Scand. J. Immunol., Vol. 6, 1977.
Migration Inhibition Produced by Sodium Periodate Oxidation of the Macrophage Membrane, and Reversal by Sodium Borohydride R. A. FOX, L. A. FERNANDEZ & R. RAJARAMAN Departments of Medicine and Microbiology. Dalhousie University and Camp Hill Hospital, Halifax, Nova Scotia, Canada
Fox, R. A., Fernandez, L. A. & Rajaraman, R. Migration Inhibition Produred by Sodium Periodate Oxidation of the Macrophage Membrane, and Reversal by Sodium Borohydride. Scaad. / . Immunol. 6, 1151-1157, 1977. Guinea pig peritoneal exudate cells were harvested 3 to 4 days after the intraperitoneal injection of Marcol oil. The washed ceils were exposed to various concentrations of sodium periodate in phosphate-buffered saline (PBS) at pH 7A for 10 min at + 4 ° C . The cells were then used in the in vitro migration assay, and migration was consistently inhibited at concentrations from 10~^ to 10"^M. The viability of the macrophages was not affected by this treatment. Sodium borohydride {10"^ to 10"^M) in PBS for 10 min at pH 7.4 reversed the periodate effect. Experiments with purified macrophages showed that sodium periodate has a direct effect on macrophage function rather than an indirect effect via the potentiation of migration inhibition factor. In support of this, the in vitro spreading of macrophages on glass substrate for 1 h has been shown to be inhibited. This spreading inhibition can also be reversed by treatment with sodium borohydride. These results provide a new approach to understanding the biological significance and role of macrophage migration inhibition. R. A. Pox, Department of Medicine, Building 7, Camp Hill Hospital, 1763 Robie St., Halifax, Nova Scotia, Canada BJH 3G2
Sodium periodate oxidation of rat lymph node (18) and human peripheral blood lymphocytes (20) results in blast formation. The conditions used Co achieve blastogenesis indicated that only the surface glycoproteins were being oxidized, and it was suggested that aldehyde groups are formed on sialo glycoproteins (4, 26). These aldehyde groups can be reduced back to an alcohol by treatment with sodium borohydride. Blastogenesis is blocked or reversed by sodium borohydride treatment (1). Apart from lymphocyte transform^ion, other biological effects are produced by periodate treatment; for example, cytotoxicity is induced and can be demonstrated by chromium release from labeled tumor cells (17). Surface modification by
periodate has been shown to inhibit the homing of CBA/J lymphocytes to lymph nodes (26). It has been known for some time that macrophages play an essential role in lymphocyte transformation in response to various stimuli such as antigens (19) and mitogens (13)Macrophages are also necessary for transformation in response to periodate oxidation (3). Removal of glass-adherent cells from lymphocyte suspensions results in a significant reduction in transformation. A further significant observation was that normal lymphocytes could be stimulated, and transformation induced, by exposure to a macrophage monolayer treated with sodium periodate (3). This observation has led to speculations with regard to the
1152 R- A. Pox, L. A. Pernandez & R. Rajaraman
mechanism of action of sodium periodate in inducing lymphocyte transformation. The situation is complicated by the ohser%'ation thai periodate oxidation of one population of lymphocytes, also treated with mitomyein, will stimulate another, untreated, autologous population in a one-way mixed lymphocyte reaction (2). Thus lymphocyte transformation might result from any of the following three possibilities: direct stimulation of the lymphocyte that ultimately transforms; antigenic alteration of one population of lymphocytes, which then act as stimulator cells; or alteration of macrophages (monocytes), which then stimulate the lymphocytes. We found this latter possibility' intriguing but, apart from this observation using macrophage monolayers (3), were unable to find any other studies on the interaction of periodate with guinea pig peritoneal exudate macrophages and their subsequent behavior modification in vitro.
MATERIALS AND METHODS Adult guinea pigs, multicolored and albino, weighing between 250 and 400 g, were iniected intraperitoneally with 20-30 ml sterile Marcol 22 oil (Imperial Oil, Canada). There to four days later the peritoneal cavity wa.s drained and washed through with culture medium. The cells were separated from the oil by centrifugation and were then washed thoroughly with medium. After the third wash in tissue culture medium the cells were washed in ice-cold phosphate-buffered saline (PBS) (O.OOlM sodium phosphate, 0,14M sodium chloride, pH 7.4) and then resuspended in PBS. Sodium periodate was made up fresh on the day of the experiment, at the appropriate concentration in PBS. The pH of these solutions were carefully adjusted to 7.4 after equilibration at +4°C. Test aliquots of macrophages were resuspended in the appropriate-strength sodium periodate/PBS solution for 10 min at + 4°C; control cells were resuspended in PBS alone. At the end of this time the cells were washed three times and then, if no further treatment was required, prepared for assay or, alternatively, subjected to further treatment. In
tlie experiments on sodium borohydride rever sal, sodium borohydride was made up fresli each day and the pH adjusted to 7.4 after temperature equilibration. Cells were exposeti to sodium borohydride at +4*C for 10 min followed by thorough wa.shing; borohydridt treatment was controlled for in the migratioi assay by using buffer alone. Once the initial experiments were performeti and optimal concentrations had been deter mined, a protocol was .set up and strictly ad hered to for five successive experiments. Al' cell populations were subjected to two lO-mii treatments on ice. The first treatment was either PBS alone or sodium periodate in PBS at concentrations of lO-^M, IO-*M, lO'^M, and 10"*M. After thorough washing, each of the five populations was exposed to PBS alone oi sodium borohydride at three concentrations 10--3M, lO-iM. and lO'^M. Thus the periodate treated cells were controlled for with cells exposed to PBS only. The