Cloning, sequencing and analysis of cDNA encoding bovine intercellular adhesion molecule-1 (ICAM-1)

Veterinary

Veterinary Immunology and Immunopathology 59 (1997) 121-129

ELSEVIER

immunology arKI immunopathology

Cloning, sequencing and analysis of cDNA encoding bovine intercellular adhesion molecule(ICAM-11 Em-kyung a Department

Lee ‘, Sang-Gu Kang b, Marcus E. Kehrli Jr. ‘. *

of Microbiology,

Immunolo,qy,

and Prewntiw

IA 50010, ’ Department

’

Metabolic

1

ofZoology

and Genetics. lonu

Diseases and Immunology

Medicine.

Iowa State Unic,ersir\

Ame.\.

USA State Uniwrsi~.

Research Unit, NADC-USDA-ARS,

Ames. IA 50010.

USA

lo,vu State Unic,er+y

Ame.s.

IA 50010. USA

Accepted I I March 1997

Abstract Intercellular adhesion molecule-l (ICAM-1) is an inducible glycoprotein that interacts with the leukocyte Pr-integrins, LFA-1 and Mac-l. We have isolated and analyzed a cDNA clone coding for the putattve bovine ICAM- gene and compared it with known comparative sequences from other species as well as bovine ICAM-3. The 339%bp bovine ICAM- cDNA sequence codes for 53.5 amino acids and shows 57% homology with human ICAM- and 47% homology with bovine ICAMat the amino acid levels. The predicted number and positions of cysteine residues in bovine ICAM-I are all conserved among species including bovine ICAM-3. It has two arginineglycine-aspartate (RGD) sites in the extracellular region and a serine residue in the cytoplasmic tail. Northern blot results show that the bovine ICAM-I gene is expressed in stimulated leukocytes whereas bovine ICAMis expressed predominantly in resting neutrophils. 0 1997 Elsevier Science B.V. Kq\lwrd.v

Intercellular adhesion molecule; &-integrins; Leukocytes: Inflammation; mRNA: cDNA sequence

1. Introduction Intercellular immunoglobulin

adhesion (I&like

molecule (ICAM)-l is a cell surface glycoprotein with five domains. It plays an important role in interactions between

* Corresponding author. Tel.: + 1 515 239 X462: fax.: + 1 515 239 8458; e-mail: [email protected]. usda.gov. 0165.2427/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PI1 SOl65-2427(97)00051-2

122

E-K. Lee et al./

Veterinary

Immunology

and Immunopathology

59 (1997) 121-129

leukocytes and in transendothelial migration of leukocytes through its expression on vascular endothelium by interacting with the /3,-integrins, LFA-1 (CD1 la/CD181 and Mac-l (CD1 Ib/CDl8) on leukocytes (Marlin and Springer, 1987; Dustin and Springer, 1988). ICAM- is constitutively expressed on hematopoietic cells, vascular endothelium, fibroblasts and some epithelial cells, and its expression is unregulated in inflammatory responses while ICAMexpression is restricted to cells from lymphoid and myeloid lineages (de Fougerolles and Springer, 1992; Cordell et al., 1994). It has been reported that Ig domains 1 and 2 of ICAM- 1 mediate binding to LFA-I and Ig domain 3 of ICAMis involved in binding to Mac-l (Staunton et al., 1990; Diamond et al., 19911. Leukocyte emigration from blood to sites of inflammation involves the sequential interaction by adhesion molecules expressed by both leukocytes and endothelial cells. Transendothelial migration of leukocytes begins with leukocyte rolling, which is mainly dependent on selectins, followed by activation of integrins. firm attachment to endothelium, migration across the endothelial surface to intercellular junctions and the subsequent migration between endothelial cells (Kishimoto, 19911. Integrin binding to ICAM-I is particularly important for firm attachment and migration across endothelium. It has been postulated that ICAM- is potentially the most important ligand for LFA- 1 in the initiation of the immune response because the expression of ICAMon resting leukocytes is low and ICAMis high (de Fougerolles and Springer, 1992; Fawcett et al., 1992). The purpose of this paper is to report the cloning and sequencing of a cDNA encoding bovine ICAM-I and comparative sequence analysis with other species (Simmons et al., 1988; Horley et al., 1989; Kita et al., 1992; Manning et al., 19951. Gene expression of bovine ICAMand ICAMis also studied in resting and stimulated leukocytes.

