Immunology 1979 36 323
The metabolism of nucleic acids in mice*
D. CHIA, CAROLE A. DORSCHt, L. LEVY & E. V. BARNETT Department of Medicine, Division of Rheumatology, UCLA School of Medicine, Los Angeles, California 90024
Received 18 April 1978; acceptedfor publication 2 June 1978
INTRODUCTION
Summary. The metabolism of three forms of nucleic acid, native-DNA (N-DNA), single strand DNA (ss-DNA), and polyinosinic-polycytidylic acid (poly I: C), was investigated in vivo in randomly bred Swiss-Webster mice. Clearance of these substances from the circulation and tissue localization were determined at selected time intervals following the intravenous injection of 1251-labelled compounds. N- and ss-DNA were removed from the circulation more rapidly than was poly I: C. All three materials localized principally in reticuloendothelial-rich organs, i.e. liver and spleen. N-DNA was degraded by the liver more slowly than was poly I: C or ss-DNA. At 4 h following injection, the liver contained 26%, 13%, and 10% of the injected doses, respectively. Three days after injection, 4*5% of the N-DNA persisted in the liver, as compared to only 0-6% of the poly I: C and 0-2% of the ss-DNA. The possibility that these differences in metabolism of N-DNA, poly I: C, and ss-DNA may be related to their differing immunogenic potentials in experimental systems is discussed.
The literature contains many examples of compounds, similar in chemical structure, which differ in their immunogenicity. Attempts have been made to correlate the metabolic fate and tissue localization of these compounds with their immunogenic potential (Unanue, 1972). The nucleic acids, native DNA (N-DNA), single strand DNA (ss-DNA), and polyinosinic-polycytidylic acid (poly I C) represent a similar series of compounds which, although chemically alike, differ markedly in their ability to induce antibody formation in experimental animals. Thus, despite numerous attempts, N-DNA is non-immunogenic (Stollar, 1973). ss-DNA alone is not immunogenic but, when coupled covalently (Halloran & Parker, 1966) or electrostatically (Plescia, Braun & Pelczuk, 1964) to a carrier protein and administered in Freund's complete adjuvant antibodies can be induced. Poly I: C can be immunogenic when administered in saline alone and does not require carrier or Freund's adjuvant (Parker & Steinberg, 1973). In contrast to these observations, patients with systemic lupus erythematosus (SLE) and New Zealand Black/White (NZB/W) F1 mice, who develop an autoimmune disease similar to SLE, form antibodies against all three of these materials, including N-DNA (Arana & Seligman, 1967; Thoburn, Koffler & Kunkel, 1971). It is possible that differences in the immunogenicity of nucleic acid antigens may be related to
*Supported in part by grant number GM-15757 from The United States Public Health Service. tPresent address: Connective Tissue Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Correspondence: Dr David Chia, UCLA Rehabilitation Center, 1000 Veteran Avenue, Los Angeles, California 90024. 00 1 9-2805/79/0200-0323 $02.00 (O 1979 Blackwell Scientific Publications
323
D. Chia et al.
324
differences in their metabolism. If this is true, alterations in these metabolic pathways in disease states, along with other suggested defects of the immune system, could contribute to the production of antinucleic acid antibodies. The present study was designed to investigate the metabolism of these three different forms of nucleic acid in the same in vivo system. MATERIALS AND METHODS Mice 6-8 week old male Swiss-Webster mice, 25-30 g, were obtained from Hilltop Laboratories (Scottdale, Penn.). Mice were fed Purina Laboratory Chow and received water ad libitum.
Nucleic acids Calf thymus DNA was obtained from Worthington Biochemicals (Freehold, New Jersey). T-7 bacteriophage DNA was a gift from Dr V. N. Schumaker (University of California, Los Angeles, Calif.). Poly I: C was obtained from Miles Laboratories (Elkhart, Indiana), and tetradeoxycytidine (dCp)4 was obtained from P-L Biochemicals, Inc. (Milwaukee, Wisconsin).
Table 1. Clearance of N and ss-DNA from the blood
Dose/total blood volume* (Mean+ S.D.)
Time (min)
N-DNA
3 7 15 30 60 240 1440
15-78+ 7-91 8-73+1-25 9-26+0-62 8-72+ 152 5-36+0-78 2-31+0-74 0-29+0 14
ss-DNA
17-23+ 8-44 9-08+1 10
13-03+2-07t 10-35+ Illt 7-35+1-16t 4 70+1-81t 0 34+0-33
*Blood volume assumed to be 10% of body weight.
tP< 0-01.
using a Beckman model L2-65B ultracentrifuge. Molecular weights were calculated from their sedimentation coefficient (Burgi & Hershey, 1963). Animal studies Mice were injected intravenously via the lateral tail vein with 2-3 pg of '25I-N-DNA or ss-DNA or 0-6 pg of 1251-poly I: C. Three to six animals were killed at 3, 7, 15, 30, 60 and 240 min after injection. Blood and various organs were counted for 1251 in a gamma scintillation counter (Nuclear Chicago).
Radiolabelled nucleic acids Nucleic acids were labelled with l25l by the method of Commerford (1971). Specific activities of calf thymus N-DNA, T-7 DNA, poly I: C and dCp4 were 0 05 ,uCi/pg, 0 02 ,pCi/,ug, 0 25 ,pCi/pug and 05 ,pCi/,ug respectively. For certain experiments '25I-N-DNA was sonicated for 10 min using a Sonifier Cell Disruptor (model 140D, Heat Systems -Ultrasonics, Inc., Plainview, New York). 1251.sS_ DNA was prepared by heating the '25I-N-DNA at 100° for 10 min, followed by rapid cooling in ice. A deoxyribonuclease (DNase) digest was prepared by incubation of 25I-N-DNA (4 pg/ml) with 20 ,ug/ml of DNase I (Worthington Biochemicals, Freehold, New Jersey) in pH 7-4 phosphate buffered saline, 0005 M magnesium chloride, for 30 min at 370. Residual DNase activity was destroyed by heating at 1000 for 10 min and then cooling in ice.
