Lipopolysaccharide induces activation of CD14-associated protein ...

THEJOURNAL OF BIOLCOICAL CHEMISTRY Val. 268,No. 28,Issue of October 5 , pp. 20725-20728, 1993 Printed i n U.S.A.

Communication Lipopolysaccharide Induces Activation of CD14-associated Protein Tyrosine Kinase p53/56lyn* (Received for publication, April 27, 1993, and in revised form, J u l y 15, 1993)

Irena &.efanova$, Marta L. CorcoranP, Eva M. Horak, Larry M. WahlP, Joseph B. Bolenl, and Ivan D. Horak

t o be the c e n t r a l e v e n in t the pathophysiology of septicemia (7). The mechanism of cell activation initiated by the binding of bacterial LPS t o CD14, glycophosphatidylinositol (GPII-anchored protein, is u n k n o w n (8). However, recently described co-precipitation of GPI-linked molecules, including CD14, and protein tyrosine kinases (PTK) (9) and phosphorylation of several proteins on tyrosine induced by LPS in murine macrop h a g e s ( 1 0 )suggest an involvement of PTK in the signal transd u c t i o n p a t h w a y s triggered by LPS. Here we report that PTK p53/56'Yn co-immunoprecipitates with CD14, and LPS transiently stimulates activity of PTKs p53/56'Yn, ~ 5 8 / 6 4 ~and '~, p59c-ffl i n human monocytes.

From the Metabolism Branch, Division of Cancer Biology, Diagnosis, a n d Centers, National Cancer Institute, National Institutesof Health, Bethesda, Maryland 20892, the §Laboratory of Immunology, of Health, Dental Research Institute, National Institutes Bethesda, Maryland 20892, and the IDepartmentof Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey08543-4000

EXPERIMENTAL PROCEDURES

Cells-Peripheral blood monocytes were isolated by elutriation (11). Antibodies-The anti-CD53 (MEM-53) andanti-CD14 (MEM-18) monoclonal antibodies (mAb) were from Dr. Vaclav Horejsi (Instituteof Molecular Genetics, Prague, Czech Republic). The anti-pp60""" mAb (327) (12) was a gift from Dr. Joan S. Brugge (Ariad Pharmaceutical Inc.,Cambridge, MA). Polyclonal antipeptide antibodies to the Srcrelated PTKs were obtained from rabbits immunized with synthetic of Bacterial lipopolysaccharide(LPS)induces a pleiotro- peptide corresponding to aminoacid sequences in the unique domain pic activation of the immune system which might sub- each individual kinase (13).The mAb 4G10 (Upstate Biotechnology) sequently result in septic shock. One of the cell surface was used to detect phosphotyrosine on immunoblots. Cell Activation-Purified monocytes (5 x 106/ml) were stimulated receptors for LPS is the glycophosphatidylinositol-an- with LPS from Escherichia coli 055:B5 (Difco Laboratories) (1ng/ml) at chored protein CD14. Binding of LPS to CD14 induces 37 "C in RPMI 1640 medium with 10% human AB serum for indicated production of lymphokines such as tumor necrosis fac- periods of time, spun down by high speed centrifugation in an Eppentor-cu (TNF-a), interleukin-1 (IL-l), IL-6, and IL-8, and dorf centrifuge for 8 s, and the pellet was immediately solubilized in CD14 is subsequently released from the cell surface. ice-cold lysis buffer (10 m~ Tris-HC1 (pH 8.21, 140 mM NaCl, 2 m~ However, the mechanism of signaling via CD14 is still EDTA, 5 m~ iodoacetamide, aprotinin (10 pg/ml), leupeptin (10 pg/ml), not known. We report here that protein tyrosine kinase 0.1 m~ quercetin, 0.1 mM tosylphenylalanyl chloromethyl ketone, 0.1 (PTK) p561Yn is coupled to the LPS receptor CD14 m~ N=-p-tosyl-L-lysine chloromethyl ketone, 0.1 mM N-benzyloxycarin human monocytes. LPS rapidly activates CD14-asso- bonyl-L-phenylalanine chloromethyl ketone, and1 mM Na3V0, (Sigma). by SDS-polyacrylciated p5B1yn simultaneously with PTKs p5Shck and Lysates weremixed with sample buffer