Immunology 1977 32 843

Leucocyte migration inhibitory activity in supernatants of cultured human mononuclear cells stimulated with mitogens or C3- and Fc-receptor reactions LIISA RXSXNEN* & H. ARVILOMMI Department of Cell Biology University ofJyvdskyld, Jyviskyli, and Public Health Laboratory, Jyvaskyld, Finland

Received 5 July 1976; acceptedfor publication 18 November 1976

Summary. We studied whether reactions at the lymphocyte membrane receptors for complement, immunoglobulin or mitogens would induce lymphokine production. Human peripheral blood mononuclear cells were stimulated by C3- or Fc-receptor reactions and with phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) and cultured for 3 or 4 days. C3- and Fc-receptor reactions were brought about by rosette formation with red cells coated with human complement or antibody. Culture supernatants were assayed for migration inhibitory activity with human leucocytes by the leucocyte-migrationin-agarose test. On the average, no migration inhibitory activity could be detected in cultures stimulated by C3- or Fc-rosette formation, mean migration indices (MI) being 99 2 and 96 1, respectively. Of the mitogens, PHA induced distinct lymphokine synthesis (mean MI 60 2) while the mean MI with LPS varied from 97 9-69 8, depending on the mitogen preparation used and the conditions of culture. We conclude that PHA and LPS are able to activate human lymphocytes into elaboration of migration inhibitory factors whilst reactions at C3- or Fc-receptors fail to do so.

INTRODUCTION Lymphocytes, after stimulation with antigens, mitogens or various other agents, may be activated to proliferative responses, to antibody synthesis or to the elaboration of other soluble factors termed lymphokines. Stimulation of human cells with phytohaemagglutinin (PHA) leads to a proliferative response and to the elaboration of various lymphokines (Pick & Turk, 1972). With bacterial lipopolysaccharide (LPS), however, controversial results have been obtained by utilizing the lymphocyte transformation test (Chess, MacDermott & Schlossman, 1974; Dimitriu, Dy & Bona, 1974; Greaves, Janossy & Doenhoff, 1974; Ivanyi & Lehner, 1974). As lymphocyte proliferative responses and lymphokine production seem to be independent phenomena (Mackler, Altman, Rosenstreich & Oppenheim, 1974; Wahl, Iverson & Oppenheim, 1974), study of the induction of lymphokine activity might help in answering the question whether LPS activates human lymphocytes or not. In recent years considerable interest has been focused on lymphocyte membrane receptors for antibodies and complement components. The functional significance of Fc- or C3-receptors is obscure although some intriguing indications have been recently reported as reviewed by Nussenzweig (1974) and Warner (1974). It is possible that one of the functional consequences of the reactions at these

* Present address: Institute of Biomedical Sciences, School of Medicine, University of Tampere, SF-33101 Tampere 10, Finland. Correspondence: Dr Liisa Rdsdnen, Institute of Biomedical Sciences, School of Medicine, University of Tampere, SF-33101 Tampere 10, Finland.

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receptors is the induction of lymphokine activities and in fact reports have been recently published demonstrating the production of a mitogenic factor and a chemotactic factor by guinea-pig and human cells after stimulation of these receptors (Mackler et al., 1974; Wahl et al., 1974; Sandberg, Wahl & Mergenhagen, 1975). We decided to study whether reactions at the lymphocyte membrane receptors for complement, immunoglobulin or mitogens would activate human mononuclear cells into lymphokine production and if so, what cells are responsible for this function. In this first paper we report the presence of migration inhibitory activity in supernatants of human mononuclear cell cultures after stimulation with PHA and LPS but not after C3- or Fc-receptor reactions.

MATERIALS AND METHODS

Isolation of blood mononuclear cells Mononuclear cells from healthy individuals were obtained by Ficoll-Isopaque centrifugation of heparinized peripheral blood (Boyum, 1968). The cells were washed three times with Hanks's balanced salt solution (HBSS) by centrifuging at 200 g for 10 min. The final cell suspension contained 7-20% monocytes identified by nonspecific esterase activity (Yam, Li & Crosby, 1971) and less than 0.5% granulocytes as judged by morphological criteria. Cell cultures Culture medium. If not otherwise stated cells were cultured in Medium 199 (Grand Island Biological Co., Grand Island, New York), supplemented with 10% heat-inactivated horse serum (Flow Laboratories, Irvine, Scotland). The medium also contained 70 u/ml penicillin and 70 4ug/ml streptomycin (Difco Laboratories, Detroit, Michigan). Sodium bicarbonate (final concentration 0-1 M) and HEPES-buffer (10 mM) (Flow Laboratories) were added to the medium and its pH was adjusted with sodium hydroxide to 7 4. C3- and Fc-receptor reactions. Human 0 erythrocytes were coated with complement by incubating with fresh human serum at low ionic strength, as described earlier (Arvilommi, 1974) and the control erythrocytes were treated with heat-inactivated serum. For making antibody coated red cells 1 ml of

