Scand. J. Immunol. 8, 127-134, 1978
P.-Microglobulin from Normal and Leukaemic Guinea-Pig Lymphocytes F. K. STEVENSON, M. W. J. CLEETER & G. T. STEVENSON Tenovus Research Laboratory, General Hospital, Southampton, UK
Stevenson, F.K., Cleeter, M.W.J. & Stevenson, G.T. Pj-Microglobulin from Normal and Leukaemic Guinea-Pig Lymphocytes. Scand. J. Immunol. 8, 127-134, 1978. Pu-Microglobulin has been isolated in useful quantities from the urine of strain-2 guinea-pigs after either treatment with sodium chromate or induction of the h^C leukaemia. Antibodies raised against the Pj-microglobulin were used to set up a radioimmunoassay which measured its export into culture fluid by normal and leukaemic lymphocytes. Material containing p^-microglobulin was also obtained by digestion of the lymphocytic surfaces with papain; fractionation demonstrated both free and combined forms, with no qualitative difference between those from normal and those from leukaemic cells. F. K. Stevenson, Tenovus Research Laboratory, General Hospital, Southampton SO9 4XY, UK.
p2-Microglobulin was first isolated from the urine of patients with tubular proteinuria in 1968 by Berggard & Beam [5]. It was described as a protein of low molecular weight (11,600 daltons) with an electrophoretic mobility in the P2 region. Although these workers considered that it might form part of a larger molecule and suggested a possible analogy with the light chains of immunoglobulin molecules, it was not until 1972 that its broader significance emerged. At this time it was reported that Pj-microglobulin was present in lymphocytic plasma membranes [6, 20, 22], and its association with histocompatibility antigens was recognized shortly afterwards [12, 17]. There is good evidence of non-covalent bonding between Pa-microglobulin and the HL-A alloantigens in human cell membranes, and such an association has been demonstrated recently in the guinea-pig lymphocytic membrane [7]. However, it should be noted that spontaneous non-covalent association of l with other molecules can occur [24].
Another problem is whether or not the association with histocompatibility antigens accounts for all the pj-rficroglobulin in the plasma membrane. 'Capping' studies on human lymphocytes have shown that not all is associated with HL-A antigens [18, 25], although in mouse splenocytes it has been claimed that all the surface Pj-microglobulin can be accounted for by association with H-2, Tla (thymusleukaemia) [29] and Qa-2 antigens [16]. Biosynthetic studies have demonstrated in several systems that free Pj-microglobulin is produced by lymphocytes in culture, but in neither human nor mouse lymphocytes [30] was it possible to demonstrate histocompatibility antigens in the culture supernatant. This was in spite of the fact that for human lymphocytes both Pa-microglobulin and histocompatibility antigens appeared to have similar turnover rates on the cell surface. Our findings in the guinea-pig are similar to those in man and mouse in that under the conditions used, both normal and leukaemic
0300-9475/78/0800-0127 $02.00 © 1978 Blackwell Scientific Publications
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F. K. Stevenson, M. fV. J. Cleeter & G. T. Stevenson
lymphocytes give rise to free P2-microglobulin in the culture medium, but not to Pj-microglobulin-associated material of high molecular weight. Papain digestion of the cell surfaces, however, gave rise to two Pa-microglobulincontaining fractions, one corresponding to the free molecule and the other with a molecular weight of approximately 46,000 daltons.
MATERIALS AND METHODS Isolation of f^^-microglobulin. Isolation of guinea-pig Pj-microglobulin from urine of sodium chromatetreated animals was essentially as described by Berggird [3] for rabbit Pj-m'croglobulin. Briefly, eight strain-2 guinea-pigs were injected subcutaneously with sodium chromate (10 mg per kg body weight), and 24 h specimens of urine were collected into a few drops of toluene by means of metabolic cages. Tests for protein with Albustix (Ames Company) became strongly positive on day 3 after injection, and urines were collected from days 3 to 7 inclusive. Pooled urines were centrifuged and dialysed into 0.1 M Tris-HCI-1.0 M NaCl, pH 8.0. Analysis for protein by the Lowry method [15] showed excretion of 120 mg/24 h per guinea-pig, whereas normal urine showed 9 mg/24 h. The urine was then concentrated in an Amicon ultrafiltration apparatus using a UM-2 membrane (quoted lower exclusion limit, 1000 daltons), and 500 mg of protein was loaded onto a column of Sephadex G-150 equilibrated with the Tris buffer. The low molecular weight fraction from this column was collected and fractionated further on DEAE-cellulose (Whatman DE32) using a starting buffer of 0.01 M Tris-HCl, pH 7.8, followed by a gradient to 0.4 M NaCl. It was possible to follow purification of the PJmicroglobulin by means of its ability to block the reactivity of rabbit anti-human Pj-microglobulin (Dakopatts) with guinea-pig LjC leukaemia cells as assessed by indirect immunofluorescence. The peak with most of this activity was collected and used for immunization. Guinea-pig Pj-microglobulin was also prepared from the urine of strain-2 guinea-pigs in the terminal stage of the LjC leukaemia. It is known that during the development of human leukaemia or lymphoma quite large amounts of Pj-m'croglobulin are produced [reviewed in Ref. 19]; we therefore hoped to prepare P^-microglobulin from animals without recourse to sodium chromate. The excretion of monoclonal X light chain by these guinea-pigs has been reported previously [26]. It was necessary to remove this before further processing the urine. This was done by passing urine which had been dialysed into 0.02 Tris-HCl-0.1 M NaCl-0.02% NaN;,, pH 8, through an immunosorbent consisting of Sepharose 4B to which an IgG fraction from a sheep anti-guinea-pig X chain serum had been covalently coupled [1]. Aliquots of 25 mg urinary protein were passed through a column of 50 ml; the yield of urinary protein after immunosorption was 35%. The protein not bound by the immunosorbent was
