CELLULAR
IMMUNOLOGY
Evidence
37, 298-307 (1978)
for a Surface Factor
Receptor
for Human
on a B-Lymphoid
Migration
Inhibitory
Cell Line1
THOMAS F. MCLEOD, PETER G. CORDEIRO, SUSAN K. MELTZ, AND PHILIP R. GLADE Department
of Pediatrics, Division of Pediatric Infectious Diseases and Immunology, University of Miami School of Medicine, Miami, Florida 33125 Received
August
31,1977
Reception by PGLC-33H target cells for the migration inhibitory factor (MIF) produced by this established line has been investigated by pulse time and temperature dependence, MIF absorption, and abrogation by trypsinization. PGLC-33H supernatants containing MIF were concentrated 5X with Carbowax and dialyzed against serum free RPMI-1640 before use. Prior to standard capillary migration assay a minimum 30 min pulse of MIF at 37°C is required for significant migration inhibition (MI > 20%). No significant MI is observed when cells are pulsed at 4°C for up to 2 hr. Preincubation with PGLC-33H for 1 hr at 37°C reduces activity of supernatants from 38 to 13% MI; at 4°C to 27% MI. Trypsinization of target cells for 30 min at 25°C abrogates response to MIF (43 to - 14% MI). Trypsinized cells did not reduce activity of supernatants. MIF activity is abolished (32 to 3% MI) in samples preincubated with supernatants of the trypsinized cells inactivated with serum. These data suggest that cells from the human B-lymphoid cell line PGLC-33H have a surface receptor for human MIF.
INTRODUCTION Although the conditions influencing the elaboration of MIF by various short term and long term cell cultures have received considerable attention in the decade following its’ discovery, the mechanisms of interaction between MIF and target cells remain largely unknown. Studies from several laboratories (2-S) have demonstrated that established human cell lines of lymphoid and of non-lymphoid origin have the capacity to elaborate migration inhibitory factor (MIF) with inhibitory activity for the migration of guinea pig peritoneal macrophages and of target cells from human B-lymphoid cells lines. This heat stable, trypsin sensitive, non-dialyzable material has an estimated MW between 14,300 and 25,700 and co-elutes from Sephadex G-75 with MIF produced by phytomitogen and antigen stimulated human peripheral lymphocytes (6). In the present series of investigations we docu1 This work was supported by the United States Public Health Service Research Grant No. AI 12475 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health and a research grant from the Upjohn Company, Kalamazoo, Michigan. Portions of the work were presented at the Annual Meeting of the American Association of Immunologists (FASEB) April 7, 1977, Chicago, Illinois (1). 298 0008-8749/78/0372-0298$02.00/O Copyright 0 1978by AcademicPress,Inc. All rights of reproductionin any form reserved.
MIF
RECEPTOR
ON
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ment the possibility that the PGLC-33H target cell interacts derived MIF by means of a cell surface receptor. MATERIALS
AND
LINE
with
299 PGLC-33H
METHODS
Maintenance of the lymfihoid cell line. The methods employed in the establishment and continuous culture of human B-lymphoid cell lines and a description of their characteristics have been reported previously (7). PGLC-33H is a well-characterized human B lymphoid cell line derived from the peripheral blood of an adolescent female with acute infectious mononucleosis and has been maintained in continuous suspension culture since 1967 (8). This cell line was utilized for the production of migration inhibitory factor (MIF) and also served as the target cells in the MIF capillary assay. PGLC-33H cells were maintained at 37°C in roller bottles at 2 rpm (Bellco Roller Apparatus, Vineland, N.J.) in complete RPM1 1640 medium containing 100 pg/ml streptomycin, 100 units/ml penicillin, 2.92 mg/ml L-glutamine and 17% (v/v) heat inactivated fetal bovine serum (FBS) (Associated Biomedics Systems, Inc., Buffalo, N.Y.) . The cells were passaged routinely at 72 to 96 hr intervals by gravity sedimentation of the cells, removal of l/2 of the supernatant culture medium, and replacement with fresh complete medium. Viability was estimated by exclusion of 0.1% trypan blue dye. Maintenance of HeLa cells. The HeLa cell line was maintained in continuous monolayer culture in RPM1 1640 supplemented with 100 fig/ml streptomycin, 100 units/ml of penicillin, 2.92 mg/ml L-glutamine, and 10% (v/v) heat inactivated fetal bovine serum. The line was passaged twice weekly by trypsinization with 0.201, trypsin for 3-5 min at 20°C. Prior to study the cells were seeded into sterile plastic 75 cm* culture flasks (Falcon, Oxnard, Calif.). Pre$wation of MIF. PGLC-33H cells in the log phase of growth (within 24 hr of the addition of fresh medium) were collected by centrifugation at 150g for 20 min at 20°C and resuspended to a final cell concentration of 4-6 x lo6 cells/ml in fresh serum free RPM1 1640 supplemented with antibiotics and glutamine. Following incubation at 37°C in the roller apparatus for 24 hr the PGLC-33H cells were separated from the medium by centrifugation at 150g for 20 min at 20°C. The supernatant medium was filtered with a Millipore filter (0.45 p) and stored at - 20°C until use. Serum-free medium without PGLC-33H cells was handled in an identical fashion and served as control in all investigations. For routine studies of MIF activity 50 ml aliquots of thawed supernatants were each placed in dialysis tubing (A. H. Thomas, Philadephia, Pa., M.W. cut off 12,000; boiled in three changes of distilled water) and concentrated room temperature to 10 ml (5~ concentration) with 100 g of polyethylene glycol, average MW 20,000 (Fisher Scientific Company, Fairlawn, N.J.). Following a change of dialysis tubing, the 5X concentrated supernatants were each dialyzed at 4°C for 24 hr against serum-free RPM1 1640 with antibiotics and L-glutamine. Although the change of tubing was used to decrease the chance of contamination of samples by the polyethylene glycol, studies have shown that polyethylene glycol has no effect upon the migration of PGLC-33H cells or upon MIF at concentrations as high as 1 mg/ml. Samples (5X MIF or 5X Control) were utilized immediately or stored in 5.0 ml aliquots at - 20°C. Capillary migration assay for MIF. The inhibition of migration of PGLC-33H
300
MCLEOD
ET AL.