2. Materials

and methods

2.1. Preparation

of cDNA probe for screening

the librar?,

One pair of primers, S-CCTCTCCGTGGTGCTGCTC-3’ and S-GCAGTTAACAGGCCAGGAG-3’, from Ig domains 2 and 3 of the cDNA encoding bovine ICAM- was designed (Lee et al., 1996) to amplify 280 bp of DNA, which has high homology with cDNA of human ICAMbased on sequence analysis. The PCR product was labeled with 3’P-dCTP using random priming method (Pharmacia, Piscataway, NJ) and used as a probe to screen a bovine endothelial cell cDNA library. 2.2. cDNA library screening

and clone isolation

A Uni-ZAP’” XR cDNA library (Stratagene, La Jolla, CA) of bovine endothelial cells treated with recombinant human tumor necrosis factor-a was used for screening. Plaque lifts and phagemid in vivo excision were carried out according to manufacturer’s protocols. Plaque hybridization was performed overnight at 42°C in a solution containing 50% formamide, 6 X SSC, 3.3 X Denhardt’s solution, 25 mM Na,HPO,/NaH,PO,

E-K. Lee et al. / Veterinar?, ImmunrAog~~ and Immunopathology

59 (1997) 121-1119

123

solution, 0.1 mg/ml sonicated salmon sperm DNA, and 0.4% SDS. Filters were washed twice in 2 x SSC/O.l% SDS for 5 min at room temperature CRT). once in 0.2 X SSC/O.l% SDS for 10 min at RT, twice in 0.1 X SSC/O.l% SDS for 5 min at RT. and final washing in 0.1 X SSC/O.l% SDS for 5 min at 60°C then exposed at - 70°C for 18 h to X-ray film for autoradiography. Three rounds of plaque hybridization were done to enrich for positive clones. After the third screening, the A clones were subjected to in vivo excision. The pBluescript phagemids excised from the A clones were purified and insert sizes were evaluated by restriction enzyme digestion analysis. One of eleven positive clones, containing a cDNA insert that was approximately 3.4 kb, was selected for further characterization. 2.3. DNA sequencing and computer analysis Double-stranded DNA sequencing was performed by the dideoxy-nucleotide chain termination method @anger et al., 1977) using manual (USB, Cleveland, OH) and automated dideoxy sequencing at the Iowa State University DNA Sequencing Facility. DNA and deduced amino acids were analyzed using the programs in the CCG software package (University of Wisconsin Genetics Computer Group. Madison, WI, USA). 2.4. Leukocyte isolation and actiuation Mononuclear cells (MNC) and polymorphonuclear cells (PMN; neutrophils) were separated as described (Roth and Kaeberle, 1981; Kehrli et al., 1989). The isolated neutrophils were suspended to 5 X 10’ cells/ml in Earle’s Balanced Salt Solution (EBSS). Cell suspensions were incubated with opsonized zymosan (OZ; 10 mg/ml) for 3 h and platelet activating factor (PAF; 1 pg/ml) for 0.5 h, 1.5 h, 3 h, and 18 h along with equal volume of medium. Mononuclear cells were enriched by buoyant-density gradient centrifugation on percoll gradients (1.084 sp. gr., 400 X g for 40 min). harvested. and washed in phosphate buffered saline (PBS). The washed mononuclear cells were resuspended to 1 X lo* cells/ml in RPMI-1640 medium containing 10% fetal bovine serum with antibiotics and antimycotic, and stimulated with pokeweed mitogen (PWM: 10 pg/ml) and concanavalin A (Con A; 1 p.g/ml) for the same intervals as neutrophils. 2.5. Preparation

Qfgene spec$c cDNA probes and northern blot analysis

Two sets of PCR primers were designed from cDNAs of bovine ICAMand -3 genes. A DNA fragment (307 bp) from bovine ICAM- using 5’-TGGAGATAGCTGGGCAGGT-3’ (at 1671 bp) and 5’-GTGGAGCACGTTCAGGAC-3’ (at 1978 bp) primers was amplified and a 353 bp fragment from bovine ICAMprimers, S-GCACCGTAATAGGAGCATCCAGT-3’ (at 1269 bp) and 5’-GCAGCGAGAGTCACCAAGCCC3’ (at 1613 bp). The amplified fragments were used as hybridization probes for the following experiments. Total RNA from PMN and MNC extracted using Trizol’” (Gibco BRL, Gaithersburg, MD) and chloroform, precipitated with isopropanol, and washed with 75% ethanol. Twenty micrograms of total RNA from each sample were