further purified by ion-exchange chromatography on DEAE-ce!lulose, as for sodium chromate-induced Pjmicroglobulin, and again monitored by blocking activity using L^C leukaemic cells. The two preparations were compared by electrophoresis on polyacrylamide gel and by crossreactivity with an antiserum raised against rabbit p^-microglobulin kindly provided by Dr I. BerggSrd. Immunization of a sheep and preparation of antibody. The p2-microglobulin obtained from the sodium chromate-treated guinea-pigs was used to raise an antiserum in a sheep. A Clun cross-bred sheep was immunized with 1 mg p2-microglobulin in complete Freund's adjuvant (CFA) in four intramuscular sites. A second injection of antigen in CFA was given 5 weeks later in the back, and blood was taken 1, 2 and 3 weeks after this. Blood was collected onto glass beads for defibrination and the serum prepared by centrifugation. The antiserum reacted with pj-microglobulin by immunodiffusion, and detected it thus in normal urinary protein prepared by 50-fold concentration of normal urine through an Amicon UM-2 membrane. In fact it was present in the latter in suflicient quantity to yield an immunosorbent (Sepharose 4B conjugated to normal urinary protein, 1 ml to 10 mg) suitable for preparing purified antibody. Antiserum (10 ml) was passed through the immunosorbent (30 ml) and the column washed thoroughly with 0.02 Tris-HCl-0.1 M NaCl0.02% NaNj. The absorbed antibody was then eluted with 0.5 M NH4OH and immediately dialysed into cold Tris buffer. Further analysis of the purified antibody by immunodiffusion showed reactivity with the immunogen (two lines) and with the Pj-microglobulin obtained from leukaemic urine (one line). There was thus evidence of a second (unidentified) component in the immunogen, not detectable in the preparation from leukaemic urine. It was therefore decided to set up the radioimmunoassay using purified antibody and to use the Pj-microglobulin from leukaemic urine as the labelled antigen. Radioimmunoassay (RIA). Radioimmunoassay for guinea-pig Pj-microglobulin was carried out by allowing the test antigen and a radioactive standard antigen to compete for antibody coupled to beads of Sephadex G-25 superfine [9]. The coupled antibody was the purified sheep anti-Pn-microglobulin, and the radiolabelled antigen, at a specific activity of "^I of approximately 85 Ci/g, was P^-microglobulin obtained from leukaemic urine. Radioiodination was by the use of chloramine-T, and labelled protein was always purified from possible breakdown products by passage through a column of Sephadex G-25 (fine). Radiolabelled antigen (450 pg) in 0.1 ml was added to 1.0 ml of a solution of the test antigen; 1 ml of immunosorbent at a concentration of solid phase which bound 8-10% of labelled antigen when this alone was present was then added. All dilutions were with assay buffer (0.05 M phosphate buffer containing NaCl, 0.08 M, and sodium azide, 0.05%, pH 7.3) to which horse haemoglobin (Miles) had been added to give a concentration of 2 mg/ml. After incubation for 48 h the solid phase was separated by centrifugation, washed and counted. Preparation of cells. Normal nodal lymphocytes were
^2-Microglobutin in Leukaemia obtained from strain-2 guinea-pigs by chopping the dissected inguinal, cervical and mesenteric lymph nodes with scissors in the cold. The tissue was then gently rubbed through a nylon sieve and the resulting suspension passed through a glass wool plug to remove tissue debris. The cells were then washed four times with cold Eagle's minimal essential medium (MEM). Leukaemic lymphocytes were prepared from the peripheral blood of strain-2 guinea-pigs in the final phase of the L^C leukaemia (leucocyte count ^ 200,000 per [JLI). The washing procedure was as described previously [27]. For experiments to examine production of Pa-microglobulin in culture, cells were suspended in MEM containing 1% non-essential amino acids, 2 mM L-glutamine and 100 IU/ml of both penicillin and streptomycin. The cell suspension (2x 10' cells per ml) was swirled gently at 37''C. At intervals, aliquots were removed and centrifuged to remove cells, and the supernatants assayed for Pj-microglobulin by RIA. Papain digestion of celts. This was carried out as described previously [10] using a cell suspension of 10' cells per ml, for 30 min at 37°C in the presence of deoxyribonuclease (0.2 mg/ml), dithiothreitol (0.25 min), and Mg++ (5 mM). The concentration of papain (Worthington) used was 0.5 mg/ml. Controls were cell suspensions treated in exactly the same way but in the absence of papain. Gel chromatography of ^^'"'''^''oglobulin from eells. To assess the molecular size of the p^-microglobulincontaining-material produced during either cell culture of papain digestion, columns of Ultragel AcA 44 (LKB) or Sephadex G-lOO (Pharmacia) were used. The columns (1.8x71 cm) were equilibrated in assay buffer containing horse haemoglobin (20 (xg/ml) and were calibrated by using standards of human IgG, human transferrin, bovine serum albumin (BSA), ovalbumin (OA), and bovine ribonuclease A (RNAse A). From the elution profiles obtained a graph was constructed of log molecular weight versus elution volume (point of elution of 0.6 x peak height), from which the molecular weight of the unknown could be estimated. The supernatants obtained from either cell culture or papain digestion were concentrated (2.5 times) in an Amicon ultrafiltration apparatus using a UM-2 membrane, and an aliquot of 1 ml from each was applied to the column. Aliquots of the column efiluent were then assayed for p2-™icroglobulin.
RESULTS Validation of the antigen It was first necessary to establish the authenticity of the isolated Pj-microglobulin and its antibody, particularly with regard to the RIA. Dr I. Berggird kindly supplied us with a sample of goat antiserum raised against rabbit Pa-microglobulin which he had found to react in immunodiffusion with his guinea-pig
129
globulin. It was found to react also with the preparations described, both when using sodium chromate and L2C leukaemia induction. However, as this is a cross-reaction, only weak precipitin lines could be obtained. Electrophoresis of the two preparations on 5% polyacrylamide gel showed a single band from the material isolated from leukaemic urine. This moved a distance of 0.42 times that of guinea-pig serum albumin after electrophoresis for 2 h. The product from sodium chromate treatment showed a band with the same electrophoretic mobility but there was evidence also of material moving more slowly. Dr Berggdrd also used our sheep antibody with his purified guinea-pig Pj-microglobulin and obtained a good precipitin line (personal communication). A more definitive comparison of Dr Berggard's guinea-pig Pj-microglobulin and the product from leukaemic urine was made by radioimmunoassay. Binding curves are shown in Fig. 1, where it is clear that the pamicroglobulin samples from the two laboratories are reacting in a parallel fashion and that Dr Berggard's Pa-microglobulin (at a concentration of 25 ng/ml) is capable of causing dissociation of 85% of the labelled antigen from the immunosorbent—that is, the product from leukaemic urine and that from Dr BerggSrd's
0-8
0-6
04
0-2
ng/ml
100
FIG. 1. Comparison of antigenic properties of Pa-microglobulin prepared in two different laboratories. Samples of pj-microglobulin from two laboratories ( l = D r Berggard's laboratory in Sweden, 2=this laboratory) compared in a RIA, where the two preparations were each allowed to compete with labelled antigen for binding to a solid-phase immunosorbent carrying antibody against p2-'T''croglobulin. BIB° is the fraction of labelled antigen bound at that particular antigen concentration.
