Immunology 1978 34 815

Cellular collaboration in the production of human leucocyte migration inhibition factor

R. H. WEISBART, D. T. Y. YU, R. BILLING, P. T. FAN, P. J. CLEMENTS & H. E. PAULUS Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024, U.S.A.

Received 23 May 1977; acceptedfor publication 4 October 1977

MATERIALS AND METHODS

Summary. The participation of cell subpopulations in the expression of leucocyte migration inhibition factor (LMIF) in response to Concanavalin A and Protein A was evaluated for cells isolated from the peripheral blood of five healthy subjects. LMIF activity could not be attributed to the function of T cells, B enriched cells, or monocytes acting alone, or to a combination of B enriched cells and monocytes. The LMIF response was the result of a collaborative event that occurred between T cells and B enriched cells, or between T cells and monocytes.

Subjects We studied mononuclear cells that were isolated from the peripheral blood of five healthy volunteers. Isolation and identification of T cells, B cells and monocytes Lymphocytes were isolated from peripheral blood

by Ficoll-Hypaque gradient centrifugation (Boyum, 1968). T cells rosetted with sheep red blood cells were separated from B cells on Ficoll-Hypaque gradients, and monocytes were removed from B cell enriched preparations with a magnet after the cells had been incubated with carbonyl iron (Yu, Ramer & Kacena, 1977). Monocytes were isolated by adherence to glass, and they were recovered by treating them with 2mM EDTA in saline (Yu, 1977). B lymphocytes were identified by the presence of surface immunoglobulin (SIg) detected by fluorescein-conjugated antisera to human immunoobulins after a 30 min incubation at 370 in medium free of human serum (Horowitz & Lobo, 1976). T lymphocytes were identified as those cells which

INTRODUCTION Leucocyte migration inhibition factor (LMIF) is a soluble mediator produced by stimulated lymphocytes (Rocklin, 1974). Recent work implicates the participation of both T and B lymphocytes in the expression of LMIF (Chess, Rocklin, MacDermott, David & Schlossman, 1975). The relationship of cell subpopulations in the expression of LMIF, however, has not been clearly defined. We therefore, studied the limiting concentrations of T cells, B cells, and monocytes that influence LMIF activity, and the effect of combining different proportions of these cell types.

formed rosettes with neuraminidase treated SRBC, and monocytes were recognized as mononuclear cells that stained for the presence of peroxidase (Yu et al., 1977). Cells binding aggregated human IgG were identified by incubating them with heat aggregated human gamma globulin followed by washing and

Correspondence: Richard H. Weisbart, Department of Medicine, 1115 Veterans Administration Hospital, 16111 Plummer Street, Sepulveda, California 91343. 0019-2805/78/0500-0815 $02.00 (C) 1978 Blackwell Scientific Publications

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incubation with fluorescein conjugated antisera to human immunoglobulins (Horowitz & Lobo, 1976). Lymphocyte culture techniques Cell preparations were washed in Hanks's Buffered Salt Solution (HBSS), and 2000 to 75,000 cells were cultured in Micro Test II plates (Falcon Plastics, Oxnard, California) containing 150,ul of Medium 199 with 25 mm HEPES Buffer, 2-2 g NaHCO3 per litre (Grand Island Biological Company, Berkeley, California), 10% foetal calf serum, penicillin 100 units per ml and streptomycin 100,ug per ml. All cultures were established in pairs, with a stimulated culture that contained 25 ug/ml of Con A or 20 ug/ml of Protein A (Pharmacia, Piscataway, New Jersey) and a non-stimulated culture with an equivalent amount of Hanks's BSS; these concentrations were found to be optimum in preliminary studies and did not produce non-specific inhibition of polymorphonuclear leucocyte migration. Cultures were incubated at 370 in the presence of 5 % CO2. After 4 days, 50 pl of cell-free supernatant were removed, and the non-stimulated culture supernatants were reconstituted with the same concentration of Con A or Protein A as used in the stimulated cultures. The supernatants were stored at - 200 in Micro Test II plates sealed with pressure sensitive film until assayed. The cultured cells were viable as determined by trypan blue exclusion. Agarose gel technique for the detection of LMIF The agarose technique used was previously described (Weisbart & Mickey, 1977). The agarose medium contained 1 % agarose, 10% horse serum, penicillin 100 units/ml and streptomycin 100,ug/ml. Thirty ml of agar were dispensed in 150 mm round tissue culture dishes (Integrid, Falcon Plastics, Oxnard, California). 128 1-5 mm diameter wells were cut in each agar plate; the agar plugs were aspirated, and the plates were covered with 15 ml of light weight mineral oil. Purified polymorphonuclear leucocytes (PMN) (95-99%) were isolated from peripheral blood by Ficoll-Hypaque gradient centrifugation and the red blood cells were removed by dextran sedimentation (Weisbart & Mickey, 1977). All tests were done with PMN from two donors who were previously studied and whose PMN gave comparable results. Target cell PMN were not obtained from the same donors who provided lymphocytes for the produc-

