- Email: [email protected]
Enhancing effect of α-interferon on the C2 synthesis by cultured human monocytes
Immunology Letters, 15 (1987) 41-44 Elsevier IML 00870
Enhancing effect of c -interferon on the C2 synthesis by cultured human monocytes E. Ujhelyi, K. O n o d y , D o Q u a n g M i n h , B. Sarkadi a n d G. FiJst National Institute o f Haematology and Blood Transfusion, Budapest, Hungary (Received 16 September 1986) (Revision received 15 January 1987) (Accepted 20 January 1987)
I. Summary C2 synthesis by human peripheral blood monocytes cultured in the presence of human ainterferon (IFN-a) was studied. IFN-o~ was added in different amounts (1-1000 IU/ml) to the cultures on day 3 and was removed on day 7. As control, mock interferon (m-IFN) was also tested. C2 content of the culture supernatants was measured by immunohaemolytic method. IFN-c~ was found to enhance C2 synthesis in a dose-dependent way. The enhancing effect could be observed even after the removal of IFN-a. C2 production by the cultured monocytes was increased by m-IFN as well; the extent of enhancement however, was found to be significantly lower than that induced by the corresponding amount of IFN-ot. In contrast to the enhancing effect on C2 production, IFN-a did not influence total protein synthesis in the cultures, suggesting a selective stimulatory action on the C2 gene.
2. Introduction Interferon has multiple effects (increased spread-
Key words." Complement; C2; Interferon; Monocyte Correspondence to: Dr. George Fiist, National Institute of Haematology and Blood Transfusion, Budapest, Dar6czi u 24. P.O.B. 44, H-1502 Hungary.
ing, tumoricidal activity, Fc receptor expression) on monocytes and macrophages. The in vitro administration of interferon-c~ (IFN-o0 to cultured monocytes dose-dependently inhibits the increase in size of the cells as well as the development of three maturation markers: the lysosomal enzyme acid phosphatase, the membrane-associated leucine aminopeptidase and the ability of the cultured cells for phagocytosis [1]. On the other hand, IFN-a treatment prevents the loss of cytotoxic activity of the monocytes occurring during culturing [I]. According to the experiments of Lee and Epstein [2], in vitro administration of IFN-o~ to monocytemacrophage cultures delays the maturationdependent loss of peroxidase positive granula in monocytes. One of the most significant immunological functions of monocytes-macrophages is the synthesis of proteins of the complement system [3]. According to Beatty et al. [4] normal monocytes produce functionally active C2, factor B, and D for up to 6 wk in culture. There are several substances which can stimulate or inhibit complement protein production by cultured monocytes [ 4 - 8 ] . Lym£ phokines belong to most potent biological products affecting complement synthesis. Unfractionated lymphokines stimulate the production of C2 [9, I0] and 7-interferon increases synthesis of C3, C2, and factor B by monocyte cultures [11, 12]. In the present work we have studied the effect of purified IFN-c~ on the production of haemolytically active C2 by cultured peripheral blood human monocytes
0165-2478 / 87 /$ 3.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
41
and compared it to the effect of the same substance on total protein synthesis. IFN-c~ markedly increased C2 production but had no effect on the rate of total protein synthesis.
3. Materials and Methods
3.1. Isolation and culturing of monocytes Monocytes were isolated from blood mononuclear ceils separated on Ficoll-Uromiro gradient followed by a 60 min adherence to 0.28 cm 2 Linbro tissue culture wells at 37°C. 200/~1 of cell suspension ( 4 - 5 x 106 cells/ml) was added to each well. Nonadherent cells were removed by washing with R P M I 1640. For long-term culturing of monocytes 200 tzl of R P M I 1640 containing 20°7o heat inactivated (120 min, 56°C) AB + serum, 100 IU/ml penicillin and 10/zg/ml streptomycin were added to the ceils. The cultures were kept in 37 °C in a humidified 5°70 CO 2 and air atmosphere for 17 days. Sampling and change of the medium was at 3 - 4 - d a y intervals.
