Intercellular adhesion molecule 1 (ICAM-1) has a ... - Europe PMC

Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3095-3099, May 1988 Immunology

Intercellular adhesion molecule 1 (ICAM-1) has a central role in cell-cell contact-mediated immune mechanisms (lymphocyte interactions/T-cell/B-cell collaboration/endothelial cell/cytotoicity)

ANDREW W. BOYD*t, STEFAN 0. WAWRYK*, GORDON F.

BuRNSt, AND JOHN V. FECONDO*

*Lions Clinical Cancer Research Laboratory, Cellular Immunology Unit, Walter and Eliza Hall Institute, Royal Melbourne Hospital, 3050, Australia;

and

tDivision of Immunology, Institute of Medical and Veterinary Science, Box 14, Rundle Mall Post Office, Adelaide, 5000, Australia

Communicated by G. J. V. Nossal, December 22, 1987 (received for review December 7, 1987)

esis in the early embryo (13). In postembryonic life, specific groups of CAMs appear to participate in the adhesive mechanisms that determine structure (14). Within the hemopoietic system where CAMs are not required to determine form, molecules such as those of the CD11 cluster regulate the specific interactions occurring between hemopoietic

The role of Intercellular adhesion molecule 1 ABSTRACT (ICAM-1) in immune function was probed by using the Wehi-CAM-1 (W-CAM-1) monoclonal antibody. This antibody blocks aggregation of cell lines mediated by the ICAM-1 molecule and is here shown to block homotypic binding of purified populations of activated T and B lymphocytes (blasts) and also aggregation of mixed T- and B-cell blasts. We also demonstrate that W-CAM-1 inhibited T-cell adhesion to normal human endothelial cells, the first step in lymphocyte egress into the tissues. In tests of immune function, W-CAM-1 had a modest inhibitory effect on T- and B-cell activation by potent mitogens and no effect on the response of activated lymphocytes to lymphokines. By contrast, activation induced by cell-cell contact (mixed lymphocyte reaction, T-cellmediated B-cell activation) was significantly inhibited. Moreover, the antibody was shown to block elements of both effector arms of the immune system (cytotoxic cell function and immunoglobulin production). These findings-show that the ICAM-1 molecule is a central component of the mechanism of lymphocyte-endothelial cell adhesion. The studies of Iymphoid function demonstrate a pivotal role for this molecule in both the T-cell/T-cell and T-cell/B-cell interactions, which underpin the regulation of the immune response, and in the mechanism of cell-mediated cytotoxicity.

cells. We report the effect of a monoclonal antibody, WehiCAM-1 (W-CAM-1), which recognizes intercellular adhesion molecule 1 (ICAM-1) (15). This antibody will be shown to inhibit a wide range of in vitro assays of immune functions. We also show that W-CAM-1 inhibits lymphocyte-endothelial cell adhesion. These findings suggest a central role for ICAM-1 in regulating cell-cell interactions within the immune system and, therefore, of ultimate effector functions.

MATERIALS AND METHODS Normal Tissue Specimens. Normal human peripheral blood, lymph node, bone marrow, and spleen were obtained from biopsy material. Human endothelial cells were generously provided by B. Maloney (Commonwealth Serum Laboratories, Parkville, Victoria, Australia). All specimens were obtained according to protocols approved by Institutional Ethics Committees. Monoclonal Antibodies. W-CAM-1 antibody. W-CAM-1 was obtained from a fusion between NS-1 hypoxanthine/ aminopterin/thymidine-sensitive myelona cells and splenocytes from BALB/c mice immunized With Raji cells. The immunization protocol and technique of cell fusion were according to standard methods (16). The antibody detected a 90-kDa protein under both reduced and unreduced conditions. Biosynthetic labeling experiments demonstrated a biosynthetic precursor of =70 kDa. In tunicamycin-treated Raji cells, a single band at 55 kDa was precipitated. These characteristics and the functional properties described below indicate that this antibody recognizes ICAM-1 (15, 17). Within normal tissues, the molecule was virtually undetectable on peripheral blood leukocytes but was expressed on endothelial cells, tissue histiocytes, and large germinal center B cells. W-CAM-1 is an IgG2b antibody and was purified on protein A-Sepharose 4B CL (Pharmacia, Uppsala) and eluted from the column with 0.1 M acetic acid (pH 3). The antibody was digested with papain (16) to yield F(ab)1 and Fc fragments. The latter were removed by absorption to protein A-Sepharose and purity of the recovered F(ab), fragment was confirmed by NaDodSO4/PAGE. F(ab), was found to give peak binding at 30 ug/ml, compared with 1 pug/ml for the whole antibody. Experiments described use these and

