Immunology 1990 71 442-448

Lymphocyte activation and expression of the human leucocyte-endothelial cell adhesion molecule 1 (Leu-8/TQ1 antigen) C. BOHRER, C. BERLIN, H.-G. THIELE & A. HAMANN Department of Medical Immunology, Eppendorf University Hospital Hamburg, FRG Acceptedfor publication 19 June 1990

SUMMARY The Leu-8/TQ I antigen, recently shown to be the human equivalent ofthe murine MEL- 14 lymph node homing receptor and now designated as leucocyte-endothelial cell adhesion molecule, LECAM-1, has been used to dissect T, B and natural killer (NK) cells into various subpopulations with different functional properties, such as suppressor-inducer (CD4+ Leu-8/TQl +) or helper (CD4+ Leu-8/TQ1-) T lymphocytes. Diminished Leu-8/TQI antigen density has been reported after lymphocyte activation with B- or T-cell specific stimuli for several days. In the present study, we sought to determine the signal requirements for altered Leu-8/TQ1 expression. Exposure of freshly isolated peripheral blood lymphocytes to phorbol 12-myristate 13-acetate (PMA) and/or calcium ionophores was found to cause rapid down-regulation of the Leu-8 antigen, with little or no Leu-8 reactivity remaining I hr after simultaneous addition of PMA and calcium ionophores to the culture medium. TQI reactivity was changed in parallel with that of Leu-8. Leu-8 expression of T and B cells was sensitive to both PMA and calcium ionophores, whereas Leu-8 expression of NK cells was affected by PMA but not by the calcium ionophore A23187. The effect of PMA on Leu-8/TQl expression could be antagonized by pretreatment with the protein kinase C inhibitor, H7, whereas that of A23187 could not. Partial re-expression of the Leu-8 antigen was seen 3 days after addition of PMA alone but not when it was given together with calcium ionophores. After Leu-8 downregulation induced by 1 hr treatment with PMA or calcium ionophores, Leu-8 re-expression was observed within 24 hr when stimuli were removed by washing. Activation of T cells with anti-CD3 antibody resulted in a partial reduction of Leu-8/TQl expression, first detectable after 1-3 hr of stimulation and maximal at 24 hr. Fully mitogenmic concentrations of concanavalin A (Con A) did not affect Leu-8 expression within the first hours of culture. Transient reduction of Leu-8 expression was seen 24 hr after supraoptimal dosages of Con A, but Con A-stimulated lymphocytes cultured for 4-12 days with interleukin-2 (IL-2) retained Leu-8/TQl reactivity to a similar degree as fresh cells. In contrast to Leu-8/TQl, CD45RA expression was not altered after 1 hr treatment with PMA and/or calcium ionophores but gradually disappeared during 1 week after Con A stimulation. Thus, Leu-8/ TQ1 +CD45RA+ lymphocytes could be rapidly converted into Leu-8/TQ1 -CD45RA+ cells by PMA and/or calcium ionophores in vitro within 1 hr, whereas Leu-8/TQl +CD45RA- cells were generated by Con A stimulation over several days. INTRODUCTION

Functionally heterogeneous subpopulations of leucocytes may be distinguished by their expression of specific arrays of cellsurface antigens. The monoclonal antibodies (mAb) anti-Leu-8 Abbreviations: CD, cluster determinant; Con A, convanavalin A; DMSO, dimethyl sulphoxide; FCS, foetal calf serum; FITC, fluorescein isothiocyanate; IL-2, interleukin-2; mAb, monoclonal antibody; NK, natural killer; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate. Correspondence: Dr A. Hamann, 1. Medizinische Klinik, Abtl. Immunologie, Universitaitskrankenhaus Eppendorf, Martinistr. 52, D-2000 Hamburg 20, FRG.

and TQI (Gatenby et al., 1982; Reinherz et al., 1982) have been shown to react with a molecule now termed LECAM-1 (leucocyte endothelial cell adhesion molecule 1) which is present on virtually all human peripheral blood-derived granulocytes, monocytes, roughly two-thirds of T- and B-lymphocytes, and one-third of NK cells (Lanier & Loken, 1984; Kansas, Wood & Engleman, 1985a). The Leu-8/TQI cDNA has been cloned, and displays a 77% amino acid homology to the murine MEL-14-defined peripheral lymph node homing receptor (Camerini et al., 1989; Tedder et al., 1989; 1990). This 90,000 molecular weight (MW) glycoprotein is thought to mediate organ-specific interactions between lymphocytes and high endothelial venules in the process of lymphocyte extravasation into peripheral lymph nodes.

