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Hormonal regulation of complement biosynthesis in human cell lines—I. Androgens and gamma-interferon stimulate the biosynthesis and gene expression of C1 inhibitor in human cell lines U937 and HepG2
Mokulur Immunology,Vol. 27, No. 2, pp. 191-195, 1990 Printed in Great Britain.
0161-5890190 $3.00+ 0.00 Pergamon Press plc
HORMONAL REGULATION OF COMPLEMENT BIOSYNTHESIS IN HUMAN CELL LINES-I. ANDROGENS AND GAMMA-INTERFERON STIMULATE THE BIOSYNTHESIS AND GENE EXPRESSION OF Cl INHIBITOR IN HUMAN CELL LINES U937 AND HepG2 A. FALus*t,
KATALIN G. FEH~R*, ERZSBBETWALCZ*, MARTA BROZIK*, G. F&T:, TUNDE HIDV~GI~, T. FEH~~R§and KATALIN MERI?TEY*
*Department of Immunology, Division of Molecular Biology, National Institute of Rheumatology and Physiotherapy, Budapest, Hungary; :Department of Immunopathology, National Institute of Hematology and Blood Transfusion, Budapest, Hungary; and §First Department of Medicine, Semmelweis University, Medical School, Budapest. Hungary (First received 11 April 1989; accepted in ret’ised form27 July 1989)
Abstract-Cl
inhibitor (Clinh), a member of the serine protease inhibitor gene superfamily, is a glycosylated plasma protein inhibiting the proteolytic activities of Clr and Cls and involved in the regulation of coagulation, fibrinolysis and kinin-releasing systems. In this study, the in citro effect of androgen hormones, dehydroepiandrosterone (DHEA), testosterone (TEST) and recombinant human y-interferon (y-IFN), has been determined on the production of Clinh in human cell lines. In both human monocytoid,/histiocytoid cell line U937 and in hepatoma derived cell line HepG2, DHEA and TEST upregulated the gene expression and secretion of Clinh. The most pronounced effect was detected in the concn range 10-7~10-9 M of the hormones. Under the same conditions DHEA and TEST had no detectable effeit on the biosynthesis of C3, C2 and factor B by these cells. but DHEA at higher concn (10m4 M) slightly increased that of C4 in HepG2 cells. Both in U937and in HepG2 cells recombinant y-IFN markedly increased the gene expression and secretion of Clinh. This effect of y-IFN was abolished by histamine.
INTRODUCTION
(Donaldson and Evans, 1963; Rosen et al., 1965; Cohen, 1987). Hereditary angioneurotic edema (HAE) is a dominantly inherited disease evoked by apparent heterozygous deficiency of Clinh. There are two types of HAE: either a diminished plasma level of normal Clinh protein (type I), or a dysfunctional Clinh molecule (type II) in various concns (from 30 to 400% of normal 15-35 mg/dl level) can be detected. The molecular background of the disease at the Clinh gene level is still not completely clarified (Cicardi et al., 1987; Stoppa-Lyonnet et al., 1987; Colten, 1987). Treatment of HAE involves the application of an anabolic steroid, Danazol (Gelfand et al., 1976), testosterone and dehydroepiandrosterone (DHEA) (Ko6 et al., 1983). In this study we investigated the in vitro effect of two androgenic hormones, DHEA and testosterone (TEST), as well as human recombinant y-IFN on the production of Clinh by human monocytoid cell line U937 and hepatomaderived cell line HepG2. Both androgens and y-IFN revealed a marked upregulation of the gene expression and biosynthesis of Clinh. These model systems are convenient and valuable for in vitro studies of the effects of pharmacological agents as well as biological factors on the production of Clinh.
Cl inhibitor (Clinh) (mol. wt 105,000) is one of the most heavily glycosylated plasma proteins (Harrison, 1983). The bulk of the plasma Clinh is most likely synthesized by hepatocytes (Johnson et al., 1971); however, activated monocytes (Bensa et al., 1983; Laiwah et al., 1985) and the monocyte-like cell line U937 (Randazzo et al., 1985) are also capable of generating Clinh. Human recombinant y -1FN was found to be a highly potent stimulus for the production of Clinh in peripheral monocytes (Lotz and Zuraw, 1987). Clinh regulates the activity of the classical activation pathway of complement by reducing the proteolytic activity of activated Clr and Cls, the subcomponents of Cl, thus preventing the activation of C4 and C2 by Cls (Ratnoff er al., 1969). Clinh rapidly dissociates the activated Clr and Cls by the formation of stable Clr-Cls-(Clinh), complexes (Ziccardi and Cooper, 1979). This deficiency of the functionally active Clinh is the most common isolated genetic defect of the complement system tAuthor to whom correspondence should be addressed at: Department of Immunology, National Institute of Rheumatology and Physiotherapy, Budapest 114, P.O. Box 54, H-1525, Hungary. 191
A.