periodate- and borohydride-treated cells were controlled for with the PBS- and borohydride-treated cells. The conditions of periodate treatment were specifically altered for one experiment, in which the macrophages were exposed to the various concentrations of sodium periodate al room temperature for 30 min (24). In seven experiments the peritoneal exudate cells were collected in the usual manner am! washed. The cells were then layered ontc^ Ficoll-Isopaque (9%-34%) and centrifuget at 2500 rpm for 20 min. The purified mono nuclear population was then removed an*,i washed twice in RPMI 1640. The cells were washed twice, resuspended in medium witl 10% FCS, and allowed to settle in Petri dishes for 1 h at 37°C. The nonadherent cells wert washed off and the adherent populations col lected after removal with a rubber spatula. The purified macrophage population was then sub jected to either sodium periodate (lO^^M) or PBS for 10 min at +4°C and allowed to migrate in Che usual manner. Macrophage migration of guinea pig peritoneal exudate cells was measured by a capillarT, tube assay as previously described (9-12)
Periodate
Inhibits
Macrophage
Migration
1153
Tahie 1. Macrophage migration inhibition with sodium periodate* Concen(M)
Experiment 1
10"3
10-* 10-^ 10^"
0.52 0.75 0.66 0.70
•y
0.^1 0.69 0.77 0.85
3
-t
5
0.49 0.68 0.77 0.92
0.45 0.66 0.68 0.72
0.58 0.56 0.80 0.87
Mean
Standard deviation
0.51 0.67 0.74 0.81
0.05 0.07 0.06 0.10
* Guinea pig peritoneal macrophages art exposed to sodium periodate on ice for 10 min. The control ctUs are exposed to phosphate-buffered saline for the same length of time at the same pH and temperature. The results are expressed as migration index, and an index of 0.80 or less is indicative of significant inhibition.
The results are expressed as migration index area of test migration "
•
area of control migration'
hi vitro spreadmg assay. The peritoneal exudate cells were collected and washed three times in RPMl 1640 medium. The cells were divided into several aliquots and suspended at a concentration of 10-16 X 10^ cells/ml in PBS and sodium periodate at various concentrations for 10 min on ice. After the treatments the ceils were washed in PBS, and 2.5 X IO^^ cells in 2 mi of medium were added to clean glass coverslips in Petri plates and kept at room temperature for 1 h. Duplicate plates were prepared for each treatment schedule. After this the supernatant was poured off, the plate was washed twice with PBS, and the cells were then mijced with 2% glutaraldehyde in O.lM phosphate buffer at pH 7.2. Under these conditions 95% of the cells adhere and spread on the coverslip (22). The degree of spreading was assessed with a Zeiss phase contrast microscope with 40 X 15 magnification. The percentage of well-spread cells in stage 4 (wellspread cytoplasm with flattened nucleus) was taken as a measure of the degree of cell deformation and spreading (22, 23). RESULTS Macropbage migration Exposure of macrophages to sodium periodate for 10 min on ice results in inhibition of migration. In the early experiments it was
found that a concentration of between 10 ^M and 10"^M was optimal to produce inhibition. There appeared to be some variation in the early experiments with regard to the degree of inhibition. This was overcome by strictly controlling the pH and using freshly made solutions for each experiment. Using the protocol described in Materials and Methods, we consistently obtained macrophage migration inhibition with lO'^M sodium periodate, with a mean M.i. of 0.51 and a standard deviation ± 0.05 (Table I). It can be seen that inhibition was also found consistently at 10-* and lO^^M concentrations. At lO'^M, significant inhibition occurred twice out of five experiments. The treated cell populations were assessed for viability, and approximately 95% of the cells excluded vital dye. At each concentration of sodium periodate, reversal or blocking of inhibition was attempted with the three concentration.s of sodium borohydride. The results with macrophages exposed to 10"^ periodate are shown in Fig. 1; at this concentration the mean M.I. was 0.51. Succeeding exposure to lO'-'^M sodium borohydride resulted in a marked decrease in the degree of inhibition—M.I., 0.80. At the two lower concentrations of borohydride, 10"^M and lO'^M, migration inhibition was less effectively reversed, with migration indices of 0.61 and 0.69, respectively. At the next lower concentration of sodium periodate, 10 '*M, which resulted in significant migration inhibition (M.I., 0.67), complete reversal occurred at each concentration of sodium borohydride—
1154 R- A. Fox, L. A. Fernandez & R. Rajaraman
ipilluiB
Fig. 1. Peritoneal exudate cells were suspended in tO"'''M sodium periodate for 10 min on ice. At the end of this time the cells were washed in phosphatebuffered saline (PBS) and then resuspended in PBS or sodium borohydride at various concentrations for 10 min on ice. The cells were then washed and prepared for the capillary tube migration assay. The cells were allowed to migrate in RPMl t(J40 medium with 25'yf normal guinea pig serum (heat-inactivated). The results are expressed as migration index, and a value of 0.80 or less indicates significant inhibition of migration.
0.85 (10-3M), 0.99 CIO"''M), and 0.94 (IO"*M). This was also true with lO^^M sociiiun periodate. Thus optimum concentration for sodium borohydride reversal is from 10"-'* to 10"^'M, but the effectiveness varies with the concentration of sodium periodate previously used. We performed a further experiment, duplicating the conditions used by Remold (25; see Addendum), in which peritoneal macrophages
Pnrfotfal*
Cooctniiation
(Metarl
Fig. 2. Peritoneal exudate cclLs wcrt; suspended in phosphate-buffered saline (control) or sodium periodate at various concentrations, at room temperature for 30 min. After these treatments the cells were washed and prepared for the capillary tube migration assay. The cells were allowed to migrate in RPMI 1640 medium containing 2 5 % normal guinea pig serum (heat-inactivated). The results are expressed as migration index, and a value of 0.80 or less indicates significant inhibition of migration.
