a novel anti-rat cd18 monoclonal antibody triggers lymphocyte homotypic aggregation and granulocyte...

1640 M. D. Pavlovid, M. ColiC. N. Pejnovid et al. Eur. J. Immunol. 1994. 24: 1640-1648

Milo; D. PavloviZ.", Miodrag Calif", Nada PejnoviCv, Takuya Tamatanie, Masayuki Miyasakae Aleksandar DujiCv

A novel anti-rat CD18 monoclonal antibody triggers lymphocyte homotypic aggregation and granulocyte adhesion to plastic: different intracellular signaling pathways in resting versus

and

Institute of Medical Research, activated t hymocyt es Military Medical Academyv, Belgrade and Department of Immunology, Tokyo Metropolitan Institute of Medical Science*, . Tokyo

We have raised a monoclonal antibody (mAb), NG2B12, directed against rat CD18, capable of inducing lymphocyte homotypic adhesion and granulocyte adherence to plastic. NG2B12-induced aggregation is temperature sensitive and requires metabolic energy, an intact cytoskeleton and the presence of Mg2+, but is independent of protein synthesis. Ca2+is not only dispensable but exerts a suppressive effect on the NG2B12-induced adhesion. The adhesion is readily observed in thymocytes and concanavalin A blasts of thymocytes and splenocytes but is very weak in resting spleen and lymph node cells. NG2B12 also enhances phorbol 12-myristate 13-acetate (PMA)-induced aggregation in an additive fashion. The NG2BlZinduced homotypic adhesion is mediated by LFA-1. mAb against ICAM-1 completely inhibited the induced adhesion of activated cells but inhibited only partially and in a time-dependent manner the adhesion of resting thymocytes.The activation of protein phosphatases 1 and 2A (as assessed by the use of okadaic acid) is necessary for the NG2BlZinduced adhesion of both resting and activated thymocytes. In contrast, H-7 (an inhibitor of protein kinase C and A), substantially suppressed the adhesion of resting thymocytes, whereas W-7 (an inhibitor of calmodulin-dependent protein kinase) inhibited the adhesion of activated thymocytes. NG2B12 induces both adherence to plastic and homotypic aggregation of granulocytes; the events being blocked by anti-CD18 (WT.3) and anti-CDllb/CDllc (OX-42) mAb, augmented by okadaic acid and not modified by H-7 and W-7. Additionally, we have demonstrated that NG2B12 and PMA employ distinct intracellular signaling pathways in inducing adhesion of both thymocytes and granulocytes.

1 Introduction

The family of leukocyte cell adhesion molecules, LeuCAM (P2-integrins), comprises three members which share a common fi chain (CD18) but have distinct a chains, LFA-1 (CDlla/CD18), CR3 (CDllb/CD18) and p150,95 (CDllc/CD18) [l] . These glycoproteins are widely distri- buted on leukocytes and, mediating a variety of cell-cell interactions, play an important role in many immunologic reactions (reviewed in [2]). Intercellular adhesion must be tightly regulated to ensure a timely and adequate immune response while avoiding an undesirable cellular aggregation which might bring about tissue damage [3].The function of the fi2-integrins may be controlled on at least three levels. Apart from short- and long-term changes in their surface expression and modulation of distribution in the plane of the plasma membrane, an avidity for their respective ligands is subject to swift alteration [4-61. A rapid transi-

[I 124571

Correspondence: Miodrag colic, Institute of Medical Research, MMA. Crnotravska 17. 11002 Belgrade, Yugoslavia

Abbreviations: LFA-1: Lymphocyte function-associated anti- gen-1 ICAM-1,2,3: Intercellular adhesion molecules- 1.2,3 CR3: Complement receptor 3 PP: Protein phosphatase

Key words: Antibody / CD18 / Adhesion I Signaling I Leuko- cyte

tion from a nonadhesive to an adhesive state mostly involves a shift in ligand avidity, the mechanism of which is incompletely understood. An intracellular activation signal is a necessary step in the acquisition of the high-avidity state. The LFA-1-dependent adhesion can be induced by phorbol esters which activate PKC or by stimulation of a number of cell surface Ag such as CD2, CD3, CD14, CD15, CD16, CD19, surface Ig, CD39, CD40, CD43, CD44. CD45, and MHC class I1 [7-91. Moreover, engagement of CDl la and CD18 themselves can activate the LFA- 1-dependent adhesion pathway [ 10-131. This is in line with numerous findings confirming that LFA-1 can generate intracellular signals which contribute to lymphocyte activation and proliferation [ 14-16]. Although the precise mechanisms inducing a high-avidity state of the P;?-integrins remain to be elucidated, the direct phosphorylation of one or both chains and/or the action of a regulatory protein(s) or lipid mediators may be involved [ 17-20]. In any event, it seems that the mode of avidity regulation differs among different cell types [19,21, 221.

Homotypic adhesion, originally reported to be induced by phorbol esters [7], represents a suitable model for exploring mechanisms of leukocyte aggregation. In the present study, using this model, we describe the first mAb directed to rat CD18 that promotes adhesion of lymphocytes and granulocytes through an LFA-1- and CR3-dependent adhesion pathway, respectively. The strength of the induced aggregation depends upon the type and activation state of the examined cells. Furthermore, we provide evidence that this

0014-298019410707-160$10.00 + ,2510 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1994

Eur. J. Immunol. 1994. 24: 1640-1648 Anti-rat CD18 mAb promotes lymphocyte and granulocyte adhesion 1641

mAb potentiates PMA-induced leukocyte adhesion and utilizes distinct intracellular signaling routes, depending on the type and activation state of the examined cells. In addition these routes are different from those involved in PMA-induced leukocyte adhesion.

