳ60 kb of mouse genomic DNA, including the gene as indicated. B, Southern blot analysis of F1 CD14 heterozygote intercross encoding CD14, was obtained from Genome Systems (St. Louis, MO) after mating. Mouse tail DNA from the intercross of F CD14 heterozygotes was screening a genomic library using a combination of PCR and Southern 1 digested with SacI and analyzed by Southern blot as described above. A hybridization as previously described (20). The genomic structure of the ϩ ϩ CD14 locus was mapped by a combination of restriction digest and South- band of 6 kb is detected in wild-type ( / ) mice, bands of 6 and 17 kb in ϩ Ϫ ern blotting. A targeting vector, KO3CD14 (Fig. 1A), was generated that heterozygous ( / ) mice, and a single band of 17 kb in homozygous removed the entire coding sequence of the CD14 gene. KO3CD14 con- mutant (Ϫ/Ϫ) mice. C, Western blot confirming the absence of CD14 tained the 4 kb of sequence immediately upstream of the start site of trans- protein in CD14-deficient mice. Cell lysates from peritoneal macrophages lation of the CD14 coding sequence, an intervening neor cassette derived derived from CD14ϩ/ϩ, CD14ϩ/Ϫ, and CD14Ϫ/Ϫ mice (n ϭ 2) were ana- from PGKneopA that replaced all CD14 coding sequences, and the 2.5 kb lyzed by Western blot for CD14 and the macrophage marker F4/80. No of sequence immediately downstream of the coding region. A thymidine CD14 protein was detected in CD14-deficient mice confirming the deletion kinase gene was also incorporated into the plasmid as a negative selectable of this gene. However, the macrophage-specific F4/80 Ag was detected in marker to reduce the frequency of nonhomologous recombinant transfor- cell lysates from all genotypes (ϩ/ϩ, ϩ/Ϫ, and Ϫ/Ϫ). mants. The linearized vector was electroporated into J1 embryonic stem (ES) cells derived from 129/SvEv mice, and 200 G418/gancyclovir resis- tant clones were selected. Genomic DNA from selected colonies was di- Backcrossing of the CD14-deficient mice into the C57BL/6J strain has gested with SacI and analyzed by Southern blotting. Southern blots were been continued for four generations in a pathogen-free facility, but the hybridized with a 32 P-labeled NheI-NcoI fragment located outside of the macrophages used for the experiments described in this work were derived targeting vector, washed in 2 ϫ SSC at 55°C and 65°C, and exposed to from animals that were backcrossed for one or two generations. For ex- BioMax-MR film (Kodak, Rochester, NY). The NheI-NcoI probe recog- periments requiring autologous serum, CD14 wild-type, -hemizygous, and nizes a 6-kb fragment in the endogenous CD14 locus and a 17-kb fragment -null mice were bled by retro-orbital puncture using heparinized micro- in the targeted locus. capillary tubes. Blood was pooled within genotypes and centrifuged at Correctly targeted ES cell lines were expanded and microinjected into 5000 rpm for 10 min, then serum was isolated. blastocysts. Chimeric offspring were bred to mice of the C57BL/6J strain (The Jackson Laboratory, Bar Harbor, ME), and germline transmission of Isolation of peritoneal macrophages the targeted allele was confirmed by Southern blotting of tail DNA as described above (Fig. 1B). CD14-deficient mice were obtained by inter- Peritoneal macrophages were isolated from 12-wk-old mice 4 days follow- cross of mice hemizygous for CD14 with the expected Mendelian ratio. ing i.p. injection of 1 ml of 3% thioglycollate broth (Difco, Detroit, MI) as 4274 BACTERIAL STIMULATION OF CD14-NULL MACROPHAGES

we previously described (21). Unless otherwise stated, wild-type and hemi- 15 min before stimulation with E. coli bioparticles. Inhibitors were main-

