Vol.
183,
No.
February
1, 1992
BIOCHEMICAL
RESEARCH
COMMUNICATIONS Pages
1992
28,
CLONING Catherine
OF
MURINE AND RAT VASCULAR
Hession,
Cindy
Pamela
Williams,
'Oklahoma
May,
Mark
Wysk,
Paul
Received
BIOPHYSICAL
AND
Inc.,
Medical
Research
January
8,
Richard Linda
Kincade',
Biogen,
CELL ADHESION Tizard,
MOLECULE-l
Patricia
Burkly,
163-169
Kensuke
Chisholm, Miyake',
and Roy Lobb
14 Cambridge
Center,
Foundation,
MA, 02142
Oklahoma
City,
73104
OK,
1992
Vascular cell adhesion molecule-l (VCAMl) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen 4 (VLA4). We have cloned the cDNAs for both murine lung libraries. Both sequences and rat VCAMl from endotoxin-treated encode proteins with seven extracellular Ig-like domains, which show respectively, with human VCAMl. Both murine 75.9% and 76.9% identity, show VLA4-dependent binding to COS cells and human cell lines Two mAbs, M-K/l and Mtransiently expressing murine and rat VCAMl. an antigen on murine bone marrow stromal cell K/2, which recognize lines, bind to murine VCAMl expressed in COS cells and block VCAMlconfirming that these mAbs recognize murine dependent adhesion, VCAMl. 0 1992Academic mess,Inc.
VCAMl is activated
endothelium
endothelium, in both 8-l
an adhesion
as well
normal
integrin
and signal
and
(l),
and
transduction
expression
in
tissue
VLA4 on leukocytes
role
to sites
expressed
(6). both
on
(l-3).
of the
cDNAs for
and mediates
vascular cell
(2,6).
and
Here we report
and rat
types
with
both
interaction
immune responses
murine
inflamed
VCAMl interacts
(4,5),
of inflammation
on cytokine-
and dendritic
The VCAMl/VLA4 in
in vitro
vivo
as on macrophage-like
and inflamed
pathophysiologic emigration
molecule
the
adhesion
may play in
a
leukocyte the cloning
VCAMl.
MATERIALS AND METHODS Librarv construction and screening. FVB female mice (Taconic Farms) were treated i.p. with lipopolysaccharide (LPS) (Difco) 4.5 mg/kg, or F344/NTAC FBR male rats (Taconic) were treated i.p. with 2.5 mg/kg LPS, for 6-8 hours, then animals sacrificed and organs frozen in 0006-291X/92
163
$1.50
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
183,
No.
1, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
liquid nitrogen. Lung and kidney RNA were isolated as described (7). Polyadenylated RNA was prepared by oligo(dT) cellulose chromatography (Pharmacia PL type7). Double stranded cDNA was synthesized essentially as described (8) except that MMLV reverse transcriptase (BRL) was used to obtain the first strand. Double stranded cDNA was ligated into a Notl-EcoRl adaptor, purified on a 5%-20% potassium acetate gradient (9), ligated to EcoRl-digested X-gtl0 arms, packaged and plated on E. coli BNN102 cells (10). To obtain murine VCAMl clones 1~10~ phage were plated, and replicate lifts on Genescreen Plus filters were hybridized with a 32P-labelled 30mer (NEN) oligonucleotideprobe (5'-AGTCTCCCCCTTCAGTAATTCAATCAATCTCCAG-3')obtained from the sequence of a partial murine VCAMl genomic clone (L. Burkly et al., unpublished). This sequence is highly conserved (29/30 nucleotides) between human and murine VCAMl. Filters were hybridized overnight at 65OC in plaque screen buffer (NEN) containing 10% dextran sulfate and 100 pg/ml tRNA. They were subsequently washed at 65'C in lXSCC, 1% SDS, and exposed to film. After purification of hybridizing plaques and analysis of insert size, the two longest clones were subcloned into pNNl1 for DNA sequence analysis. To screen for rat VCAMl, 2~10~ phage were screened with the same probe, using the above methods, except that hybridization was performed at 50°C and washing was carried out at 50 C in 4XSCC, 1% SDS. Cell lines and antibodies. Ramos and 7OZ/3 cells were obtained from the ATCC, and grown in RPMI1640/10% fetal bovine serum. Monoclonal antibodies (mAbs) HP1/2 (IgGl) to VLA4, M-K/l and M-K/2 (IgG2b's) to murine stromal cell antigen, PS/2 (IgG2b) to murine VLA4, and KM201 (IgGZb) to murine CD45, have been described (11-13). COS cell transfections. Transient expression of VCAMl cDNAs was performed as described (1,14). Briefly, 20 pug VCAMl cDNA in pCDM8, or vector alone as control, was electroporated into 1x10' COS7 cells and plated in 48-well plates at lo5 cells/well for adhesion assays, which were performed as described (15) about 65 hr post transfection. For inhibition of adhesion, either COS cells (mAbs M-K/l, M-K/2, KM201) or lymphoid cells (mAbs HPl/2, PS/2) were incubated for 30 min at 5 pg/ml prior to the assay. Northern analvses. 5 ,ug polyA+ mRNA was electrophoresed through formaldehyde agarose gels and blotted as described (1). Probes were prepared by random priming (16). The murine VCAMl probe contained nt 1-2115 of the sequence. Pyruvate kinase was used as a control housekeeping gene for mRNA quantitation. RESULTS AND DISCUSSION
Ch aactr
eriz
XgtlO
constructed clones
from
of murine
sequence
of
unpublished sequence. insert into
size. pNNl1
a
the VCAMl, partial
lungs
of
phage
LPS-treated
mice
and
were screened
with
a probe
murine
VCAMl
information),
highly
17 hybridizing
plaques
The two longest for
DNA sequence
libraries
genomic
clone
homologous were
clones, analysis. 164
each
to
purified
kb,
To obtain based (L.
the and
of 2.1 The
rats.
were
on the Burkly,
human
cDNA
analysed
for
were subcloned
cDNA sequence
is
the
Vol.
