In Vitro Cell. Dev. Biol. 30A:875-880, December 1994 © 1994 Society for In Vitro Biology 1071-2690/94 $02.50+0.00

A TRUNCATED RECOMBINANT INTERCELLULAR ADHESION MOLECULE-1 INHIBITS ADHESION OF LEUKEMIC CELL LINES

TO UPREGULATED ENDOTHELIAL CELLS

LISA ROSS, CYNTHIA M. DAVIS, ~ o LESLIE MOLONY

Department of Cell Biology, Biology Division, Glaxo Inc. Research Institute, 5 Moore Drive, Research Triangle Park, North Carolina 27709

(Received 13 January 1994; accepted 16 May 1994)

SUMMARY

Intercellular adhesion molecule-l, a member of the immunoglobulin supergene family, is the ligand for the integrin lymphocyte function associated antigen-1. Intercellular adhesion molecule-1 and lymphocyte function associated antigen- 1 binding interactions mediate leukocyte adherence and migration. Previous work has shown that the adherence of lymphocyte function associated antigen-1 is directed to the first immunoglobulinlike domain of the endothelial cell surface protein intracellular adhesion molecule-1. We have constructed a truncated intercellular adhesion molecule-1 gene encoding the first 185 amino acids from the amino terminus and overexpressed it in Escherichia coll. The recombinant protein was purified from insoluble inclusion bodies and refolded into an active conformation by a denaturation/renatura- tion cycle. The identity of the protein was confirmed by microsequencing and by Western blot analysis using a polyclonal antibody to ICAM-1. We have demonstrated that this soluble region of the otherwise membrane-bound ligand is an inhibitor of Molt or HL-60 cell adhesion to cytokine-stimulated endothehal cells.

Key words: endothelial cells; ICAM-1; leukocyte adhesion; LFA-1; pET vectors; recombinant DNA.

IrCrRODUCTIOr~

Intercellular adhesion molecule-1 (ICAM-1) and its receptor lymphocyte function associated antigen-1 (LFA-1) are cell surface molecules that bind to one another to promote many immunological responses such as T lymphocyte specific responses and leukocyte binding to endothelial cells followed by emigration into inflamma- tory sites (Dustin et al., 1986; Marlin and Springer, 1987). ICAM- 1 is normally expressed at low levels on a wide variety of tissues, but cytokines and bacterial products, released at sites of inflammation, produce elevations in the level of both mRNA and protein expres- sion in these tissues (Pober et al., 1986; Rothlein et al., 1988). ICAM-1 protein levels have been shown to be elevated in several immune disorders, including arthritis, psoriasis, and graft rejection (Hale et al., 1989; Griffiths and Nickoloff, 1989; Nickoloff et al., 1990; Adams et al., 1990). Monoclonal antibodies produced against ICAM-1 have been shown to inhibit T lymphocyte mediated injury in vivo (Cosimi et al., 1990). ICAM-1 has also recently been identified as the cellular receptor for the rhinovirus from the major subgroup (Marlin et al., 1990). ICAM-1 is a 90 000 molecular weight single chain cell surface molecule composed of five im- munoglobuhnlike domains, a hydrophobic transmembrane domain, and a short cytoplasmic domain (Staunton et al., 1990).

It has been demonstrated that when ICAM-1 protein molecules were mutated at specific sites within their first or second domains, the ability of these molecules to bind LFA-1 was greatly reduced (Staunton et al., 1990). Amino acid substitutions within the first 90 amino acids of ICAM-1 produced the most loss of binding. In order

to design a therapeutic agent to inhibit LFA-1/ICAM-1 interac- tions, we produced a soluble protein containing 185 amino acids from the first immunoglobulinlike domain of the ICAM-1 molecule. This protein could function as an effective inhibitory molecule.

The aim of this study was to determine whether a soluble portion of the ICAM-1 protein could function as an effective in vitro inhibi- tor of lymphocyte (Moh-4) cell or myeloid (HL-60) cell adhesion to cytokine stimulated human umbilical vein endothelial cells (HU- VEC), by interfering with LFA-1/ICAM-I binding. The E. coil ex- pression vector containing the truncated ICAM-1 gene was con- structed to allow further fusion with other gene products and to keep the expression products in the bacterial periplasm.