5 % human 0 Rh-positive erythrocytes in saline was spun down and the supernatant discarded. One drop of human incomplete anti-RhD-serum (Finnish Red Cross Transfusion Service, Helsinki) was added to the packed cells and the suspension was incubated at 370 for 30 min. After incubation the erythrocytes were washed three times with saline and finally suspended in 2 ml HBSS. The control cells were similarly treated with normal human serum. C3- and Fc-reactions were brought about by forming rosettes by centrifuging 2 x 106 mononuclear cells (in 1 ml culture medium) with 0 2 ml of suspension of complement or antibody coated or control red cells for 5 min at 200 g. The cells were gently resuspended by a Pasteur pipette and a drop of the suspension was removed for counting the rosettes. Finally, half the cells were cultured for 4 days at 370 and the other half at 4°.

Stimulation with mitogens 2 x 106 mononuclear cells in 1 ml culture medium were given a pulse by incubating with PHA (Bacto Phytohaemagglutin P, Difco Laboratories) at a concentration of 50 ug/ml (this refers to the concentration of the desiccated preparation) for 30 min at 37°. The control cells were incubated in the medium alone. The cultures were then washed twice with HBSS and the cells resuspended in 1 ml culture medium. Half the cultures were incubated for 3 days at 370 and the other half at 4°. After culturing the suspensions were centrifuged at 2500 g for 15 min and the cell-free supernatants collected for migration inhibition assay. If not tested immediately the supernatants were stored at - 20°. We tested two different LPS preparations. In preliminary experiments concentrations of 1, 10 and 100 ,ug/ml were tested and 100 jig/ml was chosen for use in further experiments. When LPS W E. coli 055 Bs (Difco Laboratories) was used the cells were treated as above. The other LPS preparation was a gift from Dr M. Sarvas and was prepared from wild type Salmonella typhi murium with hot phenol extraction as described by Yuasa, Lewinthal & Nikaido (1969). In experiments with this LPS the mitogen was present throughout the culture. Cultures containing 2 x 106 cells/ml were set up and the cells were incubated in culture medium supplemented with 10% fresh or heatinactivated human AB serum for 3 days at 37° or 40. Before testing migration inhibitory activity the

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non-LPS-containing supernatants were reconstituted with LPS. Leucocyte migration inhibition assay The method used in the present study was the leucocyte migration agarose test of Clausen (1971). 9 ml of HEPES-buffered gel (pH 7 4) containing 1%Y agarose (Indubiose A37 agarose, L'industrie biologique frangaise S.A., Gennevilliers, France), 10% Medium 199 (ten-fold), 10% horse serum, penicillin 70 u/ml and streptomycin 70 mg/ml was poured into disposable petri dishes (0 9 cm, A/S Nunc, Roskilde, Denmark) and left to solidify. The plates were kept at 40 and used the same day. Nine volumes of heparinized blood were sedimented with one volume of 5 % dextran T 150 (Pharmacia Fine Chemicals, Uppsala, Sweden) in HBSS at 370 for 30-45 min. Serum and buffy coat leucocytes were collected and the leucocytes washed with HBSS twice. The cells were counted and transferred into 0 8 ml disposable tubes (Sterilin Ltd, Richmond, Surrey). After centrifuging the cells were suspended in the culture supernatants to be assayed. The leucocyte concentration was adjusted to 1 x 106/5 Md. These tubes were incubated at 370 for 30 min before the cell suspension was pipetted into the wells in the agarose plates. Wells with a diameter of 3 0 mm were punched in the agarose layer. Leucocyte suspension, 5 ,ul, was pipetted into each well. Each supernatant was tested with four to six replicate determinations. The plates were incubated in humidified atmosphere at 370 overnight, fixed with 10% formalin for 60 min and stained with 01 %Y crystal violet. The migration patterns were enlarged by an overhead projector and diameters were measured for calculating the migration area. The bottom of the well was not included in the migration area. Migration index (MI) was calculated as follows: area of migration in the presence of active supernatant , X 100. Ml = area of migration in the presence of control supernatant