2 Materials and methods

2.1 mAb and reagents

NG2B12 mAb was produced at the Institute of Medical Research, MMA, Belgrade,Yugoslavia. Briefly, fusion was performed with P3X-Ag.8 myeloma cells and splenocytes of a BALB/c mouse immunized with rat inflammatory granulocytes. This mAb was purified by salt fractionation and its isotype is IgGI. In Western blot, NG2B12 reacted with a molecule of 90-95 kDa from rat thymocyte and granulocyte lysates, and immunoprecipitation studies on biotin-labeled cells confirmed that this mAb recognizes an epitope of rat CD18. NG2B12 binds almost all rat leukocytes but the expression of its epitope was always lower in comparison with the CD18 epitope recognized by WT.3 mAb. mAb WT.3 (anti-rat CD18; IgGI), WT.l (anti-rat CDlla; IgG2,) and 1A29 (anti-rat CD54; IgGI) were produced at the Department of Immunology, Metropolitan Institute of Medical Science, Tokyo, Japan [23,24]; ED7 (IgG1) and ED8 (IgG1) specific for the rat CDl lb were kindly provided by Dr. C. Dijkstra (Free University of Amsterdam, The Netherlands) [25]; OX-42 (anti-rat CDllb/CDllc) (IgG2,) and OX-8 (anti-rat CD8; IgGI) were purchased from Serotec, GB.

The reagents used were PMA (Sigma, St. Loius, MO), sodium azide (Merck, Darmstadt, FRG), cytochalasin B (Sigma), cycloheximide (Sigma), 1-(5-isoquinolinylsuIfo- nyl)-3-methyl-piperazine (H-7; Sigma), N-(6-aminohexyl)- 1-naphthalenesulfonamide (W-7; Sigma), genistein (ICN, Cleveland, USA), okadaic acid (Sigma), sodium orthovanadate (Sigma), human recombinant (hr) IL-2 (Amersham International, GB) and Con A (Pharmacia LKB, Uppsala, Sweden).

2.2 Cells and cell cultures

A 0 rats, female, 6-8 weeks old, bred at the Farm for Experimental Animals, MMA (Belgrade, Yugoslavia), were used as a source of cells. Thymocytes were isolated from thymuses by teasing the organs against a steel mesh to obtain a single-cell suspension. Spleen and lymph node cells were collected in the same way from spleens and axillar and cervical lymph nodes. Peripheral blood granulocytes were isolated from buffy coats by centrifugation on Nyco- Prep Animal 1007 (Nycomed, AS, Oslo, Norway). Pelleted cells were mixed with isotonic solution of ammonium chloride to lyse erythrocytes, extensively washed by centrifugation and were > 95 % granulocytes as estimated by May-Gruenwald-Giemsa staining. Inflammatory granulocytes were obtained by the use of polyvinyl sponges inserted under the animals' skin as previously described by Middleton and Campbell [26].

Con A blasts were obtained by culturing thymocytes or splenocytes (1 x lo7 - 1.5 x 107/ml) at 37°C in complete

medium supplemented with 1 pg/ml Con A and 3 U/ml hrIL-2 for 3 days. On the 3rd day, the cells were collected, extensively washed, resuspended in fresh medium and viable cells were separated by centrifugation on NycoPrep Animal 1007 (Nycomed).

2.3 Adhesion assays

2.3.1 Semiquantitative adhesion assay

To measure homotypic adhesion we used the modified method described by Rothlein and Springer [7]. Cells were resuspended in RPMI 1640 medium with 10 YO FCS at 5 x lo6 cells/ml (resting thymocytes) or at 2 x lo6 cells/ml (Con A blasts) and placed in 96-well flat-bottom microtiter plates. The cells were incubated at 37 "C in the absence or presence of mAb and/or PMA. Where indicated, different reagents and blocking mAb were preincubated with the cells at 37 "C for 30 min. To down-regulate PKC, cells were incubated with PMA (100 ng/ml) for 18 h at 37°C then viable cells were separated on NycoPrep Animal 1007, washed and used in the adhesion assay [27].The plates were read after various time periods and scores ranged from 0 to 5 : 0, no aggregation; 1, less than 10% of the cells in aggregates; 2,lO-50 YO of the cells formed small clusters; 3, 50-80 YO of the cells in small loose and/or compact clusters; 4, > 80% of the cells formed large lose and/or compact clusters; 5 , nearly 100 YO of the cells in large very compact aggregates. All cells and mAb were tested three times and scored by two independent observers.