MATERIALS AND METHODS

Bacterial strains and culture. The bacterial strain used for expression of the recombinant ICAM-1 was the lysogen E. coil strain BL21 (DE3), which contains in its chromosome a single copy of the T7 RNA polymerase gene under the control of the inducible lac uv5 promoter. The BL21 (DE3) strain also contained the chloramphenicol resistance plasmid pLysS, which has a T7 lysozyme gene in a reverse orientation to the T7 RNA polymerase ¢3.8 promoter such that a low level of lysozyme is produced (Studier et al., 1990). PLysS has little effect on the growth rate of the bacteria or on the expression of the target genes on induction of the T7 RNA polymerase by the addition of 0.4 mM isopropylthio-~-galactoside (IPTG) to the media. T7 lysozyme is a natural inhibitor of the T7 RNA polymerasc and is used to increase the tolerance of BL21 (DE3) for plasmids expressing toxic proteins by inhibiting the basal activity of the T7 RNA polymerase produced in the uninduced cell. Bacteria were cultured in LB medium with 30 ttg/ml chlor- amphenicol with or without 100 gg ampiciilin (amp)/ml when necessary for plasmid culture.

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Construction of the pET3a4c recombinant plasmid containing the trun- cated ICAM-1 gene. A plasmid containing the cDNA for the intact ICAM- 1 molecule (pICAM-1) was generously provided by Dr. Brian Seed (Harvard Medical School, Boston, MA). To construct the fragment containing the first 185 amino acids of the mature, translated ICAM-1 gene, synthetic oligonu- cleotides complementary to the ends of the target sequence were produced• These oligonucleotides contained a unique Nde I restriction site immedi- ately prior to the beginning of the translated portion of the ICAM-1 mole- cule and following the end of the desired translated region. They were hybridized to the plasmid plCAM-1 to amphfy that specific segment of the DNA by polymerase chain reaction, using the protocol supplied by Perkin Elmer Cetus. The 27 residue signal peptide coding sequence was not amph- fled because the protein was to be expressed in bacteria.

The amplified fragment was digested with Nde I restriction enzyme and purified by agarose gel electrophoresis, followed by electroelution. The amphfied segment was then ligated using T4 DNA ligase into an Nde 1 restricted, dephosphorylated pET3a plasmid. The pET3a plasmid (obtained by licensing arrangement from Associated Universities, Inc.) contains a strong T7 promoter derived from the ~bl0 promoter inserted into the BAMH I site of pBR322, opposite to that of the te t promoter. In the absence of T7 RNA polymerase, transcription of the target gene by E. coli RNA polymer- ase is very low. Transcription of the T7 RNA polymerase gene is under the control of the lacUV5 promoter, and addition of 0.4 mM IPTG to a growing culture of BL21 (DE3) will induce the T7 RNA polymerase, which will then transcribe the target DNA in the plasmid.

Insertion of the amplified ICAM-1 segment directly into the gene ~ 10 initiation codon via the unique Nde I site allowed for the production of ICAM-1 protein with only an additional methionine on the amino terminal. Twenty-two additional amino acids from the vector sequence were added at the carboxy terminal (Fig. 1).

Truncated ICAM expression in E. coll. E. coli BL21 (DE3) carrying plasmids pLysS and pET3a4c from an overnight culture were diluted 100- fold in Luria broth containing 100 #g/ml ampicillin. When the Absor- banceroo reached 0.6-1.0, isopropyl-thio-galactosidase was added to a final concentration of 0.4 mM to induce expression of the recombinant ICAM-1. Cells were harvested by centrifugation 15-18 h after induction and washed in Tris buffered saline (TBS) containing 0.1% sodium azide, after which the cells were lysed by freeze thawing and the lysate incubated with DNAse I for approximately 30 min or until the lysate was no longer viscous. The lysate was then pelleted at 10,000 ×g for 15 min, washed in TBS (pH 8.8) and resuspended in the same buffer. The inclusion bodies were then solubilized in TUE buffer, a solubilizing buffer containing 20 mM Tris (pH 8.8), 8 M urea, 0.1% B-mercaptoethanol (B-ME), 0.1% sodium azide, 0.001% phenylmethylsulfonyl fluoride, 2 mM ethylenediaminetetraacetic acid. The material was then centrifuged to remove insoluble debris, and loaded onto a DE-52 column equilibrated with the same buffer. The ICAM-1 truncated protein did not bind to the DE-52. Refolding was accomplished by initial dilution with 20 mM Tris (pH 8.8) to 4 M urea, followed by gradual dialysis performed by stepwise dilution of the dialysis buffer with 20 mM Tris (pH 8.8) containing 0.1% B-ME for 1 wk. Once a concentration of 0.1 M urea