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F. K. Stevenson, M. W. J. Cleeter & G. T. Stevenson
although viability by trypan blue exclusion at 6 h was more than 90%; viability at 20 h had decreased to 76%. The production of Pa-microglobulin by normal lymph node lymphocytes was less than that by the leukaemic lymphocytes: at 2 h the amount of Pj-microglobulin in the supernatant from the normal lymphocytes was 27% of that Production of ^^^-microglobulin by cells in culturefrom the L2C cells. However, the significance of this comparison is difficult to assess owing to the The results of measuring Pa-m'croglobulin in mixed cell population in the lymph nodes and the supernatant obtained from LjC cells in to differences in the rates of cell death. culture for various times are shown in Fig. 2. It is clear that the rate of production of p2Gel chromatography of ^^-microglobulin promicroglobulin falls off after about 6 h, showing duced in culture and by papain digestion a similar pattern to that which we have observed In order to determine the molecular sizes for the incorporation of (^H-leu). Presumably of the Pj-microglobulin yielded by cells in the cells have diminished metabolic vigour, culture or upon treatment with papain, fractionation of supernatants was carried out on ^ • calibrated columns of Ultragel AcA 44 or 120 Sephadex G-lOO as indicated in Materials and Methods. The results of RIA carried out on the papain digest of L^C cells and the corresponding Q 80 control are shown in Table I. First, it is clear OJ • that there is more material reacting as Pr a> a. • microglobulin in the assay after treatment of =' 40 the cells with papain than in the control. A proportion (~30%) of material from both digest and control is lost as a result of con1 1 1 1 centration. However, during the subsequent 12 Time (h) fractionation on AcA 44 there is very little loss. Separation of the digest was very similar on both F r c 2. Production of Pj-microglobulin by LjC AcA 44 and Sephadex G-lOO, although the leukaemic cells in culture. Leukaemic lymphocytes were latter was marginally more satisfactory for prepared from fresh blood of guinea-pigs in the terminal phase of leukaemia. After being washed, cells were determination of molecular weight. The suspended in medium at'2x 10' cells per ml and swirled separation of p^-microglobulin-containing gently at 37°C. Aliquots were withdrawn at intervals, material on the G-lOO is shown in Fig. 3, where centrifuged and assayed for pj-microglobulin. cell;
laboratory are antigenically very similar. However, the fact that the binding curve obtained with the latter lies to the left of that of the former must mean that Dr BerggSrd's material is either more pure or more reactive because of its state of dispersion. Assay results have been expressed in terms of this product.
TABLE I. The effect of papain on the Pj-microglobulin of leukaemic lymphocytes Pj-Microglobulin (ng per 2 x 10' cells) Separation on Ultragel Aca 44 Before concentration Control Test
7.1 15.9
After concentration 5.5 10.8
High molecular fraction weight
Low molecular weight fraction
% recovery
-3.4
4.8 7.5
87.9 100
Leukaemic lymphocytes at lO'/ml were digested for 30 min with papain at 0.5 mg/ml. Supernatants from test and control were concentrated x 2.5 and 1 ml aliquots of each were applied to a column of Ultragel AcA 44 (1.8 x 71 cm). Aliquots of the column effluents were then examined forPa-microglobulin by RIA.
^i-Microglobulin in Leukaemia
131
40
30
20
10 -
S---8Vol. effluent (mM
Fio. 3. Separation on Sephadex G-lOO of Pa-microglobulin-eontaining supernatants from LjC leukaemic cells after incubation in the presence or absence of papain. LjC leukaemic cells (10* cells per ml) were incubated for 30 min at 37°C either in medium or in the presence of papain (0.5 mg/ml). Supernatants were collected by centrifugation, concentrated and applied to a column of Sephadex G-lOO (1.8 X 71 cm). Aliquots of the effluent were then assayed from pj-microglobulin: • , (Jj-microglobulin after incubation with papain; O, in the absence of papain. Bovine RNAse A was eluted at between 97 and 106 ml of effluent volume.
it can be seen that papain had produced a high molecular weight fraction from the cells. The production of free Pa-microglobulin appears to have been little affected by the presence of papain; there is no evidence of degradation and there may in fact have been an increase in the amount secreted. A similar experiment was carried out on normal nodal lymphocytes. It was necessary first to remove dead cells by treatment with a medium of low ionic strength [31]. The results obtained after digestion with papain are shown in Fig. 4. The number of cells digested was the same as for LjC cells and it is evident that much less Pa-ni'croglobulin has been produced. Quantitation in this case, however, is difficult owing to the mixed cell population present in lymph nodes. The main point emerging is that the same high molecular weight P2-microglobulin-containing fraction has been obtained from the normal cells. Furthermore, there seems to be relatively less free Pj-microglobulin produced than by leukaemic lymphocytes. Estimation of the molecular weight of the 'combined' form of Pa-microglobulin was 46,000
daltons (elution volume = 72.4 ml) when standards of human IgG, transferrin, BSA and OA were used on G-lOO (Fig. 5). The same value was found for the material derived from both leukaemic and normal lymphocytes. The 'free' Pa-microglobulin was always eluted a little later than bovine RNAse A (molecular weight, 13,750 daltons), suggesting a molecular weight of approximately 12,000.
DISCUSSION The Pa-microglobulin of guinea-pigs has been described by Berggard [4] and appears to be structurally similar to that of human and rabbit preparations. Tt has been possible to use described methods of damaging the proximal tubules of the kidney in order to cause excretion of low molecular weight proteins in the urine. After purification, a fraction which consists predominantly of Pa-microglobulin was isolated and used to immunize a sheep. However, it was found also that during development of the LaC leukaemia, strain-2 guinea-pigs excrete
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F. K. Stevenson, M. W. J. Cleeter & G. T. Stevenson 20
c'
10
100
120
Vol. effluenf (ml)
FIG. 4. Separation on Sephadex G-lOO (3a-microglobulin-containing supernatants from normal lymph node lymphocytes after incubation in the presence or absence of papain. Normal lymph node lymphocytes from strain-2 guinea-pigs were incubated in the presence or absence of papain exactly as for leukaemic lymphocytes. Supernatants were concentrated and applied to a column of Sephadex G-lOO (1.8 x 71 cm). Aliquots of the effluent were then assayed for Pj-microglobulin: • , Pa-microglobulin after incubation with papain; O, in the absence of papain. Bovine RNAse A was eluted at between 97 and 106 ml of effluent volume.
1-6 -
50
60
70
Elution volume (ml)
FIG. 5. Gel chromatography on Sephadex G-lOO (1.8x71 cm) of glycoproteins of known molecular weights. Elution volumes were taken at the point of elution of 0.6 x peak height.