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Leu-8/TQI expression Recently, additional mAb have been raised and shown to react with the Leu-8/TQ I antigen (Kishimoto, Jutila & Butcher, 1990; Tedder et al., 1990). Some antibodies were found to inhibit human lymphocyte binding to high endothelial venules of murine peripheral lymph nodes, indicating that the human homologue of the MEL-14 antigen may function as a homing receptor as well. Subpopulations of T and B cells defined on the basis of low or high Leu-8/TQl expression differ in their functional characteristics (Gatenby et al., 1982; Kansas et al., 1985a, b; Reinherz et al., 1982; Damle, Mohagheghpour & Engleman, 1984; Takada, Koide & Engleman, 1989). This is consistent with the finding that the expression of Leu-8 antigen changes upon activation of lymphocytes: treatment of Leu-8+ B cells with the combination of rabbit anti-human p-chain and B-cell growth factor, but not with either agent alone, over 48-72 hr has been demonstrated to cause the loss of Leu-8 expression in addition to causing these cells to proliferate (Kansas et al., 1985b). Leu-8 expression of unfractionated peripheral blood lymphocytes (PBL) when activated with phytohaemagglutinin (PHA), antiCD3 plus phorbol 12-myristate 13-acetate (PMA) or ionomycin plus PMA for 3 days has been demonstrated to be strongly reduced; the marked decline was reported to occur between 8 and 24 hr (Kanoff & James, 1988). In contrast, CD4+Leu-8+ cells cultured continuously with irradiated allogeneic stimulator cells for up to 5 weeks were shown to display only a minor reduction of Leu-8 antigen density and clearly remained Leu-8 +. In the present study, we sought to determine signal requirements for alterations of Leu-8 expression. Our results suggest that extremely rapid Leu-8 down-regulation can be elicited with agents known to directly activate protein kinase C (PKC), such as PMA and/or calcium ionophores, increasing intracellular Ca2+ levels. Recently, further evidence for rapid down-regulation of the molecule by PMA has been published (Kishimoto et al., 1990; Tedder et al., 1990). Slow and partial down-regulation was found to be induced by activation via anti-CD3, whereas only supraoptimal concentrations of the lectin concanavalin A (Con A) were able to reduce Leu-8/TQl expression transiently after 24 hr.

MATERIALS AND METHODS Cell culture Human peripheral blood mononuclear cells (PBMC) were obtained from healthy human volunteers (laboratory personnel or blood donors) by Ficoll-Hypaque (Pharmacia, Freiburg, FRG) density gradient centrifugation. Cells were resuspended in RPMI-1640 with 25 mm HEPES,2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% foetal calf serum (FCS; Pan Systems, Aidenbach, FRG) and cultured in 12-well plastic plates (Costar, Cambridge, MA). Phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, the PKC inhibitor H7 (1-(5-isoquinolylsulphonyl)-2-methylpiperazine) (all from Sigma, Deisenhofen, FRG), and ionomycin (CalbiochemBehring, Marburg, FRG) were kept frozen in DMSO and diluted just prior to use. Monoclonal anti-CD3 antibody BMA 030 (Behring, Marburg, FRG) was used at a concentration of 50 ng/ml. Concanavalin A (Con A) was purchased from Serva, Heidelberg, FRG. Human recombinant interleukin-2 (IL-2;

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Cetus, Emeryville, CA) was used at a final concentration of 10 U/ml. Immunofluorescence analysis Mouse anti-human mAb anti-Leu-8, anti-Leu-4 (CD3), antiLeu-3a (CD4), anti-Leu-2a (CD8a), anti-Leu-M3 (CD 14), antiLeu-l la (CD16), anti-Leu-16 (CD20), anti-Leu-18 (CD45RA), and isotype-matched controls directly coupled to FITC or phycoerythrin (PE) were purchased from Becton-Dickinson, Heidelberg, FRG. mAb TQl was purchased from Coulter Electronics, Krefeld, FRG, and FITC-coupled mAb IOT14 (CD25) was from Immunotech, Marseille, France. For immunofluorescence staining, non-adherent cells (less than 0-5% anti-Leu-M3+) were harvested at the time-points indicated, and dead cells were removed by metrizamide (Nycomed, Oslo, Norway) cushion centrifugation. Subsequently, all incubations were carried out on ice in the presence of 0 1 % NaN3 and 1 mg/ ml human immunoglobulin. Cells were incubated with mAb for 30 min, washed three times, and incubated for further 30 min with FITC-conjugated goat anti-mouse immunoglobulin (Becton-Dickinson), unless directly labelled mAb had been used. After washing as above, cells were fixed with 1 % paraformaldehyde and analysed on a FACSCAN flow cytometer (BectonDickinson, Heidelberg, FRG). For double marker analysis, cells were stained simultaneously with anti-Leu-8-PE and FITC-coupled anti-Leu-l la, anti-Leu-16 or anti-Leu-18, respectively, and analysed with appropriate compensation and gate setting. In some experiments, B cells were labelled with antiLeu-12-PE (Becton-Dickinson) and gated out upon analysis. RESULTS