and analyzed amide gel electrophoresis (SDS-PAGE) andimmunoblotting or sub~ 5 9 ~ - Inhibition ~*. of PTKS by herbimycin A completely jected to immunoprecipitation followed by in vitro kinase assay or imblocks LPS-induced down-modulation of CD14 and pro- munoblotting. In someexperimentsthe cells werepretreatedwith duction of l " - a and IL-1. These datasuggest a critical herbimycin A before LPS stimulation. Monocytes (5 x 106/ml) were role of PTKs in the LPS/CD14-mediatedsignal transduc- incubated 4 h at 37 "C in RPMI 1640 medium with 10% human AB tion pathway in human monocytes. serum in the presence or absence of 10 PM herbimycin A(Life Technologies Inc.). Prior to stimulation,cells were centrifuged and resuspended A. in fresh medium with or without herbimycin Zmmunoprecipitation and Kinase Assay"CD14 was immunoprecipiOne of the initial steps of immune response to bacterialen- tated by the solid phase immunoisolation technique a s described (14). dotoxin is the binding of lipopolysaccharide (LPS)I to oneits of Briefly, 96-well U-bottom plastic plates (Falcon Labware, Oxnard,CAI cell surface receptor CD14 e x p r e s s e d o n the surface of monowere coated with goat anti-mouse immunoglobulin (Sigma) (0.1 mg/ml) at 37 "C for 2 h. After washing with PBS, 50 pl of mAb (20 pg/ml) cytes and macrophages. CD14 recognizes complexes of opsoa t 4 "C for 4 h.Unoccupied nized LPS, LPSiLPS-binding protein (1) and LPS/septin (2). solution in PBS were applied and incubated This interaction inducesan oxidative burst (3), enhancement of binding siteson the plastic wereblocked by 0.2% gelatin, 1%glycine in on ice for 4 h. PBS a t 37 "C for 1h. Lysates were applied and incubated adhesivity of monocytes (41, and release of lymphokines such as After washing with lysis buffer, the contentsof wells were either eluted tumor necrosis factor-a(TNF-a) ( l ) , interleukin-1 (IL-1) (3, 5), with SDS-PAGE sample buffer or subjected to in vitro kinase assay. interleukin-6 (IL-6), and interleukin-8 (IL-8) (6), which seems Contents of wells were incubated with50 pl of 25 m~ HEPES (pH7.2) containing 3 mM MnCl,, 20 mM MgC12, 0.1% Nonidet P-40, and1pCi of [ Y - ~ ~ P J A(specific TP activity, 3000 Ci/mM) (DuPont NEN). Kinasebuffer * The costs of publication of this article were defrayed in partby the for enolase phosphorylation contained1~ M A T(Sigma) P andexogenous payment of page charges. This article must therefore be hereby marked substrate rabbit muscle enolase(100pg/ml) (Sigma). After incubation "uduertisement" in accordance with 18 U.S.C. Section 1734 solely to for 5 min a t 25 "C, proteins were eluted withSDS-PAGE sample buffer indicate this fact. and subjected to SDS-PAGE and autoradiography. Src-related PTKs $ On leave from the Institute of Molecular Genetics,Czech Academy of Sciences, Prague, Czech Republic. To whom correspondence shouldbe were immunoprecipitated by a n incubation of lysates with optimized addressed: Bldg. 10/4N115, National Cancer Institute, NIH, Bethesda, amounts of various polyclonal antisera or mAb on ice for 4 h. Immune complexes were collected with Staphylococcus aureusproteinA MD 20892. Tel.: 301-402-1895; Fax: 301-402-3647. (Pansorbin, Calbiochem) coupled to the appropriate second antibody, if The abbreviations used are: LPS, lipopolysaccharide; TNF-a, tumor buffer, the proteins were subjected necrosis factor-a; IL, interleukin; GPI,glycophosphatidylinositol;PTK, needed. After three washings in lysis TP above, except that 5 PCi of [ Y - ~ ~ P I Awas protein tyrosine kinase; mAb, monoclonal antibody; PAGE, polyacryl- for in vitro kinase assay (see amide gel electrophoresis; A P T , anti-phosphotyrosine; PBS, phosphate- used per sample). After washing with buffer lysis without Nonidet P-40, buffered saline. proteins were subjected to SDS-PAGE and autoradiography.For second ~