TCA (trichloroacetic acid) precipitable secreted proteins after metabolic labelling with [35S]methionine (Amersham) in primary monocytemacrophage cultures. [35S]Methionine in Dulbecco medium at a final concentration of 15 tzCi/ml was added to monocyte cultures on day 7 and day 14. After 24 h, media were harvested, centrifuged to remove cellular debris, and stored at - 70 °C. Incorporation of radiolabelled amino acids into secreted protein was estimated using TCA precipitation of extracellular media according to a modification of the method of Hall and Colten [16]. The tissue culture medium was added to an equal volume of 50 mM Tris-HC1, p H 7.5, containing 1% sodium deoxycholate, 1°70 Triton X-100, and 100 mM KC1. 20 #1 of 50°70 TCA were added to 180 tzl of the medium detergent solution and the mixture was kept for 10 min in ice, and centrifuged for 10 min at 1800xg. The pellet was washed twice with 5°70 TCA. The precipitate was dried and then solved in 200 ~1 0.1 N a O H ; 5 ml alcoholic scintillation solution was added, and then radioactivity was counted in Beckman 7500 liquid scintillation equipment.
Interferon
IFN-e¢ preparations purified according to Cantell et al. [13] with a specific activity of 1xl06 I U / m g protein were used in the experiments. As control, mock interferon (m-IFN) purified exactly as interferon from leukocytes not treated with interferon inducers was applied.
3.2. Sampling of cultures and storage of samples IFN-c~ and m - I F N were added to monocyte cultures on day 3, and the supernatants were removed on day 7. Medium not containing IFN-o~ or m - I F N was added, and was changed at 3 - 4 - d a y intervals. The supernatants were stored in liquid nitrogen until tested. For the D N A measurements, the monolayer was washed with ice-cold PBS, and the cells were removed by vigorous scratching. The monocytes-macrophages were stored at - 2 0 °C until their D N A content was assayed by spectrofluorimetry as described by Blackburn et al. [14] and Einstein et al. [15]. 3.3. Radiolabeling of newly synthesized proteins in
culture The newly synthesized proteins were identified as 42
3.4. C2 functional assays C2 was measured by the standard stoichiometric haemolytic assay using EAC14 and rat complement EDTA as described by Opferkuch et al. [17].
3.5. Cell sizing Cultured monocytes were analysed for cell size in a Laborscale analyzer (Medicor, Hungary).
4. Results
4.1. Effect of a 4 day IFN-o~ treatment on the C2
production by human monocytes in culture Monocyte cultures were treated with 100 I U / m l IFN-o~ and corresponding amount of m - I F N from day 3 to day 7. Cultures were sampled on days 3, 7, 10, 13 and 17. Levels of C2 expressed in effective molecules #g D N A in untreated cultures and in cultures treated with I F N - a or m - I F N are shown in Table 1. C2 content of the supernatants markedly increased in the presence of IFN-a, and the
Table 1 Effect of IFN and m-IFN treatment on the C2 content of supernatants by cultured human monocytes expressed in proportion to the DNA content of the cultures (means of 4 experiments). Day of culture
Control
IFN-c~ 100 lU/ml
200
/
m-IFN
//
150
(eff. mol. x 10 7//xg of DNA, mean -+ SEM) 3 7 10 13 17
6.86_+2.20 18.30_+6.81 5.15-+0.69 4.04_+ 1 . 3 3 2.88_+0.73
6.86_+ 2.20 78.53_+20.0* 55.05_+15.17" 20.95_+ 3.67** 9.11_+ 2.74
6.86_+2.20 40.40_+9.10 18.05+__5.11 6.50_.+_0.49 3.04+_0.63
×
E
.
Significant difference compared to the control cultures: *P< 0.05, **P<0.01.
5C
IFN-e~ on C2 synthesis The effect of IFN-c~ on C2 production by cultured monocytes was found to be dose-dependent (Fig. 1). A small elevation in the C2 content of culture supernatants could be observed after treatment with 10 I U / m l of IFN-c~. 100 I U / m l gave a pronounced effect whereas 1000 I U / m l did not increase the C2 production further. 4.3. The influence of IFN-o~ on the size of mono-
cytes in culture Size distribution of monocytes cultured for 7 and 14 days was determined by a particular size analyzer. IFN-c~ treatment was found to inhibit in-
/
"
D. . . . . . .
/
enhancing effect was found to be the highest after its removal. Treatment with m - I F N also increased the C2 content of the culture supernatants; the effect, however, was less marked than that of the IFN-c~. 4.2. Dose-dependency of the enhancing effect of
1o0
/ /
,
Cl
..~J~------~ /
3
~
Adminlstrotlon
lb
11
17d~
of IFN-O~
Fig. 1. Cumulative C2 synthesis by cultured rnonocytes after [FN-c~ treatment. Control (X X); 1 IU/ml IFN-c~ (o---o); 10 IU/ml IFN ( t o); 100 IU/ml IFN (o--o); 1000 IU/ml IFN-c~ (A A). Results of one representative experiment out of 5.