zygous controls for CD14-null mice were F2 littermates. Mice were anes- tained in culture media for the duration of the experiment (4 h). thetized using Metofane (Union, NJ) and sacrificed by cervical dislocation. Peritoneal lavage was performed with 6 ml of HBSS (Life Technologies) containing 0.5 mM EDTA. Collected peritoneal cells were treated with Results 0.17 M ammonium chloride to disrupt contaminating RBC, washed twice Characterization of CD14-deficient mice in HBSS, and resuspended in DMEM (Life Technologies) containing 100 U/ml penicillin and 100 ␮g/ml streptomycin and supplemented with 2.5% Screening of ES cell clones by Southern blotting, as described in autologous mouse serum. Peritoneal cells were allowed to adhere to tissue Fig. 1A, revealed the correct disruption of the CD14 allele in 2 of culture plastic for 2 h, after which nonadherent cells were removed by 100 clones. Although both ES cell clones were injected into blas- rinsing the cell monolayer with media. Adherent cells were typically tocysts, only one line produced high percentage chimeras. Male Ͼ97% positive for the macrophage-specific F4/80 Ag as determined by FACS analysis. chimeras from this line were backcrossed to C57BL6J females and offspring were screened for germline transmission of the targeted

Western analysis allele using tail DNA. F1 hemizygotes were intercrossed, and For Western analysis, peritoneal macrophages were seeded at 3 ϫ 106 cells screening revealed the expected Mendelian ratio of CD14-null, per well in six-well plates. Cells were lysed in TNET buffer (50 mM Tris, -hemizygous, and wild-type offspring. A representative Southern pH 8, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100) and centrifuged blot depicting the three genotypes is shown in Fig. 1B. The wild- for 5 min at 15,000 rpm to pellet debris. To detect CD14 and phosphory- type allele is 6 kb and the targeted allele is 17 kb. CD14-null mice lated or total c-Jun N-terminal kinase (JNK), equivalent amounts of protein were heat denatured in the presence of Laemmli sample buffer containing appeared to be phenotypically normal. The deletion had no obvi- 5% 2-ME and electrophoresed on 10% SDS-PAGE gels. To detect F4/80, ous adverse effect on fertility or litter size.

samples were run on 8% polyacrylamide gels under nonreducing condi- Western blot analysis of peritoneal macrophages from CD14- Downloaded from tions. Proteins were transferred to Immobilon-polyvinylidene difluoride deficient mice showed no expression of CD14 (Fig. 1C). As ex- membrane (Millipore, Bedford, MA) and blocked in TBS-T containing 5% pected, CD14 was detected in peritoneal macrophages from hemi- nonfat milk for CD14 and F4/80, or 1% BSA in TBS-T for phosphoJNK. Primary Abs used were anti-murine CD14 rat monoclonal Ab (catalog no. zygous and wild-type mice. Western blot analysis of the same 09471D; PharMingen, San Diego, CA), anti-F4/80 cell culture supernatant lysates for the macrophage-specific Ag, F4/80, revealed similar (1:5 dilution) from the F4/80 hybridoma cell line (American Type Culture levels of this protein in CD14-null, -hemizygous, and wild-type Collection, Manassas, VA), rabbit anti-phosphoJNK Ab (Promega, Madi-