183,
No.
3,,, 1992
BIOCHEMICAL
composite
of
composite
sequence
sequence,
a
overlapping is
2217
same probe
two longest
bp in
open
for
at lower
clones,
sequence
clones,
and is
3016 bp in length,
sequences
have
is
the
frame,
clones.
57
bp of
and
753
bp
phage were
of
contains
these
GenBank
of
5'
UT
3'
UT
screened
with the
and sequenced. two
57 bp of
and 741 bp of
The
plaques
were subcloned
kb,
with
two
contains
composite
been deposited
COMMUNICATIONS
Of 4 hybridizing
stringency.
VCAMl
these
VCAMl,
of 1.7 and 2.1
a 2217 bp open reading
of
frame,
rat
The rat
RESEARCH
length,
reading
clones
but
BIOPHYSICAL
sequences
3028
bp
To obtain
sequence. the
the
AND
overlapping
5' UT sequence,
3' UT sequence.
The DNA
(Accession
MB4487,
#;s:
MB4488). Deduced amino acid sequences peptide
of of
sequence region,
murine
of rodent
and rat
24 amino acids
containing
VCAEll
(aa),
seven
(1,14,17).
(Fig.
Ig-like
Both
VCAMls.
both
a 22
as found
sequences
contain
contain
aa transmembrane
in the major four
contain
five
extracellular
potential
domains.
N-glycosylation
The sequences
are
human seven domain form of VCAMl (14,17) and 76.9% identity, By comparison, which
respectively,
ICAMl,
shares
an adhesion
many similarities
identity
between
murine
identity
between
individual
similar
throughout
Interestingly, molecules
the
the (Fig.
Northern
analvses
analyses
of
both
the
molecule with
and
aligned
the
VCAMl
domains
cytoplasmic
varying tail
that
is
the
of
the
1, and show 75.9%
(1,2),
is
and
in
of human VCAMl.
and Ig superfamily
sequences
molecule,
(1,14,17),
with
sequence
VCAMl
human
in Ig-
sites
in Figure
with
form of
cysteines
domains 1 and 4, as does the human VCAMl sequence
both
a signal
by a 674 aa extracellular
domains, tail,
The deduced amino acid
1)
followed
and a 19 aa cytoplasmic
human VCAMl like
seauences
member
shows about (18).
The
not
only
from
68%
identical
in
50%
percent high to
all
but 84%.
three
1). of
VCAMl
untreated
exoression
in
and LPS-treated 165
inflamed murine
lung. lung
Northern
tissues
are
675
Domain 7--> KDILLTaFPSeS~CDTVIISCTCGnWEtWII~eTGDtVLKSIdGaYTIRWLQLkDAGVYECESKnkVGSQLRSLTLDVqGrEnNRDYfSPELL KDIqLTvFPSkSVKEGDTVIISCTCGnVPEtWII ~TGDmVLKSVdGsYTIRqAQLqDAGIYECESKteVGSQLRSLTLDVkGkE~NKDYFSPELL KDIqLTvFPSkSVKEGDTVIISCTCGs'JPEiWII ~TGDmVLKSVnGsYTIRkAQLqDAGVYECESKteVGSQLRSLTLDVkGkEnNRYFSPBLL MI-LT-FPS-S~GDTVIISCTCG-~E-~I~-T~-~SV-G-YTIR-AQL-~AG~CKSK--VGSQ~SI.~V-G-E-N~YFSPSLL
697 Cytoplasmic-->715 vLYfASSLIIPAIGHIIYFARKANMK GSYSL-KV aLYcASSLVIPAIGHIVYF ARKANMKGSYSLVEAQKSKV aLYfASSLVIPAIGnIIYFARKANMK GSYSLVEAQKSKV -LY-ASSLVIPAI'Z'!IIYFARKANMKGSYSLVEAQKSKV
Transmembrane-->
575
AMRHEDSGIYVCSGVNLVGL~
486 Domain 6--> StQtLYVNVAPrDtTVlVSPSsrlEEGSs~ClSqGfPaPKILWSRQLpNGELQPLSeNa'PLtLisT~DSG~lCEGINqAG~SrKe~LIlQvtp SvOpL~~lcBtTIwVSPSpIlEEGSpVNLTCsSdGiPaPKILWSRQLnNGELQPLS~N~CLtFmsTXrDDSGIYvCEGINeAGiSrKsVELIIQvvy StQtLYVMTAPkEpTIwVSPSpVpEEGSpVNLTCsSdCfPtPKILUSRQ~NGELQPLSqN~fLaFmaT~DSGIY~EGINeAGiSkKs~LIIQgss S-Q-LYVNVAP-E-TI-VSPS-I-EEGS-~LTC-S-G-P-PKILWSRQL-NGELQPLS-N-'CL-F--'~K-EDSGIY-CEGIN-AG-S-K-~LIIQ---
379 Domain 5--> YSFPrDPeIsmSGgLVnG~sVTVsCkVPsVYPLOrLEIELLKGETiLenieFlEDtdSLEllRsLEmTFIPTiEDTGKaLVCqAkLHid~~~PKQhQ YSFPeDPvIlonSGpLVhGrpVTVnCtVPnVYPFDhLEIELLKGETtLmkkyFlEEmgiKSLEtKiLEtTFIPTiEDTGKsLVC1ArL~sgeME~EPKQRQ YSFPaDPeIeiSGpLVhGrpVTVnCtVPnWPFD~EIELGKGETtLlnkfLrEEigtKSLEtKsLEmTFIPTaEDTGKaLVC1AkLHssqME~EPKQhQ YSFP-DP-I--SG-LV-C--VTV-C-VP-VYPFD-LEIELET-L----F-EE---KSL~-K-LE-TFIPT-EDTGK-LVC-A-LII---ne-EPKQhQ
287 Domain 4--> rkEVELIVQEKPFtVEISPGprI~QI~SV~~s~~SPSFSWRTQiDSPLsGkVRsE~~tnSTLtLSpV~fEnEHSYLCtVTCyhkkLEKgiQVE1 kaaVELWQEKPFiVDISPGsqVAAQVGDSVvL~a~i~SPSFS~TQtDSPLnGv~El;akSTLvLSsVgfEdEHSYLC~VTClqrtLEKrLQVEv ktEVELIVQ~FtVDISPGsqVAAQVGDSVvLKaavGCSPSFSWRTQtDSPLnGeVRdEGatSTLtLSpVgvEdEHSYLCKtiQVEv --EVELIVQEKPF-VDISPG--VAAQVGDSV-LTC---GCK-.QVE-