were exposed to sodium periodate at room temperature for 30 min, and significant inhibition of in vitro migration resulted. The results artshown in Fig. 2; M.I.'s of 0.39, 0.62, and O.«2 were obtained at concentrations of lO'^M, 10"''M, and 10~^M, respectively. At lower concentrations of sodium periodate the migration was completely normal. In the seven experiments with purified macrophages sodium periodate consistently
Periodate Inhibits Macrophage Migration
1155
Table II. Macrophage spreading inhibition*
Ex-
peri-
PBS PBS
NaIO4 PBS
NalOi -t-NaBH.i
PBS NaBH*
11 34 57
4 13 40
6 28 55
27 64
ment 1 2
12
• The results of three experiments are shown, expressed as the percentage of well-spread cells. Each cell population was subjected to two 10-min treatments on ice, "> X JO'* sodium periodate. lO'^M sodium borohydride, or PBS. After treatment the cells were suspended in RPMI 1640 medium and allowed to spread on Petri plates.
•
/
/
Fig. 3. Peritoneal exudate cells were suspended in phosphate-buffered saline (control) or sodium periodate at various concentrations for 10 min on ice. After being washed the cells were allowed to spread on clean glass covcrslips at room temperature for 1 h. The degree of spreading was then assayed using a Zeiss phase contrast microscope. The results are expressed as the percentage well-spread cells, stage 1 (well-spread cytoplasm with flattened nucleus).
This effect on macrophage spreading can be blocked by sodium borohydride treatment; the results of three such experiments arc shown in Table II. The proportions of well-spread cells are significantly smaller in the periodate-treated population (P < 0.05, paired / test). There was no significant difference between the control and the periodate/borohydride-treated cells.
DISCUSSION
produced migration inhibition; the mean M.I. was 0.39, with a standard deviation of 0.19. The M.I.'s ranged from 0.09 to 0.60. /;; vitro spreading assay
Sodium periodate treatment of macrophages directly affects the macrophages, in that the .spreading on glass for 1 h is inhibited. This was a consistent obser^'ation in five experiments; the results of one experiment are shown in Fig. 3. The same concentrations of periodate were used as in the experiments on migration inhibition. There was inhibition of spreading at each concentration, with 42% well-spread cells at 10-3M. 47% at lO-^M, 5 1 % at lO-^M, imd 58% at 10-^M; of the control cells, 78% were well spread.
Sodium periodate consistently and reproducibly produces inhibition of in vitro macrophage migration. The cells remain viable and the effect can be reversed with sodium borohydride. These conditions are identical to those used for lymphocyte treatment, and in that situation it has been concluded that only surface membrane glycoproteins are being affected (26). It appears likely that the response demonstrated here is due to a direct effect on the macrophages, and specifically due to alteration of surface membrane glycoproteins, which are known to be important in the migration inhibition response (12). One might postulate that the migration inhibition is a secondary response, due to lymphocyte activation and migration inhibition factor (MIF) release. This appears unlikely in view of the consistent effect with purified
1156 R. A. Pox, L. A. Fernandez & R. Rajaraman
macrophages and the inhibition of spreading. In these spreading experiments only macrophages were counted. Remold (25) has recently published evidence that modification of the macrophage membrane can potentiate the macrophage response to MIF. He also states that sodium periodate treatment alone has no effect on macrophage migration, but no experimental results are reported. We have reproduced the periodate treatment conditions of Remold (25), and this clearly results in migration inhibition. The reasons for the discrepancy are not apparent, but we found it imperative to make fresh solutions for each experiment with carefully adjusted pH at +4°C.
is not well understood, although it has been postulated to function in a regulatory capacit)' in controlling lymphocyte activity. A possibility to be considered is that the periodate-treated (MIF-treated) macrophages may recruit virgin lymphocytes into an immune reaction, thus augmenting the response, To try to solve such a complex interplay of cellular events using crude materials with multiple or labile activities presents a horrendous challenge. Introduction of a simple analogue that successfully mimics a particular lymphokine activity would be of great advantage.
The inhibition of early spreading is perhaps a iittie surprising, since spreading has been compared to "frustrated" phagoc)tosis (16). Macrophage migration inhibition is thought to be linked to activation of the macrophage and increased phagocytosis (15). We need to look more closely at these phenomena and their mediators. There are two t)'pes of spreading behavior discussed in the literature: the fast spreading for 1 h, onto inert substrate, which is inhibited by serum, and the slow spreading occurring over longer periods of time and in media enriched with serum (21). The slow spreading is linked with a net increase in cellular protein and lysosomal enzymes (5, 6). The possible connection between these two phenomena and migration inhibition is not well understood and needs further study. It has been shown that fast spreading is inhibited by lymphokine preparations (7), and macrophagespreading inhibition has been promoted as a good in vitro correlate of delayed hypersensitivitj', very close to migration inhibition (8). Furthermore, macrophages round up over 1 h when exposed to partially purified lymphokine preparations (l4). The rounding up would fit very well with the decreased deformability and inhibition of spreading that we have found.