2.3.2 Quantitative adhesion assay

Cells were resuspended in FWMI 1640 with 10% FCS at 1 x 107/ml and placed in 2 ml plastic tubes. Stimuli were added in a final volume of 200 pYtube and cells incubated at 37 "C for 2 h. Where indicated, various mAb were preincubated with the cells at 37 "C for 30 min, whereas metabolic inhibitors were applied 30 min before, simultaneously or 30 min after the addition of NG2B12. Cells were then mildly vortexed and free cells counted in a hemacytometer. Percent of the cells in aggregates = 100 x [l- (number of free cells)/(number of free cells in control)]. Controls were cells incubated in the same conditions but without stimuli. Whenever metabolic inhibitors were used, cell viability was checked by trypan blue dye exclusion. Peripheral blood granulocytes were resuspended at 1.5 x 105 in 30 p1 5 % FCS-containing RPMI 1640 medium, placed in a Terasaki microwell plate and cultured in hanging drop at 37°C for 2 h. For the blocking experiments, the cells were preincubated with various mAb or metabolic inhibitors at 37 "C for 30 min. Counting was performed in a hemacytometer as described above.

2.4 Colorimetric assay of granulocyte adherence to plastic

We used a modified assay, initially described by Oez et al. [28]. In brief, peripheral blood granulocytes were resuspended in RPMI 1640 with 5 YO FCS and NG2B12 and/or PMA and seeded at 2 x 1@/ml in 96-well flat-bottom microtiter plates. Where indicated, the cells were preincu-

1642 M. D. PavloviC, M. colic, N. PejnoviC et al. Eur. J. Immunol. 1994. 24: 1640-1648

bated with various mAb or metabolic inhibitors at 37 "C for 30 min. Afterwards, the plates were incubated at 37°C for 1 h. Then, nonadherent cells were removed by washing three times with medium. Adherent cells were stained with 0.1 '70 methylene blue in PBS for 15 min. Thereafter, the plates were washed three times in running water and left to air-dry. To dissolve the dye 250 p1 0.1 M HCI was added to each well and absorbance was measured by an ELISA reader (Behring ELISA Processor, Behring, FRG) at 650 nm.

3 Results

3.1 NG2Bl2 promotes homotypic adhesion of rat leukocytes

While screening hybridoma supernatants from a fusion described in Sect. 2.1, a hybridoma, designated NG2B12, was selected for its ability to elicit homotypic adhesion of rat thymocytes. This mAb was shown to detect an epitope present on rat CD18 asdescribed in Sect. 2.1. Afterwards, a series of experiments was performed to explore the pro- aggregatory properties of the mAb. As seen in Fig. 1, NG2B12 induced moderate (up to 50% of the cells were aggregated) homotypic adhesion of resting thymocytes and exhibited an additive effect on the aggregation evoked by PMA. Induction of aggregation was not observed with WT.3, WT. 1 or a species-matched irrelevant (OX-8) mAb

Figure 1 . Induction of thymocyte aggregation by NG2B12 or PMA or their combination. (A) Medium; (B) NG2B12 mAb; (C) PMA; (D) NG2B12 mAb and PMA. Cells were plated at 1 X 10b/well with NG2B12 (30 kg/ml) or PMA (50 nglml) or their combination. incubated at 37°C and photographed at 80 X after

(data not shown). The time course of NG2BlZinduced thymocyte aggregation was recorded and compared to the kinetics of adhesion evoked by PMA. The NG2B12- induced aggregation started about 30 min later and lasted longer than that evoked by PMA (Fig. 2). Thymocyte aggregation was optimal using 30 pg/ml NG2B12 and was still visible at the concentration as low as 0.5 pg/ml confirming the specificity of its effect (data not shown). In contrast to thymocytes, resting spleen and lymph node cells were not able to form visible aggregates following the addition of NG2B12 but this mAb clearly enhanced PMA-induced aggregation of these cells. However, a quantitative test of homotypic adhesion showed that NG2B12 does induce the weak aggregation of resting splenocytes and lymph node cells (about 20 % of the cells found in aggregates) (data not shown). At the same time, NG2B12 readily doubled spontaneous homotypic adhesion of Con A blasts of thymocytes and splenocytes (Fig. 3A and B).

'NG2B12 * PMA *NG2812+PMA

7

0 0 . 5 1 2 3 4 5 2 4

time (houra)

Figure 2. Time course of thymocyte homotypic aggregation induced by NG2B12 or PMA or their combination. Cells (1 X 1O6/well) were aggregated with 30 pg/ml NG2B12. or 50 ng/ml PMA or their combination.

3.2 Requirements for NGZBlZinduced homotypic adhesion

We next analyzed the physiologic requirements for NG2B 12-induced homotypic aggregation. The results are presented inTable 1. NG2B12 could not induce aggregation of thymocytes and Con A blasts of thymocytes in HBSS without divalent cations or with Ca2+ only at any given concentration (1-5 mM), while the aggregation was fully restored in HBSS with Mg2+ in a dose-dependent manner. Table 1 shows that 2mM Mg2+ completely restored cell aggregation, whereas 4 mM Mg2+ increased the adhesion above a usual level seen in complete medium containing 10 '70 FCS. Interestingly, the addition of Ca2+in HBSS with Mg2+ significantly suppressed NG2B12-evoked aggregation. The aggregation was fully prevented by incubating cells at 4°C or in the presence of sodium azide or cytochalasin B, confirming its dependence upon active cell metabolism and actin cytoskeleton (Table 1). Cyclohexi- mide, an inhibitor of protein synthesis, had no effect o n the - .

2 h of culture. aggregation.