macrophages, eliminating nonspecific protein degradation as an http://www.jimmunol.org/ son, WI), and rabbit antistress-activated protein kinase/JNK (New England Biolabs, Beverly, MA). Blots were incubated with HRP-conjugated sec- explanation for the absence of CD14 in the lysates of the null ondary Ab (Sigma, St. Louis, MO) against the appropriate species and animals. These results established that the CD14-null mice were developed using an enhanced chemiluminescence detection system. homozygous for the targeted genomic allele and that they were therefore unable to produce any CD14 protein. Detection of nuclear NF-␬B EMSAs for nuclear NF-␬B were performed as previously described (22). Impaired responses of CD14-deficient macrophages to LPS Briefly, 4 ␮g of crude nuclear protein was incubated with a 32P-labeled oligonucleotide containing the consensus sequence for NF-␬B binding We evaluated pretranscriptional events, such as NF-␬B transloca- from the murine IgG chain gene enhancer. The resulting complexes tion and JNK kinase activation, as well as cytokine production, to by guest on October 1, 2021 were analyzed by nondenaturing PAGE. The gels were transferred to filter determine the consequences of the absence of CD14 on macro- paper, dried, and exposed to x-ray film. Scanning densitometry of the au- ␬ toradiographs was performed using Molecular Analyst software. phage responses to endotoxin. A loss of NF- B activation in re- sponse to LPS was observed in CD14-null peritoneal macrophages Cytokine ELISAs as assessed by an EMSA (Fig. 2A). Wild-type macrophages ex- ␬ Peritoneal macrophages were seeded in six-well plates at 5 ϫ 105 cells per hibited dose-dependent activation of NF- B in response to as little well in 0.6 ml of medium. Peritoneal macrophages were stimulated with as 1 ng/ml of LPS. In contrast, doses of up to 100 ng of LPS/ml did LPS from E. coli K235 or heat-inactivated E. coli K12 bioparticles (Mo- not induce NF-␬B activity in CD14-null macrophages. However, lecular Probes), and cell supernatants were harvested at the indicated times. IL-1␤, which acts independently of CD14, induced the same level Supernatants were centrifuged for 5 min to remove cellular debris, trans- ␬ ferred to new tubes, and stored at Ϫ70°C until analysis. TNF-␣ and IL-6 of NF- B translocation in CD14-null and wild-type macrophages, in cell supernatants were measured by ELISA using matched pair Abs from indicating that the loss of LPS-induced NF-␬B translocation in the Endogen (Cambridge, MA). null mice was a specific consequence of the CD14 deletion. In addition to NF-␬B translocation, LPS induces the rapid ac- Binding assays tivation of several MAPKs, including the c-Jun N-terminal MAPK The binding of bacterial membrane particles to peritoneal macrophages (17). Western analysis revealed the accumulation of phosphory- was assessed as previously described (14). BODIPY-labeled E. coli K12 lated JNK in wild-type macrophages within 30 min of LPS or E. bioparticles (Molecular Probes) were added to CD14 wild-type or -null coli stimulation (Fig. 2B). In contrast, LPS and E. coli activation of peritoneal macrophages (ratio of bacteria to cells was 100:1 or 10:1) in media containing 2.5% autologous serum for 5, 15, and 30 min at 37°C. To JNK was reduced in CD14-deficient macrophages. However, IL- assess binding and uptake, cells were washed in PBS and assessed by 1␤, which stimulates JNK activation independently of CD14, rap- FACS analysis. To assess uptake alone, cells were incubated in 2% trypan idly induced JNK phosphorylation in both CD14-null and wild- blue for 1 min to quench extracellular fluorescence, washed in PBS, and type macrophages. These results indicate that engagement of assessed by FACS analysis. The ability of trypan blue to quench BODIPY- labeled particles has been previously established in cells lines that cannot CD14 by either LPS or whole E. coli contributes to JNK engage in CR3-mediated phagocytosis due to the expression of mutant activation. “tailless” CD11/CD18 (12). Ten thousand gated events were recorded for Corresponding with the loss of the early steps in LPS-induced each condition, excluding nonbound bacteria and cellular debris on the , CD14-null macrophages exhibited a loss of basis of forward and side light scatter. A second gate was established cytokine production in response to LPS (Fig. 3). In wild-type mac- separating bright from nonfluorescent macrophages. Results are expressed as a percentage of cells that fall within this gate. rophages, a 5-h incubation with LPS resulted in a dose-dependent increase of both TNF-␣ and IL-6 in culture supernatants. However, Blocking studies no TNF-␣ or IL-6 were detected in culture supernatants from Cells were preincubated with 4 ␮g of NIF protein/ml to inhibit CD11b/ CD14-null macrophages stimulated with up to 100 ng/ml of LPS CD18 binding, or with 10 ␮M cytochalasin B to inhibit phagocytosis, for from E. coli K235. Similar results were obtained using LPS from The Journal of Immunology 4275 Downloaded from

FIGURE 2. A, Impaired NF␬B translocation in CD14-deficient macro- phages. Peritoneal macrophages from CD14 wild-type, -heterozygous, or -null mice were treated with the indicated concentrations of LPS, and nu- clear extracts were assayed for NF␬B content by EMSA. Nuclear extracts were incubated with a 32P-labeled NF␬B-specific probe, and complexes were resolved on a 4% native polyacrylamide gel. The band representing http://www.jimmunol.org/ nuclear NF␬B bound to the NF␬B oligonucleotide is indicated by the ar- row, whereas unbound probe migrated at the bottom of the gel. B, JNK activation is impaired in CD14-deficient macrophages. Western blot anal- ysis was performed to detect activated JNK using an Ab that recognizes the phosphorylated protein. Wild-type (upper panel) and CD14-deficient (low- er panel) peritoneal macrophages were stimulated with LPS (100 ng/ml), E. coli bioparticles (10:1) or rIL-1␤ (5 ng/ml) for the indicated times, and cell extracts were run on 10% reducing polyacrylamide gels. The phos-

phorylated forms of JNK1 and JNK2 are indicated by arrows. Reprobing of by guest on October 1, 2021 this blot with an anti-JNK Ab confirmed equal loading of total JNK protein in wild-type and null macrophage lysates (data not shown).