ACKNOWLEDGEMENTS
Tlius it would appear that periodate treatment mimics some of the changes produced with MIF preparations. Fast spreading and in vitro migration are inhibited. We also need to look at macrophage activation, phagocytosis, and 'slow' spreading. The biological role of MIF
We thank Mrs. K. Rajaraman and Ms. R. McGuire for their expert technical assistance, Miss Pat Godin for secretarial assistance, and the Department of Photograpiiy at Camp Hill Hospital for the preparation of the figures. This work was supported by M.R.C. (Canada) and Dalhousie University Internal Medicine Research Foundation, ADDENDUM In personal correspondence. Remold has informed us that he did not use PBS but balanced salt solution without glucose as buffer in liis experiments. There are also minor differences in his capillar)' tube migration assay. It is possible that these may account for the discrepant results. REFERENCES l.Abell, C. W., Fritz, R. A.. Novak, R. A. & Monahan. T. M. p. 227 in Membrane Transformations in Neof'iasia, Miami Winter Symposium, Vol. 8. Academic Press Inc., New York, 1976. 2. Beyer, C. F. & Bowers. W. E. Periodate and concanavalin A induced blast transformation of rat lymphocytes by an indirect mechanism. Proc. nut. Acad. Set. (USA) 12, 3590, 1975. }>. Biniaminov, M., Ramot, B. & Novogfodsky, A. Effect of macrophages on periodate-induced transformation of normal and chronic lymphatic leukemia lymphocytes. Clin. exp. Immunol. 16, 235, 1974. 4. Blumenfeld. O. O., Gallop, P. M. & Liao, T. S. Modification and introduction of a specific radio-
Periodate Inhibits Macrophage Migration active label into the erythrocyte membrane sialoglycoproteins. Biochem. hiophys. Kes. Commttn. 48, 242, 1973. =1. Cohn, Z. A. & Benson, B. The differentiation of mononuclear phagoc>tes. Morphology, cj'tochemistry and biochemistry. / . exp. Med. 121, 153, 1965. f.. Cohn, Z. A.. Hirsh, J. G. & Fedorko, M. E. The in vitro differentiation of mononuclear phagocytes. IV. The ultras tructure of macrophage differentiation in the peritoneal cavity and in culture. / . exp. Med. 123, 747, 1966. ^. Dekaris, D., Smerdel, S. & Veselic, B. Inhibition of macrophage spreading by supernatants of antigen-stimulated sensitized lymphocytes. Europ, J. Immunol. 1, 403, 1971. s. Fauve, R. M. & Dekaris, D. Macrophage spreading: inhibition in delayed hypersensitivity. Science 160, 795, 1968. 9- Fox, R. A., Gregory, D. G. & Feldman, J. D. Migration inhibition factor (MIF) and migration stimulation factor (MSF) in fetal calf serum. /. Imfnunol. 112. 1861, 1974. 10. Fox, R. A., Gregory, D. G. & Feldman, J. D. Macrophage receptors for migration inhibitor)' factor (MIF) migration stimulation factor (MSF) and agglutination factor. / . Immunol, 112. 1867, 1974. 11. Fox, R. A. & MacSween. J. M. The isolation of migration inhibition factor. Immunol. Commun. 3, (-1) 375. 1974. 13. Fox, R. A., MacSween, J. M. & McGuire, R. L. Potentiation of the macrophage response to migration inhibition factor from fetal calf serum by blood group substances with human H activity'. Scand. } . Immioiol. 5. 941, 1976. 13. Levis, W . R. & Robbins, J. M. Effect of glassadherent cells on the blastogenic response of purified lymphocytes to phytohemagglutinin. Exp. Cell Res. 61. 153, 1970. It. Nath, I., Poulter, L. W. & Turk, J. L. Effect of lymphocyte mediators on macrophages in vitro, a correlation of morphological and cytochemical changes. Clin. exp. Immunol. 13, 45 5, 1973. i:i. Nathan. C. F., Remold, H. G. & David, J. R. Characterization of a lymphocyte factor which alters macrophage functions. /. exp, Med. 137, 275, 1973. Received 24 June 1977 Received in revised form 15 August 1977
1157
16. North, R. J. The uptake of particulate antigens. / . reticuloendoth. Sac. 3, 203, I96S. 17. Novogrodsky. A. Induction of lymphocyte cytotoxicity by modification of the effector on target cells with periodate or with neuraminidase and galactose oxidase. /. Immunol. 114, 1089, 1975. 18. Novogrodsky, A. & Katchalski, E. Induction of lymphocyte transformation by periodate. PEBS Lett. 12, 297, 1971. 19. Oppenheim, J. J., Hersh, E. M. & Block, J. B. The response to various stimuli of human peripheral lymphocytes which have been purified by passage through glass bead column, p. 183 in Elves, M.W. (ed.) Proceedings of the Symposium on The Biological Effects of Phytohemagglutinin. The Robert Jones and Agnes Hunt Orthopaedic Hospital Management Committee, Oswestry, England, 1966. 20. Parker, J. W., O Brien, R. L.. Lukes, R. J. & Steiner, J. Transformation of human lymphocytes by sodium periodate. Lancet 1. 103, 1972. 21. Rabinovich, M. Macrophage spreading in vitro. p. 369 in van Furth, R, (ed.) Mononuclear Phagocytosis in Immunity, Infection and Pathology. Blackweli Scientific Publications, Oxford, 1975. 22. Rajaraman, R., Fox, R. A.. Vethamany, V. G., Fernande2. L. A. & MacSween, J. M. Adhesion and spreading behaviour of human peripheral blood mononuclear cells (PBMC) in ritru. Exp. Cell Res. 107, 179, 1977. 23. Rajaraman, R.. Rounds, D. E., Yen, S. P. S. & Rembaum, A. A scanning electron miaoscope study of cell adhesion and spreading in vitro, Exp. Cell Res. 88, 327. 1974. 24. Remold, H. G. Requirements for L-fucose on the macrophage membrane receptor for MIF. / . exp. Med. 138, 1065, 1973. 25. Remold, H. G. Chemical treatment of macrophages increases their responsivenc-ss to migration inhibitory factor (MIF). / . Immunol, 118, \, 1977. 26. Zatz, M. M., Goldstem, A. L, Blumenfeld. O. O., White, A. Regulation of normal and leukemic lymphocyte transformation and recirculation by sodium periodate oxidation and sodium borohydride reduction. Natttre (New Biol.) 240, 252, 1972.