Pa-microglobulin in the urine, the amount doubling in the last few days. In order to avoid undesirable interactions in the radioimmunoassay, the Pa-microglobulin obtained from leukaemic urine was used as the radiolabelled
antigen. Such an assay showed a parallel response with material from Dr Berggard's laboratory, suggesting antigenic identity. Accordingly, the assay was then used to measure production of p^-microglobulin by leukaemic and normal lymphocytes either in culture of after treatment with papain. The production of free Pa-microglobulin with a MW of approximately 12,000 daltons by human cultured cell lines has been described previously [14], and results from a whole range of cell cultures have been reviewed [19]. Production by the guinea-pig leukaemic cells in culture (approximately 13 ng/10' cells/1 h during the first 2 h) is quantitatively closest to that of the human lymphoma cell lines (12-45 ng/10' cells/ 1 h), and lower than the lymphoblastoid lines. The molecular size of the guinea-pig product as detected in the assay appears to be approximately 12,000 daltons and at the low concentrations of Pa-tnicroglobulin which exist on the column there is no tendency to form aggregates. If, however, larger amounts (300 ng) are fractionated, it is possible to detect material representing approximately 15% of the total which is reactive in the assay, with a MW of up to 50,000. It has been reported that aggregation will occur at higher concentrations of bovine and rabbit pa-microglobulins [2]. The culture medium obtained from LaC leukaemic cells contains no detectable large molecular weight form of
^^-Microglobulin in Leukaemia However, digestion of the cells with papain under conditions where the surface IgM is rapidly cleaved [27] produces a larger Pamicroglobulin-containing molecule of 46,000 daltons. This demonstrates that the assay is capable of detecting a combined form of P2microglobulin and adds significance to our failure to detect such material after cell culture alone. Similar results have been reported for human lymphocytes [8] and murine splenocytes [30] using different techniques of detection. Thus, the lack of secretion into the culture medium of the alloantigenic portion of the cell surface histocompatibility antigen appears to be a general phenomenon. Treatment with papain presumably cleaves the guinea-pig histocompatibility antigens at the cell surface and releases the combined form of j32-microglobulin into the supernatant. From the elution position of Sephadex G-lOO, the molecular weight of this fraction is 46,000 daltons, which is close to that obtained from human splenocytes (approximately 50,000) [21], from a cultured human lymphoblastoid line (47,000 daltons) [28], or from murine splenocytes [13]. The latter workers obtained, after papain treatment of cells and fractionation under non-dissociating conditions, a monomeric fragment of molecular weight approximately 50,000, which consisted of a 39,000 dalton heavy chain fragment and one non-covalently associated p^-microglobulin. Detergent extraction of cells gave rise to two species, with a molecular weight of 92,000 and 12,000 daltons, the heavier molecule apparently being produced by disulphide bond formation during the extraction procedure. In our system it would appear that the larger fragment produced by papain digestion of guinea-pig lymphocytes consists of a molecule of Pa-microglobulin together with a heavy chain of molecular weight of 34,000 daltons—that is, very similar to that of the mouse. Investigation of guinea-pig histocompatibility antigens has been carried out recently in another laboratory [23]. These workers used the technique of solubilization of labelled cells in detergent followed by immunoprecipitation with alloantisera and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. They reported production of two molecules with molecular weights of approximately 40,000 and 12,000, and these
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were found under both reducing and nonreducing conditions. An earlier paper from a related group [11] using the same technique suggested a molecular weight of 45,000 for the detergent-solubilized alloantigenic chain of the guinea-pig histocompatibility antigen. This would allow a molecule of approximately 11,000 daltons to remain in the membrane after cleavage with papain.
ACKNOWLEDGMENTS This work has been financially supported by Tenovus of Cardiff and the Wessex Regional Health Authority. We are grateful to the staff of Allington Farm, Porton Down, Wiltshire, for animal facilities.
REFERENCES 1 Ax6n, R., Porath, J. & Ernback, S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature (Lond.), 214, 1302, 1967. 2 Becker, J.W., Ziffer, J.A., Edelman, G.M. & Cunningham, B.A. Crystallographic studies of bovine P2"'"''^''oglobulin. Proc. nat. Acad. Sci. (USA), 74, 3345, 1977. 3 Berggird, I. Isolation and characteristics of a rabbit Pj-microglobulin: comparison with human p^microglobulin. Biochem. biophys. Res. Commun. 57, 1159, 1974. 4 Berggird, I.p^-Microglobulins: isolation, properties and distribution. Fed. Proc. 35, 1167, 1976. 5 Berggard, I. & Bearn, A.G. Isolation and properties of a low molecular weight pj-globulin occurring in human biological fluids. / . biol. Chem. 243, 4095, 1968. 6 Bernier, G.M. & Fanger, M.W. Synthesis of Pumicroglobulin by stimulated lymphocytes. / . Immunol. 109, 407, 1972. 7 Bjorck, L., Cig6n, R., BerggSrd, B., Low, B. & Berggird, I. Relationships between Pj-microglobulin and alloantigens coded for by the major histocompatibility complexes of the rabbit and the guinea pig. Scand. J. Immunol. 6, 1063, 1977. 8 Cresswell, P., Springer, T., Strominger, J.L., Turner, M.J., Grey, H.M. & Kubo, R.T. Immunological identity of the small subunit of HL-A antigens and Pj-microglobulin and its turnover on the cell membrane. Proc. nat. Acad. Sci. (USA), 71, 2123, 1974. 9 Eady, R.P., Chappie, J.C., Hough, D.W. & Stevenson, G.T. The specificity of a solid phase radioimmunoassay for human immunoglobulins. / . Immunol. Methods, 7, 179, 1975.
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10 Eady, R.P., Hough, D.W., Kilshaw, P.J. & Stevenson, G.T. Recovery of immunoglobulin removed from lymphocytic surfaces by proteolysis. Immunology, 26, 549, 1974. 11 Finkelman, F.D., Shevach, E.M., Vitetta, E.S., Green, I. & Paul, W.E. Guinea pig immune response-related histocompatibility antigens: partial characterization and distribution. / . exp. Med. 141, 27, 1975. 12 Grey, H.M., Kubo, R.T., Colon, S.M., Poulik, M.D., Cresswell, P., Springer, T., Turner, M. & Strominger, J.L. The small subunit of HL-A antigens is Pa-microglobulin. J. exp. Med. 138, 1608, 1973. 13 Henning, R., Milner, R.J., Reske, K., Cunningham, B.A. & Edelman, G.M. Subunit structure, cell surface orientation, and partial amino-acid sequences of murine histocompatibility antigens. Proc. nat. Acad. Sei. (USA), 73, 118, 1976. 14 Hutteroth, T.H., Cleve, H., Litwin, S.D. & Poulik, M.D. The relationship between Pj'microglobulin and immunoglobulin in cultured human lymphoid cell lines. / . exp. Med. 137, 838, 1973. 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. Protein measurement with the Folinphenol reagent. J. biol. Chem. 193, 265, 1951. 16 Michaelson, J., Flaherty, L., Vitetta, E.S. & Poulik, M.D. Molecular similarities between the Qa-2 alloantigen and other gene products of the 17th chromosome of the mouse. / . exp. Med. 145, 1066, 1977. 17 Nakamuro, K., Tanigaki, N. & Pressman, D. Multiple common properties of human p^-microglobulin and the common portion fragment derived from HL-A antigen molecules. Proc. nat. Aead. Sei. (USA), 70, 2863, 1973. 18 Neauport-Sautes, C , Bismuth, A., Kourilsky, F.M. & Manuel, Y. Relationship between HL-A antigens and Pj-microglobulins as studied by immunofluoresence. / . exp. Med. 139, 957, 1974. 19 Nilsson, K., Evrin, P.E. & Welsh. K.l. Production of p2-microglobulin by normal and malignant human cell lines and peripheral lymphocytes. Transplant. Rev. 21, 53, 1974. 20 Peterson, P.A., Cunningham, B.A., Berggard, 1. & Edelman, G.M. Pg-microglobulin—a free immunoglobulin domain. Proc. nat. Acad. Sei. (USA), 69, 1697, 1972.