~~~~

20725

LPS CDl4-associated Activates

20726

Lyn

on 15% gel) inthe discontinuous buffer immunoprecipitation, in vitrophosphorylated CD14 immunoprecipitate after V8 digestion were analyzed was first eluted by lysis buffer containing 1% SDS insteadof Nonidet system of Laemmli (15).Immunoblotting was carriedaccording to Ref. P-40 and then diluted with lysis buffer with 1% Nonidet P-40to a final 16. Briefly, blotting was performed for 1 h using transfer buffer (0.025 20% methanol. Nitrocellulose concentration of 0.1% SDS and subjected to the second immunoprecipi- M Tris, 0.19 M glycine (pH 8.4)) containing replicas after electrotransfer were incubated for 30 min in 5% bovine tation with indicated rabbit antibodiesto PTKs. V8 Digestion-Proteins phosphorylated inin vitrokinase assay were serum albumin (or 5% nonfat milk for Lyn immunoblotting), 0.05% for 1h in 0.05%Tween 20 in PBS separated by SDS-PAGE and localized in theunfixed gel by brief auto- Tween 20 in PBS and then incubated radiography. The corresponding zones were cut out and finely homog- containing anti-phosphotyrosine mAb 4G10 in a 1pg/ml concentration enized, and radiolabeled proteins were eluted by an overnight incuba- or anti-Lynrabbit polyclonal antiserumdiluted 1:500,followedby tion a t 37 "C with a 10-fold volume of a solution containing 0.1 M horseradish peroxidase-coupled goat anti-mouse or anti-rabbit immuNH4HCO3(pH 8.3). 0.1% SDS, 1mM phenylmethylsulfonyl fluoride, and noglobulin (15,000 and1:40,000, respectively). The bands were visual5 mM iodoacetamide. After addition of 10 pgof bovine serum albumin, ized using a n enhanced chemiluminescence detection system (Amersham Corp.). the supernatants werelyophilized, and SDS was extracted with methaAnalysis of Surface Expression of CD14 and Measurement of TNF-a nol. Purified phosphoproteins weredissolved in 40 pl of 0.1 M Tris-HC1, pH 6.8, and incubated for 1 h a t 37 "C with 0.5 pg ofV8 protease and ZL-Ze-Surface expressionof CD14 was analyzed by direct immunofluorescence with fluorescein conjugate of mAb to CD14 (Leu-M3) (BoehringerMannheim).Thesampleswere mixed 1:l withsample (Becton Dickinson). Concentration of TNF-a in supernatants was measbuffer and analyzed by SDS-PAGE and autoradiography. SDS-PAGE and Zmmunoblotting-Electrophoresis in thepresence of ured by enzyme-linked immunoassay (Du Pont) and concentration of SDS was performed on 10% polyacrylamide gels (exceptthat samples ILla by enzyme-linked immunoassay (ImmunotechS.A.).

-

67

45 -

45

30 -

3a

67

FIG.1. Identification of PTK pW66'~co-immunoprecipitated with CD14.A, comparison of CD14 and Src-related PTKs immunoprecipitates isolated from monocytes and phosphorylated in vitro. B , reimmunoprecipitation of CD14-associated PTK. First lane, phosphoproteins labeled in vitro in the immunoprecipitate obtainedby anti-CD14 mAb; other lanes, phosphoproteins reimmunoprecipitated from CD14 immunoPTKs. C, peptides generatedby V8 protease digestionof isolated 53/56-kilodalton phosphoproteins precipitate by antibodies against the indicated from CD14 immunoprecipitate and phosphoproteinsfrom indicated PTKs. The undigested PTKs, run on the samepolyacrylamide gel, had higher apparent molecular weights than the digested peptides (data not shown). All samples were analyzed on10% SDS-polyacrylamide gel except that peptides preparedby limited proteolysis usingV8 protease( C )were separatedon 15%SDS-polyacrylamide gel. Molecular size standards are shown in kilodaltons.