crease in volume of the cells during culturing (data not shown). 4.4. The effect of IFN-c~ on the total protein syn-
thesis by cultured monocytes Incorporation of radiolabelled methionine into acid-precipitable protein was studied in 7- and 14-day-old cultures. In contrast to the enhancement of C2 production, neither IFN-c~ nor m-IFN changed the extent of total protein synthesis in the cultures (Table 2).
Table 2 [35S]Methionine incorporation into TC A precipitable protein in monocyte cultures treated with IFN-c~ or m-IFN (means of 3 experiments). Day of culture
Control
IFN-~ 1 IU/ml
m-IFN 10 I U / m l
100 I U / m l
1000 IU/ml
28,865_+11,506 12,631_+ 5 , 6 1 3
25,269_+ 8,924 26,279+_10,813
(cpm, mean _+ SEM) 7 14
21,303_+5,143 17,505_+8,628
19,129_+2,668 15,415_+6,720
21,487_+5,160 16,073_+7,357
21,991_+8,487 19,820_+3,129
43
5. Discussion IFN-a was found to enhance dose-dependently the level of haemolytically active C2 in supernatants of cultured human peripheral blood monocytes. Since culture supernatants were completely changed at each sampling, and no reduced C2 inactivation in IFN-c~ treated cultures was found (unpubl. obs.), enhancement of C2 level was most probably due to an increase in C2 synthesis in cultures treated with IFN-c~. This finding is in agreement with the most recent results of Strunk et al. [12] obtained with IFN-7. The effect of IFN-c~ was long-lasting. Increase of C2 production could be observed after removal of IFN-a from the cultures; the extent of enhancing effect was even higher than that observed in the presence of IFN-c~. Since lymphokines can increase the in vitro synthesis by monocytes of C2 and other complement proteins [9, 10], it was not surprising that m-IFN preparations were also found in the present study to slightly enhance C2 production by cultured monocytes. Since however, the enhancing effect of purified aIFN on C2 production was significantly higher than that of the m-IFN, it seems probable that the increase in C2 synthesis observed after administration of IFN-c~ was due mostly to IFN-c¢ itself. This conclusion was also supported by the finding that different IFN-c~ preparations including one highly purified preparation made in Cantell's laboratory, had the same enhancing effect on C2 synthesis. Experiments studying the effect of monoclonal antiIFN-a antibodies on C2-synthesis-enhancing effect of IFN-c~ preparations are under way in our laboratory. In contrast to C2 production, total de novo protein synthesis by cultured monocytes was not influenced by IFN-a. This observation indicates that synthesis of only some proteins was stimulated by IFN-c~, that is, effect of IFN-c~ cannot be explained by a general enhancement of protein synthesis in the cells. Similar findings were reported by Perlmutter et al. [18]. These authors observed that interleukin-1 increased factor B synthesis, but did not influence C2 production in murine fibroblasts. Similarly, Strunk et al. [12] observed increase in C2 and Bf but not in C3 production by human monocytes treated with IFN-7. These observations suggest that synthesis of some complement proteins by monocytes is independently regulated. IFN-a was found to selectively regulate other monocyte func-
tions as well. Similar to our present findings, Becker [1] observed a dose-dependent inhibition of cell volume in monocyte cultures after short-term or long-term IFN-c~ treatment. IFN-c~ also inhibited the expression of three maturation markers (acid phosphatase, leucine aminopeptidase and phagocytosis). By contrast, the same treatment prevented the loss of cytotoxic activity of the monocytes in culture. Selective regulation of C2 synthesis by IFN-a can occur in two different ways: IFN-a either increases the rate of C2 synthesis by monocytes producing C2 under normal culture conditions or induces new monocytes to produce this complement component. Experiments studying this problem are in progress in our laboratory.
Acknowledgement The authors thank Miss Aranka Pfinya for excellent technical assistance.
