Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 3197-3200, August 1975

Immunology

Leukocyte migration inhibitory factor (LMIF) induced by concanavalin A: Standardized microassay for production in vitro (cell-mediated immunity/immunodeficiency/lymphocyte transformation in vitrolymphokines)

ANDRZEJ J. G6RSKI, Bo DUPONT, JOHN A. HANSEN, AND ROBERT A. GOOD Clinical Immunology Laboratory, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, N.Y. 10021

Contributed by Robert A. Good, April 28,1975 A standardized microculture system has ABSTRACT been developed to assess the ability of lymphocytes to secrete leukocyte migration inhibitory factor (LMIF) in response to the nonspecific mitogen concanavalin (Con A). LMIF-rich supernates collected from stimulated Iymphocytes cultured in plastic microtiter plates are assayed by pulse exposure of purified human granulocytes and inhibition of their migration in agarose medium. LMIF activity in this system is suppressed by the protein synthesis inhibitor puromycin, but not by inhibition of lymphocyte proliferation by irradiation. It is demonstrated that normal lymphocytes stimulated with mitogen elaborate LMIF activity, while lymphocytes from malignant lymphoma patients are frequently unable to produce it. Thus. mitogen-induced mediator production may be a useful parameter in further characterization of primary and secondary immunodeficiencies.

Lymphocytes stimulated in vitro with appropriate antigens and mitogens release factors called lymphocyte effector molecules or lymphokines, which have various biological properties in vivo and in vitro (1). One of the most important and best characterized mediators, migration inhibitory factor (MIF) probably represents two different molecules: one of them acting on macrophages (MMIF) and the other on peripheral leucocytes (LMIF). It has been shown that the synthesis and secretion of MIF precedes lymphocyte transformation and is dissociable from cell proliferation (2). It has also been shown by Rocklin et al. that both T (thymus-derived) and B (bone-marrow-derived) lymphocytes produce MIF (3). In some forms of immunodeficiency lymphocytes are able to transform in vitro but fail to release MIF (4). Recently, the presence of MIF in serum has been described in animals exhibiting delayed hypersensitivity reactions (5), in patients with lymphoproliferative malignancies (6), and in patients with hepatitis (7). In addition, Yoshida et al. (8) have shown that lymphocytes from patients with Sezary syndrome spontaneously release MIF in culture. The presently available methodologies for in vitro detection of MIf are not well standardized. Techniques vary in the number of cells used for the assay (1 X 106 per ml to 1.5 X 107 per ml), in the period of culture, culture conditions, and in cell separation procedures. The number of cells necessary to perform the test has been a limiting factor in the practical application of the MIF assay. Recently, a convenient agarose migration method has been introduced by Clausen (10, 9). This method has been modified to a two-step, or so-called indirect, MIF assay. Abbreviations: LMIF, leukocyte migration inhibitory factor; MMIF, macrophage migration inhibitory factor; MIF, migration inhibitory factor; Con A, concanavalin A; PHA, phytohemagglutinin.

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First, lymphocytes are stimulated with antigen and then the supernatant is transferred to a target cell culture to measure for MIF activity. This is regarded as a more reliable measure of cell-mediated immunity than the direct, one-step MIF assay (10, 11). A significant limitation of this modification is that the investigators use "buffy coat" cells as migratory target for LMIF. Skin-negative blood donors are a prerequisite to ascertain that the LMIF activity measured is due to the response of the test lymphocytes and not the response of immunocompetent cells in the "buffy coat" to antigen (12). Even if it was possible to make certain that the blood donor is not sensitized to the antigen tested, the lymphocytes present among the migratory cells might themselves respond to lymphokines, and secrete other factors. "Buffy coat" cells also contain a significant number of red cells. This is an additional hazard, since erythrocytes have receptors for MIF and can absorb the mediators from supernates (13). The indirect MIF assays available have been developed to assess antigen-induced LMIF production to a variety of specific antigens. A reliable assay for evaluation of mitogeninduced LMIF has not yet been developed. In this study we present data on a standardized microculture system for the measurement of mitogen-induced LMIF production in vitro. We also demonstrate that the lymphocytes from most of the patients with lymphoproliferative malignancies lack the ability to elaborate LMIF following mitogen stimulation.