A r:

LPS

94-

6

94. 67.

FIG.2. Time course of tyrosine phosphorylation and activation of CDlCassociated p53/56'm in h u m a n peripheral blood monocytes after stimulation with LPS.A, APT immunoblot of lysates of LPS-stimulated monocytes; €?, in vitro kinase assay on CD14 immunoprecipitates; C, in vitrokinase assay on CD14 immunoprecipitates withexogenous substrate enolase; D, APT immunoblotting of CD14 immunoprecipitates; E , detection of p53/56'Y" in CD14 immunoprecipitates by immunoblotting.The positions of enolase ( E )and p53/56'Y" are indicated. Molecular size standards are shown in kilodaltons.

LYN =I 45-

45-

30-

D

30-

30-

LPS

E

,. >

4 L

LPS

LPS CDl4-associated Activates

20727

Lyn

To elucidate an active role of the PTK p53/56'Y" coupled to RESULTS AND DISCUSSION To identify the PTK co-precipitated with CD14 from human CD14 in response to LPS, changes of tyrosine phosphorylation monocytes, in vitro phosphorylated CD14 immunoprecipitate in monocytes during LPS triggering were analyzed. Tyrosine was compared with those of all known members of the phosphorylation of multiple proteins induced by LPS at a nanogram per milliliter concentration was detected as early as 1 Src-related F'TK family (Fig. LA). p53/56'Yn, ~ 5 8 / 6 4 ~ 'and ~, min after stimulation and continued to increase over 30 min ~59"'- and a trace amountof p60fYnwere detected.The doublet (Fig. 2 4 ) . Analysis of CD14-immunoprecipitated complexes reof phosphoproteins in the CD14 immunoprecipitate was remarkably similar to the profile ofp53/56'Y". CD14-phospho- vealed LPS induced an increase of kinase activity of CD14rylated immunoprecipitate was therefore subjected to the sec- associated p53/56'Y" (Fig. B),as well as an increase of phosond immunoprecipitation with antibodiesto themost abundant phorylation of PTK substrate enolase (Fig. 2C) and anincrease F'TKs in monocytes ~53/56~Y", ~ 5 8 / 6 4 ~and "~, (Fig. 1B 1. of protein tyrosine phosphorylation detected by anti-phospho&immunoprecipitation of CD14-associated phosphoproteins tyrosine (APT) immunoblotting (Fig. 2 0 ) . No change of the by anti-p53/56'Y" antibodies confirmed the PTK associated with amount of p53/56'Ynwas detectedin CD14 immunoprecipitates CD14 as p53/56'Yn. Furthermore, digestion of phosphoproteins by immunoblotting withanti-p53/56'" antibodies at early time co-precipitated with CD14 and p53/56'Yn with protease from S. points (Fig.2E). The relative decline of CD14-precipitated p53/ aureus strain V8 (V8 protease) yielded identical peptides (Fig. 56'Y" detected after 15 min of activation is probably caused by E ) , different from those derived from ~ 5 8 / 6 4 and ~ " ~~ 5 9 " ~ P . already described shedding of CD14 from the surface of actiObserved co-precipitation of CD14 and ~53/56~Y" could not be vated monocytes (17,181. All these datasuggest that LPS bindcaused by cross-reactivity of used antibodies. Polyclonal anti- ing to CD14 induces an increase of specific activity of CD14Lyn antibody was obtained from rabbits immunized with syn- associated p53/56'p. In orderto determine if p53/56'Y" is the only PTK involved in thetic peptides correspondingto theamino acid sequence in the LPS-induced activation of monocytes, PTK activity and the unique domain of p53/56'Yn. Immunoprecipitation of p53/56'" by this antibody was blocked by specific peptides, and preim- APT profile of immunoprecipitates of total cellular p53/56'p, ~ ~ ~ 5, 9 ~ " were w analyzed aRer LPS stimulation. mune rabbit serum did not immunoprecipitate p53/56'yn (13). ~ 5 8 / 6 4 and The anti-CD14 d b did not immunoprecipitate any protein tyrosine kinase activity from B cell leukemia cell line NALMS, which is CD14 negative. However, p53/56'Y" can be immunoprecipitated from this cell line using polyclonal anti-Lyn antibody (data not shown). A

LPS

30-

B

LPS

30-

C

LPS

30-

B

30

30

30-

0

c

.