Eur. J. Immunol. 1094. 24: 1640-1648

A

1,MEDIUM; 2,wT.l; 3.wT.3; 4 , l m 5.NG2612; 6,NG2612+W.l; 7,NG2612+W.S; 8,NG2812+1A20; 9 , P W lO,PMA+wT.l; ll.pMA+wT.S; 12,PMA+IA20: 13.NG261 P+PMA; 14,NG2612+ PMA+WTl; 15,NQ2612+ PMA+wT3; 16,NG2812+PMA+lA29;

Anti-rat CD18 mAb promotes lymphocyte and granulocyte adhesion 1643

B

A g g r e g a t i o n

Figure 3. mAb inhibition studies of NG2B12- or PMA-induced cell adhesion. (A) Con A blasts of thymocytes; (B) Con A blasts of splenocytes; cells were seeded at 4 x 105lwell and stimuli (30 pg/ml NG2B 12 or 50 nglml PMA or their combination) were added alone or simultaneously with inhibitory concentrations of the mAb WT. 1 (CDlla) (10 pglml); WT.3 (CD18) (10 pglml); 1A29 (CD54) (10 pglml). Aggregation was scored after 2 h of incubation.

To determine the intracellular signaling pathways involved in NG2B12-induced aggregation, we preincubated cells with different concentrations of H-7 (an inhibitor of protein kinase A and C), W-7 (an inhibitor of calmodulin-dependent protein kinase), genistein (an inhibitor of tyrosine kinases), okadaic acid [an inhibitor of protein phosphatases (PP)l and 2A] and sodium orthovanadate (an inhibitor of protein tyrosine phosphatases). Table 1 and Fig. 4 show that H-7, W-7 and okadaic acid significantly suppressed NG2B12-induced homotypic adhesion of thymocytes, while genistein and orthovanadate had no visible influence on the adhesion. PMA-evoked aggregation was similarly suppressed by these agents (Table 1). To reveal potential differences in intracellular mechanisms of the PMA- and NG2B12-induced aggregation, we down-regulated PKC activity by a prolonged treatment of thymocytes with PMA (18 h). Thereafter, while newly added PMA did not evoke aggregation, NG2B12 did induce an aggregation response of these thymocytes (data not shown), suggesting that it could act through PKC isoenzymes insensitive to PMA (most likely the aPKC family) [29]. Con A blasts of thymocytes behaved differently in response to the inhibitors. The aggregation induced by NG2B12 or PMA was inhibited in the presence of W-7 or okadaic acid but not H-7 (Table 1). For a better assessment of the effects of all the metabolic inhibitors, these agents were applied to cells 30 min before, simultaneously or 30 min after the addition of NG2B12 and the adhesion was estimated quantitatively. In parallel, the aggregation was monitored semiquantitatively in microtiter plates. H-7 partially inhibited the adhesion of resting thymocytes, showing maximal efficacy when preincubated with the cells. As expected, H-7 had no influence on the aggregation of Con A blasts (Table 2). Okadaic acid had a more conspicuous inhibitory effect on the adhesion of both resting and activated thymocytes regardless of the time of its addition. Orthovanadate, like genistein, did not modify the adhesion of the cells, although it slightly and variably increased the aggregation of resting thymocytes (Table 2). Surprisingly, disparate findings were obtained when W-7 was applied to resting thymocytes in microtiter plates vs. plastic tubes. As seen in Fig. 4, W-7 almost completely prevented the cluster formation of

Figure 4. Effects of various metabolic inhibitors on NG2B12-induced homotypic adhesion of resting thymocytes. Cells were plated at 1 x l0blwell in medium only (a) or NG2B12 (30 pg/ml) was added alone (b) or following the 30-min preincubation of the cells at 37°C with 80 pM H-7 (c), 1 pM okadaic acid (d), 20 pM W-7 (e), 100 pM orthovanadate (g) or 50 pglml genistein (h). (f) W-7 (20 p ~ ) was added 30 min after the addition of NG2B12. The cells were photographed after 2 h at 80 X .

1644 M. D. PavloviC, M. ColiC, N. PejnoviC et al. Eur. J. Immunol. 1994. 24: 1640-1648

Table 1. Requirements for NG2B12- and PMA-induced cell adhesiona)

Chemicakondition Resting thymocytes Activated thymocytes NG2B12 PMA NG2B12+PMA NG2B12 PMA NG2B12+PMA

Medium 2 3 4 3 4 5 EDTA 0 0 0 0 0 0 MgZ + Ca2 +- f ree HB SS 0 ND ND 0 ND ND Mg2+ (2 mM) 2 ND ND 3 ND ND Mg2+ (3 mM) 3 ND ND 4 ND ND Mg2+ (4 mM) 4 ND ND 5 ND ND Ca2+ (4 mM) 0 ND ND 0 ND ND Mg2+ (4 mM)+Ca2+(1 mM) 2 ND ND 2 ND ND Mg2+ (4 mM)+Ca2+(2 mM) 0 ND ND 0 ND ND Sodium azide (0.2 %) 0 0 0 2 4 5 Cytochalasin B (5 pglml) 0 0 0 0 1 0 Cycloheximide (10 pglml) 2 3 4 3 4 5 H-7 (80 pM) 1 1 2 3 4 5 W-7 (20 PM) 0 0 0 1 2 3 Genistein (50 pglml) 2 3 4 3 4 5

Orthovanadate (100 pM) 2 3 4 3 4 5 Okadaic acid (1 pM) 0 0 0 0 ND ND

a) Cells were cultured in the presence of NG2B12 (30 pglml) and/or PMA (50 nglml). Where indicated, they were preincubated with inhibitors at 37°C for 30 min. For studies of divalent cation dependence, the cells were washed and resuspended in Mg2+, CaZ+-free HBSS medium and various concentrations of Mg2+ and Ca2+ were added as denoted in theTable. Aggregation was scored after 2 h of incubation.