lymphoid cell line target cells was assayed by a modification of the capillary migration inhibition assay of George and Vaughan (9) as previously described ( 10). Forty-eight hours prior to assay cells were passaged into fresh complete RPM1 1640 medium as described. Twenty-four hours prior to assay the cells were collected by centrifugation at 15Og for 10 min at 20°C and resuspended at a concentration of 0.5 X lo6 viable cells/ml in complete RPM1 1640 medium in roller bottles. After incubation for 16 hr at 37°C in the roller apparatus at 2 rpm, the cell suspension was centrifuged at 15Og for 10 min and the cells were resuspended in fresh complete medium to a concentration 3-4 x lo7 cells/ml. The cells were then packed in microhematocrit tubes with a microhematocrit centrifuge (International Microhematrocrit Centrifuge Model MB, Meedham Heights, Mass.). The capillaries were cut at the cell-liquid interface, placed in Lexy culture chambers (Mini-lab Co., Quebec, Canada) and the chambers sealed with cover glasses. The chambers were filled with complete RPM1 1640 medium, with control material or with experimental material with FBS-added to a concentration of 17% (v/v) and incubated for 18 to 24 hr. The areas of cell migration were determined by projection of migration patterns with a photoenlarger, tracing the images, and measurement of areas by planimetry. All studies were performed in triplicate and the inhibition of migration (% MI) was calculated by the formula : y0 migration
inhibition
=
1 - are~~on~rol)
x 100
Inhibition of 20% or more is considered significant. Variation among triplicates was no more than -C 10%. Pulse exposure of PGLC-33H target cells to MIF. Prior to pulse exposure studies, samples of each lot of 5X MIF were subjected to serial twofold dilutions in 5X Control and assayed for MIF activity as described. The MIF activity of 5~ MIF was invariably reduced below significant levels of dilution to 1: 4. Pulse exposure of target cells to PGLC-33H MIF was therefore conveniently terminated by addition of complete medium to a final dilution of the MIF to 1: 10 or greater. Twenty mls of the PGLC-33H target cell suspension containing 0.8-1.0 X lo6 viable cells/ml were added to multiple sterile plastic conical centrifuge tubes and centrifuged at 150g for 10 min at 20°C. The supernatants were decanted and the target cells were resuspended in 2 ml of either 5~ MIF or 5X Control, each supplemented to 17% (v/v) with FBS. The tubes were incubated for varying periods of time in a 37°C water bath with frequent gentle agitation. The pulse was terminated by the addition of 20.0 ml of complete medium to the cell suspensions followed by centrifugation at 150g for 2 min at 20°C. The supernatant fluids were decanted, the cell pellets were resuspended in 0.5 ml of complete medium and the cells were assayed in the capillary migration test as described. Percent migration inhibition was calculated using cells pulsed with 5X Control as the denominator in the equation. Treatment of target cells with trypsin. Target cells passaged as for use in the capillary assay were treated with 0.2% trypsin as previously described (11). The PGLC-33H target cells (0.8-1.0 x lo6 viable cells/ml) were washed four times in sterile H.anks Balanced Salt Solution (HBSS) and resuspended at a concen-
MIF
RECEPTOR
ON
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301
tration of 2 x lo6 cells/l.0 ml of 0.2% trypsin in HBSS. After l/2 hr at room temperature the trypsin was then inactivated by addition of an equal volume of complete RPM1 1640 medium (17% PBS v/v). Tl le cells were removed by centrifugation at 15Og for 10 min at 2O”C, washed in complete RPM1 1640 medium and resuspended in complete medium to a final concentration of 1 X lo6 cells/ml. Control target cells were processed in a similar manner without trypsin. Viability of both sets of target cells (trypsin treated and control) was 99% as determined by trypan blue dye exclusion. Thirty milliliter aliquots of each set of target cells were then centrifuged at 150g for 10 min. 5~ MIF or 5X Control was added to the cell pellets and a pulse exposure study was performed as described. The $&MI was calculated using the results of the trypsin or non-trypsin treated cells pulsed with 5 X Control in the denominator. Removal of MIF activity with PGLC-33H cells. Target cells passaged as for use in the capillary migration assay were collected by centrifugation at 150g for 10 min at 20°C and washed two times in HBSS. Following resuspension to a concentration of 1 x lo6 cells/ml in HBSS, 20 ml aliquots were again centrifuged at 150~ for 10 min, the supernatants decanted and 3.0 ml of 5 X hlIF or 5X Control supplemented to 17% v/ v with FBS were added to the cell pellets. The cells were thoroughly resuspended in the samples and incubated with frequent agitation for lhr in a 37°C water bath or in a 4°C ice bath. The suspensions were then centrifuged at 25Og for 10 min, the supernatants drawn off and assayed for h,lIF activity in the capillary migration assay. Removal of MIF activity with HeLa. Monolayer cultures of HeLa cells grown to confluency in 75 cm2 culture flasks (17-20 x lo6 cells) were washed three times with HBSS. Three ml samples of 5X MIF or 5~ Control supplemented to 17% v/v with FBS were added to the flasks and were incubated for 1 hr at 37°C or at 4°C. The samples were then removed from the monolayers and assayed for MIF activity as described. Production of tryfsinixed cell supernatants. PGLC-33 cells (12.5 x 108) xvere washed three times in 250 ml volumes of HBSS and incubated at 20°C for 30 min in 100 ml of 0.2% trypsin. The trypsin was inactivated by the addition of 10 ml FBS and the suspension was then centrifuged at 250g for 10 min at 20°C. The supernatant was drawn off, passed through a 0.45 p Millipore filter (Millipore Corp., Bedford, Mass.) and used immediately, since storage or freezing resulted in irreversible precipitation. Control supernatant consisted of 1.0 ml FBS added to 10 ml of 0.2% trypsin. Trypsinized cell supernatants were obtained from monolayer cultures of HeLa cells washed three times in HBSS by the addition at room temperature of 5.0 ml of 0.2% trypsin in HBSS to the 75 cm2 culture flasks. When the cells began to dislodge from the culture flask surface (approximately 3-5 min) 0.5 ml of FBS was added to inactivate the trypsin and the supernatant fluid was decanted. This material was centrifuged at 25Og for 10 min at 20°C and passed through a 0.45 ,,, Millipore filter. Control supernatant consisted of 1.0 ml FBS added to 10 ml of 0.2% trypsin. Blockade of MIF activity zwith trypsinized cell supernatants, The ability of the trypsinized cell supernatants to block the effect of MIF on PGLC-33H target cells was examined by mixing control supernatants (trypsin + FBS) or trypsinized cell supernatants + FBS with 5~ MIF or 5 x Control samples supplemented to 15% (v/v) FBS, incubating for 1 hr at 37°C in a water bath, and assaying mixtures for MIF activity by the capillary migration assay.
302
MCLEOD
ET
AL.
MRC.
OURA?-1Oh' OF PUS& FIG. 1. The temperature/time dependence of the inhibition of migration of PGLC-33H target cells induced by 5X MIF. The area of migration of cells pulsed with 5X Control was used for calculation of percent migration inhibition.
RESULTS P&e
Exposure of Target Cells to MIF
PGLC-33H cells (approximately 20 X 106) were exposed to 2.0 ml of 5~ MIF or to 5~ Control for periods of time up to 3 hr at 37°C and up to 2 hr at 4°C prior to testing in the capillary migration assay (Fig. 1). By 30 min of pulse exposure to 5~ MIF at 37”C, target cells would subsequently demonstrate significant migration inhibition in the capillary assay (MI = 29%). No significant migration inhibition could be demonstrated for target celIs exposed to 5X MIF at 4°C for periods up to 2 hr. These studies suggest that interactions between MIF and PGLC-33H target cells which result in significant migration inhibition (> 20%) 18 hr later can occur in a relatively short pulse exposure time and require actively metabolizing cells. Response of Trypsinized
Target Cells to MIF
If the interaction between 5~ MIF and PGLC-33H target is dependent upon a surface receptor, it should be possible to abolish the interaction by mild pretreatment of target cells with trypsin. In this series of experiments non-trypsin treated control PGLC-33H target cells pulsed with 5X MIF for I hr demonstrated a migration inhibition of 22% at 18 hr (Table 1). PGLC-33H target cells trypTABLE
1
Response of Trypsinized PGLC-33H Target Cells to 5 X MIF Cell treatmenta cells + 5 X M IFb trypsinized cells + 5X MIF”
To Migration inhibition (To MI) 22 -14
(A 164%)
a Trypsinization with 0.2y0 trypsin at 20°C for 3 hr; MIF pulse and assays at 37°C. Results record the average of triplicate studies. b ‘% MI = (1 - area cells + 5X MIF/area cells + 5X Control) X 100. c % MI = (1 - trypsinized cells + 5X MIF/trypsinized cells + 5X Control) X 100.
