Lipopolysaccharide binding protein and CD14 in LPS dependent macrophage activation

Immunobiol., vol. 187, pp. 227-232 (1993) The Scripps Research Institute, La Jolla, CA, USA

Lipopolysaccharide Binding Protein and CD14 in LPS Dependent Macrophage Activation PETERS. TOBIAS and RICHARD]' ULEVITCH

Abstract The activation of monocytes, macrophages, and neutrophils by lipopolysaccharides (LPS) involves a serum protein, LPS binding protein (LBP), and a membrane protein, CD14. Evidence to date suggests a pathway in which serum LPS first binds to LBP. The LPS-LBP complexes then interact with CD14, leading to cellular activation. This scheme is supported by experiments in which either LBP or CD14 are blocked by antibodies as well as experiments in which LBP is added to serum free media and CD14 is expressed on cells which normally lack CD14. Discovery of this pathway suggests novel approaches to anti-LPS therapy.

Several proteins with high affinity for the lipid A moiety of LPS are known. Some of them are cell associated while others are plasma proteins. However, only two have a demonstrated role in mammalian cellular activation, these are LPS binding protein (LBP) and CD14. What is known about these two proteins and their role in LPS dependent cellular activation will be summarized here. Two recent reviews covering this subject have been published (1, 2). LBP was discovered as a component of acute phase serum capable of regulating the binding of LPS to high density lipoproteins. The phenomenon was noted for acute phase sera from rabbits (3), humans (4), rats, mice, pigs, and sub-human primates (P. S. TOBIAS et aI., unpublished results). The protein has subsequently been isolated and cloned from rabbit and human sera (5). When the activity of LBP was recognized, it was thought that normal sera had essentially no LBP (3). More recent analyses, made possible by monoclonal antibodies, reveal that human LBP is present at 3-10 flg/ml, rising to as much as 200 Ilg/ml or more after an acute phase response (6). While the role of LBP in cellular activation is now fairly clear, its role as an acute phase reactant and whether the LBP in normal serum does more than aid in the presentation of LPS to cells remains a matter of conjecture. The amino acid sequences of rabbit and human LBP as discerned from cDNA clones (3) are shown in Figure 1; the two sequences are 69 % identical. The protein is synthesized in the liver (7). While Kupffer cell mRNA has not been screened for LBP message, alveolar macrophage mRNA does not hybridize with probes for LBP message (T. MARTIN, unpublished result) suggesting that it is the hepatocytes which synthesize

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LBP in the liver. The amino acid sequence of rabbit LBP contains only two cysteines which are presumably disulfide bonded. Human LBP contains four cysteines, two in the same positions as the rabbit cysteines and two additional residues, suggesting that the human protein has two disulfide loops. Both LBPs contain sites for N-linked carbohydrate at Asn residues 276, 327, and 363. In the rabbit protein, these appear to be biantennary