LVN:

3

30-

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Time course of activation of p63/53'~, and p6Wp' in human peripheral blood monocytes after stimulation with LPS.In vitro kinase assayon immunoprecipitates of: A, p53/56'yn; B , ~ 5 8 1 6 4 ~C, ' ~ p5Q"m. ; APT immunoblotting of immunoprecipitates of: I), p53/56'P; E , ~58/64~'"; F, p5Wfv. G, reimmunoprecipitation of PTKS from phosphorylated CD14 immunoprecipitatesderived from LPSstimulated monocytes. All of the controls were from the same experiment at time zero. In other experiments no activation of FTKs was detected aRer adding the medium withoutLPS to the cells (data not shown). Positions of F T & are indicated. Molecular size standards are shown in kilodaltons. FIG.3.

C

Herblmycln TNF-n1570

IL-1

(1

+

+

I LPS 1915

31 214

+ 8

75

'

103 13

FIG.4. Effect of herbimycin A on LPS-induced tyroeine phosphorylation,down-modulation of CD14, andproduction of TNF-a and &la. Surface expressionof CD14 and tyrosine phosphorylation in monocytes after LPS stimulation without (A) or with ( E ) pretreatment of cells with herbimycin A. C, production of TNF-(r and ILla by LPS-stimulated monocytes.

20728

LPS Activates CD14-associated Lyn

Surprisingly, an increase of activity and proportional changes these receptors are infact GPI-linked proteins without a cytoof phosphorylation of all three PTKs were observed (Fig. 3, plasmic domain. The exact mechanism of the association of A-F). APT immunoblotting revealed that changes of tyrosine GPI-anchored proteins with various Src-related PTKs is still phosphorylation of these PTKs paralleled their kinase activi- not known.However, our results demonstrate a correlation ties. The increase of tyrosine phosphorylation of p53/56IP and between the LPS activation of CD14-associated PTK p53/56IP, ~ 5 8 / 6 4 ~was ' ~ detectable by 1 min after stimulation, and shedding ofCD14, and the release of some cytokines. Thus, was involved later after 15min of treatment with LPS. these data first show natural communication between GPI"~ Observation of an involvement of the other PTKs ~ 5 8 / 6 4 ~ linked receptor and intracellular PTK and support the crucial and ~ 5 9 " - ~in- the LPS activation pathway led us to evaluate role of PTKs in pleiotropic response of monocytes to endotoxin. the possibility of an activation-inducible association of these Acknowledgments-We thank V. Horejsiand I. Hilgert for kindly PTKs and CD14 after LPS triggering. P53/56lP, ~ 5 8 / 6 4 ~and '~, of monoclonalantibodies; F. Hartmann for help with the ~59'-~-were reprecipitated from CD14 immunoprecipitates providing measurement of cytokines; and T.A. Waldmann, E. S. Gershon, andR. prepared from monocytesin different stages of LPS stimulation Gress for helpful suggestions and critical comments. and phosphorylated in in vitro kinase assay (Fig. 3G). ~53/56~W REFERENCES was the only PTK detected in all CD14 immunoprecipitates from resting and activated cells. While ~ 5 8 / 6 4 and ~ " ~p59c-fV 1. Wright, S . D.,Ramos, R. A,, Tobias, P. S., Ulevitch, R. J., and