MATERIALS AND METHODS Human lymphocytes are isolated from heparinized blood by gradient centrifugation on Ficoll-Isopaque ("Lymphoprep", Nyegaard & Co., Oslo, Norway) as described by B6yum (14) and the cells are washed three times in Hanks' buffered salt solution without calcium and magnesium (GIBCO). The cells are resuspended at a cell concentration of 2 X 106 cells per ml in medium RPMI 1640 (GIBCO) containing 10% heat-inactivated fetal calf serum (GIBCO), L-glutamine (1.6 mM), penicillin (100 IU/ml) and streptomycin (100 ,gg/ml). Lymphocyte culture is performed in sterile disposable Ushaped microtiter plates. (Cooke Laboratory Products, Catalog no. 1-221-24-1, Alexandria, Va.). The cultures consist of 100 p1 of cell suspension with 25 ,l of concanavalin A (Con A) solution (Difco) giving a final Con A concentration of 4 ,gg/ml. This concentration of Con A was found to have no direct effect on the migration of granulocytes after they were pulse exposed to the same dose of the mitogen. Supernates from nonstimulated lymphocyte cultures were "reconstituted" with the same amount of mitogen and found not to affect cell migration as compared to the non-"reconstituted" supernates.

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G6rski et al.

Experiments were also performed with phytohemagglutinin (PHA-P, Difco) as the mitogen in a concentration of 2.5 ug/ml. When stimulated lymphocyte cultures are performed in triplicate, a total of 1.2 X 106 cells are needed from each individual tested: 600,000 cells for triplicate control cultures and 600,000 cells for triplicate stimulated cultures. After 3 days of incubation at 370 in a humid atmosphere containing 5% CO2 the microtiter plates are centrifuged at 3000 rpm (GLC-1 centrifuge, I. Sorvall, Inc.) for 15 min and the supernates are collected from each individual culture and then tested for LMIF activity in the indirect assay using purified human granulocytes. Granulocytes are obtained by sedimentation of whole human blood with 6% dextran solution in normal saline and then purified by removal of lymphocytes by centrifugation on Ficoll-Hypaque for 30 min at 2000 rpm. It is also possible to recover the granulocytes from diluted blood after centrifugation on Ficoll-Hypaque by resedimentation with dextran and collection of "buffy coat" cells freed of lymphocytes. Granulocytes containing residual red cells are incubated with Tris-buffered 0.83% ammonium chloride for 20 min at 370 and washed three times with Hanks' solution. The granulocytes, usually 99% purified, are finally suspended in medium RPMI 1640 with 10% heat-inactivated fetal calf serum. The agarose medium is prepared according to Clausen (10, 9). A 1.5% solution of agarose (Indubiose A45, l'Industrie Biologique Francgaise, distributed by Fisher Scientific Co.) is prepared daily with distilled water and mixed with medium 199 (GIBCO) 1OX concentrated, heat-inactivated fetal calf serum, 10% solution of bicarbonate (Difco), and penicillin-streptomycin for a final concentration of 10% serum in 0.75% agarose. Aliquots (4.5 ml) are poured into 60 X 15 mm plastic tissue culture plates (Falcon Plastic, no. 3002) and allowed to gel. When gel has formed, eight wells per plate are cut with a 2.5 mm stainless steel punch. One hundred microliters of granulocyte suspension is mixed with 100,Ml of control or test supernates and the combined suspension is incubated for 30 min at 370 in plastic tubes (Falcon no. 2003). Following the incubation the cell concentration is adjusted to 3 X 108/ml and 5 ,l aliquots are added into each agarose well. The plates are incubated for 18 hr at 370 in a humid atmosphere containing 5% CO2. The total radial area of granulocyte migration is outlined with the aid of projection microscopy. The final results are expressed as Migration Index (Me) and calculated according to the formula: mean migration area in test supernate Mi = mean migration area in control supernate R2 7rR t2 where Rt is the radius of the test migration and R, is the radius of the control migration. The final radius value is calculated by subtracting the radius of the well from the total radius of migration. Each mean value expressed is obtained from four to eight replicate migration patterns. Experiments were performed with the inhibitor of protein synthesis puromycin to evaluate whether or not LMIF activity is dependent on the production of the mediator during the period of culture. Lymphocyte cultures were performed with 4 gg of puromycin per ml (puromycin dihydrochloride, Sigma Chemical Co.) added to Con-A- or PHA-P-stimulated cultures. This concentration of puromycin has been shown to not influence the migration of cells (11).