Clearance of DNA from the circulation After the intravenous administration of 1251-N-DNA or 125I-ss-DNA, radiolabel disappeared rapidly from the blood (Table 1). Rates of disappearance were comparable following N or ss-DNA. Less than 25 % of the injected dose remained after 3 min, and less than 10% after 60 min. At 15 min there was a consistently observed rise in blood radioactivity for both N and ss-DNA. This was more pronounced for ss-DNA and from 15 to 240 min blood levels remained significantly higher than those for N-DNA. By 24 h approximately 0 3 % of administered radioactivity from either material was detectable in the blood.
Determination of molecular weights Sedimentation rates for the various nucleic acid preparations were determined by ultracentrifugation on a linear sucrose density gradient of 5 to 20%.,
Tissue localization of DNA Radioactivity from both N- and ss-DNA was rapidly taken up by the liver and, to a lesser extent,
RESULTS
Metabolism of nucleic acids
325
Table 2. Uptake of N and ss-DNA by liver and spleen
% Dose/Organ (Mean+ S.D.) Liver
Spleen
Time (min) 3 7 15 30 60 240 1440
N-DNA
ss-DNA
N-DNA
ss-DNA
56-76+ 12 19 70-15+4-98 52-92+ 8-39 42-12+ 6-73 29-60+ 5-08 2602+ 3 15 7-78+1-11
59*05+6-34 54-45+ 8.18* 30-92+ 2-29* 16-12+2 10* 13-48+1-65* 10-12+0-77*
1 98+0-67 2-40+0 70 2-30+0-70 2-48+0 91 1-53+0-61 1-92+0-66 0-42+0 11
1-77+1-06 188+ 034 1-88+ 0-35 1-32+ 0.57* 1-22+0 12 0-90+ 0-27* 0.16+0.04*
0-88+0-32*
*P< 0-01.
by the spleen, as can be seen in Table 2. In the liver maximum amounts were found at 3-7 min. There was then a slow decline in hepatic radioactivity. However, this disappearance from the liver was slower for N than for ss-DNA. From 7 min on the liver contained a significantly greater fraction of the administered N-DNA than of ss-DNA. Thus after 60 min, the amount of N-DNA remaining was 2-2 times that of ss-DNA and after 24 h 8-8 times as much. At 3 days following injection, 4-5% of the dose of N-DNA remained in the liver as compared to 0-2% for ss-DNA. In the spleen the amount of N and ss-DNA remained relatively constant for the first 60 min and then fell slowly. At 240 min and 24 h levels of N-DNA were significantly higher than those for ss-DNA at P < *01. Comparable results were obtained when data were expressed as % dose/100 mg of tissue. Effect of molecular size on blood clearance and tissue localization Although the N- and ss-DNA used were derived from the same preparation, their molecular weights differed markedly, as can be seen in Table 3. The N-DNA had an average molecular weight (mol. wt.) of 2-6 x 106 daltons (15S) and the ss-DNA obtained after heat denaturation of the N-DNA preparation had a mol. wt of 1-2 x 105 daltons (6 4S). This is probably the result of the presence of single-strand nicks in the N-DNA preparation so that after heatdenaturation and subsequent strand-separation, the resultant ss-DNA pieces are of smaller size than
Table 3. Nucleic acid antigens
Type of nucleic acid
Calf thymus N-DNA Sonicated calf thymus N-DNA T-7 Bacteriophage ss-DNA Calf thymus ss-DNA DNAase digested calf thymus ss-DNA Tetradeoxycytidine Poly rI: rC
Sedimentation
Molecular
coefficient
weight
15
2-6 x 106
7.9
4-2x 105
30 5 6-2
2-0x 106 1 2x 105
2-3 7-6
1-8 x 104 1-4 x 103
would be expected. Because of the possibility that these size differences were responsible for the differences in organ retention observed for N- and ss-DNA, studies were undertaken utilizing N and ss materials of comparable size. Sonicated calf thymus N-DNA (mol. wt. 4-2 x 10s daltons) served as an example of low MW N-DNA comparable in size to CT-ss-DNA. Heat denatured T-7 bacteriophage DNA of mol. wt. 2-0 x 106 daltons was used as an example of high mol. wt. ss-DNA. Clearance studies were performed comparing high mol. wt. N- and ss-DNA, i.e., CT-NDNA and T-7 ss-DNA, and low mol. wt. N- and ss-DNA, i.e. sonicated CT-N-DNA and CT-ssDNA. In comparing N and ss-preparations of the same size, the pattern of disappearance from the blood was unchanged from that described above. Likewise, after 15 min blood levels were consistently
D. Chia et al.
326
5
- I
I(a)
II
( b)
_
4-
0 a%
,,
E
~~~~~(c)
b
0
2 5
(d)
en
03
0
4~~~~~ 20
40
A
60
/
240 0 Time (r
20
40
60
240
Figure 1. Influence of molecular weight on the levels of N-and ss-DNA in liver: (a) calf thymus N-DNA, (b) sonicated calf thymus N-DNA, (c) T-7-ss-DNA, (d) calf thymus ss-DNA. Each point represents the mean of six animals with standard deviation shown.