192 MATERIALS
TALUS et ai.
AND METHODS
Reagents Dehydroepiandrosterone and testosterone (The British Drug House Ltd, Poole, U.K.), phorbolmyristyl acetate (PMA), histamine, HCl, horseradish peroxidase (RZ3) (Sigma, St Louis, MO) and orthophenylene-diamine (OPD) (HUMAN, God6116, Hungary) were purchased commercially. Recombinant human y-IFN (1.37 x IO6 units/ml) was generously donated by Genetech (San Francisco, CA). Polyclonal antibodies against human C3 (Dakopatts, Copenhagen, Denmark), Clinh, C4 and factor B (Atlantic Antibodies, Scarborough, ME) were commercially available. RPM1 1640 medium and fetal calf serum (FCS) were obtained from Gibco Laboratories (Grand Island, NY). Cell culture U937 cells (Sundstrdm by 20 ng/ml of PMA human hepatoma-derived plated in 24-well Linbro cells/well, respectively). 2.5 cm Linbro plates incubated with different Measurement
and Nilsson, 1976) treated for 72 hr, and trypsinized cell line, HepG2, were plates (lo6 cells/well or lo5 For extraction of mRNA, were used. The cells were substances for 24 hr.
is shown in Fig. 1. Both DHEA and TEST significantly stimulate the production of Clinh in HepG2 at a concn range of 10~9-10~5 M, and that of U937 at 10m9 M and 10-7-10-5 M, respectively. The ratio of stimulation is slightly higher in HepG2 cells (4.5-4.6 x ) that in U937 cells (3.4-3.9 x ). We did not find a significant change in the production of C3, C2 and factor B by the same cell cultures and that of albumin in HepG2 cells after the incubation of cells with DHEA and TEST (measured by ELISA; data not shown). However, a moderate increase (approx. 15%) in the amount of C4 produced by HepG2 incubated with 10m4 M DHEA has been observed (further studies on C4A and C4B are in progress). EfSect of y-interferon
on C 1inh production
All concns of recombinant human y-IFN used ( IO-lo4 U/ml) also significantly upregulate the biosynthesis of Clinh in HepG2 and in U937 cells (except at 10 U/ml) (Fig. 2). The most pronounced effect on secreted Clinh is achieved in U937 and HepG2 cell lines by lo3 U/ml of y-IFN (5.2 x and 7.2 x increase, respectively). The change in the amount of Clinh-specific mRNAs (Table 1) indicates a pretranslational effect of DHEA and recombinant human y-IFN on Clinh in both cell lines.
of C 1inh
62
Hap
Detection of Clinh was performed by sandwich ELISA using horseradish peroxidase labelled polyclonal anti-Clinh antibodies and IgG. Detection
of C4, factor
B, C3 and C2
Measurement of C3, C4 and factor B by indirect sandwich ELISA was carried out using peroxidase labelled antibodies and OPD. Determination of C2 was performed by hemolytic assay (Opferkuch et al., 1971). Northern
CIINH nglml
q
150
The total cellular RNA was isolated by the LiCl-urea method of Auffray and Rougeon (1980). RNA was run on formaldehyde-agarose gel (Thomas, 1980) and blotted to nylon membrane (Pall, Biodyne, Glen Cove, NY). The blots were hybridized to 32P-dCTP labelled Clinh cDNA probe (Tosi et al., 1986) (kindly provided by Dr Mario Tosi, Paris, France). The blots were exposed to autoradiography films (Medifort, Budapest, Hungary) at -8OC. The intensity of Clinh mRNA bands was evaluated using a Beckman model CDS 200 densitometer.
10-l' lo-"
EfSect of DHEA of Clinh
and testosterone
on the biosynthesis
The effect of DHEA and testosterone (TEST) on the production of Clinh in HepG2 and in U937 cells
lo-"
10-O
*
lo-'
lo-' M
lo-'
1o-S tl
u937
CIINH ng/m(
medium lo-" RESULTS
DHEA Testosterone 7..
i
medium
blot analysis
cl
xx
10~”
10-l' 10-o
Fig. 1. The effect of dehydroepiandrosterone (DHEA) and testosterone (TEST) on the production of Cl inhibitor (Clinh) in human hepatoma-derived cell line HepG2 and in human monocytoid cell line U937. Means of three or four experiments and S.E.M. values are shown. Significant differences from medium control are indicated by *(P < 0.05) and **(P < 0.01) (Student’s f-test).