198 Domain 3--> IDenSSvptvRqaVkEMVYiSPkNTvISVnPSTkLqEGgs~~C~SEGLP~EIFWsk~NgnLQt~LSGNATLTLI~DSGlXVCSG~l~IGh~~ IDqiDStlkeRatVkELQWiSPrNTtIS~STrLqEGgaVTMTCsSEGLPAPEIFWgrKWNevLQ1LSGNATLTLIAHRHEDSGVYVCEGVNLIGrd IDgtDSipksRetVrELQWtSPkNTeISVhPSTrLhEGaaVTMTCaSEGLPAPEIFWskKWNgvLQlLSGNATLTLI ID--DS----R--V-ELQW-SP-NT-ISV-PST-L-EG--~~C-SEGLP~EIFW--K~lN--LQ-LSGNATLTLI~DSG~YVCEG~~.I~;~-
91 Domain 2--> Sr~kgIqVBIYSPPROPEIhLSGPLEaGICPItVRCsvaDVyPfdRLEIDLLKGDhLMksQEFleDadrKSLETKSLeVtFTPVIEDIgKvLVCKAKLh Sg~rsIhMIYSFPRDPEIqFSGPLEvGKPVtVKClapDIYPvyRLEIDLFKGDqLMnrQEFsaEamtKSLETKSLeVtFTPVIEDIgKaLVCKAI(L.i, SgllLErgIqVOIYSFP~PEIqFSGPLEvGKPVmVKClapDVYPidRLEIELFKGDrLMkkQUFvdEmaMSLETKSLEViFTPVIEDIeKaLVC~.y S-KLE--I-VDIYSFPWOEI-FSGPLE-GKPV-VKC---DVYP--RLEIDLFKGD-LM--QYV-FTPVIEDI-K-LVCHAKL-
-24 Signal sequence--> 1 Danain l--; ~gRHVvILGASnILWIMFA8SQAFRIEttPEdryLAQIGDSvsLTCSTTGCESPfFSWRTQIDSPLNgKVtnEGttStLTMnPVSFgNEHSYLCTATCa MP‘rrlMVaVtGAStVLWILFAvSQAPKIEisPEyktiAQIGDSmaLTCSTTGCESPlFSWRTQIDSPLNaKVrtEGskSvLTPVSFeNEHSYLCTATCg 13PvlMVaIP~StVLWILFAvSQAFKIEisPEyktlAQIGDSmlLTCSTTGCESPsFSWRTQIDSPLNgKVktEGakSvLTVSFeNEHSYLCTATCn HP-KMV-ILGAS-VLWILFA-SQAFKIE--PE----AQIGDS--LTCSTTGCESP-FSWRTQIDSPLN-IN--EG--S-LTH-PVSF-NEHSYLCTATC-
Vol.
183,
No.
1, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
12
25
q
n Ramos
7.5 -
7oz/3
of murine VCAMl mRNAs in LPS-treated lung. Fisure 2. Induction Five micrograms of polyA+ mRNA from control (lane I) and LPStreated (lane 2) murine lung tissue were electrophoresed through an agarose-formaldehyde gel, transferred, and probed with a radiolabelled murine VCAMl insert. Hybridizing mRNAs in the induced lung tissue are indicated with arrows. Molecular weight markers are indicated at right. Fisure 3. Adhesion of cell lines to COS cells transiently transfected with murine VCAMl cDNA. The adhesion to murine VCAMl of human Ramos cells or murine 702/3 cells was examined, in the presence or absence of anti-human (HPl/2) or anti-murine (PS/2) VLA4 mAbs, or of anti-murine VCAMl (MK-1, MK-2) or isotypematched control (KM201) mAbs. shown
in
Figure
In normal
2.
and is induced
low levels, tissue.
In the
induced
1.6 kb.
The nature
kidney (191,
(19),
Transient transiently adhesion
with
tissue
transfected (Fig.
of
been
cells
the with 3).
under seen
murine
and inflammed
exoression
assessed
also
(13). rat
murine the
Both
in LPS-treated
study.
lung
in
Such smaller
inflammed
tubular
epithelial
Similar
results
lung VCAMl
cDNA for
(not
murine cells
have been
shown).
cDNA. murine
human Ramos cells
COS cells VCAMl,
were
and cell
and murine
Figure 1. Homology between human, murine, and rat VCAMl protein sequences. The deduced amino acid sequences are aligned, along with a consensus sequence indicating amino acid residues identical in both sequences. The beginning of each of the seven Ig-like domains is indicated (17), as are the transmembrane region and the cytoplasmic tail.
167
at
mRNA can be seen at about
RNA is currently
endothelial
normal
ten-fold
another
cytokine-treated
and in murine
obtained
lung
mRNAs have in
a 3.2 kb VCAMl mRNA is present
approximately
of this
cross-hybridizing
lung
7OZ/3
Vol.
183, No. 1, 1992
cells
adhere
well
to transfected
totally
inhibited
adhesion
is
obtained
similar
transiently
BIOCHEMICAL
results
expressed
two putative
mAbs,
mAbs inhibit
the
binding
of
mAb KM201 is without adhesion
Adhesion
molecules
VLA4
on
fibroblast-like
interfere
with
B lymphocyte
marrow
cultures
(13).
the
cDNA probe,
and
human
VCAMl
(1,13),
related
to,
the
transiently
murine
strongly VCAMl.
antigen.
