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Clin. exp. Immunol. (1986) 66, 77-87 A sensitive radioimmunoassay for alpha-interferon: circulating a-interferon-like substance in the plasma of healthy individuals and rheumatoid arthritis patients S. SHIOZAWA, K. CHIHARA, K. SHIOZAWA, T. FUJITA, H. IKEGAMI, S. KOYAMA & M. KURIMOTO Department of Medicine, Third Division, Kobe University School of Medicine, Kobe, and Hayashibara Biochemical Laboratories, Okayama, Japan (Acceptedfor publication 21 May 1986) SUMMARY A radioimmunoassay for circulating a-interferon (IFNa) has been developed using lymphoblastoid IFNa. The assay was specific for IFNa, and did not cross-react with IFN#, IFNy, or ACTH, while it was specifically inhibited by recombinant IFNa. The radioimmunoassay (y) correlated linearly with the virus inhibition assay (x), with a regression line of y on x of y=0 659x+245 (u) (P<0 01). a-Interferon-like substance (IFNa-LS) was extracted and concentrated from plasma either by silicic acid or by antibody immunoadsorption. Serial dilutions of plasma and extracted samples of plasma showed dilution curves identical to those of standard IFNa, suggesting the presence of endogeneous IFNa in human plasma. The circulating IFNa-LS of healthy individuals aged 20 to 45 was 0 207 + 0 055 ng/ml in males (n = 48) and 0 172 + 0-076 ng/ml in females (n = 34). Gel filtration studies on a Sephadex G-75 column suggested that circulating IFNat-LS exists in a fragmented form, inactive in virus inhibition assays, in the plasma of healthy individuals. The finding may help explain why biological IFNa is often undetectable in the plasma of healthy donors, yet is detectable by radioimmunoassay. Circulating IFNa-LS in the plasma of healthy individuals declined gradually with age. IFNot-LS was significantly decreased in the plasma of rheumatoid arthritis patients, when compared with the value found in the age and sex-matched healthy controls and in osteoarthritis patients (P < 0 0001). The decrease was related neither to treatment nor to disease activity. IFNcx-LS was, however, not decreased in the plasma of vasculitis patients. Decreased IFNa-LS in rheumatoid arthritis may be important from pathogenetic and therapeutic standpoints. Keywords a-interferon radioimmunoassay rheumatoid arthritis ageing INTRODUCTION IFNa is thought to play an important role in the immune response of the host (Svet-Moldavsky & Chernyakhovskaya, 1967; Huang et al., 1971; Johnson & Baron, 1976; Schultz, Papamatheakis & Chirigos, 1977; Skurkovich, Skorikova & Eremkina, 1978; Trinchieri & Santoli, 1978; Schultz, Chirigos, & Heine, 1978; Herberman et al., 1980; Leanderson et al., 1982; Fradelizi & Gresser, 1982) especially in the maintenance ofnatural killer cell activities (Svet-Moldavsky & Chernyakhovskaya, 1967; Skurkovich et al., 1978; Trinchieri & Santoli, 1978; Minato et al., 1980; Ortaldo et al., 1980; Correspondence: S. Shiozawa, Department of Medicine, Third Division, Kobe University School of Medicine, 7-5 Kusunokicho, Chuoku, Kobe 650, Japan. 77 78 S. Shiozawa et al. Herberman et al., 1980; Targan & Dorey, 1980; Perussia & Trinchieri, 1981). However, in spite of circumstantial evidence (Gresser et al., 1983; Buffet-Janvresse & Hovanessian, 1984), there has been no definitive proof in support of the existence of endogenous IFNa. Circulating IFNa is often undetected in the plasma of healthy individuals when measured by virus inhibition assays (Hooks et al., 1979; 1982; Preble et al., 1982; Panem et al., 1982; Ytterberg & Schnitzer, 1982). Rheumatoid arthritis is a chronic inflammation of the synovium, and extensive efforts to find pathogenetic organisms in situ have so far been unrewarding. If rheumatoid arthritis is caused by some unknown pathogenetic organism coming into the synovium through the systemic circulation, as suggested by Zvaifler (1973), the ability of the host to handle invading organisms must have a significant contribution to the sequelae of chronic inflammation. In this regard, IFNa may play an important role, since it is necessary for continuous