Table 2. Effects of metabolic inhibitors on NG2B12-induced homotypic adhesion of resting or activated thymocytesa)

Inhibitor time of addition % inhibition of adhesion

Resting Activated thymocytes thymocytes

- 30 min 40 + 2 H-7 0 rnin 19 3

+ 30 min 3 ND - 30 min + 10 43

w-7 0 min + 20 32 + 30 min + 5 ND - 30 min 83 85

Ocadaic acid 0 min 95 78 + 30 min 78 70 - 30 min + 6 3

Orthovanadate 0 min + 11 + 6 + 30 min + 2 ND - 30 min 0 4

Genistein 0 min 2 0 + 30 min ND ND

a) The quantitative assay of homotypic adhesion was performed as described in Sect. 2.3.2. The inhibitors were added 30 rnin before (- 30 min), simultaneously (0 min) or 30 rnin after (+ 30 min) the application of NG2B12 (30 pglml) at the concentrations denoted in Table 1. Percent inhibition of aggregation was calculated as follows: % inhibition = 100 X [l -(no. of free cells in control - no. of free cells with inhibitorlno. of free cells in control - no. of free cells with NG2B12)]. The symbol "+" before some YO indicates stimulation. The results shown here are the mean of triplicates of a representative out of three experiments.

% inhibition of aggregation

" 1 2 4 1 2 4 1 2 4 m 1

NG2812 PMA NG2812+PMA

I manti-CD11a manti-CD18 manti-ICAM-1 I Figure 5. mAb inhibition studies of thymocyte homotypic adhesion induced NG2B12, PMA or their combination. Cells were resuspended in plastic tubes at 2 X lo6 in 200 pl RPMI 1640 with 10 % FCS and stimuli and inhibitory mAb (10 pglml) were added simultaneously. Aggregation was measured quantitatively as described in Sect. 2.3.2 after 1,2 or 4 h of incubation. Percent inhibition of aggregation was calculated as follows: % inhibition = 100 x [ l -(no. of free cells in control - no. of free cells with inhibitory mAblno. of free cells in control - no. of free cells with NG2B12 or PMA)].The results represent the mean of triplicates of a representative of three separate experiments.

titer plates even when it was applied 60 rnin after the addition of mAb (data not shown).

3.3 NG2Bl2 induces thymocyte homotypic adhesion resting thymocytes stimulated with NG2B12, whereas in tubes it either had no effect or slightly stimulated the cell aggregation (Table 2). NG2B 12-induced aggregation of Con A blasts was in both cases partly inhibited by W-7 (Tables 1 and 2). Moreover, W-7 was able to dissociate aggregates of Con A blasts induced by NG2B12 in micro-

through an LFA-1-dependent/ICAM-1-dependent and -independent pathway

mAb inhibition studies were performed to identify potential adhesion receptor(s) and its ligand(s) involved in NG2B12-induced lymphocyte aggregation. rnAb targeting

Eur. J. Immunol. 1994. 24: 1640-1648 Anti-rat CD18 mAb promotes lymphocyte and granulocyte adhesion 1645

CD18 (WT.3), CDl la (WT.l) and CD54(1A29) as well asa control (anti-CD8) mAb were added with NG2B12 and/or PMA and aggregation was measured either quantitatively or semiquantitatively. Similar results were obtained irre- spective of the assay used. Fig. 5 demonstrates that NG2BlZinduced thymocyte aggregation was completely blocked by WT.3 and substantially inhibited by WT. 1 mAb. However, 1A29 mAb could not inhibit the aggregation at an early time point (3 % inhibition after 1 h) but thereafter its inhibitory effect increased, reaching 43 % after 4 h. Thus, the NG2B12-induced thymocyte aggregation is LFA-1- dependent/ICAM-1-independent at the beginning of the adhesion process, but later it becomes partly ICAM- 1-dependent. In contrast, PMA-induced thymocyte aggregation is both LFA-1-dependent/ICAM-1-dependent and ICAM-1-independent throughout the course of the adhesion process. When aggregation was induced by the combination of PMA and NG2B12, the patterns of inhibition by WT.3, WT.l and 1A29 mAb resembled those seen in the case of the NG2B12-induced aggregation (Fig. 5 ) , except that the effect of WT. 1 and 1A29 mAb was lower after 2 and 4 h. Homotypic adhesion of Con A blasts triggered by NG2B12 is completely mediated by an LFA-l/ICAM-1 interaction since WT. 1 ,WT.3 and 1A29 mAb abrogated cell aggregation to the level of spontaneous adhesion or below it (Fig. 3A and B). However, PMA- and PMA+NG2B12- induced aggregation of Con A blasts was both LFA- 1-dependent/ICAM-1-dependent and ICAM-l-independent (Fig. 6A and B). Importantly, NG2B12 does not induce the surface up-regulation of LFA-1 as confirmed by the use of indirect immunofluorescence (data not shown).