(not
that In
addition, with
shown),
that
and
block
murine
stromal
indeed
murine
and M-K/2
is
VCAMl/VLA4
pathway
important
in
these
bone mAbs
it
is,
or
sequence N-terminal
is
cells,
a human
homologous
the
VCAMl
to
that
of
is
closely of murine
sequence
M-K mAbs bind
of
to
COS cells
VCAMl cDNA but not
to control
(Fig.
the
for
in long-term
to
VLAl-dependent
COS cells
by immunizing
endothelial
N-terminal
both
interact
mAbs selectively
by
that
the murine
mAbs M-K/l
is
sequence
from
closely
The
mRNA hybridizing
obtained
maturing
a counter-receptor
murine
suggesting
mAb blocks
of
prepared
recognized
The predicted
VCAMl-expressing confirm
of
3) to
isotype-matched
they
when included
an N-terminal
transfected
COS cells
results
presence
has
where
cultured
on
(Fig.
retention
(12,13).
antigen
Both
shown).
define
formation
examined
(12,13).
neither
and Pi-K/Z
lines
The
1) matches
(Fig.
murine
murine
cell
expressed with
(not for
cells
stromal
VCAMl
M-K/l
the
to
binding
cells
contrast,
important
Two mAbs,
murine
correlates
In
in the bone marrow,
cells.
constitutively
while
We have
also
VCAMl
Ram06 or 7OZ/3
effect.
are
RBL-1
We have
murine
and this
mAbs.
line
shown).
to human VCAMl
precursors
adherent
cell
to
either
COS cells,
anti-VLA4
transfectants,
VLA4-dependent
with
(not
RESEARCH COMMUNICATIONS
control
rat
and M-K/Z,
control
stromal
the
VCAMl
M-K/l
not
by blocking
rat
VCAMl-expressing
with
but
with
murine
lymphocyte
AND BIOPHYSICAL
3). cell
VCAMl,
lymphopoeisis
cell
adhesion
Taken antigen
jointly,
the
recognized
and confirm
to
that
by the
(12,13).
ACKNOWLEDGHENTS We thank Jacqueline Ashook Ellen Klein for excellent
for F.A.C.S analyses; technical assistance; 168
Janice DeNarinis and Diane Leone
and for
Vol.
183,
No.
1, 1992
help with figures. AI20069 and AI19884
BIOCHEMICAL
The (to
work PK).
AND
was
EIIOPHYSICAL
partly
RESEARCH
supported
COMMUNICATIONS
by N.I.H.
Grants
REFERENCES
1. C&born, L., Hession, C., Tizard, R., Vassallo, C., Luhowskyj, S., Chi-Rosso, G., & Lobb, R. (1989). Cell 59, 1203-1211. 2. Lobb, R., Hession, C., & Osborn, L. (1991) in: Cellular and Molecular Mechanisms of Inflammation, Volume 2, (C. G. Cochrane & eds.) Academic Press, N.Y. In press. M. A. Gimbrone,Jr., 3. Rice, G.E., Munro, J.M., & Bevilacqua, M.P. (1990). J. &&p. Med. 171, 1369-1374. 4. Elites, M-J., Osborn, L., Takada, Y., Crouse, C., Luhowskyj, S Herler, M.E., & Lobb, R.R. (1990). Cell 60, 577-584. 5:'Schwartz, B.R., Wayner, E-A., Carlos, T.M., Ochs, H.D., & Harlan, J.M. (1990). J. Clin. In--est.. 85, 2019-2022. 6. Burkly, L.C., Jakubowski, A., Newman, B.N., Rosa, M.D., ChiROSSO, G., & Lobb, R.R. (1991) Eur. J. Immunol. 21, 2871-2875. J.M., Pryzbyla, A.E., MacDonaid, R.J., & Rutter, 7. Chirgwin, W.J. (1979) Biochemistry 18, 5294-5299. 8. Gubler, U., & Hoffman, B.J. (1983) Gene 25, 263-269. 9, Maniatis, T., Fritch, E.F., & Sambrook, J. (1982) in: nolecular Cloning-A Laboratory Manual, Cold Spring Harbor, New York. T., Young, R.A., & Davis, R.W. (1985) in: DNA 10. Huynh, Cloning-a Practical Approach, (D. M. Glover, ed.), IRL Press, Oxford, Vol.1, pp. 49-78. 11. Pulido, R., Elites, M.J., Campanero, M.R., Osborn, L., Schiffer, S., Garcia-Pardo, A., Lobb, R., Hemler, M.E., & Sanchez-Madrid, F. (1991) J. Biol. Chem. 266, 10241-10245. 12. Miyake, K., Weissman, I.L., Greenberger, J.S., & Kincade, P.W. (1991). J. Exp. Med. 173, 599-607. 13. Miyake, K., Medina, K., Ishihara, K., Kimoto, M., Auerbach, R & Kincade, P.W. (1991) J. Cell Biol. 114, 557-565. 14: Hession, C., Tizard, R., Vassallo, C., Schiffer, S-G., Goff, D G., Luhowskyj, S., Lobb, R., & Osborn, L. Mq', P., Chi-Rosso, Chem. 266, 6682-6685. (i991). J. Biol. 15. Lobb, R-R., Chi-Rosso, G., Leone, D., Rosa, M., Newman, B., Luhowskyj, S., Osborn, L., Schiffer, S., Benjamin, C., Dougas, I ., Hession, C., 7 Chow, E.P. (1991) Bichem. Biophys. Res. Commun. 178, 1498-1504. 16. Feinberg, A.P., and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267. 17. Cybulsky, M., Fries, J., Williams, A., Sultan, P., Eddy, R., Byers, M., Shows, T., Gimbrone, M., & Collins, T. (1991) Proc. Nat. Acad. Sci. U.S.A. 88, 7859-7863. 18. Horley, K-J., Carpentino, C., Baker, B., & Takei, F. (1989) E.M.B.O. J. 8, 2889-2896. 19. Wuthrich, R.P., Snyder, T.S., & Jenkins, T.A. (1991) Amer. sot. Nephrol. (Abstr.) In Press.