21 Peterson, P.A., Rask, L. & Lindblom, J.B. Highly purified papain-solubilized HL-A antigens contain Pa-microglobulin. Proc. nat. Acad. Sei. (USA), 71, 35, 1974. 22 Poulik, M.D. & Motwani, N. Demonstration of a p2-microglobulin on the membrane of B lymphocytes. Clin. Res. 20, 795, 1972. 23 Schwartz, B.D., Kask, A.M., Paul, W.E. & Shevach, E.M. Structural characteristics of the alloantigens determined by the major histocompatibility complex of the guinea pig. / . exp. Med. 143, 541, 1976. 24 Seon, B.K. & Pressman, D. A spontaneously occurring complex of p^-microglobulin and a fragment of y-chain of IgG; isolation from the urine of a patient with plasma cell leukaemia and characterization. / . Immunol. 118, 1962, 1977. 25 Solheim, B.G. & Thorsby, B. Pa-microglobulin. Part of the HL-A molecule in the cell membrane. Tissue Antigens, 4, 83, 1974. 26 Stevenson, F.K., Mole, L.E., Raymont, C M . & Stevenson, G.T. A X Bence-Jones protein in guinea pigs. Biochem. J. 151. 751, 1975. 27 Stevenson, G.T., Eady, R.P., Hough, D.W., Jurd, R.D. & Stevenson, F.K. Surface immunoglobulin of guinea-pig leukaemic lymphocytes. Immunology, 28, 807, 1975. 28 Turner, M.J., Cresswell, P., Parham, P., Strominger, J.L., Mann, D.L. & Sanderson, A.R. Purification of papain-solubilized histocompatibility antigens from a cultured human lymphoblastoid line RPMI 4265. / . biol. CAcm. 250, 4512, 1975. 29 Vitetta, E.S., Poulik, M.D., Klein, i. & Uhr, J.W. Pa-microglobulin is selectively associated with H-2 and TL alloantigens on murine lymphoid cells. / . exp. Med. 144, 179, 1976. 30 Vitetta, E.S. & Uhr, J.W. Synthesis of surface H-2 alloantigens in murine splenocytes. / . Immunol. 115, 374, 1975. 31 von Boehmer, H. & Shortman, K. The separation of different cell classes from lymphoid organs. IX. A simple and rapid method for removal of damaged cells from lymphoid cell suspensions. / . Immunol. Methods, 2, 293, 1973. Received 6 March 1978 Received in revised form 3 May 1978
P.-Microglobulin from Normal and Leukaemic Guinea-Pig Lymphocytes F. K. STEVENSON, M. W. J. CLEETER & G. T. STEVENSON Tenovus Research Laboratory, General Hospital, Southampton, UK
Stevenson, F.K., Cleeter, M.W.J. & Stevenson, G.T. Pj-Microglobulin from Normal and Leukaemic Guinea-Pig Lymphocytes. Scand. J. Immunol. 8, 127-134, 1978. Pu-Microglobulin has been isolated in useful quantities from the urine of strain-2 guinea-pigs after either treatment with sodium chromate or induction of the h^C leukaemia. Antibodies raised against the Pj-microglobulin were used to set up a radioimmunoassay which measured its export into culture fluid by normal and leukaemic lymphocytes. Material containing p^-microglobulin was also obtained by digestion of the lymphocytic surfaces with papain; fractionation demonstrated both free and combined forms, with no qualitative difference between those from normal and those from leukaemic cells. F. K. Stevenson, Tenovus Research Laboratory, General Hospital, Southampton SO9 4XY, UK.
p2-Microglobulin was first isolated from the urine of patients with tubular proteinuria in 1968 by Berggard & Beam [5]. It was described as a protein of low molecular weight (11,600 daltons) with an electrophoretic mobility in the P2 region. Although these workers considered that it might form part of a larger molecule and suggested a possible analogy with the light chains of immunoglobulin molecules, it was not until 1972 that its broader significance emerged. At this time it was reported that Pj-microglobulin was present in lymphocytic plasma membranes [6, 20, 22], and its association with histocompatibility antigens was recognized shortly afterwards [12, 17]. There is good evidence of non-covalent bonding between Pa-microglobulin and the HL-A alloantigens in human cell membranes, and such an association has been demonstrated recently in the guinea-pig lymphocytic membrane [7]. However, it should be noted that spontaneous non-covalent association of l with other molecules can occur [24].
Another problem is whether or not the association with histocompatibility antigens accounts for all the pj-rficroglobulin in the plasma membrane. 'Capping' studies on human lymphocytes have shown that not all is associated with HL-A antigens [18, 25], although in mouse splenocytes it has been claimed that all the surface Pj-microglobulin can be accounted for by association with H-2, Tla (thymusleukaemia) [29] and Qa-2 antigens [16]. Biosynthetic studies have demonstrated in several systems that free Pj-microglobulin is produced by lymphocytes in culture, but in neither human nor mouse lymphocytes [30] was it possible to demonstrate histocompatibility antigens in the culture supernatant. This was in spite of the fact that for human lymphocytes both Pa-microglobulin and histocompatibility antigens appeared to have similar turnover rates on the cell surface. Our findings in the guinea-pig are similar to those in man and mouse in that under the conditions used, both normal and leukaemic
0300-9475/78/0800-0127 $02.00 © 1978 Blackwell Scientific Publications
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F. K. Stevenson, M. fV. J. Cleeter & G. T. Stevenson
lymphocytes give rise to free P2-microglobulin in the culture medium, but not to Pj-microglobulin-associated material of high molecular weight. Papain digestion of the cell surfaces, however, gave rise to two Pa-microglobulincontaining fractions, one corresponding to the free molecule and the other with a molecular weight of approximately 46,000 daltons.
