Immunobiol., vol. 180, pp. 221-234 (1990)

IDepartment of Medical and Physiological Chemistry, and 2Department of Anatomy, and 3Department of Tumor Immunology, University of Lund, Lund, Sweden

Alphal-Microglobulin is Mitogenic to Human Peripheral Blood Lymphocytes. Regulation by both Enhancing and Suppressive Serum Factors::' HASSABO BABIKER-MoHAMED I ,2, MARTIN L. OLSSON I , AKE BOKETOFT3 , LENNART LOGDBERd, and Bo AKERSTROM

I

Received March 6, 1989 . Accepted in Revised Form October 19, 1989

Abstract Human al-microglobulin (al-m), a 26 kilodalton serum glycoprotein, was found to exert mitogenic effects on human peripheral blood lymphocytes (PBL) in serum-free medium. Purified T cells, but not B cells, responded with proliferation to al-m, but only in the presence of monocytes. The mitogenic activity could be partially neutralized by a mouse monoclonal antibody against al-m. The mitogenicity was species-specific, since al-m homologues from rats, guinea pigs and rabbits had no effect on human PBL. In a previous study, no effect of al-m was seen on PBL in the presence of 20 % serum, and, therefore, we studied the influence of different concentrations of serum on the al-m-induced mitogenicity. Thus, human serum enhanced the mitogenic effects of a)-m on human PBL at 1 % concentration (v/v) and suppressed the effects at 10 %. The suppressing effect of serum at 10 %, but not the enhancing effect at 1 %, seemed to be conserved among several species. To test the effect of serum proteins of different molecular sizes, human autologous serum was separated by gel chromatography on Sephadex G-200 into four fractions. Fractions 1 and 2 (roughly containing proteins larger than 100 kilodaltons) suppressed the mitogenic effects of al-m, while fractions 3 and 4 enhanced the stimulation by al-m, at 0.5 % and concentrations above. It is concluded that the mitogenic effect of al-m on lymphocytes is regulated by several serum factors, both enhancing and suppressive, that does not have any proliferative effect of their own. It can be speculated that the balance between enhancing and suppressing co-factors in the blood determines the degree of tbe stimulation of lymphocytes by al-m. This is compatible with an immunomodulatory role for a)-m, in spite of its relatively constant plasma levels in health and disease.

Introduction The plasma protein al-microgiobulin (al-m), 26 kilodaltons (kDa) (1), has immunoregulatory properties. It has been shown to inhibit the pro"-This work was supported by grants from the Swedish Medical Research Council (projects no. 7044 and 7504), the Medical Faculty, University of Lund, the Foundations of A. Osterlund, G. and J. Kock, and O. E. and E. Johansson, Svenska, Likaresallskapet, and Gustav V:s 80-ars fond. Abbreviations: al-m = al-microglobulin; PBI = peripheral blood lymphocytes; FCS fetal calf serum; 2ME = 2-mercaptoethanol; PMA = phorbol myristate acetate.

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222 . HASSABO BABIKER-MoHAMED et al.

liferative response of lymphocytes to antigens (2, 3), inhibit the migration and chemotaxis of granulocytes (2, 4), and to have a mitogenic effect on guinea pig T-Iymphocytes (3). A receptor for al-m has been demonstrated on the human histiocytic cell-line U937 (5). aem is a glycoprotein (6), and glycopeptides from the protein, containing asparagine-linked oligosaccharides, carries at least some of the inhibitory effects on antigen-induced proliferation (7). The amino acid sequence of the protein has been determined, and is unique (8). al-m, also known as protein He (8), is synthesized by the liver (10, 11), and human and rat liver eDNA coding for the protein were recently cloned and sequenced (12, 13). The mRNA also codes for HI-30, the active domain of another plasma protein, inter-a-trypsin inhibitor (13). However, no protein carrying epitopes from both aem and inter-a-trypsin inhibitor has yet been reported to be present in plasma. Instead, a complex between al-m and IgA has been purified from human serum (14), and comprises approximately 'half of the total al-m in serum, 20-50 mg/l (1). An immunoregulatory role for al-m must be compatible with the fact that its plasma concentration is stable during a variety of malign and benign disorders, including neoplastic diseases and inflammatory conditions (15, 16). To study this, we investigated the effects of human al-m on peripheral blood lymphocytes (PBL) in the presence of various amounts and fractions of serum. Our results indicate that the protein is mitogenic to PBL, that this effect is regulated by several serum factors, and that the responding cells are T cells in the presence of monocytes. Materials and Methods Preparation of lymphoid ceJIs Human peripheral blood was obtained from healthy donors, and PBL were separated by Ficoll-Paque (Phamacia Fine Chemicals, Uppsala, Sweden) density centrifugation at 920 x g for 18 min (17). PBL were collected from the interface and washed twice with PBS solution and once with the medium used in the culture. T cells, B cells, and monocytes were purified by a procedure described by WAKASUGI et al. (18), modified by CARLSSON et al. (19). The viability of the cells was determined by trypan blue dye exclusion. Proteins and sera al-m homologues were purified from the urine of humans (1), rats (10), rabbits (20) or guinea pigs (21) as described. Mouse monoclonal antibody BN11.3 directed against human and rat al-m was prepared and purified as described (22). A mouse monoclonal antibody directed against a mouse leukemia virus antigen was obtained from THOMAS BRODIN, Department of Tumor Immunology, University of Lund, Sweden. Sera from fetal calves and horses were bought from Flow Laboratory, Irvine, Scotland, and cow, rat, rabbit, and guinea pig serum were obtained by venous bleeding of healthy animals. Reduction and alkylation of disulfide bonds Human arm was dissolved in 1 m16M guanidine-HCl, 0.2M Tris-HCl, pH 8.5, containing 0.02 M dithioerythritol. After incubation for 90 min, iodoacetamide was added to 0.05 M, and