:::: * ,o
Effect of androgens and y-IFN CllNH
193
on Clinh production
**
nglml 250-
::::
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R :.:. .:.: :::: ::::
*
150-
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kxl h :::: ?:S* .:.: :;:;
loo-
:$I
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::::
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:::: :::: :::: :::: 3-g v. :.:. ::::
0 0
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::::
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6 0
100 1000 low0
IO
mooTOOOOIIret y IFN
loo
HepG2
u 931
Fig. 2. The effect of recombinant human y-interferon (y-IFN) (IO-10,000 U/ml) on the biosynthesis of Clinh in cell lines U937 and HepG2. Means of four experiments, S.E.M. and P values (*P ~0.05; **P< 0.01) are presented.
Table I. Detection of Clinh mRNA from cultures of HepG2 and U937 cell lines incubated with dehydroepiandrosterone (DHEA) or recombinant y-IFN
Medium DHEA
y-IFN
HepG2 mRNA
u937 mRNA
Treatment alone
1.2 3.4 4.2 3.1 4.7 5.0
1.8” 6.1 2.1 2.0 4.9 5.7
(1 “M) (IOOnM) (10/W (100 U) (1000 U)
“Total cellular RNA (10 pg) was run on formaldehyde-agarose gels, blotted to nylon membrane and hybridized to a Clinh cDNA probe as described in Materials and Methods. Relative densitometric values of Clinh mRNA in U937 cells are means of two separate experiments.
The stimulating effect of y-IFN was completely (Table 2).
of 100 and 1000 U/ml abolished by histamine
DISCUSSION
The pharmacological/pathophysiological mechanisms operative in the effect of the clinical treatment of HAE patients by androgens is still not completely understood. The present results raise the possibility that the molecular mechanism for the beneficial effect of androgens on HAE patients may be in part the Table 2. Influence
of histamine
upregulation of the expression of Clinh gene, as proved in vitro in monocytoid and hepatoma-derived cells, by these hormones. The effectiveness of androgens on different elements of the immune and coagulation systems is presently being studied extensively. Opposite to this, estrogens enhance the immunoglobulin synthesis by inhibiting the suppressor T cells (Paavonen et al., 1981). Castration of the males of NZB/NZW Fl mice stimulates the production of anti-DNA autoantibodies of lupus animals, and administration of estrogens accelerates it still further (Roubinian et al., 1978). Moreover, Danazol and Stanazolol, which impede androgens and antiestrogens, are effective in some autoimmune diseases (Ahn et al., 1983). The biological relevance of the remarkable effect of testosterone on the expression of murine C4 genes, particularly that of Slp, a structurally related but hemolytically inactive plasma protein, is still obscure. The marked enhancing effect of testosterone on the biosynthesis of Slp and C4 was dependent on nonMHC, genetic factors (Bruinsten et al., 1987). In our experimental system DHEA slightly enhanced the biosynthesis of C4 in HepG2 cells, but neither DHEA nor testosterone altered the constitutive biosynthesis of C3, C2 and factor B in U937 and HepG2 cells. However, an enhancing effect
on the stimulating
effect of y-IFN
on the production
u931 Experiment Treatment Medium alone Recombinant y-IFN (100 U/ml) Recombinant y-IFN (100 U/ml) + histamine (IO-’ M) Recombinant y-IFN (1000 U/ml) Recombinant y-IFN (1000 U/ml) + histamine (10m4 M) “Clinh
measured
by ELISA
as described
I. 2. ng/lO”cells 43.1” 92.5
in Materials
of Clinh
HepG2 I. 2. “g/lo5 cells
40.3 93.7
18.4 71.8
17.6 64.3
10.6 179.3
19.4 161.3
10.6 130.3
14.3 117.4
13.2
17.3
18.8
21.4
and Methods.