Antibodies to human leukocyte cell-surface antigens have been extensively characterized, resulting in a World Health Organization classification (CD classification) of human leukocyte antigens (1). Lineage-restricted membrane antigens have been most studied, using monoclonal antibodies as "probes" of function. Studies of the T3 (CD3) molecule on T cells (2, 3) and the B-cell-restricted B1 (CD20) (4, 5) antigen have demonstrated a role for these molecules in Tand B-cell activation, respectively. However, several cellsurface molecules that are not lineage restricted have been shown to have potent immune regulatory functions. One of these is leukocyte function antigen 1 (LFA-1), an adhesion molecule that is widely distributed, being present on hemopoietic cells at all stages of their maturation. Antibodies to LFA-1 have been shown to inhibit aspects of both T-cell (6-8) and B-cell function (9). These in vitro data are supported by evidence from a human disease in which a defective ,3 chain of LFA-1 is synthesized (10), resulting in loss of expression of LFA-1, MAC-1, and pl50/95 (the CD1i clusters), all of which share the same 8 chain. The resulting clinical syndrome is marked by disturbances of all aspects of immune function. All members of this family of molecules have adhesion functions and are part of a larger family of cellular adhesion molecules, the integrins (11, 12). In a still wider context, cell adhesion molecules (CAMs) have been shown to regulate processes as fundamental as nmorphogen-

Abbreviations: EBV, Epstein-Barr virus; LFA, leukocyte function antigen; PHA, phytohemagglutinin; ICAM-1, intercellular adhesion molecule 1; IL-2, interleukin 2; MLR, mixed lymphocyte reaction. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge

payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3095

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other antibody preparations at predetermined saturation binding concentrations. Other antibodies. Wehi-B2 is an IgG2b that detects the CR2 complement receptor [Epstein-Barr virus (EBV) receptor, CD21]. With B1 (Coulter), Wehi-B2 and its F(ab)1 fragments were used as a nonspecific control of identical isotype to W-CAM-1. The CIMT antibody detects LFA-1 and was the gift of G. Pilkington (Cancer Institute, Melbourne). Additional antibodies used were OKT3, OKT4, OKT8 (Ortho Diagnostics), Mol (Coulter Immunology), and several monoclonal antibodies produced in this laboratory [3.F10 (anti-HLA DR), I.C12, and L.A10]. Flow Cytometric Analysis. Samples were stained by standard direct or indirect immunofluorescence methods. Monoclonal antibodies were prepared at optimal dilutions in 5% human AB serum in phosphate-buffered saline. Indirect fluorescence analysis was with fluorescein isothiocyanatelabeled F(ab')2 fragments of sheep anti-immunoglobulin antibody (Silenus, Melbourne). Samples were fixed in 1% formalin and analyzed on ah EPICS 752 cell sorter (Coulter). Preparation of Purified Populations. Antibody and complement lysis. Cells were incubated with complement binding antibody for 30 min at 40C. The cells were washed once and resuspended in an optimal concentration of newborn rabbit complement in Eisen's balanced salt solution. The cells were incubated for 1 hr at 370C. OKT3 rosettes. Cells were incubated with OKT3 antibody for 30 min at 4°C. The cells were washed and thoroughly mixed with rabbit anti-mouse immunoglobulin-conjugated sheep erythrocytes prepared using the CrCl3 coupling method (18). The mixture was incubated for 5 min, centrifuged at 200 x g, and incubated at room temperature for 15 min. The resuspended cells were separated on Ficoll/Hypaque. The T-cell fraction (pellet) was lysed in distilled water and transferred to fresh medium. In some cases, T cells were suspended in culture at 106 cells per ml and activated by phytohemagglutinin (PHA) (Wellcome) at 5

gg/ml.