1 hum-Ibp rab-Ibp hum-bpi

ANPGLVARIT TNPGLlTRIT VNPGVVVRIS

51

50

DKGLQYAAQE GLLALQSELL DKGLEYAARE GLLALQRKLL QKGLDYASQQ GTAALQKELK

RITLPDFTGD LRIPHVGRGR EVTLPDSDGD FRIKHFGRAQ RIKIPDYSDS FKIKHLGKGH

CELLHSALRP FELLRGTLRP FOLPSSQISM

SDSSIRVQGR SDAYIHVRGS SNANIKISGK

WKVRKSFFKL WKVRKAFLRL WKAQKRFLKM

SES.SGRPTG YCLSCSSDIA SES.SGRPTV TTSSCSSDIQ SNPTSGKPTI TCSSCSSHIN

DVEVDMS.GD NVELDIE.GD SVHVHISKSK

ESRICEMIQK SVSSDLQPYL ESKICRQIEE AVTAHLQPYL NSQVCEKVTN SVSSKLQPYF

QTLPVTTEID QTLPVTTQID QTLPVMTKID

hum-Ibp rab-Ibp hum-bpi

YEFHSLNIHS YKFYSLKIPR YSFYSMDIRE

hum-Ibp rab-Ibp hum-bpi

QGSFDVSVKG ISISVNLLLG KNSFDLYVKG LTISVHLVLG SGNFDLSIEG MSISADLKLG

hum-Ibp rab-Ibp hum-bpi

SGWLLNLFHN QIESKFQKVL LEELLNLLQS QIDARLREVL VGWLlQLFHK KIESALRNKM

100 VPGQGLSLSI LPGQGLSLDI VPNVGLKFSI

101

150

101

201 hum-Ibp rab-Ibp hum-bpi

SFADIDYSLV SFAGIDYSLM SVAGINYGLV

151

250 EAPRATAQML EVMFKGEIFH RNHRSPVTLL MVMSLPEEH EAPRATAGML DVMFKGEIFP LDHRSPVDFL APAMNLPEAH APPATTAETL DVQMKGEFYS ENHHNPPPFA PPVMEFPAAH

251 hum-Ibp rab-Ibp hum-bpi

301 hum-Ibp rab-Ibp hum-bpi

300

NKMVYFAISD YVFNTASLVY SRMVYFSISD YVFNTASLAY DRMVYLGLSD YFFNTAGLVY

HEEGYLNFSI TDDMIPPDSN IRLTTKSFRP HKSGYWNFSI TDAMVPADLN IRRTTKSFRP QEAGVLKMTL RDDMIPKESK FRLTTKFFGT

350

NMNLELQGSV PSAPLLNFSP NMNLELQGTV NSEQLVNLST NMKIQIHVSA STPPHLSVQP

GNLSVDPYME IDAFVLLPSS ENLLEEPEMD IEALVVLPSS TGLTFYPAVD VQAFAVLPNS

SKEPVFRLSV ATNVSATLTF NTSKITGFLK AREPVFRLGV ATNVSATLTL NTRKITGFLK HTTGSMEVSA ESNRLVGELK SLASLFLIGM

PGKVKVELKE SKVGLFNAEL PGRLQVELKE SKVGGFNVEL LDRLLLELKH SNIGPFPVEL

FVPRLARLYP FVPLLANLYP FLPEVAKKFP

400

351 hum-Ibp rab-Ibp hum-bpi

401 hum-Ibp rab-Ibp hum-bpi

LEALLNYYIL LEALLNYYIL LQDIMNYIVP

450 NTLYPKFNDK LAEGFPLPLL NNLYPKVNEK LAHRFPLPLL ILVLPRVNEK LQKGFPLPTP

KRVQLYDLGL RHIQLYDLLL ARVQLYNVVL

QIHKDFLFLG QTHENFLLVG QPHQNFLLFG

451 hum-Ibp rab-Ibp hum-bpi

ANVQYMRV ANIQYRRV ADVVYK*.

Figure 1. Alignment of the amino acid sequences of human and rabbit LBP with human BPI.