Migration Inhibition Produced by Sodium Periodate Oxidation of the Macrophage Membrane, and Reversal by Sodium Borohydride R. A. FOX, L. A. FERNANDEZ & R. RAJARAMAN Departments of Medicine and Microbiology. Dalhousie University and Camp Hill Hospital, Halifax, Nova Scotia, Canada
Fox, R. A., Fernandez, L. A. & Rajaraman, R. Migration Inhibition Produred by Sodium Periodate Oxidation of the Macrophage Membrane, and Reversal by Sodium Borohydride. Scaad. / . Immunol. 6, 1151-1157, 1977. Guinea pig peritoneal exudate cells were harvested 3 to 4 days after the intraperitoneal injection of Marcol oil. The washed ceils were exposed to various concentrations of sodium periodate in phosphate-buffered saline (PBS) at pH 7A for 10 min at + 4 ° C . The cells were then used in the in vitro migration assay, and migration was consistently inhibited at concentrations from 10~^ to 10"^M. The viability of the macrophages was not affected by this treatment. Sodium borohydride {10"^ to 10"^M) in PBS for 10 min at pH 7.4 reversed the periodate effect. Experiments with purified macrophages showed that sodium periodate has a direct effect on macrophage function rather than an indirect effect via the potentiation of migration inhibition factor. In support of this, the in vitro spreading of macrophages on glass substrate for 1 h has been shown to be inhibited. This spreading inhibition can also be reversed by treatment with sodium borohydride. These results provide a new approach to understanding the biological significance and role of macrophage migration inhibition. R. A. Pox, Department of Medicine, Building 7, Camp Hill Hospital, 1763 Robie St., Halifax, Nova Scotia, Canada BJH 3G2
Sodium periodate oxidation of rat lymph node (18) and human peripheral blood lymphocytes (20) results in blast formation. The conditions used Co achieve blastogenesis indicated that only the surface glycoproteins were being oxidized, and it was suggested that aldehyde groups are formed on sialo glycoproteins (4, 26). These aldehyde groups can be reduced back to an alcohol by treatment with sodium borohydride. Blastogenesis is blocked or reversed by sodium borohydride treatment (1). Apart from lymphocyte transform^ion, other biological effects are produced by periodate treatment; for example, cytotoxicity is induced and can be demonstrated by chromium release from labeled tumor cells (17). Surface modification by
periodate has been shown to inhibit the homing of CBA/J lymphocytes to lymph nodes (26). It has been known for some time that macrophages play an essential role in lymphocyte transformation in response to various stimuli such as antigens (19) and mitogens (13)Macrophages are also necessary for transformation in response to periodate oxidation (3). Removal of glass-adherent cells from lymphocyte suspensions results in a significant reduction in transformation. A further significant observation was that normal lymphocytes could be stimulated, and transformation induced, by exposure to a macrophage monolayer treated with sodium periodate (3). This observation has led to speculations with regard to the
1152 R- A. Pox, L. A. Pernandez & R. Rajaraman
mechanism of action of sodium periodate in inducing lymphocyte transformation. The situation is complicated by the ohser%'ation thai periodate oxidation of one population of lymphocytes, also treated with mitomyein, will stimulate another, untreated, autologous population in a one-way mixed lymphocyte reaction (2). Thus lymphocyte transformation might result from any of the following three possibilities: direct stimulation of the lymphocyte that ultimately transforms; antigenic alteration of one population of lymphocytes, which then act as stimulator cells; or alteration of macrophages (monocytes), which then stimulate the lymphocytes. We found this latter possibility' intriguing but, apart from this observation using macrophage monolayers (3), were unable to find any other studies on the interaction of periodate with guinea pig peritoneal exudate macrophages and their subsequent behavior modification in vitro.
MATERIALS AND METHODS Adult guinea pigs, multicolored and albino, weighing between 250 and 400 g, were iniected intraperitoneally with 20-30 ml sterile Marcol 22 oil (Imperial Oil, Canada). There to four days later the peritoneal cavity wa.s drained and washed through with culture medium. The cells were separated from the oil by centrifugation and were then washed thoroughly with medium. After the third wash in tissue culture medium the cells were washed in ice-cold phosphate-buffered saline (PBS) (O.OOlM sodium phosphate, 0,14M sodium chloride, pH 7.4) and then resuspended in PBS. Sodium periodate was made up fresh on the day of the experiment, at the appropriate concentration in PBS. The pH of these solutions were carefully adjusted to 7.4 after equilibration at +4°C. Test aliquots of macrophages were resuspended in the appropriate-strength sodium periodate/PBS solution for 10 min at + 4°C; control cells were resuspended in PBS alone. At the end of this time the cells were washed three times and then, if no further treatment was required, prepared for assay or, alternatively, subjected to further treatment. In
tlie experiments on sodium borohydride rever sal, sodium borohydride was made up fresli each day and the pH adjusted to 7.4 after temperature equilibration. Cells were exposeti to sodium borohydride at +4*C for 10 min followed by thorough wa.shing; borohydridt treatment was controlled for in the migratioi assay by using buffer alone. Once the initial experiments were performeti and optimal concentrations had been deter mined, a protocol was .set up and strictly ad hered to for five successive experiments. Al' cell populations were subjected to two lO-mii treatments on ice. The first treatment was either PBS alone or sodium periodate in PBS at concentrations of lO-^M, IO-*M, lO'^M, and 10"*M. After thorough washing, each of the five populations was exposed to PBS alone oi sodium borohydride at three concentrations 10--3M, lO-iM. and lO'^M. Thus the periodate treated cells were controlled for with cells exposed to PBS only. The periodate- and borohydride-treated cells were controlled for with the PBS- and borohydride-treated cells. The conditions of periodate treatment were specifically altered for one experiment, in which the macrophages were exposed to the various concentrations of sodium periodate al room temperature for 30 min (24). In seven experiments the peritoneal exudate cells were collected in the usual manner am! washed. The cells were then layered ontc^ Ficoll-Isopaque (9%-34%) and centrifuget at 2500 rpm for 20 min. The purified mono nuclear population was then removed an*,i washed twice in RPMI 1640. The cells were washed twice, resuspended in medium witl 10% FCS, and allowed to settle in Petri dishes for 1 h at 37°C. The nonadherent cells wert washed off and the adherent populations col lected after removal with a rubber spatula. The purified macrophage population was then sub jected to either sodium periodate (lO^^M) or PBS for 10 min at +4°C and allowed to migrate in Che usual manner. Macrophage migration of guinea pig peritoneal exudate cells was measured by a capillarT, tube assay as previously described (9-12)
Periodate
Inhibits
Macrophage
Migration
1153
Tahie 1. Macrophage migration inhibition with sodium periodate* Concen(M)
Experiment 1
10"3
10-* 10-^ 10^"
0.52 0.75 0.66 0.70
•y
0.^1 0.69 0.77 0.85
3
-t
5
0.49 0.68 0.77 0.92
0.45 0.66 0.68 0.72
0.58 0.56 0.80 0.87
Mean
Standard deviation
0.51 0.67 0.74 0.81
0.05 0.07 0.06 0.10
* Guinea pig peritoneal macrophages art exposed to sodium periodate on ice for 10 min. The control ctUs are exposed to phosphate-buffered saline for the same length of time at the same pH and temperature. The results are expressed as migration index, and an index of 0.80 or less is indicative of significant inhibition.