B-Cell Assays. Anti-inmmunoglobulin-induced mitogenesis. Responses to anti-IgM antibody-coupled beads were tested as described (19). MLA144 supernatants were prepared from phorbol ester-treated Gibbon ape leukemia MLA144 cells. The supernatant was concentrated 10-fold and a 50-80% ammonium sulfate cut was prepared. This was dialyzed against phosphate-buffered saline. EBV-induced mitogenesis. Spleen cells were depleted of T cells by antibody (OKT3) and assayed for response to EBV as described (20). ELISA assay for immunoglobulin production. Diluted supernatants (50-,ul aliquots) were added to microwells coated with sheep anti-human immunoglobulin antibody (20 ,ug/ml) (Silenus) and incubated for 4 hr. The wells were washed and horseradish peroxidase-conjugated anti-human immunoglobulin antibody was added. The plates were incubated overnight, washed extensively, and substrate (16) was added. After incubating for hr, the optical density of each well was measured in a Titertek ELISA reader. Concentrations of antibody were calculated from assay of a standard

immunoglobulin preparation. T-cell-mediated B-cell assay. Spleen cells were separated into B- and T-cell fractions by, immune rosetting with OKT3 as described above. The T-cell fraction was irradiated to 5000 rads. Meanwhile, the B cells were stained with B1 (CD20) fluorescein isothiocyanate and sorted for the positive (B) cells. The purified B-cell population was mixed in equal proportion with T cells and in the presence or absence of PHA as described (9). The cultures were pulsed with [3H]thymidine after 2 days and harvested 15 hr later. T-Cell Assays. Interleukin 2 (IL-2) response by activated T cells. Peripheral mononuclear blood cells or purified T cells

cultured at 106 cells per ml with PHA. The cultures harvested after 3 days, washed, and recultured in 96-well trays overnight. Various amounts of IL-2 (kindly provided by Cetus, Emeryville, CA) were added the next day, and after a further 24 hr the cultures were pulsed with [3H]thymidine and harvested 6 hr later. Mixed lymphocyte reaction (MLR) cultures. MLR cultures were established by mixing equal numbers of mononuclear cells from two unrelated donors and cultured at a final concentration of 2 x 106 cells per ml. The cultures were "fed" after 4 days with fresh medium and recombinant IL-2 (Cetus) at 1000 units/ml and harvested 3 days later. In some experiments, the mixed cells were cultured at 105 cells per well in 96-well trays and incubated for 6 days, and the wells were pulsed with [3H]thymidine and harvested 15 hr later. Aggregation Assays. Lymphoid aggregation. Lymphoid aggregation was performed as described (15). A cut-off for aggregate size of three cells was chosen as discriminatory based on preliminary experiments. Endothelial adhesion assay. Near confluent endothelial cultures were used. After washing the wells three times with medium, antibody diluted in medium was added. The lymphoid cells were also washed three times and then added to were were

the wells. Statistical Methods. All experiments were performed on three or more occasions. Most comparative statistics use Student's t test to determine significance.

RESULTS Intact W-CAM-1 and F~ab)j Fragments Inhibit Intercellular Adhesion. The W-CAM-1 antibody was characterized in part by the demonstration of inhibition of intercellular adhesion of human cell lines that spontaneously aggregate in tissue culture. We wished to investigate the inhibition of aggregation of normal T and B lymphoblasts and mixtures of these two populations by W-CAM-1. T-cell blasts were generated by activation of mononuclear cells from peripheral blood with PHA. B-cell blasts were generated by using Table 1. W-CAM-1 antibody inhibits intercellular aggregation % cells in Cell type Antibody added aggregates % inhibition Activated 83 ± 7 T cells* W-B2 (10 /Lg/ml) 77 ± 8 7 (NS) W-CAM-1 (10 ,g/ml) 24 ± 5 71 (P < 0.01) Activated 67 ± 4 B cellst W-B2 (10 jig/ml) 70 ± 6 - 5 (NS) W-CAM-1 (10 tg/ml) 22 ± 5 67 (P < 0.01) Activated 76 ± 5 81 ± 4 -6 (NS) splenocytest W-B2 (10 ,ug/ml) W-CAM-1 (10 ,ug/ml) 19 ± 7 75 (P < 0.05) NS, not significant. *Peripheral blood mononuclear cells were activated in the presence of PHA (5 ,g/ml). After 3-4 days, cells were harvested, separated on Ficoll/Hypaque, and dispersed into a single cell suspension. Cells were recultured in the presence of antibodies and the percentage of cells in aggregates was scored by carefully pipetting aliquots of cells into a hemocytometer and counting cells in aggregates (.3) vs. dispersed cells (85% OKT11 (CD2)-positive. tSpleen mononuclear cells were separated by OKT-3 rosetting (see Materials and Methods). Rosette-negative cells were activated for 3 days with anti-immunoglobulin beads (16) and assayed as described above at day 3. Routine staining showed >90% B1 (CD20)positive cells. tWhole spleen mononuclear cells were activated with both PHA and anti-immunoglobulin. The cells were harvested and assayed at day 3 as described above. The population consisted of -60% OKT11 (CD2)-positive cells and 30% B1 (CD20)-positive cells.