MIF
RECEPTOR
ON
A HUMAN
TABLE
A-LYMPHOID
CELL
LINE
303
2
Effect of Preincubation with PGLC-33H Cells and HeLa Cells on 5 X M IF Activity % MIa after 1 hr adsorption 37”
4”
No. 1 No. 2
No. 3
5X MIF 5X MIF (PGLC-33H)
27 Wg%b)
5X MIF 5 X MIF (trypsinized PGLC-33H) 5X MIF (PGLC-33H) 5X MIF 5 X MIF (HeLa)
38 13 @660/o) 25
20 (A20%) 12 (A=%) 25 39 @56%)
21 W6%)
0 % MI represent average of two studies each done in triplicate. The 5 X Control or 5 X Control (adsorbed with the appropriate cells) served as the denominator in the equation.
sinized for l/2 hr prior to a similar 1 hr pulse exposure showed a complete abrogation of the migration inhibitory effects of 5 x MIF. The subsequent enhanced migration pattern of - 14% represents a change in MI of 164%. Removal of 5x MIF
by PGLC-33H
Target Cells and by HeLa Cells
These experiments examined the ability of PGLC-33H target cells and of nonlymphoid HeLa cells to remove MIF activity from supernatants. Following exposure to PGLC-33H cells for 1 hr at 37”C, the migration inhibitory activity of 5x MIF for PGLC-33H target cells was reduced 66% (38% MI reduced to 13% MI) (Table 2). Following a similar exposure to PGLC-33H cells for 1 hr at 4°C the migration inhibitory activity of 5 x MIF was reduced by 28% (38% MI reduced to 27% MI). Trypsinized PGLC-33H cells reduced the migration inhibitory activity of 5x MIF by 20% (25 to 20% MI). Non-trypsinized PGLC-33H cells reduced the migration inhibitory activity of 5X MIF in simultaneous assays by 52% (from 25 to 12% MI). At 37°C HeLa cells reduced the migration inhibitory activity of 5x MIF by only 16% (2.5 to 21% MI). Similar studies with HeLa at 4°C demonstrated an increased migration inhibitory activity of the 5~ MIF (2.5 to 39%, A 56%). Efects of Trypsinized
Cell Supemmtants on 5'~ MIF Activity
Since trypsinized PGLC-33H target cells neither responded to a pulse exposure to MIF nor removed MIF from active supernatants, it was of interest to examine the trypsinized cell supernatant for its ability to directly block MIF activity in the capillary migration assay. Trypsinized supernatant from PGLC-33H cells was inactivated by addition of 10% FBS v/v and diluted 1: 10 in 5 x MIF or 5 x Control. This reduced the migration inhibitory activity of the MIF by 90% (40 to 3% MI) (Table 3). The trypsin control inactivated by 10% FBS and diluted 1: 10 in 5~ MIF or in 5~ Control reduced the activity of MIF only 20% (40 to
304
MCLEOD
ET
TABLE Blocking of 5X MIF Activity
AL.
3
by Trypsinized Cell Supernatants
(Cell supernatant)”
% MIc
5X MIF 5 X MIF (trypsin control)b 5X MIF (PGLC-33H Sup.)
40 32 (~20%) 3 (A90%)
.5X MIF 5 X MIF (trypsin control) 5 X MIF (HeLa Sup.)
39 39 (A 0%) 11 (~72%)
u PGLC-33H trypsinized cell supernatant and trypsin control were diluted 1: 10 and HeLa and its control 1: 5 in 5 X MIF or 5 X Control. b Trypsin control is 0.2% trypsin with 10% FBS. c The denominator for calculation of % MI was 5 X Control with the appropriate additives.
32% MI) indicating that the blocking was not due to residual trypsin activity or its effects. Supernatants from trypsinized HeLa cells inactivated with 10% FBS and diluted 1: 5 in 5~ MIF or 5~ Control reduced the migration inhibitory activity of 5x MIF by 72% (39 to 11% MI). The trypsin control in this experiment had no effect on the MIF activity. DISCUSSION Studies with guinea pig peritoneal macrophages and guinea pig lymphocyte derived MIF suggest that a surface receptor is involved in the response of this target cell to MIF. Supporting this conclusion are the observations that: inhibition of migration of guinea pig macrophages occurs after short pulse exposure to MIF (12, 13), the interaction is temperature dependent (12, 13), macrophages can remove MIF activity from supernatants and the removal is temperature dependent (12), and trypsinized macrophages will not respond to MIF nor remove MIF activity from active supernatants (12, 14). Although migration inhibition does not follow pulse exposure to MIF at 4°C migration inhibition will occur if these pulsed target cells are incubated at 37°C in MIF-free medium prior to capillary assay (13). In further support of the existence of surface receptors for guinea pig MIF are the observations by Remold (15) that a-L-fucose blocks the effects of MIF on guinea pig macrophages and that fucosidase treated macrophages no longer respond to MIF. Rocklin (16) has shown that a-L-fucose similarly will block the effects of MIF derived from concanavalin A stimulated human lymphocytes on human peripheral monocytes. Fucosidase treated human monocytes will not respond to MIF. Human B-lymphoid cell lines retain many of the cell surface structures found on human peripheral lymphocytes, including HL-A (17-20) and receptors for hormones (21-23) and for plant lectins (24, 25). Most human B-lymphoid cell lines display membrane associated immunoglobulins (2630), complement receptors (3133), receptors for IgG Fc (31, 33) and for viruses (34). We have recently demonstrated that the human B-lymphoid cell line PGLC-33H also possesses surface receptors, probably of IgM class, for a soluble extract from Cundida albicans (11, 35). In the present studies we have examined an aspect of the mechanism of action of PGLC-33H derived MIF on PGLC-33H target cells. We have
MIF
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shown that the migration of PGLC-33H target cells is significantly inhibited when the cells are exposed to PGLC-33H MIF at 37°C for as little as 30 min, but that they are unaffected by exposures at 4°C for periods in excess of 2 hr. That MIF need not be present during the entire assay period is consistent with a receptor mechanism. The temperature dependence of this phenomenon suggests that the surface interaction of MIF with the cell is an active metabolic process or perhaps, that migration inhibition is dependent upon a second temperature dependent event which must occur soon after exposure to MIF. PGLC-33H MIF activity, however, is removed from supernatants by PGLC-33H cells at 37”C, but not by PGLC-33H cells at 4°C. These additional data suggest that at least the binding or inactivation of PGLC-33H MIF by PGLC-33H cells is temperature dependent. The involvement of cell surface structures in the response of PGLC-33H target cells to PGLC-33H MIF is further implicated by the inability of trypsinized target cells to respond to MIF and the inability of trypsinized PGLC-33H cells to remove MIF activity from supernatants. Supernatants of trypsinized PGLC-33H cells (with trypsin subsequently inactivated with fetal bovine serum) pre-incubated with PGLC-33H MIF substantially block the inhibitory activity of the MIF. Although trypsin has been shown to alter intracellular structures (36, 37)) this treatment is commonly used to remove cell surface proteins from intact cells to elucidate surface receptor mechanisms. It is doubtful that MIF was being inactivated by trypsin since the trypsin controls show essentially the same migration inhibitory activity with MIF as did MIF with no additions. Unfortunately, we were unable to examine the trypsinized cells for the reappearance of response to MIF and for the return of the ability to remove MIF from active supernatants. These cells have a mean generation time of 10-12 hr and therefore, double their original numbers within 20 hr of trypsinization. The migration inhibitory activity of PGLC-33H MIF was only slightly diminished by incubation at 37°C with the HeLa cell monolayers. These data suggest that HeLa may have few or no surface receptors for PGLC-33H MIF. Trypsinized cell supernatants from this cell line, however, significantly blocked the inhibitory activity of PGLC-33H MIF. It is possible that trypsinization of the HeLa cell releases receptors for MIF which lie hidden in the intact surface. Since the mechanism by which trypsinized supernatant from PGLC-33H cells and from HeLa cells block MIF is unknown, it is also possible that some or all of the blockade is nonspecific. Incubation of PGLC-33H MIF with HeLa cells at 4°C consistently produced an increase in the migration inhibitory activity of PGLC-33H MIF. Although it is possible that HeLa cells absorb other factors which effect the migration of target cells in the capillary assay, we have no data to help clarify this paradoxical amplified migration inhibitory activity of supernatants incubated with HeLa at 4°C. The interaction of PGLC-33H target cells with PGLC-33H MIF is considerably different from the interaction of PGLC-33H target cell with Candida antigen. Unlike its response to MIF which requires at least 30 min at 37”C, PGLC-33H cells require a pulse time of less than 2 min for Candida antigen and this response is independent of temperature. Membrane modulation studies with anti-human immunoglobulin sera demonstrate that Candida antigen reception by PGLC-33H involves an immunoglobulin surface receptor of the IgM class (35). From present data it appears unlikely that the interaction of MIF with PGLC-33H target cells is dependent upon an antibody receptor mechanism.