The results are expressed as migration index area of test migration "
•
area of control migration'
hi vitro spreadmg assay. The peritoneal exudate cells were collected and washed three times in RPMl 1640 medium. The cells were divided into several aliquots and suspended at a concentration of 10-16 X 10^ cells/ml in PBS and sodium periodate at various concentrations for 10 min on ice. After the treatments the ceils were washed in PBS, and 2.5 X IO^^ cells in 2 mi of medium were added to clean glass coverslips in Petri plates and kept at room temperature for 1 h. Duplicate plates were prepared for each treatment schedule. After this the supernatant was poured off, the plate was washed twice with PBS, and the cells were then mijced with 2% glutaraldehyde in O.lM phosphate buffer at pH 7.2. Under these conditions 95% of the cells adhere and spread on the coverslip (22). The degree of spreading was assessed with a Zeiss phase contrast microscope with 40 X 15 magnification. The percentage of well-spread cells in stage 4 (wellspread cytoplasm with flattened nucleus) was taken as a measure of the degree of cell deformation and spreading (22, 23). RESULTS Macropbage migration Exposure of macrophages to sodium periodate for 10 min on ice results in inhibition of migration. In the early experiments it was
found that a concentration of between 10 ^M and 10"^M was optimal to produce inhibition. There appeared to be some variation in the early experiments with regard to the degree of inhibition. This was overcome by strictly controlling the pH and using freshly made solutions for each experiment. Using the protocol described in Materials and Methods, we consistently obtained macrophage migration inhibition with lO'^M sodium periodate, with a mean M.i. of 0.51 and a standard deviation ± 0.05 (Table I). It can be seen that inhibition was also found consistently at 10-* and lO^^M concentrations. At lO'^M, significant inhibition occurred twice out of five experiments. The treated cell populations were assessed for viability, and approximately 95% of the cells excluded vital dye. At each concentration of sodium periodate, reversal or blocking of inhibition was attempted with the three concentration.s of sodium borohydride. The results with macrophages exposed to 10"^ periodate are shown in Fig. 1; at this concentration the mean M.I. was 0.51. Succeeding exposure to lO'-'^M sodium borohydride resulted in a marked decrease in the degree of inhibition—M.I., 0.80. At the two lower concentrations of borohydride, 10"^M and lO'^M, migration inhibition was less effectively reversed, with migration indices of 0.61 and 0.69, respectively. At the next lower concentration of sodium periodate, 10 '*M, which resulted in significant migration inhibition (M.I., 0.67), complete reversal occurred at each concentration of sodium borohydride—
1154 R- A. Fox, L. A. Fernandez & R. Rajaraman
ipilluiB
Fig. 1. Peritoneal exudate cells were suspended in tO"'''M sodium periodate for 10 min on ice. At the end of this time the cells were washed in phosphatebuffered saline (PBS) and then resuspended in PBS or sodium borohydride at various concentrations for 10 min on ice. The cells were then washed and prepared for the capillary tube migration assay. The cells were allowed to migrate in RPMl t(J40 medium with 25'yf normal guinea pig serum (heat-inactivated). The results are expressed as migration index, and a value of 0.80 or less indicates significant inhibition of migration.
0.85 (10-3M), 0.99 CIO"''M), and 0.94 (IO"*M). This was also true with lO^^M sociiiun periodate. Thus optimum concentration for sodium borohydride reversal is from 10"-'* to 10"^'M, but the effectiveness varies with the concentration of sodium periodate previously used. We performed a further experiment, duplicating the conditions used by Remold (25; see Addendum), in which peritoneal macrophages
Pnrfotfal*
Cooctniiation
(Metarl
Fig. 2. Peritoneal exudate cclLs wcrt; suspended in phosphate-buffered saline (control) or sodium periodate at various concentrations, at room temperature for 30 min. After these treatments the cells were washed and prepared for the capillary tube migration assay. The cells were allowed to migrate in RPMI 1640 medium containing 2 5 % normal guinea pig serum (heat-inactivated). The results are expressed as migration index, and a value of 0.80 or less indicates significant inhibition of migration.