TRUNCATED ICAM-1 EXPRESSION 877

was reached, the pH of the dialysis buffer was lowered by dilution to 8.2, then to 7.6 (Proudfoot et al., 1988). Aliquots of this protein were dialyzed direcdy into phosphate buffered saline (PBS) for assays. The protein was carboxymethylated as described by Crestfield et al., 1963. Some precipita- tion occurred, but approximately 90% of the protein remained soluble. This protein preparation was examined for the presence of bacterial endotoxin contamination using the ICN Endotech assay and measured below the levels of detection for this assay (0.06 ng/ml).

Lymphocyte-endothelial cell adhesion assays. Primary HUVECs were purchased from Clonetics (San Diego, CA), and plated up to Passage 6 onto 0.2% gelatin coated 48-well culture dishes in umbilical vein endothelial cell growth media purchased from Clonetics, and supplemented with 8% heat- inactivated fetal calf serum (FCS). At confluency, the cells were upregulated by incubating with fresh media containing 50 U recombinant human tumor necrosis factor a (TNFo~), (Genzyme, Boston, MA)/mt media, for 16-18 h. The next day the ceils were washed once with RPMI 1640 tissue culture media supplemented with 10% FCS, 2 mM L-glutamine, and 25 mM N-2- Hydroxyethylpiperazine-N'-2-Ethane Sulfonic Acid buffer (pH 7.4) (RPMI/HEPES). Mott 4 cells (obtained from the American Type Culture Collection, Rockville, MD) were grown in RPMI 1640 tissue culture media containing 10% FCS and 2 mM L-glutamine media, and labeled by the addition of 50 gg/ml 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxy- ftuorescein (Molecular Probes, Eugene, OR), a fluorescent dye which was dissolved in 50 t, tl dimethylsulfoxide per 30 million cells and added for a 60 to 90 min incubation (Luscinskas et al., 1989). The Molt 4 cells were

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MWA B C D FI¢. 2. Production and purification of the recombinant lCAM-l protein.

The proteins were resolved by 0.1% SDS-12.5% PAGE (Laemmli, 1970) and detected by Commassie blue staining. The lane labeled MW contains molecular weight standards (in kilodaltons). Lane A contains 20 #l of total cell extract from uninduced pET3a4c. Lane B contains 20 gl of total cell extract from induced pET3a4c. Lane C contains 20 ttl (1.6 #g) of the fraction not binding to DE-52. Lane D contains 20 ttl (16 gg) of the fraction not binding to DE-52, concentrated 10-fold.

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FIc. 3. Western blot analysis of the recombinant ICAM-1 protein using a polyclonal probe to the 40-64 region of the ICAM-1 molecule. Lane A shows the inclusion body lysate prior to reduction and carboxymethylation. Lane B shows the same preparation of material after reduction and carboxy- methylation. The size of the large band in Lane B is approximately 25 kilodaltons (kD), about the same molecular weight as the putative recombi- nant ICAM-1 band produced after IPTG induction. Lane C shows the anti- body recognition of the intact ICAM-1 molecule (molecular weight 90,000 kD), which was isolated from the K-562 cell hne.

washed extensively to remove unincorporated label, followed by resuspen- sion in the RPM1/HEPES media at 1 million cells/ml. The Molt 4 cells were then preincubated with recombinant 1CAM-1 for 15 min at RT at a final concentration of 500 000 cells/ml in RPMI/HEPES media, and then 250 ttl suspension was added to each HUVEC containing well. The cells were allowed to adhere for 1 h, after which the wells were washed three times with RPMI/HEPES, and lysed by addition of 0.5 ml/well 2% Triton X-100. Two hundred/.tl ahquots of lysate were transferred to black 96-well plates and read, in duplicate, in a fluorescence analyzer (Baxter Pandex).