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Table 2. W-CAM-1 inhibits binding of activated lymphocytes to endothelial cells Number of adherents Adhering cell per high-power field Antibody Experiment A Activated peripheral 32 ± 3 blood mononuclear W-CAM-1 (1 ug/ml) 34 ± 6 (NS) cells* W-CAM-1 (10 Ag/ml) 31 ± 4 (NS) 12 + 6 (P < 0.01) W-CAM-1/F(ab), (10 ,ug/ml) 11 ± 3 (P < 0.01) W-CAM-1/F(ab), (30 ,.g/ml) W-B2 (10 jug/ml) 28 ± 8 (NS) Experiment B Purified activated 14 ± 3 T cells* W-CAM-1 (30 ,Ig/ml) 10 ± 2 (P < 0.05) 5 ± 2 (P < 0.001) W-CAM-1/F(ab), (30 ,g/ml) W-B2 (10 ,ug/ml) 15 ± 4 (NS) *Peripheral blood mononuclear cells or OKT-3 rosette-purified T cells were activated with PHA (5 ,ug/ml) for 3 days. Both the T cells and endothelial cell monolayers were washed extensively. Medium containing optimal concentrations of antibody were added to the wells followed by carefully dispersed lymphoid cell suspensions. In experiment A, 50% of added T cells had been treated with fluorescein isothiocyanate (50 ttg/ml) for 20 min at room temperature. The brightly fluorescent cells were then easily quantitated by fluorescence microscopy and correlated with the counting by light microscopy. W-CAM-1 refers to intact purified antibody, and W-CAM-1/F(ab), refers to purified F(ab), fragments of W-CAM-1.

anti-immunoglobulin-coupled bead activation of OKT3 rosette-negative spleen cells. Mixed B- and T-cell blasts were generated by activation of whole spleen with both antiimmunoglobulin and PHA. As shown in Table 1, in each case significant inhibition of aggregation was demonstrated with W-CAM-1 antibody but not by control antibody of identical isotype. In each case, the size of aggregates was significantly smaller in W-CAM-1-treated cultures when compared with controls. In other experiments, a comparable effect was observed with both F(ab), fragments of W-CAM-1 and LFA-1 antibodies.

Another cell-cell interaction central to the normal immune is the adhesion of lymphocytes to endothelial cells. W-CAM-1 binding to endothelial cells suggested that leukocyte adhesion to endothelial cell membranes may also involve ICAM-1 and be disrupted by W-CAM-1 antibody. In Table 2, experiments are shown that demonstrate that adhesion is blocked by F(ab), fragments and less effectively by the whole antibody. W-CAM-1 Is a Weak Inhibitor of Lymphocyte Mitogens. The effect of both whole W-CAM-1 antibody and F(ab), fragments on in vitro assays of B- and T-cell mitogenesis was response

Table 3. The effect of W-CAM-1 on T- and B-cell responses to potent mitogenic signals

Mitogenic response Cell source B cell: Whole spleen mononuclear cells* CD20-positive small splenic B cellst B-cell fraction of

spleent

Mitogen Anti-immunoglobulin Anti-immunoglobulin Anti-immunoglobulin Anti-immunoglobulin Anti-immunoglobulin Anti-immunoglobulin Anti-immunoglobulin Anti-immunoglobulin Anti-immunoglobulin Anti-immunoglobulin