306
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Rocklin (38, 39) has reported that the migration inhibitory effect of human leucocyte inhibitory factor (LIF, 68,000 daltons) for polymorphonuclear cells is blocked by serine esterase inhibitors and had hydrolytic activity for some artificial substrates. He has also shown that LIF and MIF are distinct from each other (40) and that MIF appears to be devoid of esterase activity (39). Although our data suggests that PGLC-33H derived MIF reacts with there cells at the plasma membrane, it does not rule out the possibility that MIF acts enzymatically at the surface to inhibit migration. While esterase activity in our supernatants has not been extensively examined, preliminary experiments indicated that cell line MIF is insensitive to phenylmethylsulfonylfluoride. In addition, these supernatants, even when concentrated more than 100 fold, contained no fibrinolytic activity. We believe that our observations, similar to those reported for the guinea pig macrophage, suggest that the inhibition of migration of the human B-lymphoid cell line PGLC-33H cells by human MIF from PGLC-33H involves a surface receptor. Further investigation of the nature of this receptor should provide us with a greater understanding of the mechanisms of interaction between MIF and its target cells and possibly a means for the further purification and characterization of human MIF. REFERENCES 1. McLeod, T. F., Cordeiro, P. G., and Glade, P. R., Fed. Proceed. 36, 1299, 1977 (abstract).
2. Granger, G. A., Moore, G. E., White, J. G., Matzinger, P., Sundsmo, J. S., Shupe, S., Kold, W. P., Kramer, J., and Glade, P. R., J. Zmmunol. 104, 1476, 1970. 3. Papageorgiou, P. S., Henley, W. L., and Glade, P. R., J. Zmmunol. 108, 494, 1972. 4. Tubergen, D. G., Feldman, J. D., Pollack, E. M., and Lerner, R. A., J. Exp. Med. 135, 255, 1972. 5. Yoshida, T., Kuratsuji, T., Takada, A., Takada, Y., Minowada, J., and Cohen, S., J. Zmmunol. 117, 548, 1976. 6. Papageorgiou, P. A., Sorokin, C. F., and Glade, P. R., J. Zmmunol. 112, 675, 1974. 7. Glade, P. R., and Broder, S. W., In “Zn Vitro Methods in Cell-Mediated Immunity” (B. R. Bloom and P. R. Glade, Eds.), pp. 561-570. Academic Press, New York, 1971. 8. Glade, P. R., Kasel, J. A., Whang-Peng, J., Hoffman, P. F., Kammermyer, J. K., and Chessen, L. N., Nature 217, 564, 1968. 9. George, M., and Vaughn, J. H., Proc. Sot. Exp. Biol. Med. 111, 514, 1962. 10. Glade, P. R., Broder, S. W., Grotsky, H., and Hirschhorn, K., In “Zn Y&r0 Methods in Cell-Mediated Immunity” (B. R. Bloom and P. R. Glade, Eds.), pp. 307-312. Academic Press, New York, 1971. 11. Baskin, B. L., Meltz, S. K., and Glade, P. R., Cell. Zmmunol. 26, 264, 1976. 12. Leu, R. W., Eddleston, A. L. W. F., Hadden, J. W., and Good, R. A., .Z. Exj. Med. 136, 589, 1972. 13. Manheimer, S., Pick, E., Immunology 24, 1027, 1973. 14. Bartfeld, H., and Atoynatan, T., Proc. Sot. Exp. Biol. Med. 130, 497, 1969. 15. Remold, H., J. Exp. Med. 138, 1065, 973. 16. Rocklin, R. E., J. Zmmecnol. 116, 816, 1976. 17. Mann, D. L., Rogentine, G. N., Fahey, J. L., and Nathenson, S. G., I. Zmmmzol. 103, 282, 1969. 18. Rogentine, G. N., Gerber, P., Transplantation 8, 28, 1969. 19. Hardy, D. A., Ling, N. R., and Knight, S. C., Nature 223, 511, 1969. 20. Han, T., Immunology 23, 355, 1972. 21. Gavin J. R., III, Buell, D. N., and Roth, J., Science 178, 168, 1972. 22. Lesnick, M. A., Gorden, P., Roth, J., and Gavin, J. R., III, J. Biol. Chem. 249, 1661, 1974. 23, Gailani, S., Minowada, J., Silvernail, P., Mussbaum, A., Kaiser, N., Rosen, F., and Shimasha, K., Cancer Res. 33, 2653, 1973.
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24. Osunkoya, B. O., Williams, A. I. O., Adler, W. H., and Smith, R. T., Afr. J. Med. Sci. 1, 3, 1970. 25. DeSalle, L., Munahata, N., Pauli, R. M., and Strauss, B. S., Cancer Res. 32, 2463, 1972. 26. Eskeland, T., and Klein, E., Z. Zmmunol. 107, 1368, 1971. 27. Lerner, R. A., McConahey, P. J., Jansen, I., and Dixon, F. J., J. Exfi. Med. 135, 136, 1972. 28. Sherr, C. J., Baur, S., Gundhe, I., Zeligs, J., Zeligs, B., and Uhr, J. W., J. Exp. Med. 135, 1392, 1972. 29. Hutteroth, T. H., Litwin, S. D., and Cleve, H., Cell. Zmmunol. 5, 446, 1972. 30. Litwin, S. D., Hutteroth, T. H., Lin, P. K., Kennard, J., and Cleve, H., J. Zmmzlnol. 113, 661, 1974. 31. Theofilopoulos, A. N., Dixon, F. J., and Bokisch, U. A., J. Exfi. Med. 140, 877, 1974. 32. Glade, P. R., and Chessin, L. N., Znt. Arch. Allergy 34, 181, 1968. 33. Huber, C., Sundstrom, K., Nilsson, K., and Wigzell, H., Clin. Exp. Zmmwaol. 25, 367, 1976, 34. Jondal, M., and Klein, G., J. Exp. Med. 138, 1365, 1973. 35. Baskin, B. L., Meltz, S. K., and Glade, P. R., Cell. Zmmuwol. 26, 274, 1976. 36. Chefurka, W., and Hayashi, Y., Biochem. Biophys. Res. Commam. 24, 633, 1966. 37. Kaji, H., Suzaka, I., and Kaji, A., J. Mol. Biol. 18, 219, 1966. 38. Rocklin, R. E. Cell Zmmunol. 27, 338, 1976. 39. Rocklin, R. E., and Rosenthal, A. S. 1. Zmmunol. 119, 249, 1977. 40. Rocklin, R. E. J. Zmmmzol. 112, 1461, 1974.