Serum Factors Regulate arm Mitogenicity . 223 incubated for 30 min in the dark. The solution was then dialysed exhaustively against water and freeze-dried.

Gel chromatography of human serum Human serum was fractionated by gel chromatography on Sephadex G-200 (Phamacia Fine Chemicals, Uppsala, Sweden). The column was equilibrated and eluted with 20 mM Tris-HCI, pH 8.0 + 0.15 M NaCI and 0.02 % NaN3 at 4°C and a flow rate of about 1.5 % of the total column volume per hour. The serum sample was centrifuged for 4 h at 50,000 x g, and the fatty coat discarded, before application to the column. The total proteins in the eluted fractions were determined by reading the absorbance at 280 nm and then pooled into four fractions. Each of these were concentrated by ultrafiltration as described (23) through a membrane with a cut-off at approximately 3 kDa, to the double sample volume, which later was compensated for in the cell cultures. The concentrated fractions were dialysed against PBS and sterile filtrated before having been added to the cell cultures.

Culture media Cell cultures were performed in RPMI 1640 or the serum-free Iscove's medium, an enriched modification of Dulbecco's modified Eagle's medium where serum is totally replaced by albumin, transferrin, and soybean lipid (Flow Laboratory) (24). RPMI was supplemented with L-glutamine (300 f!g/ml), 50 f!M 2-mercaptoethanol (2 ME) and 10 % fetal calf serum (Flow). Iscove's medium used in the cultures was with or without 2 ME, and with or without sera from humans, fetal calves, cows, horses, or human serum fractions (see above). al-m or monoclonal anti-al-m was dissolved in the appropriate medium before addition to the cultures.

Lymphocyte cultures and assay of DNA-synthesis The conditions for cultivation of PBL has been outlined earlier (7). PBL were cultured in RPMI or in serum-free Iscove's medium in 96-well microtiter plates (flat bottom plates from Nunc A/S Roskilde, Denmark), in a final volume of 0.2 ml medium and a cell concentration of 106/m!. After 3 days of culture, each culture was pulsed for 6h with 1 f!Ci of tritiated thymidine (Amersham, Buckinghamshire, U.K.). The cells were then collected with a semiautomated microharvesting apparatus, and the amount of radioactivity incorporated into cellular DNA was determined in a beta-liquid scintillation counter. All cultures were run in triplicates, and the results are given as mean cpm per culture. The SEM of triplicate samples seldom exceeded 10 % of the mean.

Results

Mitogenic effects of al-m on PEL in serum free medium It was found earlier that the addition of ul-m to human PBL in RPMI supplemented with 20 % human serum did not increase the subsequent eH]-thymidine uptake into the cells at any time during seven days of culture (2). However, a marked increase in the thymidine uptake was noted when the cells were cultured in serum-free medium for three days in the presence of ul-m. Table 1 shows a comparison between the effects of ul-m in the presence of 10 % serum and in serum-free medium. For the serumfree cultivation Iscove's medium was used, since only a small fraction of the cells survived in RPMI -medium without serum supplementation. The mitogenic effects of ul-m are dose dependent: the degree of stimulation increases with higher concentrations beyond the normal serum levels of

224 . HASSABO BABIKER-MoHAMED et a!. Table 1. Mitogenic effects of human arm (0.50 mg/ml) on human PBL, cultured in Iscove's medium with no serum addition or in RPMI medium with 10 % fetal calf serum eHJ-thymidine uptake a Iscove's medium