Exposure of freshly obtained PBMC to PMA results in rapid down-regulation of the Leu8/TQl antigen in a time- and dosedependent manner When PBMC were treated with PMA at I ng/ml for 10 min, Leu-8 expression was found to be substantially reduced (Fig. lb). The degree of Leu-8 down-regulation increased with time and PMA dosage (Fig. I c, ld). One hour after lO ng/ml PMA or 3 hr after I ng/ml there was virtually no anti-Leu-8 reactivity detectable by flow cytometric analysis. PMA dosages at or below 0-3 ng/ml had no effect on Leu-8 expression while still being partly blastogenic, as measured by increased forward scatter at 48 hr. Reactivity with mAb anti-TQ1 always paralleled that of mAb anti-Leu-8 (data not shown).

Calcium ionophores can substitute for PMA in inducing Leu-8/ TQ1 down-regulation and show marked synergism with PMA Calcium ionophores A23187 or ionomycin were also found to elicit Leu-8 down-regulation. A23 187 at I Mg/ml or more

resulted in Leu-8 down regulation (Fig. I e), plateauing 1 hr after addition of the calcium ionophore to the culture medium. Leu-8 down-regulation was more pronounced at 2 yg/ml, whereas A23187 at 0 5 pg/ml was not effective by itself. However, the latter dosage was found to augment PMA (1 ng/ml)-induced Leu-8 down-regulation (Fig. lf). A23187 (1 pg/ml) plus PMA (I ng/ml) lead to almost complete loss of Leu-8/TQI reactivity on the vast majority of lymphocytes within 1 hr, as measured by FACS analysis. Similar results were obtained with ionomycin instead of A23187 (data not shown).

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Figure 1. Effects of PMA and/or calcium ionophore A23187 on Leu-8 ) IgG2) Leu-8-FITC, ( expression of human lymphocytes. ( FITC control. (a) unstimulated cells; (b) PMA 1 ng/ml, 10 min; (c) PMA 1 ng/ml, I hr; (d) PMA lOng/ml, 0min; (e)A23187 2Mg/ml, 0min; (f) A23187 1 pg/ml+PMA 1 ng/ml, 10 min. .

In addition to down-regulation of Leu-8, culture with PMA but not A23187 for 1 hr reduces mean surface expression of CD3 and CD4 (but not CD8)

The median fluorescence of freshly obtained lymphocytes stained with FITC-coupled anti-CD3 or anti-CD4 mAb was found to be slightly but measurably reduced after 1 hr treatment with PMA but not with A23187 (Fig. 2). CD8 expression was not affected at that time-point, nor were the CD4/CD8 or CD8/ CD3 ratios. Mean fluorescence of CD16 (Leu-I la) and CD20 (Leu-16) was also invariant. NK, T, and B cells differ in their responsiveness to PMA and calcium ionophores with respect to Leu-8/TQ1 expression Lymphocytes stimulated as above were stained simultaneously with phycoerythrin (PE)-conjugated anti-Leu-8 and FITCcoupled anti-Leu-1 la (CD 16) or anti-Leu-16 (CD20), respectively, and CD16- or CD20- cells were gated out. CD16+ lymphocytes showed marked decline of Leu-8 expression when stimulated with PMA but not with A23187 (Fig. 3a), whereas CD20+ cells had decreased Leu-8 expression in response to both PMA and A23187 similarly to CD20- lymphocytes (Fig. 3b). The PKC inhibitor, H7, antagonizes the effect of PMA but not that of calcium ionophores When cells were treated with 100 pM H7 (1-(5-isoquinolylsulphonyl)-2-methylpiperazine) for 20 min at 370 prior to addition

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of PMA (3 ng/ml) or A23 187 (2 jg/ml) for another hour, Leu-8 expression was only slightly affected by the presence of PMA (Fig. 4a). However, A23187 reduced Leu-8 expression regardless of whether PBMC had been treated with H7 or not (Fig. 4b).