IMMUNOLOGY
Evidence
37, 298-307 (1978)
for a Surface Factor
Receptor
for Human
on a B-Lymphoid
Migration
Inhibitory
Cell Line1
THOMAS F. MCLEOD, PETER G. CORDEIRO, SUSAN K. MELTZ, AND PHILIP R. GLADE Department
of Pediatrics, Division of Pediatric Infectious Diseases and Immunology, University of Miami School of Medicine, Miami, Florida 33125 Received
August
31,1977
Reception by PGLC-33H target cells for the migration inhibitory factor (MIF) produced by this established line has been investigated by pulse time and temperature dependence, MIF absorption, and abrogation by trypsinization. PGLC-33H supernatants containing MIF were concentrated 5X with Carbowax and dialyzed against serum free RPMI-1640 before use. Prior to standard capillary migration assay a minimum 30 min pulse of MIF at 37°C is required for significant migration inhibition (MI > 20%). No significant MI is observed when cells are pulsed at 4°C for up to 2 hr. Preincubation with PGLC-33H for 1 hr at 37°C reduces activity of supernatants from 38 to 13% MI; at 4°C to 27% MI. Trypsinization of target cells for 30 min at 25°C abrogates response to MIF (43 to - 14% MI). Trypsinized cells did not reduce activity of supernatants. MIF activity is abolished (32 to 3% MI) in samples preincubated with supernatants of the trypsinized cells inactivated with serum. These data suggest that cells from the human B-lymphoid cell line PGLC-33H have a surface receptor for human MIF.
INTRODUCTION Although the conditions influencing the elaboration of MIF by various short term and long term cell cultures have received considerable attention in the decade following its’ discovery, the mechanisms of interaction between MIF and target cells remain largely unknown. Studies from several laboratories (2-S) have demonstrated that established human cell lines of lymphoid and of non-lymphoid origin have the capacity to elaborate migration inhibitory factor (MIF) with inhibitory activity for the migration of guinea pig peritoneal macrophages and of target cells from human B-lymphoid cells lines. This heat stable, trypsin sensitive, non-dialyzable material has an estimated MW between 14,300 and 25,700 and co-elutes from Sephadex G-75 with MIF produced by phytomitogen and antigen stimulated human peripheral lymphocytes (6). In the present series of investigations we docu1 This work was supported by the United States Public Health Service Research Grant No. AI 12475 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health and a research grant from the Upjohn Company, Kalamazoo, Michigan. Portions of the work were presented at the Annual Meeting of the American Association of Immunologists (FASEB) April 7, 1977, Chicago, Illinois (1). 298 0008-8749/78/0372-0298$02.00/O Copyright 0 1978by AcademicPress,Inc. All rights of reproductionin any form reserved.
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ment the possibility that the PGLC-33H target cell interacts derived MIF by means of a cell surface receptor. MATERIALS
AND
LINE
with
299 PGLC-33H
METHODS
Maintenance of the lymfihoid cell line. The methods employed in the establishment and continuous culture of human B-lymphoid cell lines and a description of their characteristics have been reported previously (7). PGLC-33H is a well-characterized human B lymphoid cell line derived from the peripheral blood of an adolescent female with acute infectious mononucleosis and has been maintained in continuous suspension culture since 1967 (8). This cell line was utilized for the production of migration inhibitory factor (MIF) and also served as the target cells in the MIF capillary assay. PGLC-33H cells were maintained at 37°C in roller bottles at 2 rpm (Bellco Roller Apparatus, Vineland, N.J.) in complete RPM1 1640 medium containing 100 pg/ml streptomycin, 100 units/ml penicillin, 2.92 mg/ml L-glutamine and 17% (v/v) heat inactivated fetal bovine serum (FBS) (Associated Biomedics Systems, Inc., Buffalo, N.Y.) . The cells were passaged routinely at 72 to 96 hr intervals by gravity sedimentation of the cells, removal of l/2 of the supernatant culture medium, and replacement with fresh complete medium. Viability was estimated by exclusion of 0.1% trypan blue dye. Maintenance of HeLa cells. The HeLa cell line was maintained in continuous monolayer culture in RPM1 1640 supplemented with 100 fig/ml streptomycin, 100 units/ml of penicillin, 2.92 mg/ml L-glutamine, and 10% (v/v) heat inactivated fetal bovine serum. The line was passaged twice weekly by trypsinization with 0.201, trypsin for 3-5 min at 20°C. Prior to study the cells were seeded into sterile plastic 75 cm* culture flasks (Falcon, Oxnard, Calif.). Pre$wation of MIF. PGLC-33H cells in the log phase of growth (within 24 hr of the addition of fresh medium) were collected by centrifugation at 150g for 20 min at 20°C and resuspended to a final cell concentration of 4-6 x lo6 cells/ml in fresh serum free RPM1 1640 supplemented with antibiotics and glutamine. Following incubation at 37°C in the roller apparatus for 24 hr the PGLC-33H cells were separated from the medium by centrifugation at 150g for 20 min at 20°C. The supernatant medium was filtered with a Millipore filter (0.45 p) and stored at - 20°C until use. Serum-free medium without PGLC-33H cells was handled in an identical fashion and served as control in all investigations. For routine studies of MIF activity 50 ml aliquots of thawed supernatants were each placed in dialysis tubing (A. H. Thomas, Philadephia, Pa., M.W. cut off 12,000; boiled in three changes of distilled water) and concentrated room temperature to 10 ml (5~ concentration) with 100 g of polyethylene glycol, average MW 20,000 (Fisher Scientific Company, Fairlawn, N.J.). Following a change of dialysis tubing, the 5X concentrated supernatants were each dialyzed at 4°C for 24 hr against serum-free RPM1 1640 with antibiotics and L-glutamine. Although the change of tubing was used to decrease the chance of contamination of samples by the polyethylene glycol, studies have shown that polyethylene glycol has no effect upon the migration of PGLC-33H cells or upon MIF at concentrations as high as 1 mg/ml. Samples (5X MIF or 5X Control) were utilized immediately or stored in 5.0 ml aliquots at - 20°C. Capillary migration assay for MIF. The inhibition of migration of PGLC-33H
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lymphoid cell line target cells was assayed by a modification of the capillary migration inhibition assay of George and Vaughan (9) as previously described ( 10). Forty-eight hours prior to assay cells were passaged into fresh complete RPM1 1640 medium as described. Twenty-four hours prior to assay the cells were collected by centrifugation at 15Og for 10 min at 20°C and resuspended at a concentration of 0.5 X lo6 viable cells/ml in complete RPM1 1640 medium in roller bottles. After incubation for 16 hr at 37°C in the roller apparatus at 2 rpm, the cell suspension was centrifuged at 15Og for 10 min and the cells were resuspended in fresh complete medium to a concentration 3-4 x lo7 cells/ml. The cells were then packed in microhematocrit tubes with a microhematocrit centrifuge (International Microhematrocrit Centrifuge Model MB, Meedham Heights, Mass.). The capillaries were cut at the cell-liquid interface, placed in Lexy culture chambers (Mini-lab Co., Quebec, Canada) and the chambers sealed with cover glasses. The chambers were filled with complete RPM1 1640 medium, with control material or with experimental material with FBS-added to a concentration of 17% (v/v) and incubated for 18 to 24 hr. The areas of cell migration were determined by projection of migration patterns with a photoenlarger, tracing the images, and measurement of areas by planimetry. All studies were performed in triplicate and the inhibition of migration (% MI) was calculated by the formula : y0 migration