3.4 NG2Bl2 stimulates both adherence to plastic and homotypic aggregation of granulocytes

Using a simple colorimetric assay, we showed that NG2B12 had the ability to increase granulocyte adhesion to plastic, although it was less prominent than that seen with PMA. Again, NG2B12 enhanced the adherence induced by PMA in an additive manner (Table 3). WT.3 (anti-CD18) mAb almost completely blocked NG2B12- and/or PMA-induced adhesion of granulocytes to plastic, whereas WT.l (anti- CDlla) mAb was without any significant effect. 1A29

(anti-ICAM-1) mAb was not inhibitory in this system (data not shown).This could have been anticipated since ICAM-1 is not expressed on rat granulocytes as shown previously [23]. Interestingly, OX-42 (anti-CDllblCDllc) mAb was able to decrease the NG2B12- but not PMA-induced adhesion to plastic. Most metabolic inhibitors, apart from okadaic acid which acted stimulatory, were inefficient in modulating NG2BlZinduced granulocyte adherence to plastic (Table 3). The adherence triggered by PMA was almost equally suppressed by H-7 and W-7 (Table 3), whereas other inhibitors had no effect. NG2B12 did not induce visible homotypic aggregation of granulocytes in a microtiter plate unless it was applied together with PMA

Table4. Effect of mAb or metabolic inhibitors on granulocyte homotypic adhesion induced by NG2B12 or NG2B12 + PMAa)

m Ablinhibitor Stimuli NG2B12 NG2B12 + PMA

SQ % AC Yo Ib) SQ % AC % Ih)

None 0 48 0 0 82 0 WT. 1 0 4 6 3 0 82 0 WT.3 0 10 80 2 12 85 OX-42 0 8 8 4 1 58 30 H-7 0 45 4 0 78 5 w-7 0 52 + 10 0 70 15 Okadaic acid 0 62 + 31 0 84 + 3 Orthovanadate 0 4 4 6 0 81 1 Genistein 0 4 6 3 0 83 + 1 WT.3+W-7 0 6 87 2(3)") 11 86

a) The semiquantitative assay was performed essentially as described for the colorimetric assay of granulocyte adherence to plastic in Table 3. Homotypic adhesion was scored after 2 h of incubation. In parallel, a quantitative assay was carried out in Terasaki plates as described in Sect. 2.3.2. Percentage of inhibition of granulocyte homotypic adhesion was calculated as described in Table2. The results are given as the mean of triplicates of three independent experiments.

b) SQ: Semiquantitative assay; % AC: percentage of aggregated cells; YO I: percentage of inhibition of aggregation.

c) The enhancement in the size of the aggregates observed when W-7 was added to the combination of NG2B12, PMA and WT.3 mAb was registered in four out of five independent experiments.

Table 3. Modulation of NG2B12 and/or PMA-induced adherence of granulocytes to plastic by mAb or various metabolic inhibi- torsa)

m Ablinhibitor Absorbance: stimuli addedb) None NG2B12 PMA NG2B12 + PMA

None .024 f .007 .063 f .022 .234 f .044 .310 f .045 WT. 1 ,021 f .005 .059 f .024 .229 f .031 .301 f .024 WT.3 .009 f .005 .012 f .006 .019 f .007 .046 f .017 OX-42 .019 f .002 .023 f .010 .223 f .030 .287 f .032 H-7 .025 f .007 .064 f .026 .136 f .030 .231 f .049 w-7 .031 f .009 .054 f .019 .111 k .019 ,140 f .069

.024 f .003 .lo0 f .016 ,250 f ,012 .358 f ,016 Okadaic acid Orthovanadate ,021 f .002 .059 f .013 .218 k .015 .303 f .021 Genistein .026 f .010 ,066 ? .026 ,240 f .061 ,315 f .035

a) The test was performed as described in Sect. 2.4. Granulocytes were preincubated with mAb or inhibitors at 37°C for 30 min at the concentrations described in lbble 1. Then, NG2B12 (30 pglml) and/or PMA (250 nglml) were added and adherence to plastic was measured after 1 h incubation at 37°C.

b) The results shown here represent the mean (k SD) absorbance of triplicates of four or six independent experiments.

1646 M. D. PavloviC, M. colic, N . PejnoviC et al. Eur. J. Immunol. 1994. 24: 1640-1648

and WT.3 or OX-42 mAb (Table 4). In contrast to other inhibitors which did not modify granulocyte aggregation occurring in the presence of NG2B12, PMA and WT.3,W-7 caused a slight increase in the size of clusters changing their morphology - they became confluent and irregular in shape (Table 4 and Sect. 4). To separate the phenomena of adherence to plastic and homotypic aggregation, we measured the aggregation in the hanging-drop culture in Terasaki plates to avoid the effects of plastic adherence.The results (Table 4) show that NG2B12 promoted substantial granulocyte aggregation (48 %) further potentiated by PMA (82 %). Like the plastic adherence, NG2B12-induced granulocyte aggregation was significantly inhibited by WT.3 and OX-42 mAb and moderately potentiated by okadaic acid. Other agents were all inefficient. The aggregation induced in the presence of NG2B12 and PMA was blocked by WT.3 and to a lesser degree by OX-42 mAb, possibly reflecting the inability of OX-42 to block the aggregation triggered by PMA. None of the inhibitors, including W-7 (Table 4), substantially modified granulocyte aggregation promoted in the presence of NG2B12 + WT.3 or NG2B12 + PMA + WT.3 (data not shown).