Salmonella minnesota R595 (data not shown). Although the pre- cise level of CD14 expression required for sensitive responses to LPS is unknown, an intermediate phenotype in cytokine produc- FIGURE 3. CD14-deficient macrophages demonstrate impaired cyto- tion was observed in LPS-stimulated hemizygous macrophages. kine response to LPS. Peritoneal macrophages derived from CD14 wild- The reduction of TNF-␣ (Fig. 3A) and IL-6 (Fig. 4A) production in type, -heterozygote, or -null F2 littermate mice were stimulated with E. coli ␤ CD14-hemizygous macrophages, as compared with wild-type LPS (0.1–100 ng/ml) or 5 ng/ml IL-1 , and cell culture supernatants were harvested after 5 h. TNF-␣ and IL-6 in culture supernatants were analyzed cells, clearly demonstrates a CD14 gene dosage effect. Levels of Ϯ ␣ by ELISA. Data are expressed as the mean of triplicate samples SD and TNF- and IL-6 were undetectable in unstimulated macrophages are representative of three separate experiments. from mice of all genotypes. Taken together, these data demonstrate that CD14 appears to be critical to the activation of several signal transduction pathways involved in macrophage activation by LPS. autologous and CD14-null serum, we demonstrate the important Contribution of serum-sCD14 to macrophage responses to LPS contribution of serum sCD14 in mediating signaling in response to sCD14 is believed to enhance delivery of LPS monomers to mem- low doses of LPS. At 1 ng/ml of LPS, serum sCD14 concentration brane-bound CD14 at the cell surface, as well as to enable delivery significantly enhanced cytokine production by membrane CD14- of LPS to additional components of the LPS signal transduction bearing cells (Fig. 4B). Wild-type macrophages produced 10-fold apparatus in cells lacking surface CD14 (23). Therefore, macro- more TNF-␣ when cultured in 2.5% autologous serum than in phages and sera from CD14-hemizygous and -null mice were used 2.5% CD14-deficient serum. A similar effect was observed when to clarify the relative contributions of sCD14 and membrane CD14 CD14-hemizygous macrophages were cultured in autologous se- to cytokine responses to LPS. Both CD14-null and -hemizygous rum vs CD14-deficient serum. The degree of potency of sCD14 macrophage cytokine production levels were restored to wild-type proved to be unexpected; simply culturing cells from knockout values by the addition of sCD14 to the medium (Fig. 4A). These mice in FCS for several days was sufficient to restore near normal results are similar to those found by Haziot et al. (15) despite our LPS responses even after these cells were extensively washed in se- use of concentrations of sCD14 that more closely mimic the phys- rum-free medium (data not shown). This implies that residual levels iologic levels of that protein (ϳ2 ␮g/ml) as opposed to the 10-fold of sCD14, originally provided by culture in 5% FCS, were capable of higher concentration (20 ␮g/ml) used in the previous study. Using restoring LPS responsiveness to CD14-null macrophages. These data 4276 BACTERIAL STIMULATION OF CD14-NULL MACROPHAGES