Antibody added W-CAM-1 (10 ,ug/ml) W-CAM-1 (30 ,ug/ml) W-CAM-1 (10,ug/ml) 3F.10 (10 ,ug/ml) LFA-1 (5 I.g/ml) W-CAM-1 (10 ,ug/ml) LFA-1 + W-CAM-1

EBV

EBV EBV T cell: Whole spleen*

E-rosette-positive peripheral blood

W-CAM-1 (10,ug/ml) W-B2 (10 ,g/ml)

PHA PHA PHA PHA

W-CAM-1 (10 t.g/ml) W-CAM-1 (10 ,g/ml)

([3Hlthymidine uptake) 34,712 21,291 25,795 12,273 11,222 8,523 4,556 2,097 3,769 1,352 3,603 3,839 1,561

± ± ± ± ± ± ± ± ± ± ± ± ±

1843 1971 1679 569 897 614 219 164 185 148 213 188 114

43,359 31,530 14,452 10,737

± ± ± ±

2814 1756 (P < 0.05) 619 834 (P < 0.05)

(P < 0.01)

(P < 0.05)

(P < 0.05) (P < 0.01)

(P < 0.05)

mononuclear§ *Whole spleen mononuclear cells were prepared on Ficoll/Hypaque gradients. The cells were cultured at 50,000 cells per well (10,000 cells per well for PHA activation) in 96-well trays with mitogen and antibodies. The cultures were pulsed with [3H]thymidine after 2 days and incubated for a further 15 hr before harvesting. tWhole spleen mononuclear cells were stained with B1(CD20) fluorescein isothiocyanate and the positive cells were sorted. These cells were cultured as described above. tWhole spleen cells were treated with OKT3 and Mol and then lysed with baby rabbit complement. The resulting population was >90% CD2O-positive. The cells were cultured as described above except that EBV cultures were incubated for 4 days before being pulsed with [3H]thymidine. §Peripheral blood mononuclears were E-rosetted and the rosetting fraction [>90%o OKT3(CD3)-positive] was cultured with PHA and/or antibody for 3 days.

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tested. B-cell responses were tested on whole spleen mononuclear cells and on enriched splenic B-cell populations. T-cell mitogenesis was tested on whole spleen and on E-rosette-purified peripheral blood T cells. Representative results of these experiments are shown in Table 3. Inhibition of B-cell mitogenesis by W-CAM-1 was observed, although in the most highly purified B-cell population, cell sorterpurified small B cells, this effect was minimal. By contrast, anti-Ia(3.F1O) antibody induced significant inhibition of mitogenesis. Similarly, EBV-induced mitogenesis was little affected by W-CAM-1, although anti-EBV receptor antibody was able to block mitogenesis (20). Comparable inhibition was also observed with LFA-1 antibody, and some additive effect was noted when both antibodies were present. T-cell mitogenesis was also inhibited to some degree by W-CAM-1. In other experiments, F(ab)1 fragments were tested on both PHA and anti-CD2 antibody-induced activation with no greater effect than that shown in Table 3. Lymphokine Action Is Not Affected by W-CAM-1 Antibody. The effect of W-CAM-1 on growth factor responses was tested! Activated B cells were prepared by adding anti-immunoglobulin beads to antibody (OKT3 and Mol) and complement-lysed splenic mononuclear cells, and the cells were cultured for 3 days. Indirect immunofluorescence analysis showed that >95% large CD20-positive cells were detectable and most of these stained strongly with W-CAM1. The cells were recultured at 10,000 cells per well in the presence of partially purified growth factors from MLA144 Table 4. Effect of W-CAM-1 antibody on proliferation assays requiring cell-cell contact Cell source

Antibody added

[3H]Thymidine uptake, cpm x 10-3

MLR

Peripheral blood mononuclear cells (two way)*

Peripheral blood mononuclear cell (one way)* T-cell-mediated B-cell mitogenesis

34.2 ± 1.8 W-CAM-1 (10 ,g/ml) 15.6 ± 0.9 (P < 0.01) W-CAM-1 (30 jg/ml) 12.0 ± 2.6 (P < 0.01) 36.7 ± 2.3 W-B2 (10 ,ug/ml) 21.9 ± 1.9 W-CAM-1 (10 ,ug/ml) 13.5 ± 2.4