LPS Dependent Macrophage Activation . 229

lactosaminic type glycans. Treatment of the rabbit protein with N-glycanase F removed all sugar, implying a lack of O-linked carbohydrate (B . FOURNIER and P. S. TOBIAS, unpublished results). Also indicated in Figure 1 is the sequence of human bactericidal/permeability increasing protein (BPI) from neutrophil granules (8). The amino acid sequences of human LBP and BPI are 44 % identical. While LBP and BPI share the ability to bind LPS, BPI has no ability to participate in recognition of LPS for cellular activation and actually inhibits cell activation (9). Unfortunately the identity between LBP and BPI is so high that the structural features responsible for these functional differences cannot be ascertained from the amino acid sequence data. LBP was discovered by its ability to bind LPS and the specificity of this binding has been examined with a variety of LPS types and partial structures. The conclusion from this work is that LBP binds to the lipid A moiety of LPS. The affinity of LBP for LPS from Salmonella minnesota Re595 was estimated to be near 10-9 M- 1 (10). A further examination of the functional consequences of LBP-LPS complexation led to the observation that LBP was a good opsonin for binding of LPS coated particles, either rough or smooth Salmonella typhimurium or LPS coated erythrocytes, to macrophages (11). The discovery that LBP would serve as an opsonin naturally led to the question of the identity of the receptor for the opsonized particles. In a series of experiments surveying monoclonal antibodies to macrophage surface antigens it was noted that some antibodies to CD14 would abrogate the binding of LBP opsonized particles to the cells and suggested that CD14 was the receptor for LBP opsonized particles (12). CD 14 was identified and structurally characterized as a myeloid marker antigen (13). The amino acid sequences of the lapine G. D. LEE, Genbank accession number M85233), human (14), and murine (15) proteins, as deduced from the cDNA sequences are shown in Figure 2. The three sequences share a «leucine rich motif» with several other proteins (16) but the functional significance of the leucine motif is unknown. The mature proteins are expressed on the surface of myeloid cells after processing to attach a glycerophosphorylinositol (GPI) tail which anchors the protein to the membrane without a transmembrane peptide segment (17, 18). GPI tail attachment results in removal of a c-terminal peptide of unknown length (19). CD14 is also found free in plasma at 2-6 Ilg/ml (20-22). The opsonization experiments described above suggested that functional activation of macrophages by LPS might occur by LPB and CD14 as well and this has been borne out by a considerable number of experiments. For example, immunodepletion of LBP from whole blood lowers sensitivity to LPS by at least two orders of magnitude (5). Similarly, among the proteins surveyed so far, only LBP has the ability to sensitize CD14 bearing cells for activation by LPS (23). Similar experiments with antibodies to CD14 indicate that they can be very effective at blocking the serum or LBP dependent activation of macrophages by LPS (12). Expression of CD14 on

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cells poorly responsive to LPS raises their sensitivity to LBP dependent activation by LPS by at least two orders of magnitude (24). Thus both depletion and repletion studies support the sequence of events shown in equation 1, in which LPS shed from bacterial cell walls LPS + LBP ~ (LPS-LBP) ~ (LPS-CD 14)-Macrophage ~ Activation

(1)

activates macrophages via membrane CD14 with the intermediacy of soluble LPS-LBP complexes. A recent report suggests that while the scheme of equation 1 may be correct, it is not entirely complete in that proteins other than LBP and collectively named septin, may also be able to present LPS or LPS bearing particles to CD14 for binding (25). Whether or not LBP is unique, the

1 50 hum-cd14 TIPEPCELDD EDFRCVCNFS EPQPDWSEAF QCVSAVEVEI HAGGLNLEPF rab-cd14 DTPEPCELDD DDIRCVCNFS DPQPDWSSAL QCMPAVQVEM WGGGHSLEQF ms-cd14 APPEPCELDE ES .. CSCNFS DPKPDWSSAF NCLGAADVEL YGGGRSLEYL 51 100 hum-cd14 LKRVDADADP RQYADTVKAL RVRRLTVGAA QVPAQLLVGA LRVLAYSRLK rab-cd14 LRQADLYTDQ RRYADVVKAL RVRRLTVGAV QVPAPLLLGV LRVLGYSRLK ms-cd14 LKRVDTEADL GQFTDIIKSL SLKRLTVRAA RIPSRILFGA LRVLGISGLQ 101 hum-cd14 ELTLEDLKIT rab-cd14 ELALEDIEVT ms-cd14 ELTLENLEVT

GTM.PPLPLE ATGLALSSLR GTAPPPPPLE ATGPALSTLS GTAPPPL.LE ATGPDLNILN

150 LRNVSWATGR SWLAELQQWL LRNVSWPKGG AWLSELQQWL LRNVSWATRD AWLAELQQWL

151 hum-cd14 KPGLKVLSIA rab-cd14 KPGLQVLNIA ms-cd14 KPGLKVLSIA

QAHSPAFSYE QVRAFPALTS QAHTLAFSCE QVRTFSALTI QAHSLNFSCE QVRVFPALST

200 LDLSDNPGLG ERGLMAALCP LDLSENPGLG ERG LVAALCP LDLSDNPELG ERGLISALCP

201 250 hum-cd14 HKFPAIQNLA LRNTGMETPT GVCAALAAAG VQPHSLDLSH NSLRATVNPS rab-cd14 HKFPALQDLA LRNAGMKTLQ GVCAALAEAG VQPHHLDLSH NSLRA .... D ms-cd14 LKFPTLQVLA LRNAGMETPS GVCSALAAAR VQLQGLDLSH NSLRDA .. AG 251 hum-cd14 APRCMWSSAL NSLNLSFAGL EQVPKGLPAK LRVLDLSCNR rab-cd14 TQRCIWPSAL NSLNLSFTGL QQVPKGLPAK LNVLDLSCNK ms-cd14 APSCDWPSQL NSLNLSFTGL KQVPKGLPAK LSVLDLSYNR