The Leu-8 antigen is re-expressed after removal of stimuli Leu-8 down-regulation induced by PMA and/or calcium ionophores persisted for at least 48 hr when stimuli were not removed by washing. To assess whether loss of the Leu-8 antigen was inherently permanent or potentially transient, Leu8 down-regulation was induced by treatment of cells with PMA (3 ng/ml) or A23 187 (2 pg/ml) for 1 hr. Sequentially, cells were washed once and cultured in fresh medium for an additional 23hr time period. Immunofluorescence analysis showed that Leu-8 was re-expressed thereafter regardless of the stimulus employed to induce down-regulation in the first place. Partial re-expression was also seen 72 hr after stimulation with PMA (10 ng/ ml) without deliberate removal of the stimulus, whereas reexpression could not be observed when PMA was given together with calcium ionophores.

Mitogenic stimulation with anti-CD3 or Con A does not induce the rapid and complete Leu-8/TQ1 down-regulation observed after PMA and/or calcium ionophores Activation of T cells with anti-CD3 antibody resulted in slow and only partial reduction of Leu-8. The decrease became detectable after 1-3 hr stimulation. Maximal reduction was observed at 24 hr with about 50% negative cells, whereas at 48 hr of stimulation, Leu-8 reactivity appeared to rise again (Fig. 5).

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102 101 1 104 10° Log fluorescence intensity _ Figure 4. The PKC inhibitor H7 antagonizes the effect of PMA (3 ng/ml) but not that of A23 187 (2 pg/ml) on Leu-8 expression. Fresh lymphocytes were treated either with 100 pM H7 ( ) or medium ( - - - ) for 20 min prior to addition of PMA (a), A23 187 (b), or medium (c) for ) IgG2-FITC control. another hour ( .

remaining after 12 days whereas Leu-8 was still expressed by the majority of cells (Fig. 6c). Blastogenic concentrations of Con A (1-20 Mg/ml) caused no change of Leu-8 expression by PBMC within the first hours of culture. Transiently reduced Leu-8 expression was observed 24 hr after stimulation with Con A at 16 Mg/ml but not with 4 ug/ ml. Both concentrations were equally effective in inducing CD25 (data not shown). Con A in excess of 20 pg/ml was associated with considerable toxicity, resulting in high loss of viable cells by 24 hr. After 4-12 days of culture in the presence of recombinant human interleukin-2 (rhIL-2), Con A-stimulated lymphocytes displayed similar Leu-8 reactivity as unstimulated or fresh cells (Fig. 6). Leu-8/TQ1 and CD45RA are differentially regulated by PMA/ calcium ionophores and the mitogenic plant lectin, Con A Double marker analysis showed that approximately 37% of freshly obtained lymphocytes stained double positive for Leu-8 and CD45RA (Leu-18), whereas Leu-8+ CD45RA- cells accounted for 31%, and Leu-8- CD45RA+ cells for approximately 16% (Fig. 6a). After I hr of PMA, over 90% of the lymphocytes were Leu-8-, whereas expression of CD45RA was not affected. The absolute number of cells did not change during the culture period. Thus, most lymphocytes previously displaying a Leu-8+ CD4RA+ phenotype could be converted into Leu8- CD45RA+ cells during a 1-hr incubation period with PMA in vitro (Fig. 6b). In contrast, Con A-stimulated lymphocytes gradually lost CD45RA, with less than 10% CD45RA reactivity

DISCUSSION Various cell responses are triggered by receptor-mediated hydrolysis of inositol phospholipids. This breakdown results in generation of diacylglycerol and inositol phosphates which are both thought to act as second messengers (Nishizuka, 1984, 1986). Whereas diacylglycerol directly activates PKC by increasing its affinity for Ca2 inositol phosphates cause elevation of intracellular Ca2+ levels. Experimentally, a rise of cytosolic Ca2+ levels can be achieved by calcium ionophores such as A23187 or ionomycin, whereas prolonged action of diacylglycerol can be mimicked by tumour-promoting esters like PMA (Castagna et al., 1982). In this study, PMA and calcium ionophores were used to bypass early activation steps of human lymphocytes. Both PMA and calcium ionophores elicited rapid down-regulation of the Leu-8 antigen in a time- and dose-dependent manner. Pretreatment of PBMC with the PKC inhibitor H7 abolished downregulation induced by PMA but failed to block calcium ionophore-mediated Leu-8 down-regulation. Appropriate conditions lead to almost complete disappearance of the Leu-8 antigen within 1 hr from peripheral blood-derived lymphocytes. T and B cells were sensitive to both PMA and calcium ionophores with respect to Leu-8, loss whereas NK cells (CD16+) showed reduced Leu-8 expression only after PMA. Mitogenic plant lectins like Con A or antibodies against the ,

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