MATERIALS AND METHODS Isolation of f^^-microglobulin. Isolation of guinea-pig Pj-microglobulin from urine of sodium chromatetreated animals was essentially as described by Berggird [3] for rabbit Pj-m'croglobulin. Briefly, eight strain-2 guinea-pigs were injected subcutaneously with sodium chromate (10 mg per kg body weight), and 24 h specimens of urine were collected into a few drops of toluene by means of metabolic cages. Tests for protein with Albustix (Ames Company) became strongly positive on day 3 after injection, and urines were collected from days 3 to 7 inclusive. Pooled urines were centrifuged and dialysed into 0.1 M Tris-HCI-1.0 M NaCl, pH 8.0. Analysis for protein by the Lowry method [15] showed excretion of 120 mg/24 h per guinea-pig, whereas normal urine showed 9 mg/24 h. The urine was then concentrated in an Amicon ultrafiltration apparatus using a UM-2 membrane (quoted lower exclusion limit, 1000 daltons), and 500 mg of protein was loaded onto a column of Sephadex G-150 equilibrated with the Tris buffer. The low molecular weight fraction from this column was collected and fractionated further on DEAE-cellulose (Whatman DE32) using a starting buffer of 0.01 M Tris-HCl, pH 7.8, followed by a gradient to 0.4 M NaCl. It was possible to follow purification of the PJmicroglobulin by means of its ability to block the reactivity of rabbit anti-human Pj-microglobulin (Dakopatts) with guinea-pig LjC leukaemia cells as assessed by indirect immunofluorescence. The peak with most of this activity was collected and used for immunization. Guinea-pig Pj-microglobulin was also prepared from the urine of strain-2 guinea-pigs in the terminal stage of the LjC leukaemia. It is known that during the development of human leukaemia or lymphoma quite large amounts of Pj-m'croglobulin are produced [reviewed in Ref. 19]; we therefore hoped to prepare P^-microglobulin from animals without recourse to sodium chromate. The excretion of monoclonal X light chain by these guinea-pigs has been reported previously [26]. It was necessary to remove this before further processing the urine. This was done by passing urine which had been dialysed into 0.02 Tris-HCl-0.1 M NaCl-0.02% NaN;,, pH 8, through an immunosorbent consisting of Sepharose 4B to which an IgG fraction from a sheep anti-guinea-pig X chain serum had been covalently coupled [1]. Aliquots of 25 mg urinary protein were passed through a column of 50 ml; the yield of urinary protein after immunosorption was 35%. The protein not bound by the immunosorbent was
further purified by ion-exchange chromatography on DEAE-ce!lulose, as for sodium chromate-induced Pjmicroglobulin, and again monitored by blocking activity using L^C leukaemic cells. The two preparations were compared by electrophoresis on polyacrylamide gel and by crossreactivity with an antiserum raised against rabbit p^-microglobulin kindly provided by Dr I. BerggSrd. Immunization of a sheep and preparation of antibody. The p2-microglobulin obtained from the sodium chromate-treated guinea-pigs was used to raise an antiserum in a sheep. A Clun cross-bred sheep was immunized with 1 mg p2-microglobulin in complete Freund's adjuvant (CFA) in four intramuscular sites. A second injection of antigen in CFA was given 5 weeks later in the back, and blood was taken 1, 2 and 3 weeks after this. Blood was collected onto glass beads for defibrination and the serum prepared by centrifugation. The antiserum reacted with pj-microglobulin by immunodiffusion, and detected it thus in normal urinary protein prepared by 50-fold concentration of normal urine through an Amicon UM-2 membrane. In fact it was present in the latter in suflicient quantity to yield an immunosorbent (Sepharose 4B conjugated to normal urinary protein, 1 ml to 10 mg) suitable for preparing purified antibody. Antiserum (10 ml) was passed through the immunosorbent (30 ml) and the column washed thoroughly with 0.02 Tris-HCl-0.1 M NaCl0.02% NaNj. The absorbed antibody was then eluted with 0.5 M NH4OH and immediately dialysed into cold Tris buffer. Further analysis of the purified antibody by immunodiffusion showed reactivity with the immunogen (two lines) and with the Pj-microglobulin obtained from leukaemic urine (one line). There was thus evidence of a second (unidentified) component in the immunogen, not detectable in the preparation from leukaemic urine. It was therefore decided to set up the radioimmunoassay using purified antibody and to use the Pj-microglobulin from leukaemic urine as the labelled antigen. Radioimmunoassay (RIA). Radioimmunoassay for guinea-pig Pj-microglobulin was carried out by allowing the test antigen and a radioactive standard antigen to compete for antibody coupled to beads of Sephadex G-25 superfine [9]. The coupled antibody was the purified sheep anti-Pn-microglobulin, and the radiolabelled antigen, at a specific activity of "^I of approximately 85 Ci/g, was P^-microglobulin obtained from leukaemic urine. Radioiodination was by the use of chloramine-T, and labelled protein was always purified from possible breakdown products by passage through a column of Sephadex G-25 (fine). Radiolabelled antigen (450 pg) in 0.1 ml was added to 1.0 ml of a solution of the test antigen; 1 ml of immunosorbent at a concentration of solid phase which bound 8-10% of labelled antigen when this alone was present was then added. All dilutions were with assay buffer (0.05 M phosphate buffer containing NaCl, 0.08 M, and sodium azide, 0.05%, pH 7.3) to which horse haemoglobin (Miles) had been added to give a concentration of 2 mg/ml. After incubation for 48 h the solid phase was separated by centrifugation, washed and counted. Preparation of cells. Normal nodal lymphocytes were
^2-Microglobutin in Leukaemia obtained from strain-2 guinea-pigs by chopping the dissected inguinal, cervical and mesenteric lymph nodes with scissors in the cold. The tissue was then gently rubbed through a nylon sieve and the resulting suspension passed through a glass wool plug to remove tissue debris. The cells were then washed four times with cold Eagle's minimal essential medium (MEM). Leukaemic lymphocytes were prepared from the peripheral blood of strain-2 guinea-pigs in the final phase of the L^C leukaemia (leucocyte count ^ 200,000 per [JLI). The washing procedure was as described previously [27]. For experiments to examine production of Pa-microglobulin in culture, cells were suspended in MEM containing 1% non-essential amino acids, 2 mM L-glutamine and 100 IU/ml of both penicillin and streptomycin. The cell suspension (2x 10' cells per ml) was swirled gently at 37''C. At intervals, aliquots were removed and centrifuged to remove cells, and the supernatants assayed for Pj-microglobulin by RIA. Papain digestion of celts. This was carried out as described previously [10] using a cell suspension of 10' cells per ml, for 30 min at 37°C in the presence of deoxyribonuclease (0.2 mg/ml), dithiothreitol (0.25 min), and Mg++ (5 mM). The concentration of papain (Worthington) used was 0.5 mg/ml. Controls were cell suspensions treated in exactly the same way but in the absence of papain. Gel chromatography of ^^'"'''^''oglobulin from eells. To assess the molecular size of the p^-microglobulincontaining-material produced during either cell culture of papain digestion, columns of Ultragel AcA 44 (LKB) or Sephadex G-lOO (Pharmacia) were used. The columns (1.8x71 cm) were equilibrated in assay buffer containing horse haemoglobin (20 (xg/ml) and were calibrated by using standards of human IgG, human transferrin, bovine serum albumin (BSA), ovalbumin (OA), and bovine ribonuclease A (RNAse A). From the elution profiles obtained a graph was constructed of log molecular weight versus elution volume (point of elution of 0.6 x peak height), from which the molecular weight of the unknown could be estimated. The supernatants obtained from either cell culture or papain digestion were concentrated (2.5 times) in an Amicon ultrafiltration apparatus using a UM-2 membrane, and an aliquot of 1 ml from each was applied to the column. Aliquots of the column efiluent were then assayed for p2-™icroglobulin.