HL-60 cells were purchased from the American Type Culture Collection, and grown under the same media conditions as the Molt 4 cells. The cell adhesion assay was essentially the same as the Molt 4/HUVE cell assay, except that twice as many HL-60 cells were added per well.

Fluorescence activated cell sorting analysis. Exponentially growing (fewer than 500,000 cells/ml) HL-60 or Molt cells were treated with condi- tioned media taken from endothelial cells. The endothelial cells had been upregulated with TNFct overnight, washed once as per the lymphocyte adhesion assay, and media was added and they were incubated for 90 min. The media was removed from the endothelial cells and used to resuspend the Molt or HL-60 cells to the same final concentration as in the adhesion assay. The Molt or HL-60 cells were then incubated in the conditioned media for 90 min. A second set of the same cells were incubated overnight with media containing 50 U of TNFtx/ml. Aliquots of 1 million cells were

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FIG. 4. A, Inhibition of Molt 4-HUVEC cell adhesion by recombinant ICAM-1. Molt 4 cells were incubated with TNFa stimulated HUVECs. Background binding of Molt 4 cells to unstimulated HUVE cell monolayers was also tested in each experiment, and these values (typically about 15% of the total) were subtracted prior to plotting. Bar 1: addition of 1.4 ttM recombinant ICAM-1/ml media. Bar 2: addition of 2.8 #M recombinant ICAM-1/ml media. Bar 3: addition of R 425, a rabbit polyclonal antibody made against amino acids 40-64 of the 1CAM-I molecule. B, Inhibition of HL-60 cell adhesion to TNFot upregulated HUVE cells by recombinant ICAM. HL-60 cells were incubated with TNFa upregulated HUVE cells. Background binding of HL-60 cells to unstimulated HUVE cells (approxi- mately 25% of the total binding) was determined for each experiment, and subtracted prior to plotting. Bar 1: addition of .7 #M recombinant ICAM-1. Bar 2: addition of 1.4 tiM recombinant ICAM-1/ml media. Bar 3: addition of 2.8 #M recombinant ICAM-I/ml media. Bar 4: addition of R 425, an anti-ICAM- 1 antibody.

washed twice with eoM PBS containing 0.1% sodium azide. The superna- tant was decanted and 20 gl of fiuorescendy conjugated monoclonal anti- body was added per tube and incubated for 30 min. The cells were then washed once more with cold PBS and then resuspended in cold PBS con- mining .5% paraformaldehyde and refrigerated until use. The samples were analyzed by fluorescence activated cell sorting (FACS) within 48 h. The

antibodies used for FACS were: CD54 ICAM (catalog #0726), Bear-1 (catalog #0190), and clone 25.3.1 (catalog #0157), where the antigen was LFA-1 protein, which is specific for the LFA-1 protein, and binds to the LFA-1 ot chain. All antibodies were obtained from AMAC, Inc. (Westbrook, ME).

Western blot analysis of the truncated ICAM-1 protein. Aliquots of the reduced and earboxymethylated truncated ICAM-1 protein, and the unre- duced and non-earboxy methylated form, were electrophoresed on a 7.5% polyacrylamide gel. The proteins were then electroblotted onto lmmobilon membrane (Millipore Products, Bedford, MA) and the membrane was incu- bated with a 1:500 dilution of R424, a polyclonal antibody made by immu- nizing a rabbit against the 40-64 amino acid region of the ICAM-1 mole- cule. After washing the blot, a radioactively labeled secondary goat anti-rab- bit antibody was added. The blot was incubated with the secondary antibody for 1 h and then washed. The blot was analyzed by autoradiography.