RESULTS When human mononuclear cells were cultured after C3- or Fc-rosette formation, on the whole no

migration inhibitory activity was detected in the supernatants (mean MI 99-2 and 961, respectively, Fig. 1). The proportions of C3- and Fc-rosetting cells were 11-8%, 6-3-24-0 (mean and range) and 78%, 09-13-8, respectively. In two out of nine

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Figure 1. Migration inhibitory activity in culture supernatants of cells stimulated with C3- and Fc- receptor reactions, LPS, and PHA. Each symbol represents one experiment. (0) Non-significant, (0) significant (P< 005) inhibition or stimulation of migration. Significance of inhibition or stimulation in individual experiments was calculated by comparing the experimental and control groups of replicate migration areas with Student's t-test. Horizontal bars represent the means. Experimental conditions in LPS stimulated cultures: (a) LPS difco; inactivated horse serum, (b) inactivated and (c) fresh human serum, LPS II.

experiments statistically significant inhibition was obtained in both systems. Correspondingly, significant stimulation of migration occurred in one culture of C3-, and two cultures of Fc-stimulated cells. No correlation was observed between the

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proportion of rosette-forming cells and possible inhibition or stimulation of migration. Figure 1 also shows the results of the cultures stimulated by LPS and PHA. If the cells were pulsetreated with LPS (Difco) and cultured in medium containing inactivated horse serum the mean MI was 97-9 and significant inhibition of migration occurred in only four out of eleven cultures. We also used the other preparation of LPS and changed the conditions of culture as follows: instead of pulse treatment the cells were cultured in the presence of LPS, and in addition human serum, inactivated or fresh, was used, as we thought that complement might play a role in LPS induced activation. The mean MI and the number of statistically significant inhibitions were 82-7, 5/7 and 69 8, 6/7 in heatinactivated and fresh human serum, respectively. Cells stimulated with PHA caused migration inhibition in 11/12 cases, the mean MI being 60 2. The fact that migration inhibitory activity in culture supernatants tested with this assay was due to active metabolism of cultured cells was demonstrated by blocking cellular metabolism with cold. Because it is possible that mitogens might be released from metabolically active cells at 370 but not from inactive cells at 40, we studied also the possible effect of residual PHA on leucocyte migration. The mean migration indices of five experiments with varying doses of PHA were as follows: PHA 0 5 ,ug/ml, MI 109 2; 5 ,ug/ml, 102 5 and 50 pg/ml, 92 3. To make sure that the inhibition of migration was not caused by glucose depletion we added 2 mg/ml glucose to the supernatants of two cultures. The addition of glucose did not affect migration inhibition. Heating the supernatants for 15 min at 1000 totally abolished the migration inhibitory activity. DISCUSSION We have been unable to demonstrate that reactions at C3 or Fc receptors of lymphocytes induce production of migration inhibitory activity in culture supernatants. This appears to be in contradiction to the findings reported recently by Mackler et al. (1974), Wahl et al. (1974) and Sandberg et al. (1975). They have assayed chemotactic and mitogenic lymphokines and the methods of cell stimulation were different from ours as well. Thus it is difficult to compare these results. The red cells left

over after forming the rosettes and present during culture might have hindered in some way the synthesis of migration inhibitory factors. This, however, is unlikely as in our control experiments comparable amounts of erythrocytes did not diminish or block the production of migration inhibitory factors after PHA stimulation. Even if the stimulation of B-cell receptors leads to the synthesis of a mitogenic and a chemotactic lymphokine it may not induce the production of migration inhibitory factors. Furthermore, if migration inhibitory factors are produced their low concentration may remain undetected by our assay. Recently, Taylor, Burman & Fantes (1975) have reported that phyto-mitogen-induced migration inhibition may be due to residual mitogens in the supernatants. They also state that metabolism of the cells during culture may lead to medium depletion which causes migration inhibition. In our study we did not detect any migration inhibition in culture supernatants kept at 4°. The results of direct migration inhibition tests indicated that inhibition was not due to residual PHA. Furthermore, migration inhibitory activity was not affected by the addition of glucose and was abolished by heating. LPS is generally regarded as a B-lymphocyte activator. This seems to be true in some laboratory animals but controversy exits about the mitogenity of LPS for human cells (Chess et al., 1974; Dimitriu et al., 1974; Greaves et al., 1974; Ivanyi & Lehner, 1974). The present results show that LPS is capable of activating human lymphocytes at least into lymphokine synthesis. In contrast, Dimitriu et al. (1974) found no such effect in their study. The reasons for this remain unclear but some suggestions might be found from our results. In our first experiments with LPS we could only occasionally detect migration inhibitory activity in the culture supernatants. When we changed the LPS preparation and cultured the cells in the presence of LPS in the medium supplemented with human serum instead of culturing pulse-treated cells in the medium containing inactivated horse serum more consistent results were obtained. More pronounced inhibition of migration occurred if the cells were cultured in the presence of fresh instead of heat inactivated human serum. This may be due to complement C3 which may participate in the activation of B lymphocytes by LPS. We have not analysed in detail which of the factors mentioned above was most important for lymphokine synthesis. However, a study by Pryjma,