RESULTS Validation of the antigen It was first necessary to establish the authenticity of the isolated Pj-microglobulin and its antibody, particularly with regard to the RIA. Dr I. Berggird kindly supplied us with a sample of goat antiserum raised against rabbit Pa-microglobulin which he had found to react in immunodiffusion with his guinea-pig
129
globulin. It was found to react also with the preparations described, both when using sodium chromate and L2C leukaemia induction. However, as this is a cross-reaction, only weak precipitin lines could be obtained. Electrophoresis of the two preparations on 5% polyacrylamide gel showed a single band from the material isolated from leukaemic urine. This moved a distance of 0.42 times that of guinea-pig serum albumin after electrophoresis for 2 h. The product from sodium chromate treatment showed a band with the same electrophoretic mobility but there was evidence also of material moving more slowly. Dr Berggdrd also used our sheep antibody with his purified guinea-pig Pj-microglobulin and obtained a good precipitin line (personal communication). A more definitive comparison of Dr Berggard's guinea-pig Pj-microglobulin and the product from leukaemic urine was made by radioimmunoassay. Binding curves are shown in Fig. 1, where it is clear that the pamicroglobulin samples from the two laboratories are reacting in a parallel fashion and that Dr Berggard's Pa-microglobulin (at a concentration of 25 ng/ml) is capable of causing dissociation of 85% of the labelled antigen from the immunosorbent—that is, the product from leukaemic urine and that from Dr BerggSrd's
0-8
0-6
04
0-2
ng/ml
100
FIG. 1. Comparison of antigenic properties of Pa-microglobulin prepared in two different laboratories. Samples of pj-microglobulin from two laboratories ( l = D r Berggard's laboratory in Sweden, 2=this laboratory) compared in a RIA, where the two preparations were each allowed to compete with labelled antigen for binding to a solid-phase immunosorbent carrying antibody against p2-'T''croglobulin. BIB° is the fraction of labelled antigen bound at that particular antigen concentration.
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F. K. Stevenson, M. W. J. Cleeter & G. T. Stevenson
although viability by trypan blue exclusion at 6 h was more than 90%; viability at 20 h had decreased to 76%. The production of Pa-microglobulin by normal lymph node lymphocytes was less than that by the leukaemic lymphocytes: at 2 h the amount of Pj-microglobulin in the supernatant from the normal lymphocytes was 27% of that Production of ^^^-microglobulin by cells in culturefrom the L2C cells. However, the significance of this comparison is difficult to assess owing to the The results of measuring Pa-m'croglobulin in mixed cell population in the lymph nodes and the supernatant obtained from LjC cells in to differences in the rates of cell death. culture for various times are shown in Fig. 2. It is clear that the rate of production of p2Gel chromatography of ^^-microglobulin promicroglobulin falls off after about 6 h, showing duced in culture and by papain digestion a similar pattern to that which we have observed In order to determine the molecular sizes for the incorporation of (^H-leu). Presumably of the Pj-microglobulin yielded by cells in the cells have diminished metabolic vigour, culture or upon treatment with papain, fractionation of supernatants was carried out on ^ • calibrated columns of Ultragel AcA 44 or 120 Sephadex G-lOO as indicated in Materials and Methods. The results of RIA carried out on the papain digest of L^C cells and the corresponding Q 80 control are shown in Table I. First, it is clear OJ • that there is more material reacting as Pr a> a. • microglobulin in the assay after treatment of =' 40 the cells with papain than in the control. A proportion (~30%) of material from both digest and control is lost as a result of con1 1 1 1 centration. However, during the subsequent 12 Time (h) fractionation on AcA 44 there is very little loss. Separation of the digest was very similar on both F r c 2. Production of Pj-microglobulin by LjC AcA 44 and Sephadex G-lOO, although the leukaemic cells in culture. Leukaemic lymphocytes were latter was marginally more satisfactory for prepared from fresh blood of guinea-pigs in the terminal phase of leukaemia. After being washed, cells were determination of molecular weight. The suspended in medium at'2x 10' cells per ml and swirled separation of p^-microglobulin-containing gently at 37°C. Aliquots were withdrawn at intervals, material on the G-lOO is shown in Fig. 3, where centrifuged and assayed for pj-microglobulin. cell;
laboratory are antigenically very similar. However, the fact that the binding curve obtained with the latter lies to the left of that of the former must mean that Dr BerggSrd's material is either more pure or more reactive because of its state of dispersion. Assay results have been expressed in terms of this product.
TABLE I. The effect of papain on the Pj-microglobulin of leukaemic lymphocytes Pj-Microglobulin (ng per 2 x 10' cells) Separation on Ultragel Aca 44 Before concentration Control Test
7.1 15.9
After concentration 5.5 10.8
High molecular fraction weight
Low molecular weight fraction
% recovery
-3.4
4.8 7.5
87.9 100
Leukaemic lymphocytes at lO'/ml were digested for 30 min with papain at 0.5 mg/ml. Supernatants from test and control were concentrated x 2.5 and 1 ml aliquots of each were applied to a column of Ultragel AcA 44 (1.8 x 71 cm). Aliquots of the column effluents were then examined forPa-microglobulin by RIA.
^i-Microglobulin in Leukaemia
131
40
30
20
10 -
S---8Vol. effluent (mM
Fio. 3. Separation on Sephadex G-lOO of Pa-microglobulin-eontaining supernatants from LjC leukaemic cells after incubation in the presence or absence of papain. LjC leukaemic cells (10* cells per ml) were incubated for 30 min at 37°C either in medium or in the presence of papain (0.5 mg/ml). Supernatants were collected by centrifugation, concentrated and applied to a column of Sephadex G-lOO (1.8 X 71 cm). Aliquots of the effluent were then assayed from pj-microglobulin: • , (Jj-microglobulin after incubation with papain; O, in the absence of papain. Bovine RNAse A was eluted at between 97 and 106 ml of effluent volume.
it can be seen that papain had produced a high molecular weight fraction from the cells. The production of free Pa-microglobulin appears to have been little affected by the presence of papain; there is no evidence of degradation and there may in fact have been an increase in the amount secreted. A similar experiment was carried out on normal nodal lymphocytes. It was necessary first to remove dead cells by treatment with a medium of low ionic strength [31]. The results obtained after digestion with papain are shown in Fig. 4. The number of cells digested was the same as for LjC cells and it is evident that much less Pa-ni'croglobulin has been produced. Quantitation in this case, however, is difficult owing to the mixed cell population present in lymph nodes. The main point emerging is that the same high molecular weight P2-microglobulin-containing fraction has been obtained from the normal cells. Furthermore, there seems to be relatively less free Pj-microglobulin produced than by leukaemic lymphocytes. Estimation of the molecular weight of the 'combined' form of Pa-microglobulin was 46,000
daltons (elution volume = 72.4 ml) when standards of human IgG, transferrin, BSA and OA were used on G-lOO (Fig. 5). The same value was found for the material derived from both leukaemic and normal lymphocytes. The 'free' Pa-microglobulin was always eluted a little later than bovine RNAse A (molecular weight, 13,750 daltons), suggesting a molecular weight of approximately 12,000.