Mathison, J. C. (1990) Science '249,1431-1433 undergo changes of phosphorylation after LPS triggering and 2. Wright, S . D., Ramos, R.A,, Patel, M., and Miller, D.S . (1992)J. Erp. Med. 176, might be involved in activation response of monocytes we did 719-727 3. Schutt, C., Ringel, B., Nausch, M., Bazil, V., Horejsi, V., Neels, P., Walzel, H., not prove their direct contact to LPS receptor CD14. Jonas, L., Siegl, E., Friemel, H., and Platnikow, A. (1988) FEBS Lett. 19, PTKs activated in LPS-triggered monocytes might be in321328 4. Lauener, R.P.,Geha, R. S., and Vercelli,D. (1990)J. Immunol. 146,1390-1394 volved in modulation of the response of stimulated monocytes. 5. Couturier, C., Jahns, G., Kazatchkine, M. D., and Haeffner-Cavaillon, N. Activation of monocytes via CD14 is followed by transient re(1992) Eur. J. Immunol. 22,1461-1466 lease of this receptor from the cell surface (17, 18) and produc6. Dentener, M. A., B a d , V., Von Asmuth, E. J. U., Ceska, M., and Buurman, W. A. (1993) J. Immunol. 160,2885-2891 tion of lymphokines such as TNF-a (1)and IL-1(3,5).'Ib evalu7. Lynn, W.A,, and Golenbock, D. T.(1992) Immuol. Today 13,271-276 ate therole of PTKs in regulation of these processes, monocytes 8. Wright, S . D. (1991) Curr. Opin. Immunol. 3,83-90 9. Stefanova, I., Horejsi,V., Ansotegui, I. J., Knapp, W.,and Stockinger, H. (1991) were preincubated with herbimycin A, an inhibitor of PTKs, Science 254,10161019 before LPStreatment. This step blocked protein tyrosine phos- 10. Weinstein, S . L., Gold, M.R., and De Fkanco,A. L. (1991) Proc. Natl. Acad. Sei. U. S. A. 88.4148-4155 phorylation induced by LPS and completely inhibited downL. M., and Smith, P. D. (1991) Current Protocols in Immunology, pp. modulation of CD14 (Fig. 4, A and B ) . This observation sug- 11. Wahl, 7.6.1-7.6.8, John Wiley & Sons, Inc., New York gests an involvement of PTKs in a feedback mechanism of 12. Lipsich, L. A,, Lewis, A. J., and Brugge, J. S.(1983) J. Virol. 48,352460 13. Bolen, J. B., Thompson, P.A., Eiseman, E., and Horak, I. D. (1991)Adu. Cancer regulation of CD14 expression on LPS-stimulated monocytes. Res. 67,103-149 To elucidate if PTKs regulate other monocyte functions, the 14. Tamura, G.S., Dailey, M. 0.. Gallatin, W. M., McGrath, M.S., Weismann, I. L., and Pillemer, E.A. (1984) Anal. Biochem. 136,458-464 level of TNF-a and IL-la was measured in supernatants of 15. Laemmli, U.K.(1970) Nature 227,680-685 monocytes preincubated with herbimycin A and thentriggered 16. Towbin, H., Staehelin.. T... and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. with LPS. Inhibition of PTKs blocked LPS-inducedproduction 76,435cr-4354 17. Bazil, V., and Strominger,J. L. (1991) J. Immunol. 147,1567-1574 of both TNF-a and IL-la (Fig. 4C). 18. Wright, S . D. (1991) Science 262,1321-1322 A growing number of T, B, and basophil cell surface receptors 19. Bolen, J. B., Rowley, R. B., Spana, C., and Taygankov,A. Y. (1992) FASEB J. 6, 34034409 have been found linked to PTKs related to Src (19). Several of