124

E-K. Lee et al./ Veterinary Immunology and Immunopathology

59 (1997) 121-129

electrophoresed through a 1% agarose gel containing formaldehyde, blotted to nylon membranes, and probed with 32P-dCTP labeled bovine ICAMand -3 probes separately. Prehybrization and hybridization conditions were the same as described for the cDNA library screening. Membranes were washed twice with 2 X SSC/O. I % SDS for 5 min at RT, once with 0.2 X SSC/O.l% SDS for 10 min at RT, once with 0.1 X SSC/O. 1% SDS for 5 min at RT, and finally with 0.1 X SSC/O. 1% SDS for 5 min at 55°C then exposed for 24 to 72 h to X-ray film for autoradiography.

3. Results 3.1. cDNA encoding bovine ICAM-I The cDNA sequence of bovine 1605 nucleotides that encode for nucleotide 554 and ending with composite cDNA sequence for

ICAM-I was 3398 bp with an open reading frame of 535 amino acids, starting with a methionine codon at a termination codon at nucleotide 2158 (Fig. 1). The the coding region of bovine ICAMshows

Fig. I. Nucleotide sequence and the deduced amino U65789). The hydrophobic putative signal peptide line. The cysteine residues are circled and N-linked for Ig domains are indicated above the nucleotide 3’.untranslated region is in bold.

acid sequence of bovine ICAM-I cDNA (Accession No. and transmembrane sequences are indicated by a dashed glycosylation sites are boxed. The proposed start points sequence. The polyadenylation signal AATAAA in the

E-K. Lee et al. / Veterinar?, Immunology

Table I Interspecies

comparison

of amino acid homology

and Immunopathology

59 (1997)

among five Ig-like domains

125

121-129

of ICAM-I

and ICAM-?

Comparison

Domain 1

Domain 2

Domain 3

Domain 4

Domain 5

dog/human” dog/mouseh human/mouaec bovine/human” bovine/human”

55% 49% 48% 52% 57%

77% 64% 64% 70% 64%

55% 39% 46% 48% 61%

66% 63% 57% 62% 72%

54% 56% 46%’ 5 1% S5%

“.h.‘.dDomain identity of ICAM- I among species. ‘Domain identity of ICAM- between bovine and human. Percent of amino acid identity between Ig domains among highest homology percent within ICAM-I and -3 domains.

species

was showed.

Bold numbers

represent

homology with human ICAM-1. The deduced amino acid sequences for bovine ICAM- I show 57% and 47% identity with human ICAMand bovine ICAM-3. respectively. The bovine ICAM- amino acid sequence contains a putative signal peptide, five Ig-like domains, a hydrophobic transmembrane region, and a cytoplasmic tail, which aligns it with the Ig gene superfamily. It also contains thirteen N-glycosylation sites indicating that the protein is heavily glycosylated. Fourteen cysteine residues in bovine ICAM-I are found at the same positions as those of human ICAMand bovine ICAM-3.

Fig. 2. Homology of bovine ICAM-I. human ICAM-1. and bovine ICAM-3. Alignment of Ig domains between bovine/human ICAMand bovine ICAM-l/bovine ICAM-3. Alignments of ICAMs were generated using program bestfit from GCG software package (University of Wisconsin Genetics Computer Group).

126

E-K. Lee et al./ Veterinary Immunology and Immunopathology

3.2. Q-like domain comparison

59 11997) 121-129

among species

The conservation of amino acid sequences is high throughout all five domains of bovine ICAMcompared to human ICAM(Table 1). Domain 2 is the most highly conserved domain among species, whereas domain 4 of ICAM- sequences (only bovine and human ICAMare known so far) is the most highly conserved domain. When comparing the domains of bovine ICAM-I and -3, the homology of domain 2 is 94% whereas the others are below 43% (Fig. 2). Bovine ICAM-I has one serine residue in the cytoplasmic tail, which neither human, canine, rat, nor mouse ICAM-I or -2 contain any serine residues in their cytoplasmic tails. Furthermore, bovine ICAM-I contains two Arg-Gly-Asp (RGD) sequences, found mostly in matrix molecules, unlike human ICAM- 1. IC,Lkl-3