higher following administration of ss-DNA as compared to N-DNA. In Fig. 1 hepatic levels of radioactivity following administration of each of the four nucleic acid preparations are shown, expressed as % of administered dose/100 mg of tissue. As can be seen, hepatic levels following high MW CT-N-DNA (Fig. la) are slightly higher than levels following low mol. wt., sonicated CT-N-DNA (Fig. lb). Levels for high mol. wt., T-7, ss-DNA (Fig. Ic) and for low mol. wt. CT-ss-DNA (Fig. Id) were similar throughout the time period studied. High mol. wt. N-DNA disappeared more slowly from the liver than did high mol. wt. ss-DNA
(Fig. la and c). Low mol. wt. N-DNA and low mol. wt. ss-DNA disappeared similarly until 60 and 240 min when levels following N-DNA were 1 5 and 1 9 those for ss-DNA respectively. Thus, the preferential retention of N-DNA persists when preparations of comparable size are used. To investigate further the role of nucleic acid size, a DNase digest of ss-DNA (mol. wt. 1 8 x 104) and tetradeoxycytidine (mol. wt. 1-4 x 103) were studied. As control Na125I was injected in a similar manner. Results are shown in Table 4. Liver and spleen levels of DNase digest and (dCp)4 were similar and much less than seen with N- or ss-DNA (Table 2). Organ
Table 4. Uptake of DNAase digested ss-DNA, tetradeoxycytidine and Na'251 by liver and spleen
% Dose/organ (Mean .S.D.) Spleen
Liver Time (min) 3 7 15 30 60 240
DNAase digested ss-DNA
TetradeoxyCytidine
8 94+ 1-45 7-11+ 1-66
7-92+0-77 7 70+ 1-48
6-70+1-66 4-08+0-71 2 35+ 0 23
6-08+0-80 545+1-80 2-28+0-80 085+0-30
1l20+009
DNAase
Na125I
digested
TetradeoxyCytidine
Na1251
ss-DNA
*
1-68+0-33 156+0 13 173+0-09
1l65+0-02 1*67+ 0-03
Not done.
N.D.*
0 59+0 20
0-64+0-18
0-45+0-15 0-60+0-22 0-38+ 0-09 0-43+0-20
0-48+012 0-43+0-10 0-40+0-06 0-45+0-12 0 50+ 0 15 0-33+0-15
0-12+0*00 0-18+0-03 0-14+0-02 0-16+0-05 0-15+ 0-02 N.D.*
Metabolism of nucleic acids Table 5. The rate of degradation of various nucleic acids in the liver
Nucleic acids
Rate (log % dose/min)*
T-7-ss-DNA ss-DNA N-DNA Sonicated N-DNA
-0-0182+ 001 14 -0-0215+ 00075 -0-0066+0 0043 -0-0058+ 00075
*Standard deviation of least square linear regression calculated with a confidence limit of 95%
levels of Nal25I were quite low and remained constant over a 60 min period of observation. Catabolism of DNA The catabolism of nucleic acids can also be described as the rate of disappearance of radioactivity from the liver. This was calculated for both large and small ss-DNA and N-DNA, as shown in Table 5. Both ss-DNA preparations had a faster rate of disappearance than the N-DNA preparations. Radioactivity was found in the gastric washings after the injection of nucleic acids. This radioactivity was shown to have the same mobility as free iodide on a Sephades G-25 column. Its appearance can be used as a measure of the degradation of nucleic acids. In Fig. 2, the appearance of radiolabel in the stomach after intravenous injection of various material is shown. Free iodide is found early and
persists during the observation period. Radiolabel derived from a DNase digest of DNA appears slightly faster than that derived from ss-DNA. In both of these cases maximum amount of radiolabel is found at 60 min and has decreased by 4 h. However, in the case of N-DNA, radiolabel appears more slowly and in smaller amounts in the gastric washings and is continuing to rise at 4 h. An attempt was made to determine if catabolic pathways for DNA metabolism could be saturated or, alternatively, induced to become more efficient. Groups of six mice were injected intravenously with 100 ug of N-DNA or ss-DNA daily for 5 days. On the fifth day radiolabelled homologous DNA was injected and blood and organ levels determine. No differences were observed between untreated animals and those pretreated with DNA. Metabolism of poly I: C The clearance of a third form of nucleic acid, the synthetic double-stranded RNA, poly I: C, was compared to that of N-DNA. The preparations 100 _
80 E .5 _ 60
30
327
0
0
40
0~
20 ei
0 Time (min)
Figure 2. Level of radiolabel in gastric washings for (A) calf thymus N-DNA, (0) calf thymus ss-DNA, (C1) DNAase treated ss-DNA, (0) sodium iodide. Mean for six animals.
20
40 Time (min)
60
240
Figure 3. Clearance of (a) N-DNA and (-) poly I C from the blood (assuming blood volume as 10% of body weight). The standard deviations for six animals are shown. They are significantly different where 001 >P> 0001.
D. Chia et al.
328
Table 6. Level of radiolabel in gastric washings for sonicated N-DNA and poly I: C.
Time (min)
Sonicated N-DNA
0-56±0 42 0-97+0-51
3 7 15 30 60 240
2-43±0-20 7-09+0-51 13-77±1 27 18-27+ 5-01
Poly I: C 057±0 20 0-53±0-19 1.14+0-63* 3.04±1-17* 7.30±2.28* 13-86+ 5-88
*P< 0.01.
compared were of comparable size (sonicated N-DNA 7-9S and poly I: C 7 6S). Blood clearance is shown in Fig. 3. Poly I: C disappeared from the circulation at a much slower rate than did N-DNA. Consistent with this observation are the results for tissue uptake (Fig. 4). As seen in Fig. 4a, hepatic uptake of N-DNA was maximal at 3 min, while peak uptake of poly I: C was not reached until 15 min. Both materials were (a) 4'0
4I
20 _
0
._-
E 0 0
0
0 0
0n 0
Time (min) Figure 4. Uptake of sonicated N-DNA (0) and poly I: C (@) by (a) liver, and (b) spleen. The standard deviations for 6 animals are shown. Liver is significantly different before 15 min where P< 0'001; spleen is significantly different at all points, 0'005 >P>0 001, except at 30 min.
then lost at comparable rates through 60 min. However, by 6 days following injection 2-9% of the N-DNA but only 0-6% of the poly I: C persisted in the liver. In the spleen there is a higher uptake and retention of N-DNA when compared to poly I: C (Fig. 4b). Levels of radiolabel in gastric washings following intravenous administration of sonicated N-DNA and poly I: C are shown in Table 6. Values for both materials increased slowly through 4 h but were lower for poly I: C than for N-DNA. As noted above, both of these were less than for ss-DNA (Fig. 2). These observations are consistent with a delay in catabolism resulting from a delay in hepatic intake of poly I: C.