activation of natural killer cells, the cells important for the host's front-line defence against invading organisms (Targan & Dorey, 1980; Perussia & Trinchieri, 1981). In fact, aberrant natural killer cell activities and changes in circulating IFNL% have been reported in rheumatoid arthritis (Goto et al., 1981; Neighbour & Grayzel, 1981; Dobloug et al., 1982; Faure et al., 1983; Combe et al., 1984; Goto & Zvaifler, 1985). However, with respect to the circulating level of IFNa, the results obtained using virus inhibition assays have been inconsistent so far (Hooks et al., 1979; Degr6, Mellbye & Clarke-Jenssen, 1983). In this study, we have measured circulating IFNa using a sensitive radioimmunoassay, based on a polyclonal antibody made to lymphoblastoid IFNa as immunogen, which was specific for IFN2. Circulating immunoreactive IFNx and ax-interferon-like substance (IFNa-LS), was extracted and concentrated from human plasma, and the circulating level of IFNa-LS was determined in the plasma of healthy individuals and rheumatoid arthritis patients. Gel filtration studies of IFNa-LS suggested the existence of a fragmented form of IFNa in human plasma. MATERIALS AND METHODS Production ofIFNoe. Human leukaemic lymphoblastoid BALL- I cells (Miyoshi et al., 1977) were grown up by subcutaneous transplantation into a new-born hamster which was pretreated with anti-thymocyte antisera (Tanimoto, 1982). Tumour cells (5 x 106/ml) were then primed by 100 i.u./ ml of IFN for 1 h and cultured with 500 HA/ml of Sendai virus in RPMI 1640 medium at 370C for 24 h in a spinner vessel. The supernatant was harvested, acidified to pH 2 0 using 6 M HCl, and kept at 40C for at least 24 h to inactivate viruses. Purification of IFNa. The culture supernatant, adjusted to pH 4-0 using 6 M NaOH, was applied on a SP-Sephadex C-25 column (Pharmacia Fine Chemicals Inc., Uppsala, Sweden). The eluate obtained by 0-1 M phosphate buffered saline, pH 8 0, was applied on a NK-2 Sepharose affinity column for IFNa (Celltech Laboratories, Berkshire, England) (Allen et al., 1982). A single protein peak was obtained by eluting with 0-1 M citrate buffer. The peak had specific activity of 5-4 x 108 i.U./ mg and 65% recovery. Further purification of the peak through Sephadex G-100 column (Pharmacia Fine Chemicals) gave IFNa with specific activity of 9 5 x 107 i.u./mg and 53% recovery. The final product showed a single band at mol. wt 17,000 + 1,200 daltons on SDS-polyacrylamide gel electrophoresis with isoelectric point at pH 5-4. Its anti-viral activity was neutralized by anti- IFNa (Mogensen, Pyhala & Cantell, 1975) but not by anti-IFNI sera (Mochida Pharmaceutical Company, Tokyo, Japan) (Sidwell & Huffaman, 1971). The biological activities of purified IFNa, such as species-specificity, inhibition of cellular multiplication, or activation of natural killer cells, were similar to those of leukocyte IFNoc from human peripheral blood (Green Cross Company, Osaka, Japan) (Matsuo, Hayashi & Kishida, 1974; Imanishi et al., 1980). Virus inhibition assay. The antiviral activity of IFNac was assayed on FL cells as challenged by Sindbis virus, and the result standardized according to the IFNa preparation MRC69/19B. The result correlated well with an assay employing vesicular stomatitis virus. FL cells (5 x 105/ml) were plated in a flat-bottomed multiwell microtitre plate (No. 163320, Nunc Ltd., Roskilde, Denmark) and challenged by aliquots of sample or standard reference IFNa. After incubation for 24 h at 37°C, Sindbis virus, optimally diluted to produce 50% cytotoxicity, was added to the reaction mixture, and the culture continued for additional 24 h. Neutral red dye was added 45 min before harvesting, Radioimmunoassay of circulating a-interferon 79 and the amount of dye taken up in the cell was measured under 540 nm photoabsorbance after disrupting cells with 0 07 M glycine HCI, pH 3 0, and 30% ethanol (Pidot, 1971). In our hands, 25 i.u. corresponded to 0 1 ng of IFNa protein. Experimental inducation of IFNci in the peripheral blood. To induce high titres of IFN(i in the human peripheral