were exposed to sodium periodate at room temperature for 30 min, and significant inhibition of in vitro migration resulted. The results artshown in Fig. 2; M.I.'s of 0.39, 0.62, and O.«2 were obtained at concentrations of lO'^M, 10"''M, and 10~^M, respectively. At lower concentrations of sodium periodate the migration was completely normal. In the seven experiments with purified macrophages sodium periodate consistently
Periodate Inhibits Macrophage Migration
1155
Table II. Macrophage spreading inhibition*
Ex-
peri-
PBS PBS
NaIO4 PBS
NalOi -t-NaBH.i
PBS NaBH*
11 34 57
4 13 40
6 28 55
27 64
ment 1 2
12
• The results of three experiments are shown, expressed as the percentage of well-spread cells. Each cell population was subjected to two 10-min treatments on ice, "> X JO'* sodium periodate. lO'^M sodium borohydride, or PBS. After treatment the cells were suspended in RPMI 1640 medium and allowed to spread on Petri plates.
•
/
/
Fig. 3. Peritoneal exudate cells were suspended in phosphate-buffered saline (control) or sodium periodate at various concentrations for 10 min on ice. After being washed the cells were allowed to spread on clean glass covcrslips at room temperature for 1 h. The degree of spreading was then assayed using a Zeiss phase contrast microscope. The results are expressed as the percentage well-spread cells, stage 1 (well-spread cytoplasm with flattened nucleus).
This effect on macrophage spreading can be blocked by sodium borohydride treatment; the results of three such experiments arc shown in Table II. The proportions of well-spread cells are significantly smaller in the periodate-treated population (P < 0.05, paired / test). There was no significant difference between the control and the periodate/borohydride-treated cells.
DISCUSSION
produced migration inhibition; the mean M.I. was 0.39, with a standard deviation of 0.19. The M.I.'s ranged from 0.09 to 0.60. /;; vitro spreading assay
Sodium periodate treatment of macrophages directly affects the macrophages, in that the .spreading on glass for 1 h is inhibited. This was a consistent obser^'ation in five experiments; the results of one experiment are shown in Fig. 3. The same concentrations of periodate were used as in the experiments on migration inhibition. There was inhibition of spreading at each concentration, with 42% well-spread cells at 10-3M. 47% at lO-^M, 5 1 % at lO-^M, imd 58% at 10-^M; of the control cells, 78% were well spread.
Sodium periodate consistently and reproducibly produces inhibition of in vitro macrophage migration. The cells remain viable and the effect can be reversed with sodium borohydride. These conditions are identical to those used for lymphocyte treatment, and in that situation it has been concluded that only surface membrane glycoproteins are being affected (26). It appears likely that the response demonstrated here is due to a direct effect on the macrophages, and specifically due to alteration of surface membrane glycoproteins, which are known to be important in the migration inhibition response (12). One might postulate that the migration inhibition is a secondary response, due to lymphocyte activation and migration inhibition factor (MIF) release. This appears unlikely in view of the consistent effect with purified
1156 R. A. Pox, L. A. Fernandez & R. Rajaraman
macrophages and the inhibition of spreading. In these spreading experiments only macrophages were counted. Remold (25) has recently published evidence that modification of the macrophage membrane can potentiate the macrophage response to MIF. He also states that sodium periodate treatment alone has no effect on macrophage migration, but no experimental results are reported. We have reproduced the periodate treatment conditions of Remold (25), and this clearly results in migration inhibition. The reasons for the discrepancy are not apparent, but we found it imperative to make fresh solutions for each experiment with carefully adjusted pH at +4°C.
is not well understood, although it has been postulated to function in a regulatory capacit)' in controlling lymphocyte activity. A possibility to be considered is that the periodate-treated (MIF-treated) macrophages may recruit virgin lymphocytes into an immune reaction, thus augmenting the response, To try to solve such a complex interplay of cellular events using crude materials with multiple or labile activities presents a horrendous challenge. Introduction of a simple analogue that successfully mimics a particular lymphokine activity would be of great advantage.
The inhibition of early spreading is perhaps a iittie surprising, since spreading has been compared to "frustrated" phagoc)tosis (16). Macrophage migration inhibition is thought to be linked to activation of the macrophage and increased phagocytosis (15). We need to look more closely at these phenomena and their mediators. There are two t)'pes of spreading behavior discussed in the literature: the fast spreading for 1 h, onto inert substrate, which is inhibited by serum, and the slow spreading occurring over longer periods of time and in media enriched with serum (21). The slow spreading is linked with a net increase in cellular protein and lysosomal enzymes (5, 6). The possible connection between these two phenomena and migration inhibition is not well understood and needs further study. It has been shown that fast spreading is inhibited by lymphokine preparations (7), and macrophagespreading inhibition has been promoted as a good in vitro correlate of delayed hypersensitivitj', very close to migration inhibition (8). Furthermore, macrophages round up over 1 h when exposed to partially purified lymphokine preparations (l4). The rounding up would fit very well with the decreased deformability and inhibition of spreading that we have found.