4 Discussion

In the present study we report on an mAb (NG2B12), directed to an epitope of the rat CD18 molecule, capable of triggering LFA-1-dependent and CR3-dependent adhesion events in lymphocytes and granulocytes, respectively. Recently, two anti-human CD18 mAb (KIM127 and KIM185) have been described and some functional properties of these mAb, concerning adhesion, have been inves- tigated on lymphoid cell lines and neutrophils [12, 131. Using our mAb, we explored its pro-aggregatory effects on normal, resting or activated rat lymphocytes and peripheral blood granulocytes, tracing the intracellular signaling routes implicated in the aggregation.

NG2B12 induces homotypic adhesion of resting thymocytes and Con A blasts of thymocytes and splenocytes but cannot promote visible aggregation of resting splenocytes and lymph node cells (Figs. 1 and 3A and B). However, a quantitative adhesion assay showed that NG2B12 induces a weak aggregation response of these cells (about 20% were aggregated) (data not shown). Similarly, Koopman et al. [ 111 have found that several anti-LFA-la mAb promote the adhesion of anti-CD3-activated T cells but not resting T cells. Since the levels of LFA-1 on the peripheral blood memoryT cell subset and the activated T cells were similar, they proposed that the initial activation state of the cells was responsible for the observed differences in adhesive- ness [11].The fact that resting thymocytes readily aggregate in response to NG2B12 might suggest the existence of an inherently higher activation state, at least with respect to LFA-1. NG2B12 exerts an additive effect with PMA in inducing homotypic adhesion (Figs. 1 and 2).This observa- tion raises the possibility that NG2B12 and PMA could use distinct activation mechanisms, as we have shown. Also, it confirms that there may be multiple stages of integrin avidity, as recently documented for T cell integrins activated by cations or phorbol esters [30]. To our knowledge, there have not been reports about a similar effect of an anti-&-integrin mAb when added simultaneously with phorbol esters.

In view of its dependence on the active cell metabolism, intact cytoskeleton and divalent cations, NG2B 12-induced adhesion has characteristics of the integrin-mediated aggregation. Here, we want to point out the observed require- ment for Mg2+, whereas Ca2+ exerted an inhibitory effect on the adhesion evoked by NG2B12 in the presence of Mg2+ (Table l) , a finding consistent with the association of Ca2+ with an inactive form of LFA-1 [31]. Very recently, Shimizu and Mobley [30] have reported that Mg2+/EGTA treatment up-regulates the LFA-l/ICAM-1 interaction. However, most anti-LFA-la mAb which induce the LFA- 1-dependent adhesion have been shown to require both Ca2+ and Mg2+ [ l l , 321.

Inasmuch as there is strong evidence that lymphocyte adhesion, like activation, is regulated by an interlocking cascade of phosphokinase and phosphatase molecules, using a series of their inhibitors, we tried to define potential intracellular signaling routes involved in the adhesion evoked by NG2B12.To date, there has been no information on intracellular signaling events accompanying binding of a pro-aggregatory anti-CD18 mAb, although some data exist for a few anti-CDlla mAb [ll, 321. The results in Fig. 4, Table 1 and 2 show that activation of PKC andor A and PP1 and/or PP2A is an important event for NG2BlZinduced adhesion of resting thymocytes. Inasmuch as H-7, in contrast to okadaic acid, should be preincubated with the cells to give optimal inhibition, it might imply that the activation of PKC and A is an event earlier than the activation of PP1 and/or PP2A. Since genistein and orthovanadate had no apparent effect on the aggregation, the involvement of tyrosine phosphorylation and dephospho- rylation in the process could be excluded. Of note, the action of W-7 proved to be quite different depending upon the assay used. When aggregation was measured quantitatively in plastic tubes,W-7 was either without any effect or slightly stimulated the adhesion of resting thymocytes induced by NG2B12 (Table 2). In microtiter plates, almost complete inhibition of the adhesion was seen in the presence of W-7 (Fig. 4). Occasionally, although character- istic three-dimensional and roundish clusters were absent, we could notice the appearance of gaps among groups of the cells as if they did adhere to each other in one plane but were not able to form typical clusters. The nature of the phenomenon is not clear: neither the enhanced adherence of thymocytes to plastic nor a change in the LFA-1 surface distribution is responsible for the effect of W-7 (data not shown). Possible changes in cytoskeletal organization and/or cell motility should be taken into account. A somewhat similar effect of W-7 was observed in granulocyte homotypic adhesion taking place in the presence of NG2B12, PMA and WT.3 mAb (Table 4). These findings underscore the need of prudent evaluation of the results obtained in different test systems commonly used to study homotypic adhesion. In contrast to resting t hymocytes, the adhesion of Con A blasts induced by NG2B12 was blocked by okadaic acid and W-7 (Table 1 and 2). Collectively, NG2B12-induced adhesion of resting thymocytes requires activation of PKC and/or PKA and PP1 and/or PP2A, while in Con A blasts the latter and calmodulin-dependent protein kinase are involved.The results obtained extend the recent data on differential regulation of the avidity of PZintegrins in different leukocytes [ 19-22]. While NG2BlZinduced homotypic adhesion of Con A blasts of thymocytes and splenocytes is fully ICAM-1