169
183,
No.
February
1, 1992
BIOCHEMICAL
RESEARCH
COMMUNICATIONS Pages
1992
28,
CLONING Catherine
OF
MURINE AND RAT VASCULAR
Hession,
Cindy
Pamela
Williams,
'Oklahoma
May,
Mark
Wysk,
Paul
Received
BIOPHYSICAL
AND
Inc.,
Medical
Research
January
8,
Richard Linda
Kincade',
Biogen,
CELL ADHESION Tizard,
MOLECULE-l
Patricia
Burkly,
163-169
Kensuke
Chisholm, Miyake',
and Roy Lobb
14 Cambridge
Center,
Foundation,
MA, 02142
Oklahoma
City,
73104
OK,
1992
Vascular cell adhesion molecule-l (VCAMl) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen 4 (VLA4). We have cloned the cDNAs for both murine lung libraries. Both sequences and rat VCAMl from endotoxin-treated encode proteins with seven extracellular Ig-like domains, which show respectively, with human VCAMl. Both murine 75.9% and 76.9% identity, show VLA4-dependent binding to COS cells and human cell lines Two mAbs, M-K/l and Mtransiently expressing murine and rat VCAMl. an antigen on murine bone marrow stromal cell K/2, which recognize lines, bind to murine VCAMl expressed in COS cells and block VCAMlconfirming that these mAbs recognize murine dependent adhesion, VCAMl. 0 1992Academic mess,Inc.
VCAMl is activated
endothelium
endothelium, in both 8-l
an adhesion
as well
normal
integrin
and signal
and
(l),
and
transduction
expression
in
tissue
VLA4 on leukocytes
role
to sites
expressed
(6). both
on
(l-3).
of the
cDNAs for
and mediates
vascular cell
(2,6).
and
Here we report
and rat
types
with
both
interaction
immune responses
murine
inflamed
VCAMl interacts
(4,5),
of inflammation
on cytokine-
and dendritic
The VCAMl/VLA4 in
in vitro
vivo
as on macrophage-like
and inflamed
pathophysiologic emigration
molecule
the
adhesion
may play in
a
leukocyte the cloning
VCAMl.
MATERIALS AND METHODS Librarv construction and screening. FVB female mice (Taconic Farms) were treated i.p. with lipopolysaccharide (LPS) (Difco) 4.5 mg/kg, or F344/NTAC FBR male rats (Taconic) were treated i.p. with 2.5 mg/kg LPS, for 6-8 hours, then animals sacrificed and organs frozen in 0006-291X/92
163
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AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
liquid nitrogen. Lung and kidney RNA were isolated as described (7). Polyadenylated RNA was prepared by oligo(dT) cellulose chromatography (Pharmacia PL type7). Double stranded cDNA was synthesized essentially as described (8) except that MMLV reverse transcriptase (BRL) was used to obtain the first strand. Double stranded cDNA was ligated into a Notl-EcoRl adaptor, purified on a 5%-20% potassium acetate gradient (9), ligated to EcoRl-digested X-gtl0 arms, packaged and plated on E. coli BNN102 cells (10). To obtain murine VCAMl clones 1~10~ phage were plated, and replicate lifts on Genescreen Plus filters were hybridized with a 32P-labelled 30mer (NEN) oligonucleotideprobe (5'-AGTCTCCCCCTTCAGTAATTCAATCAATCTCCAG-3')obtained from the sequence of a partial murine VCAMl genomic clone (L. Burkly et al., unpublished). This sequence is highly conserved (29/30 nucleotides) between human and murine VCAMl. Filters were hybridized overnight at 65OC in plaque screen buffer (NEN) containing 10% dextran sulfate and 100 pg/ml tRNA. They were subsequently washed at 65'C in lXSCC, 1% SDS, and exposed to film. After purification of hybridizing plaques and analysis of insert size, the two longest clones were subcloned into pNNl1 for DNA sequence analysis. To screen for rat VCAMl, 2~10~ phage were screened with the same probe, using the above methods, except that hybridization was performed at 50°C and washing was carried out at 50 C in 4XSCC, 1% SDS. Cell lines and antibodies. Ramos and 7OZ/3 cells were obtained from the ATCC, and grown in RPMI1640/10% fetal bovine serum. Monoclonal antibodies (mAbs) HP1/2 (IgGl) to VLA4, M-K/l and M-K/2 (IgG2b's) to murine stromal cell antigen, PS/2 (IgG2b) to murine VLA4, and KM201 (IgGZb) to murine CD45, have been described (11-13). COS cell transfections. Transient expression of VCAMl cDNAs was performed as described (1,14). Briefly, 20 pug VCAMl cDNA in pCDM8, or vector alone as control, was electroporated into 1x10' COS7 cells and plated in 48-well plates at lo5 cells/well for adhesion assays, which were performed as described (15) about 65 hr post transfection. For inhibition of adhesion, either COS cells (mAbs M-K/l, M-K/2, KM201) or lymphoid cells (mAbs HPl/2, PS/2) were incubated for 30 min at 5 pg/ml prior to the assay. Northern analvses. 5 ,ug polyA+ mRNA was electrophoresed through formaldehyde agarose gels and blotted as described (1). Probes were prepared by random priming (16). The murine VCAMl probe contained nt 1-2115 of the sequence. Pyruvate kinase was used as a control housekeeping gene for mRNA quantitation. RESULTS AND DISCUSSION
Ch aactr
eriz
XgtlO
constructed clones
from
of murine
sequence
of
unpublished sequence. insert into
size. pNNl1
a
the VCAMl, partial
lungs
of
phage
LPS-treated
mice
and
were screened
with
a probe
murine
VCAMl
information),
highly
17 hybridizing
plaques
The two longest for
DNA sequence
libraries
genomic
clone
homologous were
clones, analysis. 164
each
to
purified
kb,
To obtain based (L.
the and
of 2.1 The
rats.
were
on the Burkly,
human
cDNA
analysed
for
were subcloned
cDNA sequence
is
the
Vol.