DISCUSSION The Pa-microglobulin of guinea-pigs has been described by Berggard [4] and appears to be structurally similar to that of human and rabbit preparations. Tt has been possible to use described methods of damaging the proximal tubules of the kidney in order to cause excretion of low molecular weight proteins in the urine. After purification, a fraction which consists predominantly of Pa-microglobulin was isolated and used to immunize a sheep. However, it was found also that during development of the LaC leukaemia, strain-2 guinea-pigs excrete
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F. K. Stevenson, M. W. J. Cleeter & G. T. Stevenson 20
c'
10
100
120
Vol. effluenf (ml)
FIG. 4. Separation on Sephadex G-lOO (3a-microglobulin-containing supernatants from normal lymph node lymphocytes after incubation in the presence or absence of papain. Normal lymph node lymphocytes from strain-2 guinea-pigs were incubated in the presence or absence of papain exactly as for leukaemic lymphocytes. Supernatants were concentrated and applied to a column of Sephadex G-lOO (1.8 x 71 cm). Aliquots of the effluent were then assayed for Pj-microglobulin: • , Pa-microglobulin after incubation with papain; O, in the absence of papain. Bovine RNAse A was eluted at between 97 and 106 ml of effluent volume.
1-6 -
50
60
70
Elution volume (ml)
FIG. 5. Gel chromatography on Sephadex G-lOO (1.8x71 cm) of glycoproteins of known molecular weights. Elution volumes were taken at the point of elution of 0.6 x peak height.
Pa-microglobulin in the urine, the amount doubling in the last few days. In order to avoid undesirable interactions in the radioimmunoassay, the Pa-microglobulin obtained from leukaemic urine was used as the radiolabelled
antigen. Such an assay showed a parallel response with material from Dr Berggard's laboratory, suggesting antigenic identity. Accordingly, the assay was then used to measure production of p^-microglobulin by leukaemic and normal lymphocytes either in culture of after treatment with papain. The production of free Pa-microglobulin with a MW of approximately 12,000 daltons by human cultured cell lines has been described previously [14], and results from a whole range of cell cultures have been reviewed [19]. Production by the guinea-pig leukaemic cells in culture (approximately 13 ng/10' cells/1 h during the first 2 h) is quantitatively closest to that of the human lymphoma cell lines (12-45 ng/10' cells/ 1 h), and lower than the lymphoblastoid lines. The molecular size of the guinea-pig product as detected in the assay appears to be approximately 12,000 daltons and at the low concentrations of Pa-tnicroglobulin which exist on the column there is no tendency to form aggregates. If, however, larger amounts (300 ng) are fractionated, it is possible to detect material representing approximately 15% of the total which is reactive in the assay, with a MW of up to 50,000. It has been reported that aggregation will occur at higher concentrations of bovine and rabbit pa-microglobulins [2]. The culture medium obtained from LaC leukaemic cells contains no detectable large molecular weight form of
^^-Microglobulin in Leukaemia However, digestion of the cells with papain under conditions where the surface IgM is rapidly cleaved [27] produces a larger Pamicroglobulin-containing molecule of 46,000 daltons. This demonstrates that the assay is capable of detecting a combined form of P2microglobulin and adds significance to our failure to detect such material after cell culture alone. Similar results have been reported for human lymphocytes [8] and murine splenocytes [30] using different techniques of detection. Thus, the lack of secretion into the culture medium of the alloantigenic portion of the cell surface histocompatibility antigen appears to be a general phenomenon. Treatment with papain presumably cleaves the guinea-pig histocompatibility antigens at the cell surface and releases the combined form of j32-microglobulin into the supernatant. From the elution position of Sephadex G-lOO, the molecular weight of this fraction is 46,000 daltons, which is close to that obtained from human splenocytes (approximately 50,000) [21], from a cultured human lymphoblastoid line (47,000 daltons) [28], or from murine splenocytes [13]. The latter workers obtained, after papain treatment of cells and fractionation under non-dissociating conditions, a monomeric fragment of molecular weight approximately 50,000, which consisted of a 39,000 dalton heavy chain fragment and one non-covalently associated p^-microglobulin. Detergent extraction of cells gave rise to two species, with a molecular weight of 92,000 and 12,000 daltons, the heavier molecule apparently being produced by disulphide bond formation during the extraction procedure. In our system it would appear that the larger fragment produced by papain digestion of guinea-pig lymphocytes consists of a molecule of Pa-microglobulin together with a heavy chain of molecular weight of 34,000 daltons—that is, very similar to that of the mouse. Investigation of guinea-pig histocompatibility antigens has been carried out recently in another laboratory [23]. These workers used the technique of solubilization of labelled cells in detergent followed by immunoprecipitation with alloantisera and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. They reported production of two molecules with molecular weights of approximately 40,000 and 12,000, and these
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were found under both reducing and nonreducing conditions. An earlier paper from a related group [11] using the same technique suggested a molecular weight of 45,000 for the detergent-solubilized alloantigenic chain of the guinea-pig histocompatibility antigen. This would allow a molecule of approximately 11,000 daltons to remain in the membrane after cleavage with papain.
ACKNOWLEDGMENTS This work has been financially supported by Tenovus of Cardiff and the Wessex Regional Health Authority. We are grateful to the staff of Allington Farm, Porton Down, Wiltshire, for animal facilities.
REFERENCES 1 Ax6n, R., Porath, J. & Ernback, S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature (Lond.), 214, 1302, 1967. 2 Becker, J.W., Ziffer, J.A., Edelman, G.M. & Cunningham, B.A. Crystallographic studies of bovine P2"'"''^''oglobulin. Proc. nat. Acad. Sci. (USA), 74, 3345, 1977. 3 Berggird, I. Isolation and characteristics of a rabbit Pj-microglobulin: comparison with human p^microglobulin. Biochem. biophys. Res. Commun. 57, 1159, 1974. 4 Berggird, I.p^-Microglobulins: isolation, properties and distribution. Fed. Proc. 35, 1167, 1976. 5 Berggard, I. & Bearn, A.G. Isolation and properties of a low molecular weight pj-globulin occurring in human biological fluids. / . biol. Chem. 243, 4095, 1968. 6 Bernier, G.M. & Fanger, M.W. Synthesis of Pumicroglobulin by stimulated lymphocytes. / . Immunol. 109, 407, 1972. 7 Bjorck, L., Cig6n, R., BerggSrd, B., Low, B. & Berggird, I. Relationships between Pj-microglobulin and alloantigens coded for by the major histocompatibility complexes of the rabbit and the guinea pig. Scand. J. Immunol. 6, 1063, 1977. 8 Cresswell, P., Springer, T., Strominger, J.L., Turner, M.J., Grey, H.M. & Kubo, R.T. Immunological identity of the small subunit of HL-A antigens and Pj-microglobulin and its turnover on the cell membrane. Proc. nat. Acad. Sci. (USA), 71, 2123, 1974. 9 Eady, R.P., Chappie, J.C., Hough, D.W. & Stevenson, G.T. The specificity of a solid phase radioimmunoassay for human immunoglobulins. / . Immunol. Methods, 7, 179, 1975.
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