Eur. J. Immunol. 1994.24: 1640-1648 Anti-rat CD18 mAb promotes lymphocyte and granulocyte adhesion 1647

dependent (Fig. 3A and B), the adhesion of resting thymocytes is characterized by a time-dependent usage of ICAM- 1 as a ligand for LFA-1. At the very start, the adhesion is completely ICAM-1-independent but, after 2 and 4 h, it is both ICAM-1 dependent and independent (Fig. 5 ) . It is known that ICAM-1 is expressed on about 20% of rat thymocytes [33] and since the enhanced expression of ICAM-1 on thymocytes after 2- or 4-h incubation with NG2B12 is unlikely, a putative explanation might involve a change in the ligand specificity of the activated LFA-1 molecule. Other possible ligands are ICAM-2 [34] or ICAM-3 [35]. Koopman et al. have shown that a few anti-LFA-la mAb induced the LFA-1-dependent aggregation of anti-CD3-activated T cells but the aggregation was not inhibited by mAb to ICAM-1 or ICAM-2 [ l l ] . Our assumption could not be tested at present since there are no available mAb directed to rat ICAM-2 or ICAM-3.

We have shown that NG2B12 augments granulocyte adhesion to plastic (Table 3). I t is well documented that the adhesion of granulocytes to plastic critically depends on the function of CR3 (CDllbKD18) [36]. Similar anti-human CD18 mAb (KIM127 and KIM185) enhanced the adhesion of PMN to protein-coated glass or plastic by activating CR3 [12, 131. We have demonstrated that the addition of an anti-CD18 mAb (WT.3) completely abolished NG2B12- and/or PMA-induced granulocyte adhesion to plastic, whereas an anti-CDllb/CDllc mAb (OX-42) blocked only the adherence triggered by NG2B12 (Table 3). This is indirect evidence that, first, NG2B12 stimulates granulocyte adhesion to plastic through CR3 and, second, that NG2B12 and PMA probably impose distinct conformational changes in the CR3. The involvement of CDlldCD18 could not be formally excluded at present since two other anti-rat CDl lb mAb (ED7 and ED8) were not inhibitory for NG2B12 or PMA-induced adhesion (data not shown).

mAb detaches granulocytes from plastic (adhered in the presence of NG2B12 and PMA), it does not inhibit their homotypic aggregation completely, allowing the appearance of small cell clusters in a microtiter plate (Tables 3 and 4).

In conclusion, NG2B12 is the first mAb directed to rat CD18 which triggers lymphocyte homotypic adhesion and granulocyte adherence to plastic. NG2B12 appears different from other anti-&-integrin mAb in several aspects. First, it potentiates PMA-induced adhesion in an additive manner and activates distinct PKC isozymes in comparison to PMA; second, NG2B12 seems to be able to change the ligand specificity of LFA-1 on resting thymocytes; third, LFA-1, activated by NG2B12, requires the presence of Mg2+, but not Ca2+ which even has a suppressive effect; fourth, NG2B12 activates distinct intracellular enzymes in resting vs. activated thymocytes; fifth, the mAb triggers granulocyte adhesion through a distinct intracellular route and probably induces different conformational change in CR3 comparing to PMA. We hope that NG2B12 will be of great help in further elucidating the precise mechanisms of fJ2-integrin activation and signaling through these molecules.

We kindly thank Dr. C. Dijkstra forproviding us with ED7and ED8 mAb, D. Kosec for help with FACS analysis and D. VuCeviC, D. PrediC and N. StojanoviC for excellent technical assistance.

Received November 3, 1993; in final revised form April 11, 1994; accepted April 14, 1994.

5 References

While assessing the role of different intracellular enzymes, we found that okadaic acid has a stimulatory effect on NG2BlZinduced granulocyte adhesion to plastic (Ta- ble 3). In contrast, adhesion evoked by PMA was not modified by okadaic acid but was suppressed by H-7 and W-7 (Table 3). Merrill et al. [21] have reported that phorbol ester-induced aggregation of human neutrophils critically depends on PKC, whereas aggregatation triggered by a chemoattractant, formyl-Met-Leu-Phe, was actually enhanced in the presence of staurosporine, a PKC inhibitor. Similarly, Hedman and Lundgren [19] have described the opposing effects of staurosporine and okadaic acid on the LFA-1-mediated adhesion of B cells. In our experiments, okadaic acid had the contrasting effects on NG2B12- induced adhesion of thymocytes and granulocytes, suggesting distinct mechanisms operating in the regulation of &-integrin avidity in these cells (Tables 2 and 3). In addition, it seems obvious that NG2B12 and PMA employ different intracellular patways in activating granulocyte CR3 (Table 3). As seen in Table 4, a quantitative test of granulocyte homotypic aggregation, performed in hanging drop, gave essentially the same results confirming the involvement and identical regulation of CR3 in both processes triggered by NG2B12. Additionally, the assay provided the evidence that the absence of cluster formation in microtiter dates was a conseauence of the increased plastic adhesick of granulocytes. While WT.3 (anti-CD18) n v ,

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a novel anti-rat cd18 monoclonal antibody triggers lymphocyte homotypic aggregation and granulocyte...

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