183,
No.
3,,, 1992
BIOCHEMICAL
composite
of
composite
sequence
sequence,
a
overlapping is
2217
same probe
two longest
bp in
open
for
at lower
clones,
sequence
clones,
and is
3016 bp in length,
sequences
have
is
the
frame,
clones.
57
bp of
and
753
bp
phage were
of
contains
these
GenBank
of
5'
UT
3'
UT
screened
with the
and sequenced. two
57 bp of
and 741 bp of
The
plaques
were subcloned
kb,
with
two
contains
composite
been deposited
COMMUNICATIONS
Of 4 hybridizing
stringency.
VCAMl
these
VCAMl,
of 1.7 and 2.1
a 2217 bp open reading
of
frame,
rat
The rat
RESEARCH
length,
reading
clones
but
BIOPHYSICAL
sequences
3028
bp
To obtain
sequence. the
the
AND
overlapping
5' UT sequence,
3' UT sequence.
The DNA
(Accession
MB4487,
#;s:
MB4488). Deduced amino acid sequences peptide
of of
sequence region,
murine
of rodent
and rat
24 amino acids
containing
VCAEll
(aa),
seven
(1,14,17).
(Fig.
Ig-like
Both
VCAMls.
both
a 22
as found
sequences
contain
contain
aa transmembrane
in the major four
contain
five
extracellular
potential
domains.
N-glycosylation
The sequences
are
human seven domain form of VCAMl (14,17) and 76.9% identity, By comparison, which
respectively,
ICAMl,
shares
an adhesion
many similarities
identity
between
murine
identity
between
individual
similar
throughout
Interestingly, molecules
the
the (Fig.
Northern
analvses
analyses
of
both
the
molecule with
and
aligned
the
VCAMl
domains
cytoplasmic
varying tail
that
is
the
of
the
1, and show 75.9%
(1,2),
is
and
in
of human VCAMl.
and Ig superfamily
sequences
molecule,
(1,14,17),
with
sequence
VCAMl
human
in Ig-
sites
in Figure
with
form of
cysteines
domains 1 and 4, as does the human VCAMl sequence
both
a signal
by a 674 aa extracellular
domains, tail,
The deduced amino acid
1)
followed
and a 19 aa cytoplasmic
human VCAMl like
seauences
member
shows about (18).
The
not
only
from
68%
identical
in
50%
percent high to
all
but 84%.
three
1). of
VCAMl
untreated
exoression
in
and LPS-treated 165
inflamed murine
lung. lung
Northern
tissues
are
675
Domain 7--> KDILLTaFPSeS~CDTVIISCTCGnWEtWII~eTGDtVLKSIdGaYTIRWLQLkDAGVYECESKnkVGSQLRSLTLDVqGrEnNRDYfSPELL KDIqLTvFPSkSVKEGDTVIISCTCGnVPEtWII ~TGDmVLKSVdGsYTIRqAQLqDAGIYECESKteVGSQLRSLTLDVkGkE~NKDYFSPELL KDIqLTvFPSkSVKEGDTVIISCTCGs'JPEiWII ~TGDmVLKSVnGsYTIRkAQLqDAGVYECESKteVGSQLRSLTLDVkGkEnNRYFSPBLL MI-LT-FPS-S~GDTVIISCTCG-~E-~I~-T~-~SV-G-YTIR-AQL-~AG~CKSK--VGSQ~SI.~V-G-E-N~YFSPSLL
697 Cytoplasmic-->715 vLYfASSLIIPAIGHIIYFARKANMK GSYSL-KV aLYcASSLVIPAIGHIVYF ARKANMKGSYSLVEAQKSKV aLYfASSLVIPAIGnIIYFARKANMK GSYSLVEAQKSKV -LY-ASSLVIPAI'Z'!IIYFARKANMKGSYSLVEAQKSKV
Transmembrane-->
575
AMRHEDSGIYVCSGVNLVGL~
486 Domain 6--> StQtLYVNVAPrDtTVlVSPSsrlEEGSs~ClSqGfPaPKILWSRQLpNGELQPLSeNa'PLtLisT~DSG~lCEGINqAG~SrKe~LIlQvtp SvOpL~~lcBtTIwVSPSpIlEEGSpVNLTCsSdGiPaPKILWSRQLnNGELQPLS~N~CLtFmsTXrDDSGIYvCEGINeAGiSrKsVELIIQvvy StQtLYVMTAPkEpTIwVSPSpVpEEGSpVNLTCsSdCfPtPKILUSRQ~NGELQPLSqN~fLaFmaT~DSGIY~EGINeAGiSkKs~LIIQgss S-Q-LYVNVAP-E-TI-VSPS-I-EEGS-~LTC-S-G-P-PKILWSRQL-NGELQPLS-N-'CL-F--'~K-EDSGIY-CEGIN-AG-S-K-~LIIQ---
379 Domain 5--> YSFPrDPeIsmSGgLVnG~sVTVsCkVPsVYPLOrLEIELLKGETiLenieFlEDtdSLEllRsLEmTFIPTiEDTGKaLVCqAkLHid~~~PKQhQ YSFPeDPvIlonSGpLVhGrpVTVnCtVPnVYPFDhLEIELLKGETtLmkkyFlEEmgiKSLEtKiLEtTFIPTiEDTGKsLVC1ArL~sgeME~EPKQRQ YSFPaDPeIeiSGpLVhGrpVTVnCtVPnWPFD~EIELGKGETtLlnkfLrEEigtKSLEtKsLEmTFIPTaEDTGKaLVC1AkLHssqME~EPKQhQ YSFP-DP-I--SG-LV-C--VTV-C-VP-VYPFD-LEIELET-L----F-EE---KSL~-K-LE-TFIPT-EDTGK-LVC-A-LII---ne-EPKQhQ
287 Domain 4--> rkEVELIVQEKPFtVEISPGprI~QI~SV~~s~~SPSFSWRTQiDSPLsGkVRsE~~tnSTLtLSpV~fEnEHSYLCtVTCyhkkLEKgiQVE1 kaaVELWQEKPFiVDISPGsqVAAQVGDSVvL~a~i~SPSFS~TQtDSPLnGv~El;akSTLvLSsVgfEdEHSYLC~VTClqrtLEKrLQVEv ktEVELIVQ~FtVDISPGsqVAAQVGDSVvLKaavGCSPSFSWRTQtDSPLnGeVRdEGatSTLtLSpVgvEdEHSYLCKtiQVEv --EVELIVQEKPF-VDISPG--VAAQVGDSV-LTC---GCK-.QVE-