RESULTS AND DISCUSSION

Expression of truncated ICAM-1 protein in E. cob. To isolate a recombinant plasmid containing the truncated ICAM-1 gene, BL21 (DE3) cells were transformed with the construct detailed in Figure 1, and plated onto LB plates containing 100 #g amp/ml. Several colonies were picked, and mini preparations of plasmid DNA were used to determine whether an insert was present and its orientation. One of the clones, pET3a4c, was examined for its ability to express the truncated ICAM-1 protein. Upon induction of the culture with 0.4 mM IPTG, over 20% of the total bacterial protein observed by 0.1% SDS-12.5% PAGE (Fig. 2) was a unique band present at a molecular weight of approximately 25 kD, and which was not pres- ent in uninduced cells. This protein was further purified by isolating and solubilizing the inclusion bodies.

As shown in Fig. 2, lane D, additional impurities were removed by filtration through DE-52 cellulose, which did not bind the soluble ICAM-1 fragment. Analysis of the first 20 amino acids by amino terminal microsequencing identified the protein as ICAM-1 pre- ceded by a single methionine, which is added as a result of the insertion into the pET3a vector, and is necessary for expression to occur. The elution pattern of the nonreduced, soluble protein on gel filtration on Sephacryl S-300 indicated that oligomers were formed from 4 - 1 2 molecules. Reduced material eluted from a G-50 Sepha- dex column as monomers with some higher order oligomers (data not shown). The preparation was assayed for endotoxin contamina- tion, which could also have affected the ability of the HL-60 or Molt cells to bind to the activated endothelial cells. No endotoxin was detected using the ICN Endotech assay, which has a detection limit of 0.06 ng/ml.

The orientation of the insert and surrounding region was deter- mined by dideoxy sequencing using the method of Sanger et al. (1977). As shown in Fig. 1, the ICAM-1 construct has had the signal peptide sequence deleted, is preceded by a methionine (ATG), and contains an additional 22 amino acids fused to the carboxy terminus.

Western analysis of the truncated ICAM-1 molecule. Western analysis of the reduced and carboxymethylated form of the trun- cated ICAM-1 recombinant protein confirms that the approximately 25 kilodalton protein is ICAM-1. Recognition of the truncated ICAM-1 molecule was seen using a polyclonal antibody made against amino acids 4 0 - 6 4 of the ICAM-1 molecule (Fig. 3). Lane A shows that when the expressed protein is not reduced and car- boxymethylated, ohgomers of the material are produced, and a large smear is observed. In lane B, after the protein is reduced and carboxymethylated, a large band appears at approximately 25 kilo- dahons. Lane C shows the complete ICAM-1 (Molecular weight

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FIe. 5. FACS analysis of the Molt and HL-60 ceils showing HL-60 cells (A) or Molt cells (C) after incubation in endothelial cell conditioned media; and HL-60 cells (B) or Molt cells (D) after incubation overnight in media containing 50 #g TNFa/ml.

90,000 kilodahons) isolated from the K562 cell line, which overex- presses ICAM-1.

Inhibition of binding of Moh-4 or HL-60 cells to cytokine-stimu- lated HUVE cell monolayers by truncated recombinant ICAM-1. The results shown in Fig. 4 A and B demonstrate that the truncated ICAM-1 protein had functional activity. The protein can signifi- candy inhibit Moh-4 or HL-60 adhesion to TNFo~-stimulated endo- thelial cells in a dose dependent manner. The maximal inhibition observed was 70% using HL-60 cells and 48% for Molt cells at a protein concentration of 2.8 #M, the highest dose tested. Based on titration using high concentrations of rabbit polyclonal antibody r 425, which was made against the 4 0 - 6 4 amino acid portion of the ICAM-1 molecule, we have determined that the total contribution of LFA-I and ICAM-1 association to the HL-60/endothelial cell or Molt/endothelial cell adhesion was 37% and 58%, respectively. The protein inhibited binding of Molt or HL-60 cells to the activated endothelium in a dose responsive manner, and appears to inhibit all the LFA-I and ICAM-1 interaction present in these cells under these conditions. A sham assay using material that had been iso- lated from E. coil transformed with a vector containing no insert produced no inhibition in the HL-60/TNFo~-stimulated endothelial cell assay (data not shown).