Proc. Nat. Acad. Sci. USA 72 (1975)

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FIG. 1. Concanavalin-A-induced leukocyte migration inhibitory factor (LMIF) production by normal human lymphocytes and lymphocytes from patients with malignant lymphomas. Mean migration index (Mi) for the normal material is 0.60 I 0.017 (standard error of mean); for the lymphoma group: 1.01 I 0.04.

To test whether or not the production of LMIF in microculture is dissociable from lymphocyte transformation, as is the case for MIF, lymphocytes were irradiated with total doses of 1250, 3750, or 7500 rads (at 125 rads/min) and then cultured with or without Con A. Irradiated lymphocyte cultures were also studied with the addition of puromycin to the culture medium. Lymphocyte transformation in vitro was performed in round bottom microtiter plates (Cooks no. 1-221-24-1) with 3 X 105 lymphocytes per well in a total volume of 175 Al. Lymphocyte transformation was performed using the same culture medium as in the LMIF culture system. Lymphocyte proliferation was measured by incorporation of [214C]thymidine (New England Nuclear Chemicals, specific activity: 50 Ci/mol) 0.025 ,Ci per well, and the cells were harvested on Whatman GF/C glass fiber filters after further 24 hr of culture. The filters were counted in a liquid scintillation counter (Packard Instruments, Tri-Carb no. 3390). The DNA synthesis rate was expressed as mean counts per minute (cpm) obtained from triplicate cultures. Studies were performed on the lymphocytes obtained from 29 normal blood donors. In addition the lymphocytes from 22 patients with different lymphoproliferative malignancies were studied (Hodgkin's disease and non-Hodgkin's malignant lymphoma). RESULTS Normal lymphocytes stimulated in vitro with concanavalin A produce and secrete a significant amount of LMIF compared to lymphocytes from unstimulated cultures (Fig. 1). The lymphocytes from the majority of the patients with

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Proc. Nat. Acad. Sci. USA 72 (1975)

concanavalin-A-induced lymphocyte proliferation but irradiated cells were still able to produce LMIF in stimulated cultures (Fig. 2). LMIF activity was abolished whenever normal or irradiated lymphocytes were stimulated in the presence of puromycin (Table 1).

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FIG. 2. Concanavalin-A-induced lymphocyte transformation in vitro [expressed as cpm (counts per minute) Qf incorporation of "'C-labeled thymidine into the cells] and production of leukocyte migration inhibitory factor (LMIF) expressed as migration indices (Mi). The figure demonstrates that LMIF production is unaffected by irradiation of the lymphocytes while the proliferative cell response is abolished. The data are expressed as means of triplicate cultures.

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Our preliminary results also indicate that although most of the patients' lymphocytes were nonresponsive by LMIF assay to Con A stimulation, some of these patients' lymphocytes did produce LMIF "spontaneously" (without further mitogenic or antigenic challenge). This effect could be demonstrated more readily when the lymphocyte concentration was adjusted to 5 X 106/ml. LMIF activity could be demonstrated in supernates from unstimulated lymphocyte cultures and it could also be demonstrated by the inhibition of migration of normal granulocytes by addition of lymphocytes from some patients with Hodgkin's disease directly into the granulocyte cell mixture. Increasing doses of irradiation were found to suppress