194
A. FALL’S el (I1
by androgens on the plasma proteins (including fibronectin) was also demonstrated (Owens et nf., 1987). Our data confirmed the findings of Lotz and Zuraw (1987) suggesting a central role of ;I-IFN in the regulation of Clinh synthesis. However, the magnitude of the maximal elevation of Clinh by y-IFN in U937 and HepG2 cells (5.2 x and 7.2 x , respectively) and by interleukin-6 (Falus et al., 1989a,b) was comparable with that generated by the two androgens (3.4-3.9 x or 4.5-4.6 x ), indicating the significance of androgen steroids in Clinh synthesis. In mono~ytoid cell line U937 without prior stimulation by phorbol-acetate we failed to detect Clinh protein and Clinh mRNA (results not shown), thus confirming the findings of Rensa et al. (1982). These authors demonstrated that a stimulation with conditioned media from mitogen-, antigen- or allogeneicstimulated lymphocytes increased the production of all three subcomponents of Cl and Clinh by human peripheral monocytes. Laiwah PI al. (1985) also provided the ability of monocytes to produce Clinh. The negative effect of histamine on the stimulating action of p-IFN is not unique to Clinh, since a similar result of histamine action was obtained when C3 production was tested (Falus et ul., 19896). Our data suggest an in vitro enchancing effect of androgens on the biosynthesis of Clinh in both U937 and HepG2 cell lines. Earlier studies by Kob et al. (1983) demonstrated only a slight increase of Clinh in plasma of HAE patients after DHEA treatment. Rather, an inhibition of the spontaneous and immune activation of the classical pathway of complement by DHEA has been described (Hidvegi et al., 1984). According to these data the DHEA probably interferes with the internal activation of Cl. The blood level of DHEA (Koo of al., 1983) before (4.5-33.6 nmolilf and after (18.8-33.3 nmol/l) DHEA treatment is comparable to the DHEA concns (IO-‘-lO_“M) which were effective in our in ritro system. However, due to a series of metabolic changes and unknown factors our recent in vitro results do not permit us to make any conclusion on the in l+o pharmacological effectiveness and mechanism of Clinh production by androgens. Further in vitro experiments on primary monocyte and hepatocyte cultures and in Go studies on patients are needed to prove a possible physiological relevance of androgen hormones in biosynthesis of Clinh. .4c~nowleLf~~~~2rrIrs-The authors wish to thank the excellent technical assistance of M&%.r K6kai, Franciska Varga and Katalin Patay. The work was supported by grant No. OTKA 1412711988 from the Hungarian Academy of Sciences and grant No. 704,‘1988 from the Ministry of Health. REFERENCES
Ahn Y. S., Harrington W. J., Simon S. R., Mylvaganam R., Pall 1. M. and So A. G. (1983) Danazol for the treatment of idiopathic thrombocytopenic purpura. Nrn Eng(. J. Med. 308, 1396~ 1399.
A&ray C. and Rougeon F. (1980) ~ri~cation of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Eiochem. 107, 303-314. Bensa J. C., Reroui A. and Coiomb A. G. (1982) Biosynthesis of complement subcomponents Clq, Clr, Cls and Cl inhibitor by resting and stimulated human monocytes. Biochrm. J. 216, 385.-392. Bruinsten S. M., Skamene E. and Demant P. (1987) Control of expression of MHC-linked C4 and Slp proteins by nonMHC genes. Xlltlt International Complement Workshop, Chamonix, France, Abstracts, p. 139. Cicardi M., Igarashi T.. Rosen F. S. and Davis A. E., III. (1987) Molecular basis for the deficiency of complement 1 inhibitor in type I hereditary angioneurotic edema. J. clin. Invest. 79, 698 -702. Cohen Ii. R. (1987) Hereditary angioneurotic edema, 1887..l987. Nets Eng!. J. Med. 317. 43-45. Donaldson V. H. and Evans R. R. (1963) A biochemical abnormality in hereditary angioneurotic edema:absence of serum inhibitor of Cl’ esterase. Am. J. Med. 35, 37 44. Falus A., Rokita H., Waltz E., Brozik M., F&t G., Hidvegi T. and MerCtey K. (1989a) Hormonal regulation of complement biosynthesis in human cell lines----II. Upregulation of the biosynthesis of complement components C3, factor B and Cl inhibitor by interleukin-6 and interleukin-1 in human hepatoma cell line. M&c. Iw11nun. 27, 197-201. Falus A., W&z E., Brozik M., Rokita H., Fiist G., Hajnal A. and Merttey K. (19896) Stimulation of histamine receptors of human monocytoid and hepatom~-derived cell lines as well as mouse hepatocytes modulates the production of the complement compents C3. C4, factor B and C2. &and. J. fmmun. 30, 241&248. Gelfand J. A., Sherins R. J., Alling D. W. and Frank M. M. (1976) Treatment of hereditary angioedema with danazol. New Engl. J. Med. 295, 144441448. Harrison R. A. (1983) Human Cl inhibitor: improved isolation and preliminary structural characterization. Biochemistry 22, 5001-5007. Hidvigi T., Feher G. K., FehCr T., Koo E. and Filst G. (1984) Inhibition of the complement activation by an adrenal androgen, dehydroepiandrosterone. Complenrenr 1. 