[CAM- I

A

PMN KS

PMN KS

MNC RS

PMN

MNC 1

MNC RS

2

3

4

5

6

7

8

9

IO

ICAM-

18S-

Fig, 3. Northern blot analysis of bovine ICAMs. (A) 20 pg of total RNA used and probed with bovine ICAM-I and -3. Neutrophils (PMN) were also inactivated (R) or activated (S) with 02 (10 mg/ml) for 3 h. MNC were unstimulated (R) or stimulated tS1 with PWM (10 pg/ml) for 3 h. (Bl 20 /~g of total RNA from MNC (lane l-5) and neutrophils (lane 6-10) were electrophoresed and probed with bovine ICAM- and -3 fragments. MNC were stimulated with Con A (1 *g/ml) and neutrophils with PAF (1 pg/mll for 0 (lane 1 and 61, 0.5 h (lane 2 and 7). 1.5 h (lane 3 and 81. 3 h (lane 4 and 9). and 18 h (lane 5 and 101.

E-K. Lee et al. / Veterinary Immunology and Immunopathology

3.3. Expression comparison

59 ( 19Y7) 12 I - I29

17-7

qf neutrophils and mononuclear cells

Study of bovine ICAMexpression by northern blot analysis in Fig. 3 shows that total RNA levels of ICAMin resting MNC are almost undetectable but increase after 3 h and 18 h upon stimulation with PWM and Con A, respectively. Similarly, ICAM-I total RNA in resting neutrophils was not expressed at all, but was detectable after stimulation with PAF (Fig. 3). On the other hand. bovine ICAMin neutrophils was expressed abundantly in unstimulated and activated neutrophils while ICAMexpression was very low in resting MNC and was down-regulated in activated MNC.

4. Discussion We have screened a bovine aortic endothelial cDNA library and isolated a cDNA encoding for ICAM- which is 3.4 kb in length with a 5’ untranslated region of 553 bp, a complete coding sequence of 1605 bp and a 3’ untranslated region of 1237 bp. Sequence comparison indicates that bovine ICAMis a member of the Ig gene superfamily containing five Ig-like domains as does ICAM- 1 of other spe ies. Comparison to the human ICAMsequence indicates that the overall homology ,s 69% at the nucleic acid level and 57% at the amino acid level. The homology between bovine ICAMand ICAMfor amino acid is 47% with striking identity in domain 2 (94%). postulating that ICAMand -3 may be evolved from a common ancestral ICAM gene. Intercellular adhesion molecule-l is expressed at low levels on endothelial cells, epithelial cells, dendritic cells, and leukocytes and its expression can be induced and augmented by cytokines (IFN-7, TNF-a, and IL- I), retinoic acid and lipopolysaccaride (Dustin et al., 1986; Bouillon et al., 1991; Myers et al., 1992) whereas the expression of ICAMis restricted to resting leukocyte and Langerhans cells (Fawcett et al.. 1992: Acevedo et al., 1993; Cordell et al., 1994). Elsner et al. (1995) separated resting leukocytes into neutrophils and MNC to look at the expression of human ICAM-I and showed that both neutrophils and MNC constitutively express ICAMmRNA. We could not detect the constitutive expression of bovine ICAMin either neutrophils or MNC. One possibility is that we used total RNA instead of mRNA, which gives less sensitivity. Northern analysis of bovine ICAMexpression, however, shows abundant expression in resting and to a lesser extent in activated neutrophils. This contrasts with clear detection of bovine ICAMs in unstimulated MNC. Human ICAMmolecules have been reported to be expressed at a high level Ln resting lymphocytes. monocytes. and neutrophils by flow cytometric analysis (de Fougerolles and Springer, 19921. During transendothelial migration, neutrophils adhere to ICAMs of endothelial cells by means of their /3,-integrins leading to the accumulation of neutrophils at sites of inflammation. It was postulated the ICAMis involved in the initiation of an immune response because of the observation that adhesion of resting T lymphocytes to LFA- I on other lymphocytes occurs primarily via ICAM- and its expression is much higher than other LFA-I ligands (de Fougerolles and Springer, 1992). Some ICAMmolecules were released upon cell activation by enzymatic cleavage similar to L-selectin and the remaining molecules on the neutrophil surface following activation were still capable of