trypan blue to quench bound extracellular, but not intracellular, fluorescent particles (14, 25). No significant differences in E. coli binding and uptake were observed in CD14-deficient macro- phages, as compared with wild type. As shown in Fig. 5, binding was evaluated using 100:1 E. coli per macrophage at 5-, 15-, and 30-min time points. The percentage of binding was similar in CD14-deficient and wild-type macrophages under all conditions. Similar results were obtained using 10:1 E. coli per macrophage (data not shown). No differences were observed when the presence of extracellular organisms was quenched with trypan blue (data not shown). In some experiments, a modest reduction in binding was observed in CD14-null macrophages compared with wild-type cells when bacteria were preincubated in heat-inactivated (com- plement-deficient) serum (data not shown). However, when data from five individual experiments were analyzed together, this de- crease was not statistically significant. Furthermore, bacterial bind- ing was not increased by sCD14 in either wild-type or CD14-null macrophages. Incubation of CD14-null macrophages in medium with 2% wild-type mouse serum containing endogenous sCD14 did not increase bacterial binding, nor did the addition of recom- Downloaded from binant sCD14 to either wild-type or null macrophages (data not shown). These findings suggest that CD14 is not essential for bac- terial uptake and that, in the absence of CD14, other bacterial binding receptors (e.g., CD11b/CD18, SR-A, Fc␥ receptor, or ) can compensate. http://www.jimmunol.org/ E. coli outer membranes induce cytokine production in CD14- deficient macrophages We next examined cytokine production from CD14-deficient peri- toneal macrophages stimulated with heat-killed E. coli to deter- mine the significance of the observation that bacterial particles can bind and enter macrophages via alternate receptors to CD14. In contrast to our findings with LPS, CD14-deficient macrophages stimulated with 10 heat-killed E. coli per cell for 6 h elaborated by guest on October 1, 2021 both TNF-␣ (Fig. 6A) and IL-6 (data not shown) in amounts equiv- FIGURE 4. Serum sCD14 enhances LPS responses. A, Recombinant CD14 restores LPS response in CD14-null macrophages. Peritoneal mac- alent to those produced by wild-type peritoneal macrophages. This effect was dose dependent and at bacterial concentrations Ͻ10 E. rophages derived from CD14-null, -heterozygote, and wild-type F2 litter- mate mice were stimulated with 10 ng/ml LPS in the presence or absence coli per cell, TNF-␣ production by CD14-null macrophages was of 2 ␮g/ml rCD14 for 4 h. Cell culture supernatants were analyzed for significantly reduced compared with wild-type macrophages (Fig. TNF-␣ and IL-6 by ELISA. B, Serum CD14 enhances LPS responses of 6B). Although wild-type macrophages produced TNF in response membrane CD14-bearing cells. Peritoneal macrophages from wild-type or to as little as 0.1 E. coli/macrophage, no cytokine production was CD14-heterozygous F2 littermate mice were cultured in either 2.5% autol- observed at Ͻ1 E. coli/macrophage in CD14-null macrophages. ogous or CD14-deficient mouse serum and stimulated with 1 ng/ml LPS for This result establishes that CD14 can also enhance the sensitivity ␣ 5 h. Cell culture supernatants were analyzed for TNF- and IL-6 by of macrophage responses to bacterial particles, but that this en- ELISA. All data are expressed as the mean of triplicate samples Ϯ SD and hancement is observed over a narrower range than that found with are representative of two separate experiments. Levels of TNF-␣ and IL-6 were undetectable in unstimulated macrophages. LPS. Although we did not detect cytokine responses to LPS over a dosage range of 0.1–100 ng/ml in our CD14-null animals, Haziot et al. did find some LPS responsiveness in their animals at dosages confirm that circulating sCD14 contributes significantly to macro- of 1 ␮g/ml and higher. The concentration of bacteria that gave half phage responses to LPS, presumably by enhancing delivery of LPS to maximal stimulation in CD14-null macrophages was 5 E. coli/ membrane-bound CD14 or by directly enabling LPS delivery to macrophage. Using current estimates of the amount of LPS present the TLRs. in the bacterial cell wall of Gram-negative organisms (106 LPS molecules/bacterium), this dose represents ϳ40 ng/ml of LPS in E. coli binding and uptake is not altered in CD14-deficient our experiments. This dose of LPS could not induce cytokine pro- macrophages duction in CD14-null macrophages, suggesting that other factors, As CD14 can bind whole bacteria as well as its component LPS, either intrinsic to the bacterium or to the host cell, may contribute we examined the impact of CD14 deficiency on macrophage re- to the better preserved response of CD14-null macrophages to sponses to E. coli. CD14 has been reported to mediate the binding whole organisms. of bacteria to macrophages as well as to function as a phagocytic Interestingly, a strain-dependent difference was observed in the receptor for Gram-negative organisms, despite its lack of physical amount of TNF-␣ produced in response to 10:1 heat-killed E. coli. connections to the cytoskeleton (24, 25). First, we used flow cy- When CD14-null macrophages were taken from animals that were tometry to measure binding and uptake of BODIPY-labeled E. coli 129Sv/C57BL/6J hybrids and exposed to E. coli, their TNF-␣ pro- bioparticles, a commercial preparation of fluorescently labeled duction was intermediate between that produced by macrophages heat-killed bacteria. This assay takes advantage of the ability of taken from purebred mice of the two parental strains (Fig. 6A). The The Journal of Immunology 4277