od for detection of concanavalin-A-induced LMIF production by human blood lymphocytes. The method can be used when stimulating with either Con A or PHA and thus provides an additional parameter for the evaluation of in vitro lymphocyte responsiveness. The preliminary data obtained with lymphocytes from patients with lymphoproliferative malignancies seem to indicate that this assay is sensitive enough to measure defects in immune responsiveness in patients with acquired immunodeficiency. There are several advantages to this methodology. First, the microculture system enables the use of a small number of lymphocytes (2 X 105 per culture). The establishment of this microculture test system for stimulation of LMIF response makes it possible to study in vitro lymphocyte transfor'mation and LMIF production of lymphocytes from the same individual simultaneously. The fact that LMIF production in the microculture system is dissociable from cell proliferation makes such a comparison more biologically useful. The use of purified granulocytes instead of "buffy coat" cells excludes noise from contaminating lymphocytes and eliminates the interference caused by erythrocyte contamination. Pulse exposure of migrating cells to MIF-rich supernates in the two-step technique has been shown to produce inhibition independent of antigen-antibody complexes (15). The presence of Con A in stimulated supernates does not contribute to granulocyte migration inhibition in the LMIF assay. This conclusion is supported by the following observations: (1) 30 min "pulse" exposure of granulocytes to medium containing Con A in a concentration of 4 gg/ml does not affect their migratory activity. After 30 min of incubation in serum medium containing 10 gtg/ml of Con A only 1% of the cells bind mitogen (16); (2) complete abolishment of LMIF activity in puromycin-treated cultures; (3) reproducible LMIF production by normal stimulated cells and reproducible lack of LMIF production by immunodeficient patients; (4) the evidence of stimulation of migration in some immunodeficient patients suggesting the production of migration stimulation factor. Migration stimulation factor is a recently described lymphokine that has been isolated and characterized (17, 11, 13). Our experiments with irradiation and puromycin treatment of lymphocytes stimulated with mitogen indicate that the LMIF response is similar to the MMIF response in that it is dissociable from cell proliferation but is abolished by a protein synthesis inhibitor. We were able to show that LMIF response to mitogen stimulation does not require cell proliferation, which is in accordance with the data obtained by Rocklin et al (3) using antigen activation of lymphocytes. Thus, mitogen-induced LMIF production by lymphocytes can be evaluated independent of their proliferative capacity. This may be of value in analyzing immune responses at the cellular level in patients with different forms of immune deficiency. Our preliminary data on "spontaneous" LMIF release by unstimulated lymphocytes from patients with Hodgkin's disease in culture are similar to those of Yoshida et al. (8) in pa-

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Immunology: Gorski et al.

Proc. Nat. Acad. Sci. USA 72 (1975)

Table 1. Effect of puromycin on Con-A-induced LMIF activity expressed as migration indices (Mi)

(A) Normal lymphocytes Exp. no. 1 2 3 4 5

Con A + puromycin 1.02 1.05 1.00 0.94 0.80

Con A 0.58 0.69 0.60 0.68 0.51

(B) Irradiated lymphocytes 3750 rads 1250 rads

Exp no.

0 rads Con A

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1.00 0.37 1.00 0.37 1 0.41 1.00 0.45 1.00 0.51 2 0.60 1.00 0.58 N.D. 3 0.73 0.77 1.00 0.56 N.D. 4 0.51 0.53 0.96 0.51 0.80 0.31 0.69 5 Concanavalin A and puromycin are used in concentrations of 4 Ag/ml. N.D. denotes test not done.