128

E-K. Lee et al./ Veterinay

lmmunolqqy

and Immunopathobgy

59 (19971 121-129

sustaining cell adhesion (de1 Pozo et al., 1994). The functional consequences of ICAMdown-regulation on neutrophils are not evident at present. The bovine ICAMmight play a role in more recruitment of neutrophils by means of homotypic interactions during acute inflammatory response. Also, it is possible that ICAM- might interact with other counter-ligands on the surface of endothelial cells. Bovine ICAM-1, -3 and human ICAM- have serine residues in the cytoplasmic tail, whereas neither human, canine, rat, nor mouse ICAM-I or -2 contain any serine residues in their cytoplasmic tails. It was reported that serine residues of human ICAM- became rapidly and transiently phosphorylated upon cellular stimulation (Lozano et al., 1992). The serine residue on the cytoplasmic tail of bovine ICAMin MNC might be important after activation, leading to conformational changes as well as signal transduction. Integrins (mainly pj, and several p,) appear to recognize specific amino acid sequence motifs in their ligands. Arg-Gly-Asp (RGD) is found within a number of matrix proteins including fibronectin, fibrinogen, thrombospondin, vitronectin, laminin and type I collagen although not all integrins bind to ligands via RGD-containing domains (Ruoslahti and Pierschbacher. 1986; Hynes, 19921. The RGD sequences of the filamentous hemagglutinin of Bordetella pertussis has been suggested to modulate binding of ligands to Mac-l (Relman et al., 1990; Rozdzinski et al., 1995). Bovine ICAM- contains two RGD sites and murine ICAM- has one RGD sequence. Although synthetic RGD peptides failed to inhibit human LFA-1 /ICAM- 1 mediated adhesion and the synthetic peptide LASRGDHG had no effect on LFA-1 dependent cell-cell interaction in mice (Siu et al., 19891, the importance of these RGD regions in receptor-ligand interaction can not be ruled out.

References Acevedo. A., de1 Pozo. M.A., Arroyo, A.G., Sanchez-Mateos, P.. Gonzalez-Amaro. R., Sanchez-Madrid, F.. 1993. Distribution of ICAM-3-bearing cells in normal human tissues: Expression of a novel counter-receptor for LFA-I in epidermal Langerhans cells. Am. J. Path. 143, 7744783. Bouillon, M.. Tessier. P., Boulianne. R., Destrempe. R., Audette. M., 1991. Regulation by retinoic acid of ICAM- expression on human tumor ceil lines. Biochim. Biophys. Acta. 1097. 95-102. Cordell. J.L.. Pulford, K.. Turley. H.. Jones, M., Micklem. K., Doussis, LA.. Tyler. X.. Mayne. K., Gatter, K.C.. Mason, D.Y.. 1994. Cellular distribution of human leukocyte adhesion molecule ICAM-3. J. Clin. Pathol. 47, 143-147. de Fougerolles. A.R., Springer, T.A.. 1992. Intercellular adhesion molecule 3. a third adhesion counter-receptor for lymphocyte function-associated molecule 1 on resting lymphocytes. J. Exp. Med. 175, 185- 190. de1 POZO. M.A., Pulido, R., Munoz, C.. Alvarez, V., Humbria, A.. Campanero. M.R.. Sanchez-Madrid, F.. 1994. Regulation of ICAM(CD501 membrane expression on human neutrophils through a proteolytic shedding mechanism. Eur. J. Immunol. 24. 258662594. Diamond. M.S.. Staunton, D.E.. Martin, S.D., Springer. T.A.. 1991. Binding of the integrin Mac-l (CD1 lb/CD181 to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation. Cell. 65. 961-971. Dustin, M.L., Rothlein. R., Bhan. A.K.. Dinarello. C.A.. Springer, T.A., 1986. Induction by IL-l and interferon-y: tissue distribution. biochemistry, and function of a natural adherence molecule (ICAM-I 1. J. Immunol. 137, 245-254.