tion of LMIF. Supernatants from the T, B and monocyte cultures and their various combinations from each subject were assayed simultaneously with the PMN from one donor. PMN were washed with HBSS and resuspended at a concentration of 50 x 106/ml in Medium 199 with 25 mm HEPES Buffer and 10% horse serum. Four microlitre aliquots of the cell suspension were dispensed into each well of a micro test tissue culture plate (Falcon Plastics, Oxnard, California) and mixed with 4.u1 of each lymphocyte culture supernatant. The PMN-supernatant suspensions were covered with light weight mineral oil and incubated for 30 min at 37°. Each PMN-supernatant suspension was then drawn into a 50ul syringe, and 1 p1 aliquots were dispensed into each of 4 wells in an alternating pattern between 2 rows of wells. The agar plates were incubated for 6 h at 370 in the presence of 5% CO2. The agar plates were then filled with 95 % ethylalcohol, and the agar was removed 10 h later. Data analysis The largest diameter of each migration was measured to the nearest 0-5 mm with an ocular micrometer, and the area of migration was computed. The main analysis was constructed from the ratio of the average area of the test to that of the control migrations. The data were expressed as percent inhibition in the area of migration and the standard error of the mean for test/control was computed (Dahlberg, 1940). RESULTS LMIF activity from normal peripheral blood T cells, B cells and monocytes The average compositions of enriched subpopulations of mononuclear cells from the subjects studied are shown in Table 1. The T enriched population contained 98% T cells; in the B enriched population, 48% of the cells had SIg, and 48% bound aggregated human IgG. The monocyte enriched population contained 87 % monocytes. Because the B cell preparations from normal peripheral blood contained equal numbers of B cells and aggregate binding SIg negative cells, these preparations are referred to as B enriched. The average responses to Protein A of T cells, B enriched cells, monocytes, and various combinations of these cells from 4 healthy subjects are shown in Fig. 1. As many as 60,000 T cells, 40,000 B en-

Production of human leucocyte MIF

were reduced to 20,000, 10,000 B enriched cells were needed, whereas comparable numbers of monocytes were ineffective. Forty thousand B enriched cells combined with 10,000 monocytes did not produce LMIF activity. By examining the effect of adding different numbers of B enriched cells or monocytes to 40,000 T cells, we can conclude that the number of monocytes (900) contaminating the T (40,000)+B enriched (10,000) combination could not explain the response obtained, since 40,000 T cells+2000 monocytes did not respond to Protein A. Similarly, the number of B enriched cells (1200) contaminating the T (40,000)+monocyte (10,000) combination could not account for this response, since 40,000 T cells+ 2000 B enriched cells did not show a substantial response. These results indicate that either B enriched cells or monocytes collaborate with T cells to produce LMIF activity. Similar results are shown for combinations of T cells, B enriched cells, and monocytes in the presence of Con A (Fig. 2). These data represent the

Table 1. Average composition of enriched cell subpopulations isolated from the peripheral blood of five healthy subjects

Composition of lymphocyte subpopulations ( %)

T B enriched Monocytes

T

B

SI Neg aggregate binding

98 1 5

1 48 8

0 48 0

Monocvtes 1 3 87

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Number of cells x 10- 3 Figure 1. Average responses to Protein A of T cells, B enriched cells, monocytes, and combinations of these cells isolated from the peripheral blood of four healthy individuals. A, Anticipated cell number; C, Computed cell number adjusted for purity of cell preparations. Stippled column, T; hatched column, B enriched; cross-hatched, M; open column, T + B enriched or T + M.