References [1] Becker, S. (1984) Cell. Immunol. 84, 145-153. [2] Lee, S. H. and Epstein, L. B. (1980) Cell. Immunol. 50, 177 - 190. [3] Whaley, K. (1980) J. Exp. Med. 151, 501-516. [4] Beatty, D. W., Davis, A. E., Cole, E S., Einstein, L. P. and Colten, H. R. (1981) Clin. Immunol. and Immunopathol. 18, 334-343. .[5] Colten, H . R . and Borsos, T. (1974) J. Immunol. 112, 1107-1114. [6] Kolski, G. B. and Strunk, R. C. (1981) J. Immunol. 126, 2267 - 2271. [7] Lappin, D. E and Whaley, K. (1982) Clin Exp. Immunol. 49, 623- 630. [8] Strunk, R. C., Kunke, K., Nagle, R. B., Bayne, C. M. and Harrison, H. R. (1979) Pediat. Res. 13, 188-193. [9] Littman, B. H. and Schwartz, P. (1982) Arthritis and Rheumatism 25, 288-296. [10] Littman, B. H., Hall, R. E. and Muchmore, A. V. (1983) Cell. Immunol. 76, 189-195. [11] Nichols, W. K. (1984) Complement 1, 108-115. [12] Strunk, R. C., Cole, F. S., Perlmutter, D. H. and Colten, H. R. (1985) J. Biol. Chem. 260,. 15280-15285. [13] Cantell, K., Hirvonen, S., Kauppinen, H. L. and Myllylla, G. (1981) Meth. Enzymol. 78, 29-38. [14] Blackburn, M.T., Andrews, T.M. and Watts, R. W. E. (1973) Anal. Biochem. 51, 1-10. [15] Einstein, P.E., Schneeberger, E . E . and Colten, H . R . (1976) J. Exp. Med. 143, 114-126. [16] Hall, R . E . and Colten, H. R. (1977) J. lmmunol. 118, 1903 - 1905. [17] Opferkuch, W., Rapp, H. J., Colten, H. R. and Borsos, T. (1971) J. Immunol. 106, 927-931. [18] Perlmutter, D. H., Goldberger, G., Dinarello, C. A., Mizel, S. B. and Colten, H. R. (1985) Complement 2, 62.
Enhancing effect of c -interferon on the C2 synthesis by cultured human monocytes E. Ujhelyi, K. O n o d y , D o Q u a n g M i n h , B. Sarkadi a n d G. FiJst National Institute o f Haematology and Blood Transfusion, Budapest, Hungary (Received 16 September 1986) (Revision received 15 January 1987) (Accepted 20 January 1987)
I. Summary C2 synthesis by human peripheral blood monocytes cultured in the presence of human ainterferon (IFN-a) was studied. IFN-o~ was added in different amounts (1-1000 IU/ml) to the cultures on day 3 and was removed on day 7. As control, mock interferon (m-IFN) was also tested. C2 content of the culture supernatants was measured by immunohaemolytic method. IFN-c~ was found to enhance C2 synthesis in a dose-dependent way. The enhancing effect could be observed even after the removal of IFN-a. C2 production by the cultured monocytes was increased by m-IFN as well; the extent of enhancement however, was found to be significantly lower than that induced by the corresponding amount of IFN-ot. In contrast to the enhancing effect on C2 production, IFN-a did not influence total protein synthesis in the cultures, suggesting a selective stimulatory action on the C2 gene.
2. Introduction Interferon has multiple effects (increased spread-
Key words." Complement; C2; Interferon; Monocyte Correspondence to: Dr. George Fiist, National Institute of Haematology and Blood Transfusion, Budapest, Dar6czi u 24. P.O.B. 44, H-1502 Hungary.