201-206. Johnson A. M., Alper C. A., Rosen R. S. and Craig J. M. (1971) Cl-inhibitor: evidence for decreased hepatic synthesis in hereditary angioneurotic oedema. Science 173, 553 554. Koo E., Fehdr G. K., Feher 7. and F&t G. (1983) Effect of dehydroepiandrosterone on hereditary angioedema. K/in. Wochenschr. 61, 715 717. Laiwah A. C. Y., Jones L.. Hamilton A. 0. and Whaley K, (1985) Complement-subcomponent-Cl-inhibitor synthesis by human monocytes. Biochem. J. 226, 199-205. Lotz M. and Zuraw B. L. (1987) Interferon-y is a major regulator of Cl-inhibitor synthesis by human blood monocytes. J. Immun. 139, 3382-3387. Opferkuch W,, Rapp H. J., Cohen H. R. and Borsos T. (1971) Immune hemolysis and the functional properties of the second (C2) and fourth (C4) component of complement. J. Immun. 106, 927-933. Owens M. R., Cimino C. D. and Donelly Y. J. (1987) Testosterone effects on biosynthesis of coagulation proteins. Thromh. Hoemost. 57, 259-262. Paavonen T., Andersson L. C. and Adlercreutz H. (i981) Sex hormone regulation of an in r+/ro immune response. J. asp. Med. 154, 19351944. Randazzo B. P., Daatwyler R. J., Kaplan A. P. and Ghebrehiwet B. (1985) Synthesis of Cl inhibitor (ClINH) by human monocyte-like cell line, IJ937. J. Immun. 135, 1313-1319. Ratnoff 0. D., Pensky J., Ogston D. and Naff G. D. (1969) The inhibition of plasmin, plasma kallikrein. plasma permeability factor and Clr subcomponent of the first
Effect of androgens
and y -1FN on Clinh
component by serum Cl’ esterase inhibitor. J. exp. Med. 129, 3155331. Rosen F. S., Charache P., Pensky J. and Donaldson V. (1965) Hereditary angioneurotic edema: two genetic variants. Science 148, 957-958. Roubinian J. R., Talal N., Greenspan J. S., Goodman J. R. and Siiteri P. K. (1978) Effect of castration and sex hormone treatment on survival, anti-nucleic antibodies and glomerulonephritis in NZB/NZW Fl mice. J. exp. Med. 147, 156881575. Stoppa-Lyonett D., Rosi M., Laurent J., Sobel A., Lagrue G. and Meo T. (1987) Altered Cl inhibitor genes in type I hereditary angioedema. Nen Engl. J. Med. 317, I-6.
production
195
Sundstriim C. and Nilsson K. (1976) Establishment and characterization of a human histiocytic cell line (U937). Inr. J. Cancer 17, 565-571. Thomas P. S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. natn. Acad. Sci. U.S.A. 77, 5201-5205. Tosi M., Duponchel C., Bourgarel P., Colomb M. and Meo T. (1986) Molecular cloning of human Cl inhibitor: sequence homologies with a I-antitrypsin and other members of serpins superfamily. Gene 42, 265-272. Ziccardi R. J. and Cooper N. R. (1979) Active disassembly of the first complement component, Cl, by Cl inactivator. J. Immun. 123, 788-792.
0161-5890190 $3.00+ 0.00 Pergamon Press plc
HORMONAL REGULATION OF COMPLEMENT BIOSYNTHESIS IN HUMAN CELL LINES-I. ANDROGENS AND GAMMA-INTERFERON STIMULATE THE BIOSYNTHESIS AND GENE EXPRESSION OF Cl INHIBITOR IN HUMAN CELL LINES U937 AND HepG2 A. FALus*t,
KATALIN G. FEH~R*, ERZSBBETWALCZ*, MARTA BROZIK*, G. F&T:, TUNDE HIDV~GI~, T. FEH~~R§and KATALIN MERI?TEY*
*Department of Immunology, Division of Molecular Biology, National Institute of Rheumatology and Physiotherapy, Budapest, Hungary; :Department of Immunopathology, National Institute of Hematology and Blood Transfusion, Budapest, Hungary; and §First Department of Medicine, Semmelweis University, Medical School, Budapest. Hungary (First received 11 April 1989; accepted in ret’ised form27 July 1989)
Abstract-Cl
inhibitor (Clinh), a member of the serine protease inhibitor gene superfamily, is a glycosylated plasma protein inhibiting the proteolytic activities of Clr and Cls and involved in the regulation of coagulation, fibrinolysis and kinin-releasing systems. In this study, the in citro effect of androgen hormones, dehydroepiandrosterone (DHEA), testosterone (TEST) and recombinant human y-interferon (y-IFN), has been determined on the production of Clinh in human cell lines. In both human monocytoid,/histiocytoid cell line U937 and in hepatoma derived cell line HepG2, DHEA and TEST upregulated the gene expression and secretion of Clinh. The most pronounced effect was detected in the concn range 10-7~10-9 M of the hormones. Under the same conditions DHEA and TEST had no detectable effeit on the biosynthesis of C3, C2 and factor B by these cells. but DHEA at higher concn (10m4 M) slightly increased that of C4 in HepG2 cells. Both in U937and in HepG2 cells recombinant y-IFN markedly increased the gene expression and secretion of Clinh. This effect of y-IFN was abolished by histamine.