300 LNRAPQPDEL LNRAPQPGEL LDRNPSPDEL

350 301 hum-cd14 PEVDNLTLDG NPFLVPGTAL PHEGSMNSGV VPACARSTLS VGVSGTLVLL rab-cd14 PKVVNLSLDG NPFLVPGASK LQEDLTNSGV FPACPPSPLA MGMSGTLALL ms-cd14 PQVGNLSLKG NPFL. .. DSE SHSEKFNSGV VTAGAPSSQA VALSGTLALL 351 hum-cd14 QGARGFA rab-cd14 QGARGFI ms-cd14 LGDRLFV Figure 2. Alignment of the amino acid sequences of human, rabbit and murine CD14.

LPS Dependent Macrophage Activation . 231

discovery and characterization of LBP, CD14 and other LPS cell recognition proteins offer routes to new classes of therapy for intervention in endotoxemia. Acknowledgements This work was supported by NIH grants AI22563, A132021, GM37696, A115136 and GM28485. This is publication No. 7606-IMM from The Scripps Research Institute.

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232 . P. S. TOBIAS and R. J. ULEVITCH 15. MATSUURA, K., M. SETOGUCHI, N . NASU, Y. HIGUCHI, S. YOSHIDA, S. AKIZUKI, and S. YAMAMOTO. 1989. Nucleotide and amino acid sequences of the mouse CD14 gene. Nucleic Acids Res. 17: 2132. 16. TAKAHASHI, N., Y. TAKAHASHI, and F. W. PUTNAM. 1985. Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich alpha 2-glycoprotein of human serum. Proc. Nat!. Acad. Sci. USA 82: 1906. 17. HAZIOT, A., S. CHEN, E. FERRERO, M. G. Low, R. SILBER, and S. M. GOYERT. 1988. The monocyte differentation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositollinkage. J. Immuno!. 141: 547. 18. SIMMONS, D. L., S. TAN, D . G. TEN EN, A. NICHOLSON W ELLER, and B. SEED. 1989. Monocyte antigen CD14 is a phospholipid anchored membrane protein. Blood 73: 284. 19. CARAS, I. W. 1991. Probing the signal for glycophosphatidylinositol anchor attachment using decay accelerating factor as a model system. Cell. Bio!. Int. Rep. 15: 815. 20. SCHUTT, c., T. SCHILLING , U . GRUNWALD, W. SCHOENFELD, and C. KRUGER. 1992. Endotoxin-neutralizing capacity of soluble CDI4. Res. Immuno!. 143: 71. 21. KRUGER, c., C. SCHUTT, U . OBERTACKE, T. JOKA, F. E. MULLER, J. KNOLLER, M. KOLLER, W. KONIG, and W. SCHONFELD. 1991. Serum CD14 levels in polytraumatized and severely burned patients. Clin. Exp. Immuno!. 85: 297. 22. BAZIL, V. and J. L. STROMINGER. 1991. Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes. J. Immuno!. 147: 1567. 23. MATHISON, J. c., P. S. TOBIAS, E. WOLFSON, and R. J. ULEVITCH. 1992. Plasma lipopolysaccharide (LPS)-binding protein. J. Immuno!. 149: 200. 24. LEE, J.-D., K. KATO, P. S. TOBIAS, T. N. KIRKLAND, and R. J. ULEVITCH. 1992. Transfection of CD 14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein. J. Exp. Med. 175: 1697. 25 . WRIGHT, S. D ., R. A. RAMOS, M. PATEL, and D. S. MILLER. 1992. Septin: A factor in plasma that opsonizes lipopolysaccharide-bearing particles for recognition by CD14 on phagocytes. J. Exp. Med. 176: 719. Dr. PETER S. TOBIAS, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037, USA