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of 3 subjects. The outstanding difference between the responses to Con A and Protein A was that fewer collaborating B enriched cells or monocytes were required for LMIF in response to Con A. Inhibition of migration was observed with as few as 40,000 T cells in the presence of contamination by 400 B enriched cells and 400 monocytes. With 20,000 T cells, there was no response to Con A, but it could be restored by adding 10,000 B enriched cells or 10,000 monocytes. Again. the response of 20,000 T cells combined with 10,000 B enriched cells could not be explained by contamination with monocytes (500) since 20,000 T cells + 2000 monocytes did not respond; the response obtained with 20,000 T cells and 10,000 monocytes could not be attributed to contaminating B enriched cells (1000), since 20,000 T cells + 2000 B enriched cells did not respond. Forty thousand B enriched cells combined with 10,000 monocytes did not produce LMIF activity in response to Con A. average responses

DISCUSSION

The data show that T cells

are

essential for LMIF

activity, but alone, they are not sufficient. Either B enriched cells or monocytes must be added to T cells in culture to produce LMIF activity. B enriched cells, monocytes, or combinations of these cells did not produce LMIF responses in the absence of T lymphocytes. Increasing the number of T cells decreased the requirement for added B enriched cells or monocytes. The results cannot, however, be attributed to adding a small number of T cells that contaminate B enriched or monocyte preparations to sub-threshold T cell responses. This is best shown in Fig. 1 where 40,000 T cells alone did not respond to Protein A, but LMIF activity was obtained by adding 10,000 B enriched cells or 10,000 monocytes. These B enriched and monocyte preparations contained only 100 and 500 T cells, respectively, and yet 60,000 T cells alone did not produce LMIF responses.

Moreover, the results obtained cannot be attributed to the additive effects of small numbers of cells to achieve threshold responses. For example, in Fig. I we notice that 20,000 T cells combined with 10,000 B enriched cells produced consistent responses. If we conclude that 30,000 cells are, therefore, necessary for LMIF activity, we cannot

Production of human leucocyte MIF attribute the lack of responsiveness of 40,000 and 60,000 T cells alone and 40,000 B enriched cells plus 10,000 monocytes to a sub-threshold effect. The addition of only 5000 B enriched cells or monocytes to 40,000 T cells, and the addition of 2000 B enriched cells or monocytes to 60,000 T cells resulted in substantial LMIF activity. These results do not, however, answer the question of which cell subpopulation is producing LMIF and which is providing 'help'. Furthermore, these results do not demonstrate the mechanism of the 'collaborative' phenomenon; possible mechanisms include the presentation of antigen by one cell type to another by physical contact and the elaboration of soluble intermediaries from one cell type for the activation of another subpopulation. The results of our study indicate that LMIF activity is the result of complex cellular interactions. These results are at variance with another published report (Chess et al., 1974). This discrepancy is probably a result of differences in technique, since the large numbers of cultured cells used by these investigators contained sufficient numbers of contaminating cells to support the collaborative effect. Results similar to those presented in this study for LMIF have been reported for macrophage migration inhibition factor (Pick, Godny & Gold, 1975). Our study emphasizes the need to quantify carefully the subpopulations of cells contaminating 'enriched' cell preparations, and to use limiting cell concentrations when evaluating collaborative events. ACKNOWLEDGMENTS We gratefully acknowledge the excellent technical assistance of Julia Tang and Maureen Silvers.

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This investigation was supported by a grant from the National Institutes of Health (Al 13253), and USPHS grant GM 15759. REFERENCES BOYUM A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. J. clin. Lab. Invest. 21 (suppl. 97), 77. CHESS L., ROCKLIN R.E., MACDERMOTT R.P., DAVID J.R. & SCHLOSSMAN S.F. (1975) Leukocyte inhibitory factor (LIF): Production by purified human T and B lymphocytes. J. Immunol. 115, 315. DAHLBERG G. (1940) Statistical Methods for Medical and Biological Students, p. 95. George Allen and Unwin, London. HOROWITZ D.A. & LOBO P.J. (1976) Characterization of two populations of human lymphocytes bearing easily detectable surface immunoglobulin. J. clin. Invest. 56, 1464. PICK E., GODNY Y. & GOLD E.F. (1975) Participation of immunoglobulin-bearing lymphocytes in the production of macrophage migration inhibitory factor. Eur. J. Immunol. 5, 584. ROCKLIN R.E. (1974) Products of activated lymphocytes: Leukocyte inhibitory factor (LIF) distinct from migration inhibitory factor (MIF). J. Immunol. 112, 1461. WEISBART R.H. & MICKEY M.R. (1977) A microassay for leukocyte migration: Analysis of its reproducibility. J. Immunol. Methods, 16, 269. Yu D.T.Y., RAMER S. & KACENA A. (1977) Effect of corticosteroids on the response of lymphocytes to stimulation by galactose oxidase modified lymphocytes. Immunology, 33, 247. Yu D.T.Y. (1977) Human lymphocytes subpopulations: Giant human red blood cell rosettes. J. Immunol. Methods, 15, 67.

Cellular collaboration in the production of human leucocyte migration inhibition factor.

Immunology 1978 34 815 Cellular collaboration in the production of human leucocyte migration inhibition factor R. H. WEISBART, D. T. Y. YU, R. BILLI...
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