198 Domain 3--> IDenSSvptvRqaVkEMVYiSPkNTvISVnPSTkLqEGgs~~C~SEGLP~EIFWsk~NgnLQt~LSGNATLTLI~DSGlXVCSG~l~IGh~~ IDqiDStlkeRatVkELQWiSPrNTtIS~STrLqEGgaVTMTCsSEGLPAPEIFWgrKWNevLQ1LSGNATLTLIAHRHEDSGVYVCEGVNLIGrd IDgtDSipksRetVrELQWtSPkNTeISVhPSTrLhEGaaVTMTCaSEGLPAPEIFWskKWNgvLQlLSGNATLTLI ID--DS----R--V-ELQW-SP-NT-ISV-PST-L-EG--~~C-SEGLP~EIFW--K~lN--LQ-LSGNATLTLI~DSG~YVCEG~~.I~;~-
91 Domain 2--> Sr~kgIqVBIYSPPROPEIhLSGPLEaGICPItVRCsvaDVyPfdRLEIDLLKGDhLMksQEFleDadrKSLETKSLeVtFTPVIEDIgKvLVCKAKLh Sg~rsIhMIYSFPRDPEIqFSGPLEvGKPVtVKClapDIYPvyRLEIDLFKGDqLMnrQEFsaEamtKSLETKSLeVtFTPVIEDIgKaLVCKAI(L.i, SgllLErgIqVOIYSFP~PEIqFSGPLEvGKPVmVKClapDVYPidRLEIELFKGDrLMkkQUFvdEmaMSLETKSLEViFTPVIEDIeKaLVC~.y S-KLE--I-VDIYSFPWOEI-FSGPLE-GKPV-VKC---DVYP--RLEIDLFKGD-LM--QYV-FTPVIEDI-K-LVCHAKL-
-24 Signal sequence--> 1 Danain l--; ~gRHVvILGASnILWIMFA8SQAFRIEttPEdryLAQIGDSvsLTCSTTGCESPfFSWRTQIDSPLNgKVtnEGttStLTMnPVSFgNEHSYLCTATCa MP‘rrlMVaVtGAStVLWILFAvSQAPKIEisPEyktiAQIGDSmaLTCSTTGCESPlFSWRTQIDSPLNaKVrtEGskSvLTPVSFeNEHSYLCTATCg 13PvlMVaIP~StVLWILFAvSQAFKIEisPEyktlAQIGDSmlLTCSTTGCESPsFSWRTQIDSPLNgKVktEGakSvLTVSFeNEHSYLCTATCn HP-KMV-ILGAS-VLWILFA-SQAFKIE--PE----AQIGDS--LTCSTTGCESP-FSWRTQIDSPLN-IN--EG--S-LTH-PVSF-NEHSYLCTATC-
Vol.
183,
No.
1, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
12
25
q
n Ramos
7.5 -
7oz/3
of murine VCAMl mRNAs in LPS-treated lung. Fisure 2. Induction Five micrograms of polyA+ mRNA from control (lane I) and LPStreated (lane 2) murine lung tissue were electrophoresed through an agarose-formaldehyde gel, transferred, and probed with a radiolabelled murine VCAMl insert. Hybridizing mRNAs in the induced lung tissue are indicated with arrows. Molecular weight markers are indicated at right. Fisure 3. Adhesion of cell lines to COS cells transiently transfected with murine VCAMl cDNA. The adhesion to murine VCAMl of human Ramos cells or murine 702/3 cells was examined, in the presence or absence of anti-human (HPl/2) or anti-murine (PS/2) VLA4 mAbs, or of anti-murine VCAMl (MK-1, MK-2) or isotypematched control (KM201) mAbs. shown
in
Figure
In normal
2.
and is induced
low levels, tissue.
In the
induced
1.6 kb.
The nature
kidney (191,
(19),
Transient transiently adhesion
with
tissue
transfected (Fig.
of
been
cells
the with 3).
under seen
murine
and inflammed
exoression
assessed
also
(13). rat
murine the
Both
in LPS-treated
study.
lung
in
Such smaller
inflammed
tubular
epithelial
Similar
results
lung VCAMl
cDNA for
(not
murine cells
have been
shown).
cDNA. murine
human Ramos cells
COS cells VCAMl,
were
and cell
and murine
Figure 1. Homology between human, murine, and rat VCAMl protein sequences. The deduced amino acid sequences are aligned, along with a consensus sequence indicating amino acid residues identical in both sequences. The beginning of each of the seven Ig-like domains is indicated (17), as are the transmembrane region and the cytoplasmic tail.
167
at
mRNA can be seen at about
RNA is currently
endothelial
normal
ten-fold
another
cytokine-treated
and in murine
obtained
lung
mRNAs have in
a 3.2 kb VCAMl mRNA is present
approximately
of this
cross-hybridizing
lung
7OZ/3
Vol.
183, No. 1, 1992
cells
adhere
well
to transfected
totally
inhibited
adhesion
is
obtained
similar
transiently
BIOCHEMICAL
results
expressed
two putative
mAbs,
mAbs inhibit
the
binding
of
mAb KM201 is without adhesion
Adhesion
molecules
VLA4
on
fibroblast-like
interfere
with
B lymphocyte
marrow
cultures
(13).
the
cDNA probe,
and
human
VCAMl
(1,13),
related
to,
the
transiently
murine
strongly VCAMl.
antigen.