INTRODUCTION
(Donaldson and Evans, 1963; Rosen et al., 1965; Cohen, 1987). Hereditary angioneurotic edema (HAE) is a dominantly inherited disease evoked by apparent heterozygous deficiency of Clinh. There are two types of HAE: either a diminished plasma level of normal Clinh protein (type I), or a dysfunctional Clinh molecule (type II) in various concns (from 30 to 400% of normal 15-35 mg/dl level) can be detected. The molecular background of the disease at the Clinh gene level is still not completely clarified (Cicardi et al., 1987; Stoppa-Lyonnet et al., 1987; Colten, 1987). Treatment of HAE involves the application of an anabolic steroid, Danazol (Gelfand et al., 1976), testosterone and dehydroepiandrosterone (DHEA) (Ko6 et al., 1983). In this study we investigated the in vitro effect of two androgenic hormones, DHEA and testosterone (TEST), as well as human recombinant y-IFN on the production of Clinh by human monocytoid cell line U937 and hepatomaderived cell line HepG2. Both androgens and y-IFN revealed a marked upregulation of the gene expression and biosynthesis of Clinh. These model systems are convenient and valuable for in vitro studies of the effects of pharmacological agents as well as biological factors on the production of Clinh.
Cl inhibitor (Clinh) (mol. wt 105,000) is one of the most heavily glycosylated plasma proteins (Harrison, 1983). The bulk of the plasma Clinh is most likely synthesized by hepatocytes (Johnson et al., 1971); however, activated monocytes (Bensa et al., 1983; Laiwah et al., 1985) and the monocyte-like cell line U937 (Randazzo et al., 1985) are also capable of generating Clinh. Human recombinant y -1FN was found to be a highly potent stimulus for the production of Clinh in peripheral monocytes (Lotz and Zuraw, 1987). Clinh regulates the activity of the classical activation pathway of complement by reducing the proteolytic activity of activated Clr and Cls, the subcomponents of Cl, thus preventing the activation of C4 and C2 by Cls (Ratnoff er al., 1969). Clinh rapidly dissociates the activated Clr and Cls by the formation of stable Clr-Cls-(Clinh), complexes (Ziccardi and Cooper, 1979). This deficiency of the functionally active Clinh is the most common isolated genetic defect of the complement system tAuthor to whom correspondence should be addressed at: Department of Immunology, National Institute of Rheumatology and Physiotherapy, Budapest 114, P.O. Box 54, H-1525, Hungary. 191
A.
192 MATERIALS
TALUS et ai.
AND METHODS
Reagents Dehydroepiandrosterone and testosterone (The British Drug House Ltd, Poole, U.K.), phorbolmyristyl acetate (PMA), histamine, HCl, horseradish peroxidase (RZ3) (Sigma, St Louis, MO) and orthophenylene-diamine (OPD) (HUMAN, God6116, Hungary) were purchased commercially. Recombinant human y-IFN (1.37 x IO6 units/ml) was generously donated by Genetech (San Francisco, CA). Polyclonal antibodies against human C3 (Dakopatts, Copenhagen, Denmark), Clinh, C4 and factor B (Atlantic Antibodies, Scarborough, ME) were commercially available. RPM1 1640 medium and fetal calf serum (FCS) were obtained from Gibco Laboratories (Grand Island, NY). Cell culture U937 cells (Sundstrdm by 20 ng/ml of PMA human hepatoma-derived plated in 24-well Linbro cells/well, respectively). 2.5 cm Linbro plates incubated with different Measurement
and Nilsson, 1976) treated for 72 hr, and trypsinized cell line, HepG2, were plates (lo6 cells/well or lo5 For extraction of mRNA, were used. The cells were substances for 24 hr.