ACKNOWLEDGEMENTS
Tlius it would appear that periodate treatment mimics some of the changes produced with MIF preparations. Fast spreading and in vitro migration are inhibited. We also need to look at macrophage activation, phagocytosis, and 'slow' spreading. The biological role of MIF
We thank Mrs. K. Rajaraman and Ms. R. McGuire for their expert technical assistance, Miss Pat Godin for secretarial assistance, and the Department of Photograpiiy at Camp Hill Hospital for the preparation of the figures. This work was supported by M.R.C. (Canada) and Dalhousie University Internal Medicine Research Foundation, ADDENDUM In personal correspondence. Remold has informed us that he did not use PBS but balanced salt solution without glucose as buffer in liis experiments. There are also minor differences in his capillar)' tube migration assay. It is possible that these may account for the discrepant results. REFERENCES l.Abell, C. W., Fritz, R. A.. Novak, R. A. & Monahan. T. M. p. 227 in Membrane Transformations in Neof'iasia, Miami Winter Symposium, Vol. 8. Academic Press Inc., New York, 1976. 2. Beyer, C. F. & Bowers. W. E. Periodate and concanavalin A induced blast transformation of rat lymphocytes by an indirect mechanism. Proc. nut. Acad. Set. (USA) 12, 3590, 1975. }>. Biniaminov, M., Ramot, B. & Novogfodsky, A. Effect of macrophages on periodate-induced transformation of normal and chronic lymphatic leukemia lymphocytes. Clin. exp. Immunol. 16, 235, 1974. 4. Blumenfeld. O. O., Gallop, P. M. & Liao, T. S. Modification and introduction of a specific radio-
Periodate Inhibits Macrophage Migration active label into the erythrocyte membrane sialoglycoproteins. Biochem. hiophys. Kes. Commttn. 48, 242, 1973. =1. Cohn, Z. A. & Benson, B. The differentiation of mononuclear phagoc>tes. Morphology, cj'tochemistry and biochemistry. / . exp. Med. 121, 153, 1965. f.. Cohn, Z. A.. Hirsh, J. G. & Fedorko, M. E. The in vitro differentiation of mononuclear phagocytes. IV. The ultras tructure of macrophage differentiation in the peritoneal cavity and in culture. / . exp. Med. 123, 747, 1966. ^. Dekaris, D., Smerdel, S. & Veselic, B. Inhibition of macrophage spreading by supernatants of antigen-stimulated sensitized lymphocytes. Europ, J. Immunol. 1, 403, 1971. s. Fauve, R. M. & Dekaris, D. Macrophage spreading: inhibition in delayed hypersensitivity. Science 160, 795, 1968. 9- Fox, R. A., Gregory, D. G. & Feldman, J. D. Migration inhibition factor (MIF) and migration stimulation factor (MSF) in fetal calf serum. /. Imfnunol. 112. 1861, 1974. 10. Fox, R. A., Gregory, D. G. & Feldman, J. D. Macrophage receptors for migration inhibitor)' factor (MIF) migration stimulation factor (MSF) and agglutination factor. / . Immunol, 112. 1867, 1974. 11. Fox, R. A. & MacSween. J. M. The isolation of migration inhibition factor. Immunol. Commun. 3, (-1) 375. 1974. 13. Fox, R. A., MacSween, J. M. & McGuire, R. L. Potentiation of the macrophage response to migration inhibition factor from fetal calf serum by blood group substances with human H activity'. Scand. } . Immioiol. 5. 941, 1976. 13. Levis, W . R. & Robbins, J. M. Effect of glassadherent cells on the blastogenic response of purified lymphocytes to phytohemagglutinin. Exp. Cell Res. 61. 153, 1970. It. Nath, I., Poulter, L. W. & Turk, J. L. Effect of lymphocyte mediators on macrophages in vitro, a correlation of morphological and cytochemical changes. Clin. exp. Immunol. 13, 45 5, 1973. i:i. Nathan. C. F., Remold, H. G. & David, J. R. Characterization of a lymphocyte factor which alters macrophage functions. /. exp, Med. 137, 275, 1973. Received 24 June 1977 Received in revised form 15 August 1977
1157
16. North, R. J. The uptake of particulate antigens. / . reticuloendoth. Sac. 3, 203, I96S. 17. Novogrodsky. A. Induction of lymphocyte cytotoxicity by modification of the effector on target cells with periodate or with neuraminidase and galactose oxidase. /. Immunol. 114, 1089, 1975. 18. Novogrodsky, A. & Katchalski, E. Induction of lymphocyte transformation by periodate. PEBS Lett. 12, 297, 1971. 19. Oppenheim, J. J., Hersh, E. M. & Block, J. B. The response to various stimuli of human peripheral lymphocytes which have been purified by passage through glass bead column, p. 183 in Elves, M.W. (ed.) Proceedings of the Symposium on The Biological Effects of Phytohemagglutinin. The Robert Jones and Agnes Hunt Orthopaedic Hospital Management Committee, Oswestry, England, 1966. 20. Parker, J. W., O Brien, R. L.. Lukes, R. J. & Steiner, J. Transformation of human lymphocytes by sodium periodate. Lancet 1. 103, 1972. 21. Rabinovich, M. Macrophage spreading in vitro. p. 369 in van Furth, R, (ed.) Mononuclear Phagocytosis in Immunity, Infection and Pathology. Blackweli Scientific Publications, Oxford, 1975. 22. Rajaraman, R., Fox, R. A.. Vethamany, V. G., Fernande2. L. A. & MacSween, J. M. Adhesion and spreading behaviour of human peripheral blood mononuclear cells (PBMC) in ritru. Exp. Cell Res. 107, 179, 1977. 23. Rajaraman, R.. Rounds, D. E., Yen, S. P. S. & Rembaum, A. A scanning electron miaoscope study of cell adhesion and spreading in vitro, Exp. Cell Res. 88, 327. 1974. 24. Remold, H. G. Requirements for L-fucose on the macrophage membrane receptor for MIF. / . exp. Med. 138, 1065, 1973. 25. Remold, H. G. Chemical treatment of macrophages increases their responsivenc-ss to migration inhibitory factor (MIF). / . Immunol, 118, \, 1977. 26. Zatz, M. M., Goldstem, A. L, Blumenfeld. O. O., White, A. Regulation of normal and leukemic lymphocyte transformation and recirculation by sodium periodate oxidation and sodium borohydride reduction. Natttre (New Biol.) 240, 252, 1972.