(not
that In
addition, with
shown),
that
and
block
murine
stromal
indeed
murine
and M-K/2
is
VCAMl/VLA4
pathway
important
in
these
bone mAbs
it
is,
or
sequence N-terminal
is
cells,
a human
homologous
the
VCAMl
to
that
of
is
closely of murine
sequence
M-K mAbs bind
of
to
COS cells
VCAMl cDNA but not
to control
(Fig.
the
for
in long-term
to
VLAl-dependent
COS cells
by immunizing
endothelial
N-terminal
both
interact
mAbs selectively
by
that
the murine
mAbs M-K/l
is
sequence
from
closely
The
mRNA hybridizing
obtained
maturing
a counter-receptor
murine
suggesting
mAb blocks
of
prepared
recognized
The predicted
VCAMl-expressing confirm
of
3) to
isotype-matched
they
when included
an N-terminal
transfected
COS cells
results
presence
has
where
cultured
on
(Fig.
retention
(12,13).
antigen
Both
shown).
define
formation
examined
(12,13).
neither
and Pi-K/Z
lines
The
1) matches
(Fig.
murine
murine
cell
expressed with
(not for
cells
stromal
VCAMl
M-K/l
the
to
binding
cells
contrast,
important
Two mAbs,
murine
correlates
In
in the bone marrow,
cells.
constitutively
while
We have
also
VCAMl
Ram06 or 7OZ/3
effect.
are
RBL-1
We have
murine
and this
mAbs.
line
shown).
to human VCAMl
precursors
adherent
cell
to
either
COS cells,
anti-VLA4
transfectants,
VLA4-dependent
with
(not
RESEARCH COMMUNICATIONS
control
rat
and M-K/Z,
control
stromal
the
VCAMl
M-K/l
not
by blocking
rat
VCAMl-expressing
with
but
with
murine
lymphocyte
AND BIOPHYSICAL
3). cell
VCAMl,
lymphopoeisis
cell
adhesion
Taken antigen
jointly,
the
recognized
and confirm
to
that
by the
(12,13).
ACKNOWLEDGHENTS We thank Jacqueline Ashook Ellen Klein for excellent
for F.A.C.S analyses; technical assistance; 168
Janice DeNarinis and Diane Leone
and for
Vol.
183,
No.
1, 1992
help with figures. AI20069 and AI19884
BIOCHEMICAL
The (to
work PK).
AND
was
EIIOPHYSICAL
partly
RESEARCH
supported
COMMUNICATIONS
by N.I.H.
Grants
REFERENCES
1. C&born, L., Hession, C., Tizard, R., Vassallo, C., Luhowskyj, S., Chi-Rosso, G., & Lobb, R. (1989). Cell 59, 1203-1211. 2. Lobb, R., Hession, C., & Osborn, L. (1991) in: Cellular and Molecular Mechanisms of Inflammation, Volume 2, (C. G. Cochrane & eds.) Academic Press, N.Y. In press. M. A. Gimbrone,Jr., 3. Rice, G.E., Munro, J.M., & Bevilacqua, M.P. (1990). J. &&p. Med. 171, 1369-1374. 4. Elites, M-J., Osborn, L., Takada, Y., Crouse, C., Luhowskyj, S Herler, M.E., & Lobb, R.R. (1990). Cell 60, 577-584. 5:'Schwartz, B.R., Wayner, E-A., Carlos, T.M., Ochs, H.D., & Harlan, J.M. (1990). J. Clin. In--est.. 85, 2019-2022. 6. Burkly, L.C., Jakubowski, A., Newman, B.N., Rosa, M.D., ChiROSSO, G., & Lobb, R.R. (1991) Eur. J. Immunol. 21, 2871-2875. J.M., Pryzbyla, A.E., MacDonaid, R.J., & Rutter, 7. Chirgwin, W.J. (1979) Biochemistry 18, 5294-5299. 8. Gubler, U., & Hoffman, B.J. (1983) Gene 25, 263-269. 9, Maniatis, T., Fritch, E.F., & Sambrook, J. (1982) in: nolecular Cloning-A Laboratory Manual, Cold Spring Harbor, New York. T., Young, R.A., & Davis, R.W. (1985) in: DNA 10. Huynh, Cloning-a Practical Approach, (D. M. Glover, ed.), IRL Press, Oxford, Vol.1, pp. 49-78. 11. Pulido, R., Elites, M.J., Campanero, M.R., Osborn, L., Schiffer, S., Garcia-Pardo, A., Lobb, R., Hemler, M.E., & Sanchez-Madrid, F. (1991) J. Biol. Chem. 266, 10241-10245. 12. Miyake, K., Weissman, I.L., Greenberger, J.S., & Kincade, P.W. (1991). J. Exp. Med. 173, 599-607. 13. Miyake, K., Medina, K., Ishihara, K., Kimoto, M., Auerbach, R & Kincade, P.W. (1991) J. Cell Biol. 114, 557-565. 14: Hession, C., Tizard, R., Vassallo, C., Schiffer, S-G., Goff, D G., Luhowskyj, S., Lobb, R., & Osborn, L. Mq', P., Chi-Rosso, Chem. 266, 6682-6685. (i991). J. Biol. 15. Lobb, R-R., Chi-Rosso, G., Leone, D., Rosa, M., Newman, B., Luhowskyj, S., Osborn, L., Schiffer, S., Benjamin, C., Dougas, I ., Hession, C., 7 Chow, E.P. (1991) Bichem. Biophys. Res. Commun. 178, 1498-1504. 16. Feinberg, A.P., and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267. 17. Cybulsky, M., Fries, J., Williams, A., Sultan, P., Eddy, R., Byers, M., Shows, T., Gimbrone, M., & Collins, T. (1991) Proc. Nat. Acad. Sci. U.S.A. 88, 7859-7863. 18. Horley, K-J., Carpentino, C., Baker, B., & Takei, F. (1989) E.M.B.O. J. 8, 2889-2896. 19. Wuthrich, R.P., Snyder, T.S., & Jenkins, T.A. (1991) Amer. sot. Nephrol. (Abstr.) In Press.
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