A 1.C1 2 1.A1 0

B1

E

3.F1 O(la) wehi-B2

wehi-CAM1

CONTROL I

I

1000 Ig (ng/ml)

0

2000

B LFA-1/Fab 1 LFA-1 /w-CAM1

Tcellst T cells + PHAt B cellst B cells + PHAt B + T§ cells B + T + PHA§ B + T§ cells

cells (see Materials and Methods) plus or minus W-CAM-1 or control antibodies. T cells were prepared from normal peripheral blood mononuclear cells by the OKT3 rosetting method. The cells were stimulated with PHA for 2 days, by which time >95% of cells were CD2 (OKT11) positive, 49% IL-2 receptor (Tac)-positive, and 79o W-CAM-1-positive. These cells were washed twice and recultured at 105 cells per ml in fresh medium overnight. The cells were again washed and cultured at 5000 cells per well with or without recombinant IL-2 and test antibody preparations. Mitogenic responses of both B-cell and T-cell blasts were measured, and no significant difference was detected between the groups treated with W-CAM-1 or its F(ab)1 fragments and controls. The Effect of W-CAM-1 on Lymphoid Functions Requiring Cel-Cell Contact. The capacity to block cell-cell adhesion of lymphoid cells suggested that W-CAM-1 binding might block immune processes dependent on cell-cell contact. To explore this further, we tested the effects of W-CAM-1 on T cells activated in both one-way and two-way MLR and on B cells directly activated by T cells (9). Results of these studies are tabulated in Table 4 and demonstrate significant inhibition in both assays. In a variation of the T-cell-induced B-cell activation experiments, T cells activated with PHA for 3

-

0.1 0.17 0.46 0.62 1.1 3.5 0.77

± 0.02 ± 0.03 ± 0.03

± 0.1

±0.06 ±0.14

± 0.08 F(ab), (30,ug/ml) (P < 0.01) B + T cells + PHA§ F(ab), (30 gg/ml) 1.8 ± 0.11 (P < 0.02) *Peripheral blood mononuclear cells from two donors were mixed and cultured at 25,000 cells per well (50,000 cells total) for 7 days. The cultures were pulsed with [3H]thymidine and harvested 15 hr later. Experiments are shown for two-way reaction and a single one-way reaction. tT cells were prepared by OKT3 rosetting of spleen mononuclear cells. The cells were treated with mitogen C (15 ,g/ml) for 90 min. The cells were cultured at 50,000 cells per well. tB cells were prepared from the nonrosetting spleen cells described above (t). The cells were stained with B1(CD20)-fluorescein isothiocyanate and sorted. The positive cells were washed and recultured at 50,000 cells per well. §B cells and irradiated T cells were mixed in equal numbers and cultured as described above.