DISCUSSION Many studies have documented the rapid clearance of N-DNA from the blood after intravenous administration (Tsumita & Iwanaga, 1963) and its localization predominantly in liver, spleen, and, to a lesser extent, in intestine, gonadal tissue, and tumours (Ledoux, 1965; Chused, Steinberg & Talal, 1972). From the present study, it appears that ss-DNA and the synthetic RNA poly I: C are handled similarly in vivo. However, the present study also clearly demonstrates significant differences in the clearance and metabolism of N-DNA, ss-DNA, and poly I: C. Poly I: C is cleared from the blood more slowly than is either N-DNA or ss-DNA and reaches peak levels in liver and spleen more slowly. Thereafter it is eliminated from the liver at a rate comparable to N-DNA of similar size, but more slowly than is ss-DNA. N-DNA and ss-DNA are cleared from the blood at similar rates. However, peak hepatic levels are reached more slowly and are higher for N-DNA than for ss-DNA, and N-DNA disappears from liver and spleen more slowly than does ss-DNA. After 24 h 7-8% of the administered dose of N-DNA persists in the liver, as compared to only 0 9%/ of the ss-DNA (Table 2). From 24 h on, hepatic levels of N-DNA are consistently higher than levels of poly I: C which are in turn higher than ss-DNA levels. It is not wholly unexpected that a doublestranded helix would be more difficult for tissue nucleases to degrade, since degradation of such a polymer would require adjacent enzymatic nicks to occur in both strands.
Metabolism of nucleic acids It has previously been reported that hepatic levels of poly I: C are influenced by the molecular size of the administered poly I: C (Chused et al., 1972). In the present study, a similar observation was made for N-DNA. However, the molecular weight of ss-DNA did not seem important in determining tissue levels of this material. The organism is repeatedly presented with the necessity to clear or degrade nucleic acids, either exogenous, e.g. during viral infection, or endogenous, e.g. after tissue trauma. It appears from these studies as if this can be accomplished more effectively for ss-DNA than for N-DNA. The subcellular localization or the form of the persisting DNA is not known. While this may represent macromolecular DNA, the possibility that breakdown products, re-utilized by the host, are being measured cannot be totally excluded. It is intriguing to speculate that the ability of N-DNA to persist in vivo could be related to the observation that it constitutes a poorer immunogen than does ss-DNA. In other antigenic systems, relative differences in the immunogenicity of chemically similar materials have been attributed to differences in their metabolism. One such example is that of the synthetic polypeptides composed of either D or L amino acids (Gill, Papermaster & Mowbray, 1965; Janeway & Humphrey, 1968). In these systems both polymers are cleared from the circulation at comparable rates. However, the L polymer is rapidly degraded while the D polymer persists for long periods in the tissues. The L polymer is a good immunogen; the D polymer is a tolerogen or at best a weak immunogen. In the present system, N-DNA, which is nonimmunogenic except in disease states, is retained longest in the liver and is thus analogous to the tolerogenic D-amino acid polymers. ss-DNA and poly I: C which are better immunogens are more rapidly eliminated. Studies are in progress to determine if the relative immunogenicities of these materials can be altered by inducing changes in their metabolism.
L
329
ACKNOWLEDGMENTS The authors wish to thank Ms. Peggy Cohen for excellent technical assistance. REFERENCES ARANA R. & SELIGMANN M. (1967) Antibodies to native and denatured deoxyribonucleic acid in systemic lupus erythematosus. J. clin. Invest. 46, 1867. BURGI E. & HERSHEY A. D. (1963) Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3, 309. CHUSED T. M., STEINBERG A. D. & TALAL N. (1972) The clearance and localization of nucleic acids by New Zealand and normal mice. Clin. exp. Immunol. 12, 465. COMMERFORD S. L. (1971) Iodination of nucleic acids in vitro. Biochemistry, 10, 1993. GILL T. J., PAPERMASTER D. S. & MOWBRAY J. F. (1965) Synthetic polypeptide metabolism. I. The metabolic fate of enantiomorphic polymers. J. Immunol. 95, 794. HALLORAN M. J. & PARKER C. W. (1966) Preparation of nucleotide-protein conjugates: carbodiimides as coupling agents. J. Immunol. 96, 373. JANEWAY C. A. & HUMPHREY J. H. (1968) Synthetic antigens composed exclusively of L- or D- amino acids. II. Effect of optical configuration on the metabolism and fate of synthetic polypeptide antigens in mice. Immunology, 14, 225. LEDOUX L. (1965) Uptake of DNA by living cells. Adv. Nucleic Acid Res. 4, 231. PARKER L. M. & STEINBERG A. D. (1973) The antibody response to polyinosinic-polycytidylic acid. J. Immunol. 110, 742. PLESCIA O. J., BRAUN W. & PELCZUK N. C. (1964) Production of antibodies to denatured DNA. Proc. natl. Acad. Sci. 52, 279. STOLLAR B. D. (1973) Nucleic acid antigens. In: The Antigens (ed. by M. Sela) Vol. 1, 1. Thoburn R., Koffier D. & Kunkel H. G. (1971) Distribution of antibodies to native DNA, single-stranded DNA and double stranded RNA in mouse serums. Proc. Soc. exp. Biol. Med. 136, 711. TSUMITA T. & IWANAGA M. (1963) Fate of injected deoxyribonucleic acid in mice. Nature (Lond.), 198, 1088. UNANUE E. R. (1972) The regulatory role of macrophages in antigenic stimulation. Adv. Immunol. 15, 95.