blood, 5 ml of heparinized blood were incubated with 500 HA/ml of Sendai virus at 370C for various lengths of time less than 24 h, and the supernatant plasma used for assay. Radioimmunoassay of IFNcx. Antibody to IFNa was raised in a rabbit by repeated subcutaneous injection of IFNa with Freund's complete adjuvant, and was used for radioimmunoassay at a dilution of 1:40,000. The purified IFNa was radio-iodinated by lactoperoxidase; the reaction mixture contained 1-7 pg IFNcx, I mCi Na 1251 (New England Nuclear, Inc., Massachusetts, USA), 0 5 ug lactoperoxidase (Sigma Chemical Company, St Louis, USA), and 0 4 pg hydrogen peroxide in 50 lp of 0 4 M sodium acetate buffer, pH 5 6. '25I-IFNa had a specific activity of approximately 0-3 mCi/pg. This was used for radioimmunoassay immediately after repurification on a Sephadex G-75 column (0 7 x 40 cm) (Pharmacia Fine Chemicals) using 0-07 M barbital buffer as eluent. The diluent buffer for radioimmunoassay was 0-01 M phosphate buffer, pH 7 4, containing 0 16 M NaCl, 0.25% bovine serum albumin, and 10% Triton X- 100. The assay was carried out in a test tube by adding 0 1 ml sample or standard IFNa, 0 1 ml anti-IFNax antisera, and 0 3 ml of diluent buffer. To measure plasma samples, 0 1 ml of IFNax-free plasma was added in tubes containing serially diluted standard IFNa. Human plasma was used after pretreatment with 20% (w/v) Norit A charcoal power to absorb endogenous IFNa (IFNa-free plasma), since human plasma, but not sera from other species of animals such as fetal bovine serum, significantly inhibited the binding of '25I-IFNa to antibody. Separation of bound and free IFNa was carried out by adding goat anti-rabbit gammaglobulin sera. The radioactivity of precipitates was counted using autogamma counter. The results were expressed as bound per total radioactivities (B/T). Each samples were assayed in duplicate. The minimal detectable quantity of IFNa, as defined by the amount causing 10% fall from the total replaceable radioactivity (Bo), was approximately 6 pg/tube (0 06 ng/ml). Fetal bovine serum was purchased from Microbiological Associates, Maryland, USA. For specificity control, recombinant IFNaX (Ro22-8181, Roche Pharmaceuticals, New Jersey, USA), IFNJ (Kyowa Hakko Kogyo Co. Ltd, Tokyo, Japan), IFNy (Interferon Sciences, Inc., New Jersey, USA), and ACTH were used. Extraction of IFNa-LSfrom plasma. Silicic acid, 01 g, was added to 4 ml of plasma, and reacted at room temperature for 1 h with stirring. The precipitate was washed with chilled distilled water and extracted with 3 ml of 0 02 M HC1-80% acetone, and the supernatant was evaporated. After reconstitution with the assay buffer and adjusting pH to 8 0, this was used for assay or filtered through Sephadex G-75. To extract IFNat-LS by antibody immunoadsorption, gammaglobulin fraction of anti-IFNat antisera was precipitated serially by 50% and 33% ammonium sulphate solution. Affi-Gel 10 (Bio-Rad Laboratories, California, USA) was washed once with excess ice- cold isopropanol followed by three washes with ice-cold distilled water. The gel slurry was transferred to a plastic tube and briefly sedimented. Then approximately 2-5 ml bed volume of Affi- Gel 10 was reacted with 50 mg of gammaglobulin fraction of anti-IFNaX antibody in 3-8 ml of 0 1 M 3-(N-morpholino) propane sulphonic acid (MOPS) for 1 h at room temperature then at 4°C overnight. The gel was extensively washed with 0 1 M MOPS, and then reacted with plasma sample. The reaction was carried out at 4°C for 4 h or 4°C overnight, in a polystyrene tube with continuous rotation. The gel was washed with excess 0 01 M phosphate buffer, pH 7-4, containing 0 16 M NaCl and 0 1% Triton X-100, centrifuged, and reacted with aliquots of 0 2 M acetic acid, pH 2 6, containing 016 M NaCl and 01% Triton X-100 to extract IFNa. The supernatant was then harvested and lyophilized. Gel chromatography. Plasma samples, extracted either by silicic acid or antibody immunoad- sorption, were dissolved in phosphate buffer, neutralized, and immediately applied on a Sephadex G-75 column (0 9 x 44 