tients with Sezary syndrome and indicate that lymphocytes from patients with several lymphoproliferative malignancies can release mediators spontaneously. This evidence of in vivo activation suggests that a state of spontaneous activation exists in lymphocytes from patients with lymphoproliferative malignancies. This work was supported by grants from The American Cancer Society, U.S. Public Health Service Grant CA-08748-0851 NCI-CA 17404-01, National Foundation-March of Dimes, and a grant from the Foundation for the Advanced Study of Cancer. A.J.G. is supported by a Fulbright-Hays Program Fellowship. J.A.H. is a Special Fellow of Leukemia Society of America. We thank Dr. Richard O'Reilly for his help and suggestions. 1. Park, B. H. & Good, R. A., eds. (1974) in Principles of Modern Immunobiology (Lea & Fibiger, Philadelphia, Pa.), pp. 72-76. 2. Dimitriu, A., Dy, M., Thomson, N. & Bona, C. (1974) "MIF and blast transformation in human lymphoid cultures stimulated with different agents," Clin. Exp. Immunol. 18, 141148. 3. Rocklin, R. E., McDermott, R. P., Chess, L., Schlossman, S. F. & David, J. R. (1974) "Studies on mediator production by highly purified human T and B lymphocytes," J. Exp. Med. 140, 1303-1316. 4. Rocklin, R. E., Chilgren, R., Hong, R. & David, J. R. (1970) "Transfer of cellular hypersensitivity in chronic mucocutaneous candidiasis monitored in vivo and in vitro," Cell. Immunol. 1, 290-299. 5. Salvin, S. B., Youngner, J. S. & Lederer, W. H. (1973) "MIF and interferon in the circulation of mice with delayed hypersensitivity," Infect. Immun. 7,68-75. 6. Cohen, S., Fisher, B., Yoshida, T. & Bettigole, R. (1974)

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11. 12.

13. 14.

15. 16. 17.

7500 rads Con A

Con A + puromycin

0.58 0.51 0.55 0.81 0.57

1.00 0.86 0.80 N.D. N.D.

"Serum migration-inhibitory activity in patients with lymphoproliferative diseases," N. Engl. J. Med. 290, 882-886. Torisu, M., Yoshida, T. & Cohen, S. (1975) "Serum migration inhibitory activity in patients with post-transplantation hepatic dysfunction," Clin. Immunol. Immunpathol. 3,369-376. Yoshida, T., Edelson, R., Cohen, S. & Green, I. (1975) "Migration inhibitory activity in serum and cell supernatants in patients with Sezary syndrome," J. Immunol. 114, 915-918. Clausen, J. E. (1975) The agarose migration inhibition technique for in vitro demonstration of cell-mediated immunity in man. A review. Copenhagen, A/S M6llers Bogtrykkeri. Clausen, J. E. (1973) "Migration inhibitory effect of cell-free supernatants from tuberculin-stimulated cultures of human mononuclear leukocytes demonstrated by two-step MIF agarose assay," J. Immunol. 110, 546-551. Gorski, A. J. (1973) "Murine spleen leukocytes as an inhibitable target cell system for human MIF," Cell. Immunol. 12, 315-322. Weisbart, R. H., Bluestone, R., Cunningham, J. E. & Goldberg, L. S. (1973) "A modified agarose method for detection of MIF and delineation of its antigen dependency," Int. Arch. Allergy Appl. Immunol. 45,612-619. Fox, R. A., Gregory, D. S. & Feldman, J. D. (1974) "Macrophage receptors for MIF, migration stimulatory factor (MSF) and agglutinating factor," J. Immunol. 112, 1867-1872. B6yum, A. (1968) "Isolation of leucocytes from human blood. Further observations," Scand. J. Clin. Lab. Invest. 21 (suppl. 97). Kotkes, P. & Pick, E. (1975) "Studies on the inhibition of macrophage migration by soluble antigen-antibody complexes," Clin. Exp. Immunol. 19,105-120. Powell, A. E. & Leon, M. A. (1970) "Reversible interaction of human lymphocytes with mitogen Concanavalin A," Exp. Cell Res. 62, 315-325. Weisbart, R. H., Bluestone, R. & Goldberg, L. S. (1974) "Migration enhancement factor: a new lymphokine," Proc. Nat. Acad. Sci. USA 71, 875-879.

Leukocyte migration inhibitory factor (LMIF) induced by concanavalin A: standardized microassay for production in vitro.

A standardized microculture system has been developed to assess the ability of lymphocytes to secrete leukocyte migration inhibitory factor (LMIF) in ...
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