ing, tumoricidal activity, Fc receptor expression) on monocytes and macrophages. The in vitro administration of interferon-c~ (IFN-o0 to cultured monocytes dose-dependently inhibits the increase in size of the cells as well as the development of three maturation markers: the lysosomal enzyme acid phosphatase, the membrane-associated leucine aminopeptidase and the ability of the cultured cells for phagocytosis [1]. On the other hand, IFN-a treatment prevents the loss of cytotoxic activity of the monocytes occurring during culturing [I]. According to the experiments of Lee and Epstein [2], in vitro administration of IFN-o~ to monocytemacrophage cultures delays the maturationdependent loss of peroxidase positive granula in monocytes. One of the most significant immunological functions of monocytes-macrophages is the synthesis of proteins of the complement system [3]. According to Beatty et al. [4] normal monocytes produce functionally active C2, factor B, and D for up to 6 wk in culture. There are several substances which can stimulate or inhibit complement protein production by cultured monocytes [ 4 - 8 ] . Lym£ phokines belong to most potent biological products affecting complement synthesis. Unfractionated lymphokines stimulate the production of C2 [9, I0] and 7-interferon increases synthesis of C3, C2, and factor B by monocyte cultures [11, 12]. In the present work we have studied the effect of purified IFN-c~ on the production of haemolytically active C2 by cultured peripheral blood human monocytes
0165-2478 / 87 /$ 3.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
41
and compared it to the effect of the same substance on total protein synthesis. IFN-c~ markedly increased C2 production but had no effect on the rate of total protein synthesis.
3. Materials and Methods
3.1. Isolation and culturing of monocytes Monocytes were isolated from blood mononuclear ceils separated on Ficoll-Uromiro gradient followed by a 60 min adherence to 0.28 cm 2 Linbro tissue culture wells at 37°C. 200/~1 of cell suspension ( 4 - 5 x 106 cells/ml) was added to each well. Nonadherent cells were removed by washing with R P M I 1640. For long-term culturing of monocytes 200 tzl of R P M I 1640 containing 20°7o heat inactivated (120 min, 56°C) AB + serum, 100 IU/ml penicillin and 10/zg/ml streptomycin were added to the ceils. The cultures were kept in 37 °C in a humidified 5°70 CO 2 and air atmosphere for 17 days. Sampling and change of the medium was at 3 - 4 - d a y intervals.
TCA (trichloroacetic acid) precipitable secreted proteins after metabolic labelling with [35S]methionine (Amersham) in primary monocytemacrophage cultures. [35S]Methionine in Dulbecco medium at a final concentration of 15 tzCi/ml was added to monocyte cultures on day 7 and day 14. After 24 h, media were harvested, centrifuged to remove cellular debris, and stored at - 70 °C. Incorporation of radiolabelled amino acids into secreted protein was estimated using TCA precipitation of extracellular media according to a modification of the method of Hall and Colten [16]. The tissue culture medium was added to an equal volume of 50 mM Tris-HC1, p H 7.5, containing 1% sodium deoxycholate, 1°70 Triton X-100, and 100 mM KC1. 20 #1 of 50°70 TCA were added to 180 tzl of the medium detergent solution and the mixture was kept for 10 min in ice, and centrifuged for 10 min at 1800xg. The pellet was washed twice with 5°70 TCA. The precipitate was dried and then solved in 200 ~1 0.1 N a O H ; 5 ml alcoholic scintillation solution was added, and then radioactivity was counted in Beckman 7500 liquid scintillation equipment.
Interferon
IFN-e¢ preparations purified according to Cantell et al. [13] with a specific activity of 1xl06 I U / m g protein were used in the experiments. As control, mock interferon (m-IFN) purified exactly as interferon from leukocytes not treated with interferon inducers was applied.
3.2. Sampling of cultures and storage of samples IFN-c~ and m - I F N were added to monocyte cultures on day 3, and the supernatants were removed on day 7. Medium not containing IFN-o~ or m - I F N was added, and was changed at 3 - 4 - d a y intervals. The supernatants were stored in liquid nitrogen until tested. For the D N A measurements, the monolayer was washed with ice-cold PBS, and the cells were removed by vigorous scratching. The monocytes-macrophages were stored at - 2 0 °C until their D N A content was assayed by spectrofluorimetry as described by Blackburn et al. [14] and Einstein et al. [15]. 3.3. Radiolabeling of newly synthesized proteins in
culture The newly synthesized proteins were identified as 42
3.4. C2 functional assays C2 was measured by the standard stoichiometric haemolytic assay using EAC14 and rat complement EDTA as described by Opferkuch et al. [17].