cm) using 0-4 M ammonium acetate buffer, pH 4-0, as eluent. Elution was carried out at 10 ml/h at room temperature, and each 1 ml fraction was collected using a peristaltic pump system P- I (Pharmacia Fine Chemicals) and lyophilized. After reconstitution with the assay buffer, each fraction was assayed for immunoreactive IFNa. Void volume (1VO) and bed volume (V,) of the column were determined by blue dextran and saturated NaCl, respectively. Protease inhibitors. To overcome proteolytic digestion of IFNa in plasma, various protease 80 S. Shiozawa et al. inhibitors were used; the mixture of protease inhibitors used in this study contained 1 Yg/ml pepstatin A (No. 4265, Sigma), 6 pg/ml leupeptin (Protein Research Foundation, Osaka, Japan), 20 pg/ml N-ethylmaleimide (Wako Chemicals), 200 pg/ml bacitracin (Wako), 5 mM EDTA (Wako), 20 pg/ml phenylmethylsulphonylfluoride (No. 7626, Sigma), and 2000 k.i.u./ml trazirol at final concentration. The leupeptin solution was used at 250 pg/ml. To extract IFNa-LS from plasma, blood was drawn with a syringe containing the mixture of protease inhibitors and immediately centrifuged, and the supernatant plasma was stored at - 80'C until assayed. Plasma and patient profile. Human blood sample was drawn, heparinized and immediately centrifuged, and the plasma was stored at - 20'C or 80'C until assayed. Healthy donors without apparent clinical signs of disease in the present and past were used as healthy individual. The patient with systemic lupus erythematosus fulfilled the revised diagnostic criteria ofAmerican Rheumatism Association (Tan et al., 1982). Peripheral blood was obtained from 40 patients with rheumatoid arthritis (35 to 75 years old; mean 50 1) including two males, and seven patients with osteoarthritis (48 to 64 years old; mean 54). Rheumatoid arthritis patients fulfilled the diagnostic criteria of American Rheumatism Association (Ropes et al., 1958). Rheumatoid patients were considered active if the patient exhibited all of the following: (1) arthritis with pain and/or swelling in more than four joints by physician's assessment, (2) increased tendency to fatigue and more than 1 h of morning stiffness, (3) increased erythrocyte sedimentation rate exceeding 40 mm for the first hour and/or increased C-reactive protein. Patients were considered to be inactive by applying the criteria for clinical remission (Pinals et al., 1982). Patients with rheumatoid arthritis were either without treatment or had 25 mg gold thiomalate injection biweekly, plus a daily non-steroidal anti-inflammatory drug equivalent to 25-50 mg of indomethacin (shown in Fig. 6). Plasma samples of 49 rheumatoid patients (mean 45 4 years old) at A F 0 -10 _ Io . 50 P 8E -30I 0 100 1000 10000 0\ /39- interferon (iu/ml) B a30 _ 0 X0 I o -30 10~ ~ ~ N* 1 .00k < 100 1000 10000 y- inteferon ( u/ml) v 'i 0.1 0 4 2 4 ng/ml I I I I 25 100 250 1000 a-interferon (iu/ml) Fig. 1. Standard curve of a-interferon radioimmunoassay. The vertical axis gives the percentage o1 precipitated versus total 125I-a-interferon (%B/T). The horizontal axis gives the amount of serially diluted standard a- interferon. Human leukocyte BALL-1 a-interferon plasma added (0); BALL-1 a-interferon without plasma (0); recombinant a-interferon (0). Inhibition curves with fl-interferon or y-interferon are shown in panel A and B, respectively. Human plasma, added to standard dilution tubes, is preabsorbed with 20% (w/v) charcoal powder to absorb endogenous a-interferon. a-Interferon of recombinant DNA origin, Ro 22-8181, shows a similar displacement curve to BALL- 1 cell a-interferon. 