The metabolism of nucleic acids in mice*
D. CHIA, CAROLE A. DORSCHt, L. LEVY & E. V. BARNETT Department of Medicine, Division of Rheumatology, UCLA School of Medicine, Los Angeles, California 90024
Received 18 April 1978; acceptedfor publication 2 June 1978
INTRODUCTION
Summary. The metabolism of three forms of nucleic acid, native-DNA (N-DNA), single strand DNA (ss-DNA), and polyinosinic-polycytidylic acid (poly I: C), was investigated in vivo in randomly bred Swiss-Webster mice. Clearance of these substances from the circulation and tissue localization were determined at selected time intervals following the intravenous injection of 1251-labelled compounds. N- and ss-DNA were removed from the circulation more rapidly than was poly I: C. All three materials localized principally in reticuloendothelial-rich organs, i.e. liver and spleen. N-DNA was degraded by the liver more slowly than was poly I: C or ss-DNA. At 4 h following injection, the liver contained 26%, 13%, and 10% of the injected doses, respectively. Three days after injection, 4*5% of the N-DNA persisted in the liver, as compared to only 0-6% of the poly I: C and 0-2% of the ss-DNA. The possibility that these differences in metabolism of N-DNA, poly I: C, and ss-DNA may be related to their differing immunogenic potentials in experimental systems is discussed.
The literature contains many examples of compounds, similar in chemical structure, which differ in their immunogenicity. Attempts have been made to correlate the metabolic fate and tissue localization of these compounds with their immunogenic potential (Unanue, 1972). The nucleic acids, native DNA (N-DNA), single strand DNA (ss-DNA), and polyinosinic-polycytidylic acid (poly I C) represent a similar series of compounds which, although chemically alike, differ markedly in their ability to induce antibody formation in experimental animals. Thus, despite numerous attempts, N-DNA is non-immunogenic (Stollar, 1973). ss-DNA alone is not immunogenic but, when coupled covalently (Halloran & Parker, 1966) or electrostatically (Plescia, Braun & Pelczuk, 1964) to a carrier protein and administered in Freund's complete adjuvant antibodies can be induced. Poly I: C can be immunogenic when administered in saline alone and does not require carrier or Freund's adjuvant (Parker & Steinberg, 1973). In contrast to these observations, patients with systemic lupus erythematosus (SLE) and New Zealand Black/White (NZB/W) F1 mice, who develop an autoimmune disease similar to SLE, form antibodies against all three of these materials, including N-DNA (Arana & Seligman, 1967; Thoburn, Koffler & Kunkel, 1971). It is possible that differences in the immunogenicity of nucleic acid antigens may be related to
*Supported in part by grant number GM-15757 from The United States Public Health Service. tPresent address: Connective Tissue Division, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Correspondence: Dr David Chia, UCLA Rehabilitation Center, 1000 Veteran Avenue, Los Angeles, California 90024. 00 1 9-2805/79/0200-0323 $02.00 (O 1979 Blackwell Scientific Publications
323
D. Chia et al.
324
differences in their metabolism. If this is true, alterations in these metabolic pathways in disease states, along with other suggested defects of the immune system, could contribute to the production of antinucleic acid antibodies. The present study was designed to investigate the metabolism of these three different forms of nucleic acid in the same in vivo system. MATERIALS AND METHODS Mice 6-8 week old male Swiss-Webster mice, 25-30 g, were obtained from Hilltop Laboratories (Scottdale, Penn.). Mice were fed Purina Laboratory Chow and received water ad libitum.
Nucleic acids Calf thymus DNA was obtained from Worthington Biochemicals (Freehold, New Jersey). T-7 bacteriophage DNA was a gift from Dr V. N. Schumaker (University of California, Los Angeles, Calif.). Poly I: C was obtained from Miles Laboratories (Elkhart, Indiana), and tetradeoxycytidine (dCp)4 was obtained from P-L Biochemicals, Inc. (Milwaukee, Wisconsin).
Table 1. Clearance of N and ss-DNA from the blood
Dose/total blood volume* (Mean+ S.D.)
Time (min)
N-DNA
3 7 15 30 60 240 1440
15-78+ 7-91 8-73+1-25 9-26+0-62 8-72+ 152 5-36+0-78 2-31+0-74 0-29+0 14
ss-DNA
17-23+ 8-44 9-08+1 10
13-03+2-07t 10-35+ Illt 7-35+1-16t 4 70+1-81t 0 34+0-33
*Blood volume assumed to be 10% of body weight.
tP< 0-01.
using a Beckman model L2-65B ultracentrifuge. Molecular weights were calculated from their sedimentation coefficient (Burgi & Hershey, 1963). Animal studies Mice were injected intravenously via the lateral tail vein with 2-3 pg of '25I-N-DNA or ss-DNA or 0-6 pg of 1251-poly I: C. Three to six animals were killed at 3, 7, 15, 30, 60 and 240 min after injection. Blood and various organs were counted for 1251 in a gamma scintillation counter (Nuclear Chicago).
Radiolabelled nucleic acids Nucleic acids were labelled with l25l by the method of Commerford (1971). Specific activities of calf thymus N-DNA, T-7 DNA, poly I: C and dCp4 were 0 05 ,uCi/pg, 0 02 ,pCi/,ug, 0 25 ,pCi/pug and 05 ,pCi/,ug respectively. For certain experiments '25I-N-DNA was sonicated for 10 min using a Sonifier Cell Disruptor (model 140D, Heat Systems -Ultrasonics, Inc., Plainview, New York). 1251.sS_ DNA was prepared by heating the '25I-N-DNA at 100° for 10 min, followed by rapid cooling in ice. A deoxyribonuclease (DNase) digest was prepared by incubation of 25I-N-DNA (4 pg/ml) with 20 ,ug/ml of DNase I (Worthington Biochemicals, Freehold, New Jersey) in pH 7-4 phosphate buffered saline, 0005 M magnesium chloride, for 30 min at 370. Residual DNase activity was destroyed by heating at 1000 for 10 min and then cooling in ice.