is shown in Fig. 1. Both DHEA and TEST significantly stimulate the production of Clinh in HepG2 at a concn range of 10~9-10~5 M, and that of U937 at 10m9 M and 10-7-10-5 M, respectively. The ratio of stimulation is slightly higher in HepG2 cells (4.5-4.6 x ) that in U937 cells (3.4-3.9 x ). We did not find a significant change in the production of C3, C2 and factor B by the same cell cultures and that of albumin in HepG2 cells after the incubation of cells with DHEA and TEST (measured by ELISA; data not shown). However, a moderate increase (approx. 15%) in the amount of C4 produced by HepG2 incubated with 10m4 M DHEA has been observed (further studies on C4A and C4B are in progress). EfSect of y-interferon
on C 1inh production
All concns of recombinant human y-IFN used ( IO-lo4 U/ml) also significantly upregulate the biosynthesis of Clinh in HepG2 and in U937 cells (except at 10 U/ml) (Fig. 2). The most pronounced effect on secreted Clinh is achieved in U937 and HepG2 cell lines by lo3 U/ml of y-IFN (5.2 x and 7.2 x increase, respectively). The change in the amount of Clinh-specific mRNAs (Table 1) indicates a pretranslational effect of DHEA and recombinant human y-IFN on Clinh in both cell lines.
of C 1inh
62
Hap
Detection of Clinh was performed by sandwich ELISA using horseradish peroxidase labelled polyclonal anti-Clinh antibodies and IgG. Detection
of C4, factor
B, C3 and C2
Measurement of C3, C4 and factor B by indirect sandwich ELISA was carried out using peroxidase labelled antibodies and OPD. Determination of C2 was performed by hemolytic assay (Opferkuch et al., 1971). Northern
CIINH nglml
q
150
The total cellular RNA was isolated by the LiCl-urea method of Auffray and Rougeon (1980). RNA was run on formaldehyde-agarose gel (Thomas, 1980) and blotted to nylon membrane (Pall, Biodyne, Glen Cove, NY). The blots were hybridized to 32P-dCTP labelled Clinh cDNA probe (Tosi et al., 1986) (kindly provided by Dr Mario Tosi, Paris, France). The blots were exposed to autoradiography films (Medifort, Budapest, Hungary) at -8OC. The intensity of Clinh mRNA bands was evaluated using a Beckman model CDS 200 densitometer.
10-l' lo-"
EfSect of DHEA of Clinh
and testosterone
on the biosynthesis
The effect of DHEA and testosterone (TEST) on the production of Clinh in HepG2 and in U937 cells
lo-"
10-O
*
lo-'
lo-' M
lo-'
1o-S tl
u937
CIINH ng/m(
medium lo-" RESULTS
DHEA Testosterone 7..
i
medium
blot analysis
cl
xx
10~”
10-l' 10-o
Fig. 1. The effect of dehydroepiandrosterone (DHEA) and testosterone (TEST) on the production of Cl inhibitor (Clinh) in human hepatoma-derived cell line HepG2 and in human monocytoid cell line U937. Means of three or four experiments and S.E.M. values are shown. Significant differences from medium control are indicated by *(P < 0.05) and **(P < 0.01) (Student’s f-test).
:::: * ,o
Effect of androgens and y-IFN CllNH
193
on Clinh production
**
nglml 250-
::::
zoo-
R :.:. .:.: :::: ::::
*
150-
:s:
kxl h :::: ?:S* .:.: :;:;
loo-
:$I
50
.:.:
::::
:::: :::: $:i
:::: ::::
9:
:::: :::: :::: :::: 3-g v. :.:. ::::
0 0
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::::
R 3
6 0
100 1000 low0
IO
mooTOOOOIIret y IFN
loo
HepG2
u 931
Fig. 2. The effect of recombinant human y-interferon (y-IFN) (IO-10,000 U/ml) on the biosynthesis of Clinh in cell lines U937 and HepG2. Means of four experiments, S.E.M. and P values (*P ~0.05; **P< 0.01) are presented.
Table I. Detection of Clinh mRNA from cultures of HepG2 and U937 cell lines incubated with dehydroepiandrosterone (DHEA) or recombinant y-IFN
Medium DHEA
y-IFN
HepG2 mRNA
u937 mRNA
Treatment alone
1.2 3.4 4.2 3.1 4.7 5.0
1.8” 6.1 2.1 2.0 4.9 5.7
(1 “M) (IOOnM) (10/W (100 U) (1000 U)
“Total cellular RNA (10 pg) was run on formaldehyde-agarose gels, blotted to nylon membrane and hybridized to a Clinh cDNA probe as described in Materials and Methods. Relative densitometric values of Clinh mRNA in U937 cells are means of two separate experiments.