0-1 ng of a-interferon protein corresponds to 25 iu of virus inhibition activity. Radioimmunoassay of circulating a-interferon 8i the North Hyogo Medical and Orthopedic Center, drawn with EDTA, were also examined in this study. In this group of patients, approximately 80% were receiving 25 mg of gold thiomalate injection twice weekly, 30% had 125 mg D-penicillamine daily. Almost all received non-steroidal anti-inflammatory drugs, equivalent to 25-50 mg ofindomethacin daily. Rheumatoid vasculitis was diagnosed on a clinical and/or histological basis; these included the patients with cutaneous vasculitis and/or interstitial pneumonitis (Cupps & Fauci, 1981). Patients with osteoarthritis receiving non-steroidal anti-inflammatory drugs equivalent to 50 mg of indomethacin were also examined. Statistics. For calculating mean + s.d., the value of IFNa-LS less than 10% fall of sensitivity was estimated to be 0-05 ng/ml, based on the finding that, when plasma IFNx-LS of rheumatoid arthritis patient was concentrated and measured, most of the plasma level were in approximately 0 05 ng/ml (not shown). Statistical comparison was carried out using Student's t-test modified for a small sample (Hoel, 1966). RESULTS Specificity of radioimmunoassay. The standard curve of IFNa radioimmunoassay in buffer and also in human plasma is shown in Fig. 1. Human plasma, but not sera from other species of animals such as fetal bovine serum, significantly inhibited the binding of '251-IFNa to antibody, when added without pretreatment with charcoal, suggesting that endogenous IFNa may be present in human plasma. Charcoal-treated plasma however did not interfere with standard dilution curve (Fig. 1). The specificity of the radioimmunoassay was confirmed because (a) the assay did not cross-react with IFNf, IFNy, or with up to 0 34 pg of ACTH; and (b) recombinant IFN7x Ro22-8 181 inhibited the binding of '251-IFNa to a comparable level as BALL-l cell IFNcx (Fig. 1). Bioassay and characterization of IFNa-LS. Linear correlation existed between the radioimmu- noassay (y) and virus inhibition assay (x), with a regression line of y on x as y = 0 659x+ 245 (u) (P < 0-01) (Fig. 2). Intra- and inter-assay coefficients of variation of radioimmunoassay were 0-2 1% and 4-12%, respectively. The minimal detectable quantity of IFNa was approximately 0-06 ng/ml (6 pg/tube). IFNa-LS was reproducibly measured in plasma after two freeze-thaw cycles. Serial dilutions of plasma of representative individuals well coincided with the standard curve (not E . 20 5000 Xo / ~~~~~ 4000 0 0 qo / _ ~~~~~3000_ 0~s10 E~~~~~~~~~~~~ 2000 E a 500 cr0 0 5000 10000 Bioassay of a-interferon (iu/ml ) Fig. 2. Correlation between radioimmunoassay and virus inhibition assay. Heparinized whole blood (5 ml) is incubated with 500 HA/ml ofSendai virus at 37°C for a variable time less than 24 h, and the supernatant plasma is assayed simultaneously by radioimmunoassay and virus inhibition assay. The vertical axis (y) gives protein determination by radioimmunoassay (ng/ml), which is correspondingly translated into equivalent amounts of virus inhibition activity (i.u./ml), based on the correspondence of 25IU to 0 1 ng of a-interferon protein. The horizontal axis (x) gives the value of virus inhibition assay (i.u./ml). 82 S. Shiozawa et al. shown). The IFNa-LS was extracted from plasma by silicic acid, with approximately 20% yield. And the serial dilutions of the extracted sample of plasma also exactly coincided with the standard curve. Circulating IFNa-LS was also extractable by anti-IFNa antibody immunoadsorption, with approximately 90% yield. This type of extraction experiments were reproducibly repeated many times for gel filtration analysis. Gelfiltration of IFNc. To further characterize the circulating IFNa-LS, plasma from healthy donors was extracted by antibody immunoadsorption and filtered through a Sephadex G-75 column, and each fraction assayed for immunoreactive IFNa. Unexpectedly IFNa-LS eluted in a position of distinctively smaller molecular size, corresponding to fraction 16 in Fig. 3, when compared with that of standard IFNox (not shown). To rule out the possibility of artefactual degradation of IFNa during the extraction procedure, IFNa of standard molecular size was added to plasma and immediately extracted using anti-IFNa antibody-coupled Affi-GellO, with or without protease inhibitors (Fig. 3). Extraction was carried out at 40C overnight. Figure 3a shows the elution profile of IFNa filtered through a Sephadex G-75 column. While IFNa without protease inhibitors eluted in