Dustin. ML. Springer, T.A., 1988. Lymphocyte function-associated antigen (LFA-1) interaction with intercellular adhesion molecule-l (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J. Cell Biol. 107. 321-331. Elsner. J.. Sach. M., Knopf, H.-P.. Norgauer. J., Kapp. A.. Schollmeyer, P., Dobos, G.J., 19Y5. Synthesis and surface expression of ICAM- in polymorphonuclear neutrophilic leukocytes in normal subjects and during inflammatory disease. Immunobiol. 193. 456-464. Fawcett, J.. Holness. C.L.L., Needham. L.A.. Turley. H.. Gatter. K.C.. Ma&on, D.Y., Simmons, D.L.. 1092. Molecular cloning of ICAM-3. a third ligand for LFA-I. constitutively expressed on resting Iruhocyte\. Nature 360, 4X 1-488. Horley, K.J.. Carpeito. C.. Baker. B.. Takei. F., 1989. Molecular cloning trf murine intercellular adhesion molecule (ICAM- I ). EMBO J. 8, 2889-2896. Hynes, R.O.. 1992. Integrins: Versatility, modulation. and signaling in cell adhesion. Cell. 6Y. I l-25. Kehrli. M.E. Jr., Nonnecke, B.J.. Roth. J.A.. 1989. Alterations in bovine peripheral blood lymphocyte function during the periparturient period. Am. J. Vet. Res. 50, 215-220. Kishimoto. 1991. A dynamic model for neutrophil localization to inflammatory sites. NIH Reh., 3. 75-77. Kita. Y.. Takashi. T.. Iigo. Y., Tamatani, T., Miyasaka, M., Horiuchi. T.. 1992. Sequence and expressIon r)f rat ICAM-I. Biochim. Biophys. Acta. 1131, 108-110. Lee. E.-k.. Kehrli. M.E.J.. Dietz. A.B.. Bosworth, B.T., Reinhardt. T.A.. IY96. Cloning and sequencing {)t bovine intercellular adhesion molecule (ICAM)-3. Gene 174. 3 I l-3 13. Lozano. F.. Alberola-Ila. J., Places, L., Vivea. J.. 1992. Effect of protein kinase C activator\ on the phosphorylation and the surface expression of the CDwSO leukocyte antigen. Eur. J. Biochem. 103. 311-326. Manning. A.M.. Lu. H.-F., Kukielka. G.L., Oliver. M.G.. Ty. T.. ‘Toman. C.A.. Drong. R.F.. Slightom, J.L.. Ballantyne. C.M., Entman, M.L., Simth, C.W.. Anderson. D.C.. 1995. Cloning and comparative sequence analysis of the gene encoding canine intercellular adhesion moelcule-I (ICAM- ). Gene 156. 291 -Xi. Marlin. S.D., Springer, T.A.. 1987. Purified intercellular adhesion molecule-l (ICAM-I) IS a ligand tol lymphocyte function-associated antigen I (LFA- I ). Cell. 5 1. 8 13-8 I Y. Myers. C.L.. Wertheimer. S.J.. Schembri-King. J., Parks. T.. Wallace. R.W.. 1992. Induction of ICAM-I by TNF-u. lL-I /3, and LPS in human endothelial cells after downregulation of PKC. Am. J. Physiol. ~3, C767-c777. Rrlman. D.. Tuomanen. E.. Falkow. S.. Golenbock. D.T.. Saukkonen. K.. Wright. S.D.. 1990. Recognition of a bacterial adhesin by an eukaryotic integrin: CR3 (Use &, CD1 I b/CDl8) on human macrophagrs bind\ filamentouh hemaggglutinin of Bordetelkrpertussis. Cell. 61, 7007-7015. Roth. J.A.. Kaeberle. M.L.. 1981. Evaluation Immunopathol. 2. 157-174.

of bovine polymorphonuclear

leukocyte

function.

Vet. Immunol.

Rozdzinski. E.. Spellerberg. B.. van der Flier. M.. Bhattacharyya. c’.. Hoepelman. A.I.M.. Moran. M.A.. Jarpe, A.. Putney. S.D.. Starzyk. R.M.. Tuomanen. E., 1995. Peptide from a prokaryotic adhesin blocks leukocyte migration in vitro and in vivo. J. Infect. Dis. 172. 785-793. Ruoslahti. E.. Pierschbacher. 517-518.

M.D.,

1986. Arg-Gly-Asp:

Sanger. F.. Nicklen. S.. Coulson. A., 1977. DNA sequencing Acad. Sci. USA 74, 5463-5467.

A versatile

cell recognition

with chain terminating

Simmons. D., Makgoba, M.W., Seed. B., 1088. ICAM-I. an adhesion neural cell adhesion molecules NCAM. Nature 331. 624-627.

ligand of LFA-

signal.

inhibitors.

Cell. 44. Proc. Natl.

1. is homologous

10 the

Siu. G.. Hedrick. S.M.. Brian. A.. 1989. Isolation of the murine intercellular adhesion molecule I tICAM- 1 gene. ICAM-I enchances antigen-specific T cell activation. J. Immunol. 14.3, 38 13-3820. Staunton, D.E.. Dustin. M.L.. Erickson. H.P.. Springer. T.A.. 1990. The Arrangement of the immunoglobulinlike domains of ICAM-I and the binding sites for LFA-I and rhinovirus. Cell. 61. 243-754.