3.5. Cell sizing Cultured monocytes were analysed for cell size in a Laborscale analyzer (Medicor, Hungary).
4. Results
4.1. Effect of a 4 day IFN-o~ treatment on the C2
production by human monocytes in culture Monocyte cultures were treated with 100 I U / m l IFN-o~ and corresponding amount of m - I F N from day 3 to day 7. Cultures were sampled on days 3, 7, 10, 13 and 17. Levels of C2 expressed in effective molecules #g D N A in untreated cultures and in cultures treated with I F N - a or m - I F N are shown in Table 1. C2 content of the supernatants markedly increased in the presence of IFN-a, and the
Table 1 Effect of IFN and m-IFN treatment on the C2 content of supernatants by cultured human monocytes expressed in proportion to the DNA content of the cultures (means of 4 experiments). Day of culture
Control
IFN-c~ 100 lU/ml
200
/
m-IFN
//
150
(eff. mol. x 10 7//xg of DNA, mean -+ SEM) 3 7 10 13 17
6.86_+2.20 18.30_+6.81 5.15-+0.69 4.04_+ 1 . 3 3 2.88_+0.73
6.86_+ 2.20 78.53_+20.0* 55.05_+15.17" 20.95_+ 3.67** 9.11_+ 2.74
6.86_+2.20 40.40_+9.10 18.05+__5.11 6.50_.+_0.49 3.04+_0.63
×
E
.
Significant difference compared to the control cultures: *P< 0.05, **P<0.01.
5C
IFN-e~ on C2 synthesis The effect of IFN-c~ on C2 production by cultured monocytes was found to be dose-dependent (Fig. 1). A small elevation in the C2 content of culture supernatants could be observed after treatment with 10 I U / m l of IFN-c~. 100 I U / m l gave a pronounced effect whereas 1000 I U / m l did not increase the C2 production further. 4.3. The influence of IFN-o~ on the size of mono-
cytes in culture Size distribution of monocytes cultured for 7 and 14 days was determined by a particular size analyzer. IFN-c~ treatment was found to inhibit in-
/
"
D. . . . . . .
/
enhancing effect was found to be the highest after its removal. Treatment with m - I F N also increased the C2 content of the culture supernatants; the effect, however, was less marked than that of the IFN-c~. 4.2. Dose-dependency of the enhancing effect of
1o0
/ /
,
Cl
..~J~------~ /
3
~
Adminlstrotlon
lb
11
17d~
of IFN-O~
Fig. 1. Cumulative C2 synthesis by cultured rnonocytes after [FN-c~ treatment. Control (X X); 1 IU/ml IFN-c~ (o---o); 10 IU/ml IFN ( t o); 100 IU/ml IFN (o--o); 1000 IU/ml IFN-c~ (A A). Results of one representative experiment out of 5.
crease in volume of the cells during culturing (data not shown). 4.4. The effect of IFN-c~ on the total protein syn-
thesis by cultured monocytes Incorporation of radiolabelled methionine into acid-precipitable protein was studied in 7- and 14-day-old cultures. In contrast to the enhancement of C2 production, neither IFN-c~ nor m-IFN changed the extent of total protein synthesis in the cultures (Table 2).
Table 2 [35S]Methionine incorporation into TC A precipitable protein in monocyte cultures treated with IFN-c~ or m-IFN (means of 3 experiments). Day of culture
Control
IFN-~ 1 IU/ml
m-IFN 10 I U / m l
100 I U / m l
1000 IU/ml
28,865_+11,506 12,631_+ 5 , 6 1 3
25,269_+ 8,924 26,279+_10,813
(cpm, mean _+ SEM) 7 14
21,303_+5,143 17,505_+8,628
19,129_+2,668 15,415_+6,720
21,487_+5,160 16,073_+7,357
21,991_+8,487 19,820_+3,129
43
5. Discussion IFN-a was found to enhance dose-dependently the level of haemolytically active C2 in supernatants of cultured human peripheral blood monocytes. Since culture supernatants were completely changed at each sampling, and no reduced C2 inactivation in IFN-c~ treated cultures was found (unpubl. obs.), enhancement of C2 level was most probably due to an increase in C2 synthesis in cultures treated with IFN-c~. This finding is in agreement with the most recent results of Strunk et al. [12] obtained with IFN-7. The effect of IFN-c~ was long-lasting. Increase of C2 production could be observed after removal of IFN-a from the cultures; the extent of enhancing effect was even higher than that observed in the presence of IFN-c~. Since lymphokines can increase the in