Clearance of DNA from the circulation After the intravenous administration of 1251-N-DNA or 125I-ss-DNA, radiolabel disappeared rapidly from the blood (Table 1). Rates of disappearance were comparable following N or ss-DNA. Less than 25 % of the injected dose remained after 3 min, and less than 10% after 60 min. At 15 min there was a consistently observed rise in blood radioactivity for both N and ss-DNA. This was more pronounced for ss-DNA and from 15 to 240 min blood levels remained significantly higher than those for N-DNA. By 24 h approximately 0 3 % of administered radioactivity from either material was detectable in the blood.
Determination of molecular weights Sedimentation rates for the various nucleic acid preparations were determined by ultracentrifugation on a linear sucrose density gradient of 5 to 20%.,
Tissue localization of DNA Radioactivity from both N- and ss-DNA was rapidly taken up by the liver and, to a lesser extent,
RESULTS
Metabolism of nucleic acids
325
Table 2. Uptake of N and ss-DNA by liver and spleen
% Dose/Organ (Mean+ S.D.) Liver
Spleen
Time (min) 3 7 15 30 60 240 1440
N-DNA
ss-DNA
N-DNA
ss-DNA
56-76+ 12 19 70-15+4-98 52-92+ 8-39 42-12+ 6-73 29-60+ 5-08 2602+ 3 15 7-78+1-11
59*05+6-34 54-45+ 8.18* 30-92+ 2-29* 16-12+2 10* 13-48+1-65* 10-12+0-77*
1 98+0-67 2-40+0 70 2-30+0-70 2-48+0 91 1-53+0-61 1-92+0-66 0-42+0 11
1-77+1-06 188+ 034 1-88+ 0-35 1-32+ 0.57* 1-22+0 12 0-90+ 0-27* 0.16+0.04*
0-88+0-32*
*P< 0-01.
by the spleen, as can be seen in Table 2. In the liver maximum amounts were found at 3-7 min. There was then a slow decline in hepatic radioactivity. However, this disappearance from the liver was slower for N than for ss-DNA. From 7 min on the liver contained a significantly greater fraction of the administered N-DNA than of ss-DNA. Thus after 60 min, the amount of N-DNA remaining was 2-2 times that of ss-DNA and after 24 h 8-8 times as much. At 3 days following injection, 4-5% of the dose of N-DNA remained in the liver as compared to 0-2% for ss-DNA. In the spleen the amount of N and ss-DNA remained relatively constant for the first 60 min and then fell slowly. At 240 min and 24 h levels of N-DNA were significantly higher than those for ss-DNA at P < *01. Comparable results were obtained when data were expressed as % dose/100 mg of tissue. Effect of molecular size on blood clearance and tissue localization Although the N- and ss-DNA used were derived from the same preparation, their molecular weights differed markedly, as can be seen in Table 3. The N-DNA had an average molecular weight (mol. wt.) of 2-6 x 106 daltons (15S) and the ss-DNA obtained after heat denaturation of the N-DNA preparation had a mol. wt of 1-2 x 105 daltons (6 4S). This is probably the result of the presence of single-strand nicks in the N-DNA preparation so that after heatdenaturation and subsequent strand-separation, the resultant ss-DNA pieces are of smaller size than
Table 3. Nucleic acid antigens
Type of nucleic acid
Calf thymus N-DNA Sonicated calf thymus N-DNA T-7 Bacteriophage ss-DNA Calf thymus ss-DNA DNAase digested calf thymus ss-DNA Tetradeoxycytidine Poly rI: rC
Sedimentation
Molecular
coefficient
weight
15
2-6 x 106
7.9
4-2x 105
30 5 6-2
2-0x 106 1 2x 105
2-3 7-6
1-8 x 104 1-4 x 103
would be expected. Because of the possibility that these size differences were responsible for the differences in organ retention observed for N- and ss-DNA, studies were undertaken utilizing N and ss materials of comparable size. Sonicated calf thymus N-DNA (mol. wt. 4-2 x 10s daltons) served as an example of low MW N-DNA comparable in size to CT-ss-DNA. Heat denatured T-7 bacteriophage DNA of mol. wt. 2-0 x 106 daltons was used as an example of high mol. wt. ss-DNA. Clearance studies were performed comparing high mol. wt. N- and ss-DNA, i.e., CT-NDNA and T-7 ss-DNA, and low mol. wt. N- and ss-DNA, i.e. sonicated CT-N-DNA and CT-ssDNA. In comparing N and ss-preparations of the same size, the pattern of disappearance from the blood was unchanged from that described above. Likewise, after 15 min blood levels were consistently
D. Chia et al.
326
5
- I
I(a)
II
( b)
_
4-
0 a%
,,
E
~~~~~(c)
b
0
2 5
(d)
en
03
0
4~~~~~ 20
40
A
60
/
240 0 Time (r
20
40
60
240
Figure 1. Influence of molecular weight on the levels of N-and ss-DNA in liver: (a) calf thymus N-DNA, (b) sonicated calf thymus N-DNA, (c) T-7-ss-DNA, (d) calf thymus ss-DNA. Each point represents the mean of six animals with standard deviation shown.