inhibition
=
1 - are~~on~rol)
x 100
Inhibition of 20% or more is considered significant. Variation among triplicates was no more than -C 10%. Pulse exposure of PGLC-33H target cells to MIF. Prior to pulse exposure studies, samples of each lot of 5X MIF were subjected to serial twofold dilutions in 5X Control and assayed for MIF activity as described. The MIF activity of 5~ MIF was invariably reduced below significant levels of dilution to 1: 4. Pulse exposure of target cells to PGLC-33H MIF was therefore conveniently terminated by addition of complete medium to a final dilution of the MIF to 1: 10 or greater. Twenty mls of the PGLC-33H target cell suspension containing 0.8-1.0 X lo6 viable cells/ml were added to multiple sterile plastic conical centrifuge tubes and centrifuged at 150g for 10 min at 20°C. The supernatants were decanted and the target cells were resuspended in 2 ml of either 5~ MIF or 5X Control, each supplemented to 17% (v/v) with FBS. The tubes were incubated for varying periods of time in a 37°C water bath with frequent gentle agitation. The pulse was terminated by the addition of 20.0 ml of complete medium to the cell suspensions followed by centrifugation at 150g for 2 min at 20°C. The supernatant fluids were decanted, the cell pellets were resuspended in 0.5 ml of complete medium and the cells were assayed in the capillary migration test as described. Percent migration inhibition was calculated using cells pulsed with 5X Control as the denominator in the equation. Treatment of target cells with trypsin. Target cells passaged as for use in the capillary assay were treated with 0.2% trypsin as previously described (11). The PGLC-33H target cells (0.8-1.0 x lo6 viable cells/ml) were washed four times in sterile H.anks Balanced Salt Solution (HBSS) and resuspended at a concen-
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tration of 2 x lo6 cells/l.0 ml of 0.2% trypsin in HBSS. After l/2 hr at room temperature the trypsin was then inactivated by addition of an equal volume of complete RPM1 1640 medium (17% PBS v/v). Tl le cells were removed by centrifugation at 15Og for 10 min at 2O”C, washed in complete RPM1 1640 medium and resuspended in complete medium to a final concentration of 1 X lo6 cells/ml. Control target cells were processed in a similar manner without trypsin. Viability of both sets of target cells (trypsin treated and control) was 99% as determined by trypan blue dye exclusion. Thirty milliliter aliquots of each set of target cells were then centrifuged at 150g for 10 min. 5~ MIF or 5X Control was added to the cell pellets and a pulse exposure study was performed as described. The $&MI was calculated using the results of the trypsin or non-trypsin treated cells pulsed with 5 X Control in the denominator. Removal of MIF activity with PGLC-33H cells. Target cells passaged as for use in the capillary migration assay were collected by centrifugation at 150g for 10 min at 20°C and washed two times in HBSS. Following resuspension to a concentration of 1 x lo6 cells/ml in HBSS, 20 ml aliquots were again centrifuged at 150~ for 10 min, the supernatants decanted and 3.0 ml of 5 X hlIF or 5X Control supplemented to 17% v/ v with FBS were added to the cell pellets. The cells were thoroughly resuspended in the samples and incubated with frequent agitation for lhr in a 37°C water bath or in a 4°C ice bath. The suspensions were then centrifuged at 25Og for 10 min, the supernatants drawn off and assayed for h,lIF activity in the capillary migration assay. Removal of MIF activity with HeLa. Monolayer cultures of HeLa cells grown to confluency in 75 cm2 culture flasks (17-20 x lo6 cells) were washed three times with HBSS. Three ml samples of 5X MIF or 5~ Control supplemented to 17% v/v with FBS were added to the flasks and were incubated for 1 hr at 37°C or at 4°C. The samples were then removed from the monolayers and assayed for MIF activity as described. Production of tryfsinixed cell supernatants. PGLC-33 cells (12.5 x 108) xvere washed three times in 250 ml volumes of HBSS and incubated at 20°C for 30 min in 100 ml of 0.2% trypsin. The trypsin was inactivated by the addition of 10 ml FBS and the suspension was then centrifuged at 250g for 10 min at 20°C. The supernatant was drawn off, passed through a 0.45 p Millipore filter (Millipore Corp., Bedford, Mass.) and used immediately, since storage or freezing resulted in irreversible precipitation. Control supernatant consisted of 1.0 ml FBS added to 10 ml of 0.2% trypsin. Trypsinized cell supernatants were obtained from monolayer cultures of HeLa cells washed three times in HBSS by the addition at room temperature of 5.0 ml of 0.2% trypsin in HBSS to the 75 cm2 culture flasks. When the cells began to dislodge from the culture flask surface (approximately 3-5 min) 0.5 ml of FBS was added to inactivate the trypsin and the supernatant fluid was decanted. This material was centrifuged at 25Og for 10 min at 20°C and passed through a 0.45 ,,, Millipore filter. Control supernatant consisted of 1.0 ml FBS added to 10 ml of 0.2% trypsin. Blockade of MIF activity zwith trypsinized cell supernatants, The ability of the trypsinized cell supernatants to block the effect of MIF on PGLC-33H target cells was examined by mixing control supernatants (trypsin + FBS) or trypsinized cell supernatants + FBS with 5~ MIF or 5 x Control samples supplemented to 15% (v/v) FBS, incubating for 1 hr at 37°C in a water bath, and assaying mixtures for MIF activity by the capillary migration assay.
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MRC.
OURA?-1Oh' OF PUS& FIG. 1. The temperature/time dependence of the inhibition of migration of PGLC-33H target cells induced by 5X MIF. The area of migration of cells pulsed with 5X Control was used for calculation of percent migration inhibition.
RESULTS P&e
Exposure of Target Cells to MIF
PGLC-33H cells (approximately 20 X 106) were exposed to 2.0 ml of 5~ MIF or to 5~ Control for periods of time up to 3 hr at 37°C and up to 2 hr at 4°C prior to testing in the capillary migration assay (Fig. 1). By 30 min of pulse exposure to 5~ MIF at 37”C, target cells would subsequently demonstrate significant migration inhibition in the capillary assay (MI = 29%). No significant migration inhibition could be demonstrated for target celIs exposed to 5X MIF at 4°C for periods up to 2 hr. These studies suggest that interactions between MIF and PGLC-33H target cells which result in significant migration inhibition (> 20%) 18 hr later can occur in a relatively short pulse exposure time and require actively metabolizing cells. Response of Trypsinized
Target Cells to MIF
If the interaction between 5~ MIF and PGLC-33H target is dependent upon a surface receptor, it should be possible to abolish the interaction by mild pretreatment of target cells with trypsin. In this series of experiments non-trypsin treated control PGLC-33H target cells pulsed with 5X MIF for I hr demonstrated a migration inhibition of 22% at 18 hr (Table 1). PGLC-33H target cells trypTABLE
1
Response of Trypsinized PGLC-33H Target Cells to 5 X MIF Cell treatmenta cells + 5 X M IFb trypsinized cells + 5X MIF”
To Migration inhibition (To MI) 22 -14
(A 164%)
a Trypsinization with 0.2y0 trypsin at 20°C for 3 hr; MIF pulse and assays at 37°C. Results record the average of triplicate studies. b ‘% MI = (1 - area cells + 5X MIF/area cells + 5X Control) X 100. c % MI = (1 - trypsinized cells + 5X MIF/trypsinized cells + 5X Control) X 100.