FIGURE 5. E. coli binding and uptake is not altered in CD14-deficient macrophages. Binding of heat-inactivated, BODIPY-la- beled E. coli K12 bioparticles to CD14 wild- type or -null peritoneal macrophages from F2 littermate mice was assessed by FACS anal- ysis. Fluorescent bioparticles were added to cells (100:1) in media containing autologous serum (ϩ/ϩ or Ϫ/Ϫ) for 5, 15, and 30 min at Downloaded from 37°C and removed by washing in PBS; then cell fluorescence was assessed. A gate was set for fluorescence above background (R2) and 10,000 events were recorded for each condition. Data are presented as dot plots with the percentage of positive cells in the upper right corner, and are representative of http://www.jimmunol.org/ five separate experiments. by guest on October 1, 2021

CD14-null mice used in this experiment had been back-bred two worm protein NIF has been demonstrated previously to inhibit generations to C57BL/6J and thus were ϳ75% C57BL/6J. These CD11b/CD18 binding to its ligands by interfering with the func- data indicate that genetic variation at other murine loci contribute tion of the integrin metal ion-dependent adhesion site domain (19). to the macrophage cytokine response to bacterial stimulation and that We used NIF to block CD11b/CD18 binding to Gram-negative caution must be used in interpreting in vivo differences using CD14- bacteria to quantitate that portion of the E. coli-induced cytokine null mice that have not been back-bred to strain homogeneity. response that occurred via this receptor in CD14-null macro- phages. In a rosetting assay, NIF pretreatment inhibited binding of CD11b/CD18 mediates bacterial signaling in the absence iC3b-coated sheep RBC to CD14-null peritoneal macrophages, of CD14 confirming its ability to inhibit binding to CD11b/CD18 in these The CD11/CD18 family of leukocyte integrins can bind whole cells (data not shown). Pretreatment of CD14-null macrophages Gram-negative bacteria as a result of the recognition of the lipid A with NIF reduced TNF-␣ production by ϳ50% in response to 5 E. moiety in the outer membrane (9, 26). Like CD14, CD11/CD18 coli per macrophage, a dose that was determined to give half max- integrins have been reported to initiate signal transduction (11–14) imal TNF-␣ production (Fig. 7). In contrast, no difference was and are believed to enable LPS responses via the same downstream observed in TNF-␣ production by wild-type macrophages treated signaling molecules as CD14. Unlike CD14, CD11/CD18 integrins with NIF (Fig. 7, inset). These data imply that bacterial internal- do not have a high affinity for monomeric LPS (27), but avidly ization via CD11b/CD18 accounts for a substantial fraction of the interact with larger aggregates of LPS, including whole bacteria. In induction of cytokine signaling that occurs in the absence of CD14. addition to its ability to bind directly to the lipid A moiety of LPS as a constituent of the bacterial membrane, CD11b/CD18 is also a Bacterial internalization is required for induction of cytokine receptor for complement (iC3b)- and LBP-coated bacteria (28). production by CD14-deficient macrophages ␤ Although all of the 2 integrins appear to be capable of medi- Because the concentration of LPS contained in the doses of E. coli ating responses to LPS (13), mouse peritoneal macrophages ex- bioparticles used in our study is unable to initiate signal transduc- ␤ press CD11b/CD18 as the predominant 2 integrin (29). The hook- tion in CD14-null macrophages alone, we hypothesized that the 4278 BACTERIAL STIMULATION OF CD14-NULL MACROPHAGES Downloaded from

FIGURE 7. Inhibition of CD11b/CD18 reduces TNF-␣ production by

CD14-deficient macrophages. CD14-null or wild-type (inset) peritoneal http://www.jimmunol.org/ macrophages were preincubated with 4 ␮g/ml NIF protein for 15 min be- fore stimulation with E. coli bioparticles (1:1 or 5:1), and NIF was main- tained in cultures throughout the experiment. Cell culture supernatants were harvested at 4 h poststimulation and TNF-␣ was analyzed by ELISA. Data are expressed as the mean of triplicate samples Ϯ SD.

labeled E. coli bioparticles in cytochalasin B-treated cells (data not shown). These data imply that the initiation of cytokine signaling by alternative bacterial receptors requires internalization of the by guest on October 1, 2021 whole bacterium in the absence of CD14 on the cell surface.