Leucocyte migration inhibitory factor Humphrey & Klaus (1974) has shown that preparations of LPS vary regarding their ability to activate C3. In addition, Greaves et al. (1974) found differences in the proliferative response to LPS in different culture media. Thus careful selection of the mitogen and the culture conditions seems to be of importance in demonstrating the activation of human lymphocytes by LPS.

ACKNOWLEDGMENTS The authors wish to thank Dr Matti Sarvas, Central Public Health Laboratory, Helsinki, for his gift of the LPS. The work was partly supported by Emil Aaltonen's Foundation.

REFERENCES ARVILOMMI H. (1974) Capacity of complement C3 phenotype to bind on to mononuclear cells in man. Nature (Lond.) 251, 740. BOYuM A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, supplement 97. CHESS L., MACDERMOTT R.P. & SCHLOSSMAN S.F. (1974) Immunologic functions of isolated human lymphocyte subpopulations. I. Quantitative isolation of human T and B cells and response to mitogens. J. Immunol. 113, 1113. CLAUSEN J.E. (1971) Tuberculin-induced migration inhibition of human peripheral leucocytes in agarose medium. Acta Allergol. 26, 56. DIMITRIu A., DY M. & BONA C. (1974) Migration inhibition factor and blast transformation in human lymphoid cultures stimulated by different agents. Clin. exp. Immunol. 18, 141.

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GREAVES M.F., JANOSSY G. & DOENHOFF M. (1974) Selective triggering of human T and B lymphocytes in vitro by polyclonal mitogens. J. exp. Med. 140, 1. IVANYI L. & LEHNER T. (1974) Stimulation of human lymphocytes by B cell mitogens. Clin. exp. Immunol. 18, 347. MACKLER B.F., ALTMAN L.C., RoSENSTREICH D.L. & OPPENHEIM J.J. (1974) Induction of lymphokine production by EAC and blastogenesis by soluble mitogens during human B-cell activation. Nature (Lond.), 249, 834. NUSSENZWEIG V. (1974) Receptors for immune complexes on lymphocytes. Advanc. Immunol. 19, 217. PICK E. & TURK J.L. (1972) The biological activities of soluble lymphocyte products. Clin. exp. Immunol. 10, 1. PRYJMA J., HUMPHREY J.H. & KLAUS G.G.B. (1974) C3 activation and T-independent B cell stimulation. Nature (Lond.), 252, 505. SANDBERG A.L., WAHL S.M. & MERGENHAGEN S.E. (1975) Lymphokine production by C3b-stimulated B cells. J. Immunol. 115, 139. TAYLOR M.M., BURMAN C.J. & FANTEs K.H. (1975) Problems encountered in the preparation of migration inhibitory factor (MIF) and mitogenic factor (MIT) from phytomitogen-stimulated human peripheral lymphocytes and in the preparation of MIF from human lymphoid cell lines (LCL). Cell. Immunol. 19, 41. WAHL S.M., IVERSON G.M. & OPPENHEIM J.J. (1974) Induction of guinea pig B-cell lymphokine synthesis by mitogenic and nonmitogenic signals to Fc, Ig, and C3 receptors. J. exp. Med. 140, 1631. WARNER N.L. (1974) Membrane immunoglobulins and antigen receptors on B and T lymphocytes. Advanc. Immunol. 19, 67. YAM L.T., Li C.Y. & CROSBY W.H. (1971) Cytochemical identification of monocytes and granulocytes. Amer. J. clin. Pathi. 55, 283. YUASA R., LEWINTHAL M. & NIKAIDO H. (1969) Biosynthesis of cell wall lipopolysaccharide in mutants of Salmonella. V. A mutant of Salmonella typhi murium defective in the synthesis of cytidine diphosphoabequose. J. Bact. 100, 433.

Leucocyte migration inhibitory activity in supernatants of cultured human mono-nuclear cells stimulated with mitogens or C3- and Fc-receptor reactions.

Immunology 1977 32 843 Leucocyte migration inhibitory activity in supernatants of cultured human mononuclear cells stimulated with mitogens or C3- an...
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