LFA-1 4pg/ml

~-I

Fab 1 30pg/ml

w-CAM1 lOpg/ml

~~~~~~~~~~~

_ w-B2 10pg/ml

I

CONTROL 0

20

40

60

80

% cytotoxicity FIG. 1. (A) Immunoglobulin production is inhibited by W-CAM1 antibody. Whole spleen cells were cultured with anti-immunoglobulin beads and PHA with the antibodies shown. At 7 days, cultured supernatants were harvested and analyzed by ELISA. Results are tabulated in ng/ml, by reference to known standards. (B) Cell-mediated cytotoxicity is inhibited by W-CAM-1 antibody. Lymphokine-activated killer cells were generated by two-way MLR with restimulation with IL-2 3 days before assay. The cells were mixed with 51Cr-labeled K562; cytotoxicity was assayed after 4 hr and percentage specific lysis was calculated.

Immunology: Boyd et A days were irradiated to 5000 rads and added to resting B cells in the presence of IL-2 (1000 units/ml). Again, significant B-cell activation was noted, which was inhibited with both LFA-1 and W-CAM-1, the inhibition being greater when both antibodies were added. Effector lymphocyte functions known to require cell-cell contact were also tested. The first assay was immunoglobulin production by whole spleen cells activated by both anti-immunoglobulin beads and PHA, which act together to induce immunoglobulin production (19). The results of these experiments are depicted in Fig. 1A and show significant inhibition of immunoglobulin production by W-CAM-1. We also tested the cytotoxic activity of MLR cells against 51Cr-labeled K562 cells. As shown in Fig. 1B, both whole antibody and F(ab)1 fragments significantly inhibited killing of K562 cells. In this and other experiments, F(ab)1 fragments were somewhat more active in inhibiting killing. By contrast, the isotype control antibody Wehi-B2 had no effect on cytotoxicity. Levels of inhibition were comparable with LFA-1 alone and some additive effect was noted in this experiment when both antibodies were present. This additive effect was inconstant and was not evident when WCAM-1 antibody induced more profound inhibition.

DISCUSSION The W-CAM-1 detects the 90-kDa ICAM-1 glycoprotein on the basis of biochemical and functional characterization (15, 17). The antibody blocked both homotypic and heterotypic adhesion of activated T and B cells (Table 1). F(ab)1 fragments of W-CAM-1 significantly inhibited lymphocyte binding to endothelial cells (Table 2), although whole antibody was less effective, perhaps because of the design of this assay, which allows cross-linking of ICAM-1 on the two cell types by W-CAM-1 antibody. In in vitro assays of B-cell (anti-immunoglobulin) and T-cell (PHA, anti-CD2) activation (Table 3), some inhibition was noted when W-CAM-1 antibody was present in cultures of whole spleen, but more enriched populations of cells showed little or no effect. This may be due to the capacity of these stimuli to themselves cross-link cells, tending to overcome any requirement for ICAM-1-mediated adhesion to optimize facilitatory cell-cell interactions. When purified blast-cell populations were stimulated maximally with lymphokines to replace the need for T-cell help, addition of W-CAM-1 antibody was without effect. By contrast, when activation was elicited by cell-cell contact (Table 4), whether allogeneic stimulation of T cells (MLR) or T-cell-mediated B-cell activation, significant inhibition was noted. T-cell activation of resting B lymphocytes has also been shown to be inhibited by the addition of LFA-1 and CD4 monoclonal antibodies (9), suggesting that this phenomenon may involve several intercellular bonds. Inhibition of immunoglobulin production by whole spleen cell stimulated by anti-immunoglobulin was also noted (Fig. LA). In view of the dependence of this assay system on T-cell help (19), this also presumably reflects disruption of cell-cell interaction by W-CAM-1 antibody. Finally, an effector mechanism dependent on cell-cell adhesion, the cellmediated cytotoxicity assay, was examined (Fig. 1B). Both W-CAM-1 and LFA-1 antibody induced comparable levels of inhibition of cytotoxicity, but this was somewhat more marked if both antibodies were included. Taken together, these data point to a multifaceted role of the ICAM-1 molecule in immune function. The ICAM-1 antigen appears to be involved in the adhesion of lymphoid cells to endothelial cells, a crucial event in the initiation of egress from the blood and migration to sites of immune activity. With respect to events occurring at the site of immune response, ICAM-1 appears to have a role in the cell-cell interactions that control the response. The mole-

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cule also partly determines at least one effector response, cell-mediated cytotoxicity. These data parallel closely those obtained for LFA-1 (21-23), supporting the notion that LFA-1 and ICAM-1 are complementary structures (15). The discovery of a superfamily of adhesion moleculesthe integrins (11, 12), all of which are heterodimeric, with the a and P chains falling into similar size ranges-poses the question: how is ICAM-1 related to these molecules? Clearly, there is no close structural relationship between ICAM-1 and the integrins, and the ligands of the integrin family are very variable, from huge molecules such as fibronectin to the ligand for CD11b (MAC1) being C3bl fragment of complement. The most closely related molecule would seem to be the high endothelial venule (HEV) receptors (24). In humans (25), this molecule has a molecular mass identical to ICAM-1 but has a quite different distribution, being strongly expressed on peripheral blood leukocytes, whereas ICAM-1 is undetectable on these cells. Further structural analysis is required to determine whether the HEV receptors and ICAM-1 are encoded by related genes and perhaps form part of a receptor family that controls lymphocyte migration and intercellular interaction. We acknowledge the excellent technical assistance of Karen Welch and Filomena Micozzi. This work was generously supported by the Lions Fellowship Fund of the Anti-Cancer Council of Victoria, Australia.

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