Discussion The invasion of Gram-negative bacteria and the subsequent wide- FIGURE 6. CD14-deficient macrophages elaborate cytokines in re- spread activation of the has long been pre- sponse to E. coli stimulation. A, CD14-deficient macrophages stimulated sumed to be due to the interactions of bacterial LPS with mam- with 10:1 E. coli produce equivalent TNF-␣ as compared with wild-type malian cells. The study of LPS biology has been based, in part, macrophages. Macrophages derived from the 129Sv mouse strain are more responsive to E. coli than C57BL/6J macrophages. Peritoneal macrophages upon the assumption that purified preparations of LPS are valid derived from wild-type129Sv and C57BL/6J strain or the hybrid CD14- surrogates for Gram-negative bacteria. The identification of CD14 null mouse strain were stimulated with E. coli bioparticles (10:1), and cell as the binding receptor for LPS implied that CD14 was also in- culture supernatants were harvested at 2, 4, and 6 h poststimulation. TNF-␣ volved in the pathogenesis of invasive Gram-negative infections. in culture supernatants was analyzed by ELISA. Data are expressed as the However, the presence of soluble and membrane-bound CD14 mean of triplicate samples Ϯ SD. B, TNF-␣ production by CD14-deficient complicate the study of other receptors involved in macrophage macrophages stimulated with E. coli bioparticles is dose dependent. To responses to whole bacteria. Thus, we generated a CD14-null evaluate the effect of bacterial concentration on cytokine production, mac- mouse to assess both the importance of this molecule as an LPS rophages obtained from CD14-null and wild-type littermate mice were receptor, as well as to allow the exploration of alternative receptor- stimulated with 0.01–10 E. coli bioparticles per cell, and culture superna- initiated signaling pathways involved in Gram-negative infections. tants were harvested at 4.5 h poststimulation. TNF-␣ in culture superna- tants was analyzed by ELISA. Data are expressed as the mean of triplicate We generated mice in which the entire coding region of the samples Ϯ SD. CD14 gene was deleted. This strategy, unlike the gene interruption technique used in the generation of the previous CD14-null mouse, ensures that no CD14 gene product could be made (15). Despite internalization of intact bioparticles was essential for presentation this difference in our gene-targeting strategy, like Haziot et al. of the inflammatory components of the bacterial membrane to the (15), we found the phenotype of our CD14-null mouse to be char- signaling apparatus. To test whether phagocytosis of E. coli was acterized by profound hyporesponsiveness to purified endotoxin. required for TNF-␣ production, we pretreated CD14-null and wild- No response of CD14-null macrophages was observed at 100 ng of type macrophages with cytochalasin B. Cytochalasin B inhibited LPS per milliliter, whereas this concentration gave a maximal re- TNF-␣ production by CD14-deficient, but not CD14-intact, mac- sponse in wild-type cells. Addition of physiologic amounts of rophages stimulated with 1–5 E. coli per cell for 4 h (Fig. 8). sCD14 restored LPS responsiveness to the null macrophages. Ad- Inhibition of phagocytosis in both wild-type and CD14-null mac- ditionally, we confirm previous observations that two major down- rophages was confirmed by examining the uptake of fluorescently stream effectors of LPS activation, NF-␬B translocation and JNK The Journal of Immunology 4279