Presence of LFA-1 (CD 11a/CD18) on Molt-4 and HL-60 cell surfaces. While we have shown that the recombinant protein was inhibiting cell to cell adhesion in the lymphocyte/endothelial cell assay, we were interested in the effect of TNFo~ present on the endothelial cell surface, since TNFot induces CD18 mediated adhe-

sion of neutrophils (Diamond et al., 1993). The Molt and HL-60 cells were examined by FACS analysis to determine whether the LFA-1 receptor was present on the cell surface under the conditions of the lymphocyte/endothelial cell assay, and whether incubation with TNFot under extreme conditions (i.e., incubation with 50 U of TNFa /ml media overnight) would affect the amount of LFA-1 re- ceptor. As shown in Fig. 5 A and C, the LFA-1 receptor (identified using an antibody against the o 6 chain of the LFA-1 receptor, CD 1 la) is highly represented on both the HL-60 and Molt cell sur- faces. The antibody was made to the a, chain and is specific to the LFA-1 protein. While the ~2 chain (CD18) of the LFA-1 molecule can be combined with other unique a chains such as am to create MAC-I, or with a~ to create p 150, 95, the antibody made to the aj does not recognize integrins other than LFA-1. Treatment with TNF overnight does not reduce the number of LFA-] receptors on the cell surface, but causes a small increase in the number of LFA-1 receptors on the cell surface (Fig. 5 B and D). So if any residual TNFa were present on the endothelial cell surface after washing, it would not affect the ability of the Molt or HL-60 cells to bind to the upregulated endothelial cells. ICAM-1 expression on the cell sur- face of the Molt and HL-60 cells appears unaffected by TNFa treatment.

Adhesion of the LFA-1 receptor to the ICAM-1 ligand is a key event in the initiation of inflammation and in the regulation of im- mune function. Our laboratory has investigated the possible sites of interaction of these molecules. A soluble truncated form of the ICAM-1 molecule bas been expressed in E. coli, and the purified

8 8 0 ROSS ET AL.

protein has been shown to inhibit adhesion of Molt or HL-60 cells to cytokine-stimulated endothelial cells, presumably by interfering in the binding of the LFA-1 receptor to its ligand, ICAM-1. Other investigators have shown that mutating sites within the ICAM-1 mol- ecule will produce an inhibition of binding (Staunton et al., 1990). Our findings demonstrate that inhibition of binding of Molt or HL- 60 adhesion to activated endothelial cells can also be produced by addition of soluble truncated ICAM-1 molecules lacking the final three domains.

While there is soluble ICAM-1 present in the serum of patients presenting with severe inflammation, it has not been shown to pre- vent leukocyte adherence to endothelial cells. However, the level of circulating ICAM-1 in plasma of patients with melanoma increased with progression of the disease, and the authors indicate that the plasma-soluble form of ICAM-1 may play a role in host immunities and may be used as a tool for prognosis (Kageshita et al., 1992; Giavazzi et al., 1993).

To further complicate what the physiological role of soluble ICAM-1 is in vivo, after endothelial cell activation, release of ICAM- 1 increases as cell surface expression increases, and plateaus at approximately the same time that cell surface expression plateaus (Leeuwenburg et al., 1992).

Gel filtration of the pET3a expressed protein shows that there are several polymeric forms of the protein in solution, and that the percentage in the monomeric form decreases with time (data not shown) after the isolation procedure. This polymeric ICAM-1 is likely to be the effective agent causing the inhibition in the leukocyte endothelial cell interactions within our in vitro system. Soluble ICAM-1 from serum has not been isolated and characterized for the formation of oligomers. It would be interesting to determine if serum 1CAM-1 exists in muhimeric form, and whether serum ICAM-1 can inhibit leukocyte/endothelial actions in vitro in either the monomeric or multimeric forms.

ACKNOWLEDGMEI~I'S

We thank Dr. Brian Seed for providing plasmid plCAM-1, William Burk- hardt for the N-terminal sequence analysis, Dr. Bernard Allet for his help with the DNA sequencing, and Dr. David Eierman for testing the recombi- nant ICAM-1 sample for endotoxin contamination.

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