higher following administration of ss-DNA as compared to N-DNA. In Fig. 1 hepatic levels of radioactivity following administration of each of the four nucleic acid preparations are shown, expressed as % of administered dose/100 mg of tissue. As can be seen, hepatic levels following high MW CT-N-DNA (Fig. la) are slightly higher than levels following low mol. wt., sonicated CT-N-DNA (Fig. lb). Levels for high mol. wt., T-7, ss-DNA (Fig. Ic) and for low mol. wt. CT-ss-DNA (Fig. Id) were similar throughout the time period studied. High mol. wt. N-DNA disappeared more slowly from the liver than did high mol. wt. ss-DNA
(Fig. la and c). Low mol. wt. N-DNA and low mol. wt. ss-DNA disappeared similarly until 60 and 240 min when levels following N-DNA were 1 5 and 1 9 those for ss-DNA respectively. Thus, the preferential retention of N-DNA persists when preparations of comparable size are used. To investigate further the role of nucleic acid size, a DNase digest of ss-DNA (mol. wt. 1 8 x 104) and tetradeoxycytidine (mol. wt. 1-4 x 103) were studied. As control Na125I was injected in a similar manner. Results are shown in Table 4. Liver and spleen levels of DNase digest and (dCp)4 were similar and much less than seen with N- or ss-DNA (Table 2). Organ
Table 4. Uptake of DNAase digested ss-DNA, tetradeoxycytidine and Na'251 by liver and spleen
% Dose/organ (Mean .S.D.) Spleen
Liver Time (min) 3 7 15 30 60 240
DNAase digested ss-DNA
TetradeoxyCytidine
8 94+ 1-45 7-11+ 1-66
7-92+0-77 7 70+ 1-48
6-70+1-66 4-08+0-71 2 35+ 0 23
6-08+0-80 545+1-80 2-28+0-80 085+0-30
1l20+009
DNAase
Na125I
digested
TetradeoxyCytidine
Na1251
ss-DNA
*
1-68+0-33 156+0 13 173+0-09
1l65+0-02 1*67+ 0-03
Not done.
N.D.*
0 59+0 20
0-64+0-18
0-45+0-15 0-60+0-22 0-38+ 0-09 0-43+0-20
0-48+012 0-43+0-10 0-40+0-06 0-45+0-12 0 50+ 0 15 0-33+0-15
0-12+0*00 0-18+0-03 0-14+0-02 0-16+0-05 0-15+ 0-02 N.D.*
Metabolism of nucleic acids Table 5. The rate of degradation of various nucleic acids in the liver
Nucleic acids
Rate (log % dose/min)*
T-7-ss-DNA ss-DNA N-DNA Sonicated N-DNA
-0-0182+ 001 14 -0-0215+ 00075 -0-0066+0 0043 -0-0058+ 00075
*Standard deviation of least square linear regression calculated with a confidence limit of 95%
levels of Nal25I were quite low and remained constant over a 60 min period of observation. Catabolism of DNA The catabolism of nucleic acids can also be described as the rate of disappearance of radioactivity from the liver. This was calculated for both large and small ss-DNA and N-DNA, as shown in Table 5. Both ss-DNA preparations had a faster rate of disappearance than the N-DNA preparations. Radioactivity was found in the gastric washings after the injection of nucleic acids. This radioactivity was shown to have the same mobility as free iodide on a Sephades G-25 column. Its appearance can be used as a measure of the degradation of nucleic acids. In Fig. 2, the appearance of radiolabel in the stomach after intravenous injection of various material is shown. Free iodide is found early and
persists during the observation period. Radiolabel derived from a DNase digest of DNA appears slightly faster than that derived from ss-DNA. In both of these cases maximum amount of radiolabel is found at 60 min and has decreased by 4 h. However, in the case of N-DNA, radiolabel appears more slowly and in smaller amounts in the gastric washings and is continuing to rise at 4 h. An attempt was made to determine if catabolic pathways for DNA metabolism could be saturated or, alternatively, induced to become more efficient. Groups of six mice were injected intravenously with 100 ug of N-DNA or ss-DNA daily for 5 days. On the fifth day radiolabelled homologous DNA was injected and blood and organ levels determine. No differences were observed between untreated animals and those pretreated with DNA. Metabolism of poly I: C The clearance of a third form of nucleic acid, the synthetic double-stranded RNA, poly I: C, was compared to that of N-DNA. The preparations 100 _
80 E .5 _ 60
30
327
0
0
40
0~
20 ei
0 Time (min)
Figure 2. Level of radiolabel in gastric washings for (A) calf thymus N-DNA, (0) calf thymus ss-DNA, (C1) DNAase treated ss-DNA, (0) sodium iodide. Mean for six animals.
20
40 Time (min)
60
240
Figure 3. Clearance of (a) N-DNA and (-) poly I C from the blood (assuming blood volume as 10% of body weight). The standard deviations for six animals are shown. They are significantly different where 001 >P> 0001.
D. Chia et al.
328
Table 6. Level of radiolabel in gastric washings for sonicated N-DNA and poly I: C.
Time (min)
Sonicated N-DNA
0-56±0 42 0-97+0-51
3 7 15 30 60 240
2-43±0-20 7-09+0-51 13-77±1 27 18-27+ 5-01
Poly I: C 057±0 20 0-53±0-19 1.14+0-63* 3.04±1-17* 7.30±2.28* 13-86+ 5-88
*P< 0.01.
compared were of comparable size (sonicated N-DNA 7-9S and poly I: C 7 6S). Blood clearance is shown in Fig. 3. Poly I: C disappeared from the circulation at a much slower rate than did N-DNA. Consistent with this observation are the results for tissue uptake (Fig. 4). As seen in Fig. 4a, hepatic uptake of N-DNA was maximal at 3 min, while peak uptake of poly I: C was not reached until 15 min. Both materials were (a) 4'0
4I
20 _
0
._-
E 0 0
0
0 0
0n 0
Time (min) Figure 4. Uptake of sonicated N-DNA (0) and poly I: C (@) by (a) liver, and (b) spleen. The standard deviations for 6 animals are shown. Liver is significantly different before 15 min where P< 0'001; spleen is significantly different at all points, 0'005 >P>0 001, except at 30 min.
then lost at comparable rates through 60 min. However, by 6 days following injection 2-9% of the N-DNA but only 0-6% of the poly I: C persisted in the liver. In the spleen there is a higher uptake and retention of N-DNA when compared to poly I: C (Fig. 4b). Levels of radiolabel in gastric washings following intravenous administration of sonicated N-DNA and poly I: C are shown in Table 6. Values for both materials increased slowly through 4 h but were lower for poly I: C than for N-DNA. As noted above, both of these were less than for ss-DNA (Fig. 2). These observations are consistent with a delay in catabolism resulting from a delay in hepatic intake of poly I: C.