fractions 15-17, IFNa treated with the mixture of protease inhibitors (See Materials and Methods) eluted into the authentic peak of IFNa at fractions 9-11. Figure 3b shows an elution profile of IFNa treated likewise by adding 250 yg/ml of leupeptin. The addition of leupeptin to the reaction mixture effectively prevented the shift of immunoreactive peak of IFNa, suggesting that fragmentation of IFNa occured in plasma during the process of affinity extraction. Gel filtration of circulating IFNot-LS. Based on the above findings, plasma from health individuals was re-examined in the presence of a fully inhibitory dose of protease inhibitors to avoid artefactual degradation during extraction procedure (Fig. 3c). Plasma was drawn with a syringe containing the mixture of protease inhibitors and immediately processed for affinity extraction by adding protease inhibitors in an identical manner to Fig. 3a. Figure 3c shows a gel filtration profile of plasma oftwo representative healthy donors. It can be seen that the majority of circulating IFNa- LS eluted in the second peak at fractions 15-17, while a minute peak remained in fractions 9- 11 (Fig. 3c), suggesting that plasma of healthy individuals contained a predominantly fragmented form of IFNo-LS. In a preliminary experiment, plasma of an active lupus patient was filtered through Sephadex G-75 and assayed for immunoreactive IFNa. Even though plasma was extracted in the absence of protease inhibitors, a substantial part of the immunoreactive IFNa remained in the authentic peak at fractions 9-11 (Fig. 3d), suggesting that the plasma of an active lupus patient contained a certain amount of intact molecules of IFNa. This may explain why biologically detectable IFNa was present in the lupus patient's plasma (Hooks et al., 1979; 1982; Preble et al., 1982; Panem et al., 1982; Ytterberg & Schnitzer, 1982). We were unable to examine the plasma of the lupus patient in the presence of protease inhibitor. Quantification of IFNa-LS in the plasma of healthy individuals. Circulating IFNa-LS in the plasma of healthy individuals at the age 20-45 was 0207+00055 ng/ml in 48 males and 0-172+0 076 ng/ml in 34 females (Fig. 4). There were minor monthly variations in the level of circulation IFNa-LS, suggesting that there may be some seasonal variation of circulation IFNa-LS since all the assays were standardized using an internal standard; this preliminary observation requires confirmation. IFNa was not detectable, however, in the same sample of plasma when assayed by virus inhibition assays (not shown). In our hands, the minimal detectable quantity of IFNa in virus inhibition assays was 2 i.u./ml, which corresponds to 0-008 ng/ml of IFNa protein. Thus, when IFNa-LS was intact and biologically active, a circulating IFNae level of approximately 0-2 ng/ml should be detectable under virus inhibition assays. It was found that circulating IFNac-LS gradually declined with age (Fig. 5). Quantification of IFNa-LS in rheumatoid arthritis. Circulating IFNa-LS was significantly decreased in the plasma of rheumatoid arthritis patients when compared with the value found in the age and sex-matched healthy control (P< 00001) (Fig. 6). It was decreased irrespective of the disease activity. Circulating IFNcx-LS was also decreased in the active patients not treated for rheumatoid arthritis (P< 0001), whereas it was not decreased in the rheumatoid patients with vasculitis. Since rheumatoid patients commonly received high dose of non-steroidal anti- inflammatory drugs, it was necessary to examine the contribution of the drugs to circulating IFNa- LS. Circulating IFNx-LS in osteoarthritis patients receiving equivalent dose of the non-steroidal Radioimmunoassay of circulating tx-interferon 83 E c C3 I-C a) w C al 5 10 15 20 Fraction number Fig. 3. Sephadex G-75 column chromatography (0 9 x 44 cm). Elution is carried out using 0 1 M ammonium acetate buffer, pH 4 0, at room temperature at a rate 10 ml/h. Each 1 ml is fractionated, lyophilized, and reconstituted with the assay buffer. (a) Standard a-interferon is added to plasma