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2
Effect of Preincubation with PGLC-33H Cells and HeLa Cells on 5 X M IF Activity % MIa after 1 hr adsorption 37”
4”
No. 1 No. 2
No. 3
5X MIF 5X MIF (PGLC-33H)
27 Wg%b)
5X MIF 5 X MIF (trypsinized PGLC-33H) 5X MIF (PGLC-33H) 5X MIF 5 X MIF (HeLa)
38 13 @660/o) 25
20 (A20%) 12 (A=%) 25 39 @56%)
21 W6%)
0 % MI represent average of two studies each done in triplicate. The 5 X Control or 5 X Control (adsorbed with the appropriate cells) served as the denominator in the equation.
sinized for l/2 hr prior to a similar 1 hr pulse exposure showed a complete abrogation of the migration inhibitory effects of 5 x MIF. The subsequent enhanced migration pattern of - 14% represents a change in MI of 164%. Removal of 5x MIF
by PGLC-33H
Target Cells and by HeLa Cells
These experiments examined the ability of PGLC-33H target cells and of nonlymphoid HeLa cells to remove MIF activity from supernatants. Following exposure to PGLC-33H cells for 1 hr at 37”C, the migration inhibitory activity of 5x MIF for PGLC-33H target cells was reduced 66% (38% MI reduced to 13% MI) (Table 2). Following a similar exposure to PGLC-33H cells for 1 hr at 4°C the migration inhibitory activity of 5 x MIF was reduced by 28% (38% MI reduced to 27% MI). Trypsinized PGLC-33H cells reduced the migration inhibitory activity of 5x MIF by 20% (25 to 20% MI). Non-trypsinized PGLC-33H cells reduced the migration inhibitory activity of 5X MIF in simultaneous assays by 52% (from 25 to 12% MI). At 37°C HeLa cells reduced the migration inhibitory activity of 5x MIF by only 16% (2.5 to 21% MI). Similar studies with HeLa at 4°C demonstrated an increased migration inhibitory activity of the 5~ MIF (2.5 to 39%, A 56%). Efects of Trypsinized
Cell Supemmtants on 5'~ MIF Activity
Since trypsinized PGLC-33H target cells neither responded to a pulse exposure to MIF nor removed MIF from active supernatants, it was of interest to examine the trypsinized cell supernatant for its ability to directly block MIF activity in the capillary migration assay. Trypsinized supernatant from PGLC-33H cells was inactivated by addition of 10% FBS v/v and diluted 1: 10 in 5 x MIF or 5 x Control. This reduced the migration inhibitory activity of the MIF by 90% (40 to 3% MI) (Table 3). The trypsin control inactivated by 10% FBS and diluted 1: 10 in 5~ MIF or in 5~ Control reduced the activity of MIF only 20% (40 to
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TABLE Blocking of 5X MIF Activity
AL.
3
by Trypsinized Cell Supernatants
(Cell supernatant)”
% MIc
5X MIF 5 X MIF (trypsin control)b 5X MIF (PGLC-33H Sup.)
40 32 (~20%) 3 (A90%)
.5X MIF 5 X MIF (trypsin control) 5 X MIF (HeLa Sup.)
39 39 (A 0%) 11 (~72%)
u PGLC-33H trypsinized cell supernatant and trypsin control were diluted 1: 10 and HeLa and its control 1: 5 in 5 X MIF or 5 X Control. b Trypsin control is 0.2% trypsin with 10% FBS. c The denominator for calculation of % MI was 5 X Control with the appropriate additives.
32% MI) indicating that the blocking was not due to residual trypsin activity or its effects. Supernatants from trypsinized HeLa cells inactivated with 10% FBS and diluted 1: 5 in 5~ MIF or 5~ Control reduced the migration inhibitory activity of 5x MIF by 72% (39 to 11% MI). The trypsin control in this experiment had no effect on the MIF activity. DISCUSSION Studies with guinea pig peritoneal macrophages and guinea pig lymphocyte derived MIF suggest that a surface receptor is involved in the response of this target cell to MIF. Supporting this conclusion are the observations that: inhibition of migration of guinea pig macrophages occurs after short pulse exposure to MIF (12, 13), the interaction is temperature dependent (12, 13), macrophages can remove MIF activity from supernatants and the removal is temperature dependent (12), and trypsinized macrophages will not respond to MIF nor remove MIF activity from active supernatants (12, 14). Although migration inhibition does not follow pulse exposure to MIF at 4°C migration inhibition will occur if these pulsed target cells are incubated at 37°C in MIF-free medium prior to capillary assay (13). In further support of the existence of surface receptors for guinea pig MIF are the observations by Remold (15) that a-L-fucose blocks the effects of MIF on guinea pig macrophages and that fucosidase treated macrophages no longer respond to MIF. Rocklin (16) has shown that a-L-fucose similarly will block the effects of MIF derived from concanavalin A stimulated human lymphocytes on human peripheral monocytes. Fucosidase treated human monocytes will not respond to MIF. Human B-lymphoid cell lines retain many of the cell surface structures found on human peripheral lymphocytes, including HL-A (17-20) and receptors for hormones (21-23) and for plant lectins (24, 25). Most human B-lymphoid cell lines display membrane associated immunoglobulins (2630), complement receptors (3133), receptors for IgG Fc (31, 33) and for viruses (34). We have recently demonstrated that the human B-lymphoid cell line PGLC-33H also possesses surface receptors, probably of IgM class, for a soluble extract from Cundida albicans (11, 35). In the present studies we have examined an aspect of the mechanism of action of PGLC-33H derived MIF on PGLC-33H target cells. We have
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shown that the migration of PGLC-33H target cells is significantly inhibited when the cells are exposed to PGLC-33H MIF at 37°C for as little as 30 min, but that they are unaffected by exposures at 4°C for periods in excess of 2 hr. That MIF need not be present during the entire assay period is consistent with a receptor mechanism. The temperature dependence of this phenomenon suggests that the surface interaction of MIF with the cell is an active metabolic process or perhaps, that migration inhibition is dependent upon a second temperature dependent event which must occur soon after exposure to MIF. PGLC-33H MIF activity, however, is removed from supernatants by PGLC-33H cells at 37”C, but not by PGLC-33H cells at 4°C. These additional data suggest that at least the binding or inactivation of PGLC-33H MIF by PGLC-33H cells is temperature dependent. The involvement of cell surface structures in the response of PGLC-33H target cells to PGLC-33H MIF is further implicated by the inability of trypsinized target cells to respond to MIF and the inability of trypsinized PGLC-33H cells to remove MIF activity from supernatants. Supernatants of trypsinized PGLC-33H cells (with trypsin subsequently inactivated with fetal bovine serum) pre-incubated with PGLC-33H MIF substantially block the inhibitory activity of the MIF. Although trypsin has been shown to alter intracellular structures (36, 37)) this treatment is commonly used to remove cell surface proteins from intact cells to elucidate surface receptor mechanisms. It is doubtful that MIF was being inactivated by trypsin since the trypsin controls show essentially the same migration inhibitory activity with MIF as did MIF with no additions. Unfortunately, we were unable to examine the trypsinized cells for the reappearance of response to MIF and for the return of the ability to remove MIF from active supernatants. These cells have a mean generation time of 10-12 hr and therefore, double their original numbers within 20 hr of trypsinization. The migration inhibitory activity of PGLC-33H MIF was only slightly diminished by incubation at 37°C with the HeLa cell monolayers. These data suggest that HeLa may have few or no surface receptors for PGLC-33H MIF. Trypsinized cell supernatants from this cell line, however, significantly blocked the inhibitory activity of PGLC-33H MIF. It is possible that trypsinization of the HeLa cell releases receptors for MIF which lie hidden in the intact surface. Since the mechanism by which trypsinized supernatant from PGLC-33H cells and from HeLa cells block MIF is unknown, it is also possible that some or all of the blockade is nonspecific. Incubation of PGLC-33H MIF with HeLa cells at 4°C consistently produced an increase in the migration inhibitory activity of PGLC-33H MIF. Although it is possible that HeLa cells absorb other factors which effect the migration of target cells in the capillary assay, we have no data to help clarify this paradoxical amplified migration inhibitory activity of supernatants incubated with HeLa at 4°C. The interaction of PGLC-33H target cells with PGLC-33H MIF is considerably different from the interaction of PGLC-33H target cell with Candida antigen. Unlike its response to MIF which requires at least 30 min at 37”C, PGLC-33H cells require a pulse time of less than 2 min for Candida antigen and this response is independent of temperature. Membrane modulation studies with anti-human immunoglobulin sera demonstrate that Candida antigen reception by PGLC-33H involves an immunoglobulin surface receptor of the IgM class (35). From present data it appears unlikely that the interaction of MIF with PGLC-33H target cells is dependent upon an antibody receptor mechanism.