phagolysosome, in the absence of CD14. However, the preserva- tion of CD14-null macrophage cytokine stimulation in response to whole E. coli, despite the failure to phosphorylate JNK, suggests that JNK-independent signal transduction pathways are activated by bacteria that are not engaged by LPS alone. Alternatively, other proinflammatory molecules in E. coli membranes, such as lipopro- teins and peptidoglycan, may account for part of the residual re- sponses observed in the CD14-null mice. It is well established that these molecules have immunostimulatory capabilities, (33–40); however, in vitro studies have indicated that, like LPS, activation by these alternative bacterial products is also enabled by CD14 (36, 40–43). Our hypothesis that E. coli internalized via alternate receptors engage a signaling pathway within the phagolysosome is sup- ported by our finding that inhibition of bacterial phagocytosis in CD14-null macrophages completely abrogates cytokine produc- tion. One receptor that mediates bacterial signaling in the absence of CD14 appears to be CD11b/CD18. Using NIF to block this receptor, we show that CD11b/CD18 accounts for approximately half of the CD14-independent TNF-␣ production. CD11b/CD18 Downloaded from FIGURE 8. Inhibition of bacterial phagocytosis abrogates TNF-␣ pro- has been shown to selectively modulate LPS-induced gene expres- duction in CD14-deficient macrophages. CD14-null or wild-type (inset) sion, qualitatively influencing inflammatory responses to bacterial peritoneal macrophages were preincubated with 10 ␮M cytochalasin B for endotoxin (29). As a number of studies have provided evidence for 15 min before stimulation with E. coli bioparticles at the indicated doses. direct interactions between GPI-linked proteins, Fc receptors, and Cell culture supernatants were harvested at 4 h poststimulation, and TNF-␣ the integrins (44–49), it seems likely that the relationships be- was analyzed by ELISA. Data are expressed as the mean of triplicate sam- tween phagocytosis receptors and downstream signaling pathways http://www.jimmunol.org/ ples Ϯ SD. will be complex. The studies presented in this report suggest that macrophages rely heavily on CD14 to activate their arsenal of host defenses phosphorylation, require upstream activation by CD14 (16, 17). when confronting an early infection characterized by low pathogen Our data also confirm previous studies suggesting that CD14 sen- number. We demonstrate that at low bacterial inocula (1 E. coli per sitizes macrophages to the presence of purified LPS by greater than cell), the inflammatory response is severely compromised in the two orders of magnitude. Thus, it is clear from our work and from absence of CD14. One prediction of this work is that animals in- that of others, that the exquisite sensitivity of macrophage activa- oculated with low dosages of pathogens might be especially sus- by guest on October 1, 2021 tion to LPS stimulation requires CD14 (15, 16, 30). However, in ceptible to infection in the absence of CD14. For example, Jack et sharp contrast to this finding, we now demonstrate that CD14-null al. observed that LBP-null mice, when infected with small num- macrophages can be activated by heat-killed whole E. coli in a bers of Salmonella typhimurium, were hypersusceptible to infec- dose-dependent manner. At higher bacterial concentrations, the tion and thus succumbed to sepsis (50). In an animal model of stimulation of cytokines by wild-type and CD14-null macrophages human infection where a high inoculum is used, the outcome might is indistinguishable. This previously unexplored finding demon- be expected to more closely resemble endotoxin challenge. This strates that other signal transduction pathways can compensate for might explain the contradictory findings of Haziot et al., who ob- the loss in CD14 activity. served that CD14 knockout mice were resistant to Gram-negative We found no difference in the binding and uptake of E. coli in infection (15) when they used very large numbers of bacteria to CD14-null and wild-type macrophages over a range of doses (10:1, infect their CD14 knockout mice. 100:1) and conditions, including the presence of heat-inactivated It is our expectation that the widespread availability of CD14- or normal serum and wild-type vs CD14-null serum. In the absence null mice should facilitate a detailed understanding of bacterial of CD14, bacterial internalization via alternate phagocytic recep- activation of host defenses in vivo and that this work may lead to tors such as CD11b/CD18 may be a critical feature of proinflam- improved therapies for septic shock. Perhaps just as importantly, matory responses to Gram-negative bacteria. We demonstrate that there is a growing number of diseases that are thought to use the 5 E. coli per macrophage is sufficient to induce half maximal signal activation pathways used by LPS, including asthma, athero- TNF-␣ production in CD14-null macrophages. Using the estimate sclerosis, arthritis, and inflammatory bowel disease. These diseases that a single bacterium contains 106 LPS molecules (31), this E. should also benefit from the opportunity to use genetically engi- coli concentration represents ϳ40 ng of LPS per milliliter. This neered mice that serve as models of those diseases and that can concentration of purified LPS is unable to stimulate CD14-null now be rendered null for a critical component of the LPS signal macrophages, yet when presented to macrophages in the context of transduction pathway. whole bacteria, a robust inflammatory response resulted. 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