and immediately processed for extraction using antibody immunoadsorption. Extraction is carried out at 40C overnight with rotation. The mixture of protease inhibitors (see Materials and Methods) is present (a A) or absent (---- -A). (b) Standard a-interferon is added to plasma and extracted as in A, in the presence (0 O) or absence (-----0) of 250 ,ug/ml of leupeptin. (c) Plasma is drawn and affinity extracted from two healthy individuals in the presence of the mixture of protease inhibitors in a manner identical to A. (d) Plasma of an active patient with systemic lupus erythematosus is drawn and extracted without protease inhibitors (v v). The elution peak of standard a-interferon is shown ( ....... ). Each point represents the mean + s.e.m. of determinations performed in duplicate. anti-inflammatory drugs was not decreased when compared with the age and sex-matched healthy control (Fig. 6.). Plasma ofanother 49 rheumatoid arthritis patients from the North Hyogo Medical & Orthpaedic Center, drawn with EDTA, showed similar low levels of circulating IFNa-LS 0-069 + 0049 ng/ml, a significant decrease when compared with that of age-matched healthy control, 0 120 + 0050 ng/ml (P < 0005); circulating IFNa-LS was below the sensitivity limit of assay in 39 out of 49 patients examined. DISCUSSION We have detected circulating IFNa-LS in the plasma of healthy individuals using a sensitive radioimmunoassay. Since the radioimmunoassay is a specific for a-type of IFN, recognizing 84 S. Shiozawa et al. 05 r (a) ( b) Mean plasma level (age: 20-45) 0-4 H E 0 C C 0.3 [- a C 02 2- -AW Atsees -0 * -- 0*l F- I Mar Apr May Jun Male Female Fig. 4. (a) Monthly variation of circulating a-interferon-like substance in plasma of healthy individuals. Data from the same donor is connected with a line. (b) Circulating a-interferon-like substance in the plasma of healthy individuals aged between 20 and 45. Each point represents the mean of determinations performed in duplicate. 0 a 0-3 0 a a) E ri a 0 l Li- 0 0 0 1*4 A E C3 0 I. a a) 0.21- @4 C C a I- 0 0 a oI 0 0 @0 cl 0-1 40 00 00 000 I 0 o 20 30 50 60-85 Age (years) Fig. 5. Age distribution of circulating a-interferon-like substance in plasma of healthy donors. Each point represents the mean of determinations performed in duplicates. Values in each age group are given as the mean + s.e.m. Statistical significance of difference between each age group of all combinations is as follows; in males, age 30-40; P< 00001, 30-50; P < 0 0001, 30-60; P < 0-0001, 20-40; P < 0 05, 20-50; P< 0 00025, 20-60; P<001, and otherwise not significant. In females, 30-40; P<0005, 30-50; P<0005, and otherwise not significant. Radioimmunoassay of circulating ae-interferon 85 0 3 P<OOOO P<O-OOO NS NS 0 0 0-2 C 01 0~~~L00 RA RA RA OA active inactive vasculitis Fig. 6. Radioimmunoassay of circulating a-interferon-like substance in the plasma of patients with rheumatoid arthritis (RA). Each point represents the mean of determinations performed in duplicates. The values given are the mean + s.e.m. The shaded area represents the mean + s.e.m. of age and sex-matched healthy controls. Patient without drugs (0); patient treated with non-steroidal anti-inflammatory drug plus 25 mg of gold thiomalate injection biweekly (0). osteoarthritis (OA). Statistical significance by Student's t-test is given. biologically active as well as inative IFNa, it is unlikely that the assay recognized antigenically similar but unrelated peptide in plasma crossreacting with IFNa. This is supported from the following evidence: (1) serial dilutions of plasma gave identical dilution curves to that of standard IFNa; (2) circulating IFNa-LS was extracted and concentrated from plasma under acidic condition using silicic acid and HCl as well as under neutral condition using antibody immunoadsorption; (3) extracted plasma samples showed dilution curves identical to that of standard IFNa. Definitive proof for this, however, awaits the determination of the amino acid sequence of IFNaC-LS. Gel filtration studies suggested that circulating IFNa-LS appeared to be fragmented