The stimulating effect of y-IFN was completely (Table 2).
of 100 and 1000 U/ml abolished by histamine
DISCUSSION
The pharmacological/pathophysiological mechanisms operative in the effect of the clinical treatment of HAE patients by androgens is still not completely understood. The present results raise the possibility that the molecular mechanism for the beneficial effect of androgens on HAE patients may be in part the Table 2. Influence
of histamine
upregulation of the expression of Clinh gene, as proved in vitro in monocytoid and hepatoma-derived cells, by these hormones. The effectiveness of androgens on different elements of the immune and coagulation systems is presently being studied extensively. Opposite to this, estrogens enhance the immunoglobulin synthesis by inhibiting the suppressor T cells (Paavonen et al., 1981). Castration of the males of NZB/NZW Fl mice stimulates the production of anti-DNA autoantibodies of lupus animals, and administration of estrogens accelerates it still further (Roubinian et al., 1978). Moreover, Danazol and Stanazolol, which impede androgens and antiestrogens, are effective in some autoimmune diseases (Ahn et al., 1983). The biological relevance of the remarkable effect of testosterone on the expression of murine C4 genes, particularly that of Slp, a structurally related but hemolytically inactive plasma protein, is still obscure. The marked enhancing effect of testosterone on the biosynthesis of Slp and C4 was dependent on nonMHC, genetic factors (Bruinsten et al., 1987). In our experimental system DHEA slightly enhanced the biosynthesis of C4 in HepG2 cells, but neither DHEA nor testosterone altered the constitutive biosynthesis of C3, C2 and factor B in U937 and HepG2 cells. However, an enhancing effect
on the stimulating
effect of y-IFN
on the production
u931 Experiment Treatment Medium alone Recombinant y-IFN (100 U/ml) Recombinant y-IFN (100 U/ml) + histamine (IO-’ M) Recombinant y-IFN (1000 U/ml) Recombinant y-IFN (1000 U/ml) + histamine (10m4 M) “Clinh
measured
by ELISA
as described
I. 2. ng/lO”cells 43.1” 92.5
in Materials
of Clinh
HepG2 I. 2. “g/lo5 cells
40.3 93.7
18.4 71.8
17.6 64.3
10.6 179.3
19.4 161.3
10.6 130.3
14.3 117.4
13.2
17.3
18.8
21.4
and Methods.
194
A. FALL’S el (I1
by androgens on the plasma proteins (including fibronectin) was also demonstrated (Owens et nf., 1987). Our data confirmed the findings of Lotz and Zuraw (1987) suggesting a central role of ;I-IFN in the regulation of Clinh synthesis. However, the magnitude of the maximal elevation of Clinh by y-IFN in U937 and HepG2 cells (5.2 x and 7.2 x , respectively) and by interleukin-6 (Falus et al., 1989a,b) was comparable with that generated by the two androgens (3.4-3.9 x or 4.5-4.6 x ), indicating the significance of androgen steroids in Clinh synthesis. In mono~ytoid cell line U937 without prior stimulation by phorbol-acetate we failed to detect Clinh protein and Clinh mRNA (results not shown), thus confirming the findings of Rensa et al. (1982). These authors demonstrated that a stimulation with conditioned media from mitogen-, antigen- or allogeneicstimulated lymphocytes increased the production of all three subcomponents of Cl and Clinh by human peripheral monocytes. Laiwah PI al. (1985) also provided the ability of monocytes to produce Clinh. The negative effect of histamine on the stimulating action of p-IFN is not unique to Clinh, since a similar result of histamine action was obtained when C3 production was tested (Falus et ul., 19896). Our data suggest an in vitro enchancing effect of androgens on the biosynthesis of Clinh in both U937 and HepG2 cell lines. Earlier studies by Kob et al. (1983) demonstrated only a slight increase of Clinh in plasma of HAE patients after DHEA treatment. Rather, an inhibition of the spontaneous and immune activation of the classical pathway of complement by DHEA has been described (Hidvegi et al., 1984). According to these data the DHEA probably interferes with the internal activation of Cl. The blood level of DHEA (Koo of al., 1983) before (4.5-33.6 nmolilf and after (18.8-33.3 nmol/l) DHEA treatment is comparable to the DHEA concns (IO-‘-lO_“M) which were effective in our in ritro system. However, due to a series of metabolic changes and unknown factors our recent in vitro results do not permit us to make any conclusion on the in l+o pharmacological effectiveness and mechanism of Clinh production by androgens. Further in vitro experiments on primary monocyte and hepatocyte cultures and in Go studies on patients are needed to prove a possible physiological relevance of androgen hormones in biosynthesis of Clinh. .4c~nowleLf~~~~2rrIrs-The authors wish to thank the excellent technical assistance of M&%.r K6kai, Franciska Varga and Katalin Patay. The work was supported by grant No. OTKA 1412711988 from the Hungarian Academy of Sciences and grant No. 704,‘1988 from the Ministry of Health. REFERENCES
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