RPMI medium +10% FCS

Exp. 1 controlb al-m ConAe

250 4,140 23,942

173 569 8,602

Exp.2 control arm Con A

470 11,194 32,112

422 1,302 10,336

Exp.3 control arm Con A

1,154 10,470 30,471

423 1,711 9,602

Mean cpm of triplicate cultures. No addition of al-m. e The concentration of Con A was 0.5 ~g/m!. a

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Figure 1. Dose-response of human al-m in serum free medium. Human al-m was added at various concentrations to human PBL cultures in a total volume of 0.2 ml of culture medium and a cell concentration of 1 x 106/m!. [3HJ-thymidine uptake was measured after 72 h. Each point represents the mean cpm value of triplicate cultures.

Serum Factors Regulate arm Mitogenicity . 225

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Figure 2. Stimulatory effects of al-m on the proliferation of purified blood cell populations. PBL (2 x 10 5 ), purified T cells, or B cells were cultures separately or in different combinations with or without addition of purified monocytes (10 4 ). Human al-m was added to 0.5 mg/ml in a total volume of 0.2 m!. The bars represent the eHJ-thymidine uptake measured after 72 hand are expressed as the average of triplicate cultures. Con A (0.5 [,Ig/ml) was used to test functionally the puriry of purified T and B cells. T cells, B cells, or T and B cells were not stimulated by Con A, whereas addition of monocytes resulted in proliferation of T cells but not B cells. All cells were judged viable by trypan blue exclution.

al-m (Fig. 1). The results indicate a saturation of the target systems at al-m concentrations above 0.5 mg/ml. Studies on the time-course of the mitogenic effect showed that maximal effects were obtained after three days incubation with al-m (data not shown). We wanted to study the identity of the responding cell and the requirements of accessory cells. Thus, highly purified T cells or B cells were cultivated alone, in the presence of monocytes, or as a mixture of T and B cells with or without monocytes. As shown in Figure 2, al-m stimulates T cells to proliferate when monocytes are present. No effect is seen on T cells or B cells alone, or B cells with monocytes. Full proliferation is achieved in the mixture of T cells and monocytes, and, of course, T cells, B cells and monocytes. A low degree of proliferation is also obtained when T and B cells are mixed, indicating that also some B cells can function as accessory cells. It may also be explained by the presence of small amounts of monocytes in the B cell preparation (19). Table 2 shows that the addition of 2 ME enhances the mitogenic effects of al-m on human PBL. Reduction and alkylation of al-m do not promote the stimulatory effects of the protein (not shown). This,indicates that the reason

226 . HASSABO BABIKER-MoHAMED et al. Table 2. Mitogenic effects of human arm (0.50 mg/ml) on human PBL, cultured in Iscove's medium with or without 2 ME eHJ-thymidine uptake" without 2ME

with 2MEb

Con Ad

577 1,402 15,576

797 4,854 22,851

Exp.2 control al-m Con A

1,933 4,555 17,743

805 6,736 21,898

Exp.1 controlC

arm

" Mean cpm of triplicate cultures. b 50 [tM C No addition of al-m. d The concentration of Con A was 0.5 [tg/ml.

for the enhancement is not at the protein level, but probably an effect on cells or intercellular signaling. All other experiments were performed in the presence of 2 ME. al-m was clearly mitogenic to PBL from five different subjects (Table 3). Thus, the stimulation is not critically dependent on individual variation of Table 3. Mitogenic effects of human arm (0.50 mg/ml) on human PBL from five individuals, cultured in Iscove's medium eHJ-thymidine uptake"

Person no. controlb al-m ConAc

655 5,187 21,417

2

control al-m Con A

805 4,555 20,600

3

control

805 9,181 31,668

arm Con A 4

control Con A

933 4,591 21,865

control al-m Con A

311 6,983 24,628

arm 5

" Mean cpm of triplicate cultures. b No addition of al-m C The concentration of Con A was 0.5 [tg/ml.

Serum Factors Regulate al-m Mitogenicity· 227 Table 4. Mitogenic effects of 0.50 mg/ml al-m from different species on human PBL in Iscove's medium [3HJ-thymidine uptake"

arm homologue

controlb human rat rabbit gumea pIg ConAc a

b C

Exp.1

Exp. 2

Exp. 3

Exp.4

Exp. 5

797 6,802 700

2,050 11,200 2,350

933 4,591 431

530 3,817 290

919

539 21,417

1,912 31,608

580 21,860

229 14,533

1,214 634 22,558

Mean cpm of triplicate cultures. No addition of arm. The concentration of Con A was 0.5 flg/ml.

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