in human plasma; (1) circulating IFNa-LS of the healthy individuals eluted into the fraction of distinctively smaller molecular size than that of standard IFNax; ( 2) when standard IFNa was added to plasma and extracted without adding protease inhibitors, immunoreactive peak of IFNa moved from its original position to that of smaller molecular size, whereas (3) the shift was preventable by adding protease inhibitors. Since this shift was prevented by leupeptin, plasmin-like factors (Mantei et al., 1980; White & Gross, 1957) might be responsible for degradation. The existence of a fragmented and biologically inactive form of IFNax in plasma is consistent with the finding that IFNa is often undetectable in the plasma of healthy individuals when assayed by virus inhibition assays are shown in this and other studies (Hooks et al., 1979; 1982; Preble et al., 1982; Panem et al., 1982; Ytterberg & Schnitzer, 1982). Theoretically, since the plasma of a healthy individual contained approximately 0 2 ng/ml of IFNa-LS (corresponding to 50 i.u./ml of antiviral activity), it should be detectable under virus inhibition assays when the IFNa molecule was intact and biologically active. It was found in this study that circulating IFNa-LS declined gradually with age. This may be a part of the gradual fall in immune surveillance in the aged, since IFNa is important for activation of natural killer cells (Targan & Dorey, 1980; Perussia & Trinchieri, 1981). The results also showed that circulating IFNax-LS was significantly decreased in the plasma of rheumatoid arthritis patients. This is attributable to the original disease condition, as suggested by the following evidence; (1) circulating IFNat-LS was decreased in the patients untreated for rheumatoid arthritis; (2) it was decreased when compared with the value found in the osteoarthritis 86 S. Shiozawa et al. patients receiving a comparable dose of non-steroidal anti-inflammatory drugs; (3) plasma sample drawn with either heparin or EDTA gave similar results, indicating that the result was irrelevant to the method of sampling; (4) IFNa-LS was however not decreased in the rheumatoid patients with vasculitis. The latter finding might be interesting in relation to the vascular reactivity of IFNoa. Kramer et al. (1984) have shown that IFNx potentiated acute vascular rejection in the renal 'transplant recipients; IFNa appears to be related to the vascular reactivity. There have been inconsistency in results concerning the circulating level of IFNa in the rheumatoid arthritis patients; previous assays based on antiviral activities of IFNa have shown that circulating IFNa was increased in rheumatoid arthritis (Hooks et al., 1979), whereas it was later suggested to be IFNy (Degre et al., 1983). This type of uncertainty would be inevitable when the virus inhibition assays were used (Hooks et al., 1979; Preble et al., 1982). The present results utilizing radioimmunoassay seems to answer this by demonstrating that circulating IFNoc-LS is significantly decreased in rheumatoid arthritis. Decreased IFNox in rheumatoid arthritis may be important from the pathogenetic as well as therapeutic standpoints. Decreased IFNa in these patients may lead to defective functions of natural killer cells (Goto et al., 1981; Combe et al., 1984; Goto & Zvaifler, 1985), then prolong chronic inflammation, since IFNa is required for continuous activation of natural killer cells (Targan & Dorey, 1980; Perussia & Trinchieri, 1981). In-vitro production of IFNa by the patient's lymphocytes was decreased in rheumatoid arthritis (Neighbour & Grayzel, 1981). The finding of decreased IFNcx-LS may also provide a rational basis for the treatment of rheumatoid arthritis with IFNa; we have noted that, in various disease conditions, clinical efficacy of IFNox treatment appeared to be related to the level of circulating IFNcx (to be published). This work was supported in part by the Basic Researches for Joint Diseases Award and the Japan Ministry of Education & Welfare grant 59770445 to S.S. 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