DISCUSSION Many studies have documented the rapid clearance of N-DNA from the blood after intravenous administration (Tsumita & Iwanaga, 1963) and its localization predominantly in liver, spleen, and, to a lesser extent, in intestine, gonadal tissue, and tumours (Ledoux, 1965; Chused, Steinberg & Talal, 1972). From the present study, it appears that ss-DNA and the synthetic RNA poly I: C are handled similarly in vivo. However, the present study also clearly demonstrates significant differences in the clearance and metabolism of N-DNA, ss-DNA, and poly I: C. Poly I: C is cleared from the blood more slowly than is either N-DNA or ss-DNA and reaches peak levels in liver and spleen more slowly. Thereafter it is eliminated from the liver at a rate comparable to N-DNA of similar size, but more slowly than is ss-DNA. N-DNA and ss-DNA are cleared from the blood at similar rates. However, peak hepatic levels are reached more slowly and are higher for N-DNA than for ss-DNA, and N-DNA disappears from liver and spleen more slowly than does ss-DNA. After 24 h 7-8% of the administered dose of N-DNA persists in the liver, as compared to only 0 9%/ of the ss-DNA (Table 2). From 24 h on, hepatic levels of N-DNA are consistently higher than levels of poly I: C which are in turn higher than ss-DNA levels. It is not wholly unexpected that a doublestranded helix would be more difficult for tissue nucleases to degrade, since degradation of such a polymer would require adjacent enzymatic nicks to occur in both strands.
Metabolism of nucleic acids It has previously been reported that hepatic levels of poly I: C are influenced by the molecular size of the administered poly I: C (Chused et al., 1972). In the present study, a similar observation was made for N-DNA. However, the molecular weight of ss-DNA did not seem important in determining tissue levels of this material. The organism is repeatedly presented with the necessity to clear or degrade nucleic acids, either exogenous, e.g. during viral infection, or endogenous, e.g. after tissue trauma. It appears from these studies as if this can be accomplished more effectively for ss-DNA than for N-DNA. The subcellular localization or the form of the persisting DNA is not known. While this may represent macromolecular DNA, the possibility that breakdown products, re-utilized by the host, are being measured cannot be totally excluded. It is intriguing to speculate that the ability of N-DNA to persist in vivo could be related to the observation that it constitutes a poorer immunogen than does ss-DNA. In other antigenic systems, relative differences in the immunogenicity of chemically similar materials have been attributed to differences in their metabolism. One such example is that of the synthetic polypeptides composed of either D or L amino acids (Gill, Papermaster & Mowbray, 1965; Janeway & Humphrey, 1968). In these systems both polymers are cleared from the circulation at comparable rates. However, the L polymer is rapidly degraded while the D polymer persists for long periods in the tissues. The L polymer is a good immunogen; the D polymer is a tolerogen or at best a weak immunogen. In the present system, N-DNA, which is nonimmunogenic except in disease states, is retained longest in the liver and is thus analogous to the tolerogenic D-amino acid polymers. ss-DNA and poly I: C which are better immunogens are more rapidly eliminated. Studies are in progress to determine if the relative immunogenicities of these materials can be altered by inducing changes in their metabolism.
L
329
ACKNOWLEDGMENTS The authors wish to thank Ms. Peggy Cohen for excellent technical assistance. REFERENCES ARANA R. & SELIGMANN M. (1967) Antibodies to native and denatured deoxyribonucleic acid in systemic lupus erythematosus. J. clin. Invest. 46, 1867. BURGI E. & HERSHEY A. D. (1963) Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3, 309. CHUSED T. M., STEINBERG A. D. & TALAL N. (1972) The clearance and localization of nucleic acids by New Zealand and normal mice. Clin. exp. Immunol. 12, 465. COMMERFORD S. L. (1971) Iodination of nucleic acids in vitro. Biochemistry, 10, 1993. GILL T. J., PAPERMASTER D. S. & MOWBRAY J. F. (1965) Synthetic polypeptide metabolism. I. The metabolic fate of enantiomorphic polymers. J. Immunol. 95, 794. HALLORAN M. J. & PARKER C. W. (1966) Preparation of nucleotide-protein conjugates: carbodiimides as coupling agents. J. Immunol. 96, 373. JANEWAY C. A. & HUMPHREY J. H. (1968) Synthetic antigens composed exclusively of L- or D- amino acids. II. Effect of optical configuration on the metabolism and fate of synthetic polypeptide antigens in mice. Immunology, 14, 225. LEDOUX L. (1965) Uptake of DNA by living cells. Adv. Nucleic Acid Res. 4, 231. PARKER L. M. & STEINBERG A. D. (1973) The antibody response to polyinosinic-polycytidylic acid. J. Immunol. 110, 742. PLESCIA O. J., BRAUN W. & PELCZUK N. C. (1964) Production of antibodies to denatured DNA. Proc. natl. Acad. Sci. 52, 279. STOLLAR B. D. (1973) Nucleic acid antigens. In: The Antigens (ed. by M. Sela) Vol. 1, 1. Thoburn R., Koffier D. & Kunkel H. G. (1971) Distribution of antibodies to native DNA, single-stranded DNA and double stranded RNA in mouse serums. Proc. Soc. exp. Biol. Med. 136, 711. TSUMITA T. & IWANAGA M. (1963) Fate of injected deoxyribonucleic acid in mice. Nature (Lond.), 198, 1088. UNANUE E. R. (1972) The regulatory role of macrophages in antigenic stimulation. Adv. Immunol. 15, 95.