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Rocklin (38, 39) has reported that the migration inhibitory effect of human leucocyte inhibitory factor (LIF, 68,000 daltons) for polymorphonuclear cells is blocked by serine esterase inhibitors and had hydrolytic activity for some artificial substrates. He has also shown that LIF and MIF are distinct from each other (40) and that MIF appears to be devoid of esterase activity (39). Although our data suggests that PGLC-33H derived MIF reacts with there cells at the plasma membrane, it does not rule out the possibility that MIF acts enzymatically at the surface to inhibit migration. While esterase activity in our supernatants has not been extensively examined, preliminary experiments indicated that cell line MIF is insensitive to phenylmethylsulfonylfluoride. In addition, these supernatants, even when concentrated more than 100 fold, contained no fibrinolytic activity. We believe that our observations, similar to those reported for the guinea pig macrophage, suggest that the inhibition of migration of the human B-lymphoid cell line PGLC-33H cells by human MIF from PGLC-33H involves a surface receptor. Further investigation of the nature of this receptor should provide us with a greater understanding of the mechanisms of interaction between MIF and its target cells and possibly a means for the further purification and characterization of human MIF. REFERENCES 1. McLeod, T. F., Cordeiro, P. G., and Glade, P. R., Fed. Proceed. 36, 1299, 1977 (abstract).
2. Granger, G. A., Moore, G. E., White, J. G., Matzinger, P., Sundsmo, J. S., Shupe, S., Kold, W. P., Kramer, J., and Glade, P. R., J. Zmmunol. 104, 1476, 1970. 3. Papageorgiou, P. S., Henley, W. L., and Glade, P. R., J. Zmmunol. 108, 494, 1972. 4. Tubergen, D. G., Feldman, J. D., Pollack, E. M., and Lerner, R. A., J. Exp. Med. 135, 255, 1972. 5. Yoshida, T., Kuratsuji, T., Takada, A., Takada, Y., Minowada, J., and Cohen, S., J. Zmmunol. 117, 548, 1976. 6. Papageorgiou, P. A., Sorokin, C. F., and Glade, P. R., J. Zmmunol. 112, 675, 1974. 7. Glade, P. R., and Broder, S. W., In “Zn Vitro Methods in Cell-Mediated Immunity” (B. R. Bloom and P. R. Glade, Eds.), pp. 561-570. Academic Press, New York, 1971. 8. Glade, P. R., Kasel, J. A., Whang-Peng, J., Hoffman, P. F., Kammermyer, J. K., and Chessen, L. N., Nature 217, 564, 1968. 9. George, M., and Vaughn, J. H., Proc. Sot. Exp. Biol. Med. 111, 514, 1962. 10. Glade, P. R., Broder, S. W., Grotsky, H., and Hirschhorn, K., In “Zn Y&r0 Methods in Cell-Mediated Immunity” (B. R. Bloom and P. R. Glade, Eds.), pp. 307-312. Academic Press, New York, 1971. 11. Baskin, B. L., Meltz, S. K., and Glade, P. R., Cell. Zmmunol. 26, 264, 1976. 12. Leu, R. W., Eddleston, A. L. W. F., Hadden, J. W., and Good, R. A., .Z. Exj. Med. 136, 589, 1972. 13. Manheimer, S., Pick, E., Immunology 24, 1027, 1973. 14. Bartfeld, H., and Atoynatan, T., Proc. Sot. Exp. Biol. Med. 130, 497, 1969. 15. Remold, H., J. Exp. Med. 138, 1065, 973. 16. Rocklin, R. E., J. Zmmecnol. 116, 816, 1976. 17. Mann, D. L., Rogentine, G. N., Fahey, J. L., and Nathenson, S. G., I. Zmmmzol. 103, 282, 1969. 18. Rogentine, G. N., Gerber, P., Transplantation 8, 28, 1969. 19. Hardy, D. A., Ling, N. R., and Knight, S. C., Nature 223, 511, 1969. 20. Han, T., Immunology 23, 355, 1972. 21. Gavin J. R., III, Buell, D. N., and Roth, J., Science 178, 168, 1972. 22. Lesnick, M. A., Gorden, P., Roth, J., and Gavin, J. R., III, J. Biol. Chem. 249, 1661, 1974. 23, Gailani, S., Minowada, J., Silvernail, P., Mussbaum, A., Kaiser, N., Rosen, F., and Shimasha, K., Cancer Res. 33, 2653, 1973.
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24. Osunkoya, B. O., Williams, A. I. O., Adler, W. H., and Smith, R. T., Afr. J. Med. Sci. 1, 3, 1970. 25. DeSalle, L., Munahata, N., Pauli, R. M., and Strauss, B. S., Cancer Res. 32, 2463, 1972. 26. Eskeland, T., and Klein, E., Z. Zmmunol. 107, 1368, 1971. 27. Lerner, R. A., McConahey, P. J., Jansen, I., and Dixon, F. J., J. Exfi. Med. 135, 136, 1972. 28. Sherr, C. J., Baur, S., Gundhe, I., Zeligs, J., Zeligs, B., and Uhr, J. W., J. Exp. Med. 135, 1392, 1972. 29. Hutteroth, T. H., Litwin, S. D., and Cleve, H., Cell. Zmmunol. 5, 446, 1972. 30. Litwin, S. D., Hutteroth, T. H., Lin, P. K., Kennard, J., and Cleve, H., J. Zmmzlnol. 113, 661, 1974. 31. Theofilopoulos, A. N., Dixon, F. J., and Bokisch, U. A., J. Exfi. Med. 140, 877, 1974. 32. Glade, P. R., and Chessin, L. N., Znt. Arch. Allergy 34, 181, 1968. 33. Huber, C., Sundstrom, K., Nilsson, K., and Wigzell, H., Clin. Exp. Zmmwaol. 25, 367, 1976, 34. Jondal, M., and Klein, G., J. Exp. Med. 138, 1365, 1973. 35. Baskin, B. L., Meltz, S. K., and Glade, P. R., Cell. Zmmuwol. 26, 274, 1976. 36. Chefurka, W., and Hayashi, Y., Biochem. Biophys. Res. Commam. 24, 633, 1966. 37. Kaji, H., Suzaka, I., and Kaji, A., J. Mol. Biol. 18, 219, 1966. 38. Rocklin, R. E. Cell Zmmunol. 27, 338, 1976. 39. Rocklin, R. E., and Rosenthal, A. S. 1. Zmmunol. 119, 249, 1977. 40. Rocklin, R. E. J. Zmmmzol. 112, 1461, 1974.