vitro synthesis by monocytes of C2 and other complement proteins [9, 10], it was not surprising that m-IFN preparations were also found in the present study to slightly enhance C2 production by cultured monocytes. Since however, the enhancing effect of purified aIFN on C2 production was significantly higher than that of the m-IFN, it seems probable that the increase in C2 synthesis observed after administration of IFN-c~ was due mostly to IFN-c¢ itself. This conclusion was also supported by the finding that different IFN-c~ preparations including one highly purified preparation made in Cantell's laboratory, had the same enhancing effect on C2 synthesis. Experiments studying the effect of monoclonal antiIFN-a antibodies on C2-synthesis-enhancing effect of IFN-c~ preparations are under way in our laboratory. In contrast to C2 production, total de novo protein synthesis by cultured monocytes was not influenced by IFN-a. This observation indicates that synthesis of only some proteins was stimulated by IFN-c~, that is, effect of IFN-c~ cannot be explained by a general enhancement of protein synthesis in the cells. Similar findings were reported by Perlmutter et al. [18]. These authors observed that interleukin-1 increased factor B synthesis, but did not influence C2 production in murine fibroblasts. Similarly, Strunk et al. [12] observed increase in C2 and Bf but not in C3 production by human monocytes treated with IFN-7. These observations suggest that synthesis of some complement proteins by monocytes is independently regulated. IFN-a was found to selectively regulate other monocyte func-
tions as well. Similar to our present findings, Becker [1] observed a dose-dependent inhibition of cell volume in monocyte cultures after short-term or long-term IFN-c~ treatment. IFN-c~ also inhibited the expression of three maturation markers (acid phosphatase, leucine aminopeptidase and phagocytosis). By contrast, the same treatment prevented the loss of cytotoxic activity of the monocytes in culture. Selective regulation of C2 synthesis by IFN-a can occur in two different ways: IFN-a either increases the rate of C2 synthesis by monocytes producing C2 under normal culture conditions or induces new monocytes to produce this complement component. Experiments studying this problem are in progress in our laboratory.
Acknowledgement The authors thank Miss Aranka Pfinya for excellent technical assistance.
References [1] Becker, S. (1984) Cell. Immunol. 84, 145-153. [2] Lee, S. H. and Epstein, L. B. (1980) Cell. Immunol. 50, 177 - 190. [3] Whaley, K. (1980) J. Exp. Med. 151, 501-516. [4] Beatty, D. W., Davis, A. E., Cole, E S., Einstein, L. P. and Colten, H. R. (1981) Clin. Immunol. and Immunopathol. 18, 334-343. .[5] Colten, H . R . and Borsos, T. (1974) J. Immunol. 112, 1107-1114. [6] Kolski, G. B. and Strunk, R. C. (1981) J. Immunol. 126, 2267 - 2271. [7] Lappin, D. E and Whaley, K. (1982) Clin Exp. Immunol. 49, 623- 630. [8] Strunk, R. C., Kunke, K., Nagle, R. B., Bayne, C. M. and Harrison, H. R. (1979) Pediat. Res. 13, 188-193. [9] Littman, B. H. and Schwartz, P. (1982) Arthritis and Rheumatism 25, 288-296. [10] Littman, B. H., Hall, R. E. and Muchmore, A. V. (1983) Cell. Immunol. 76, 189-195. [11] Nichols, W. K. (1984) Complement 1, 108-115. [12] Strunk, R. C., Cole, F. S., Perlmutter, D. H. and Colten, H. R. (1985) J. Biol. Chem. 260,. 15280-15285. [13] Cantell, K., Hirvonen, S., Kauppinen, H. L. and Myllylla, G. (1981) Meth. Enzymol. 78, 29-38. [14] Blackburn, M.T., Andrews, T.M. and Watts, R. W. E. (1973) Anal. Biochem. 51, 1-10. [15] Einstein, P.E., Schneeberger, E . E . and Colten, H . R . (1976) J. Exp. Med. 143, 114-126. [16] Hall, R . E . and Colten, H. R. (1977) J. lmmunol. 118, 1903 - 1905. [17] Opferkuch, W., Rapp, H. J., Colten, H. R. and Borsos, T. (1971) J. Immunol. 106, 927-931. [18] Perlmutter, D. H., Goldberger, G., Dinarello, C. A., Mizel, S. B. and Colten, H. R. (1985) Complement 2, 62.