Age-related increase of autoantibodies to interleukin1α in healthy Japanese blood donors
Yasukazu Ohmotoa, Fumitaka Ogushib, Masahiro Muraguchia, Mayumi Yamakawac and Saburo Soneb

aOtsuka Pharmaceutical Co., Kagasuno, Kawauchi-cho, Tokushima, Japan; bThird Department of Internal Medicine, The University of Tokushima School of Medicine, Tokushima, Japan; and cJapan Red Cross Blood Center, Shomachi3, Tokushima, Japan

Abstract:Although autoantibodies to interleukin-1α (IL-1α autoantibodies) are known to be present in sera of apparently healthy humans, their frequency of occurrence and significance are unclear. To determine the prevalence of detectable IL-1α autoantibodies in normal human blood, we screened the plasma of blood donors (6290subjects:3977men and2313women, ages 16 to 64yr) by a radioimmunoassay which we developed using a method that could detect over5ng/ml. Moreover, we investigated immunoglobulin class of IL-1α autoantibodies and also their function. IL-1α autoantibodies were detected in 14.6% of the 6290donors. Their frequency was higher in males than females (16.6%vs.11.2%, p<0.01) and increased with age in both sexes. The proportion of subjects with a high IL-1α autoantibodies titers also increased with age. We showed that IL-1α autoantibodies were of the IgG class and that they had neutralizing function to IL-1α by receptor assay. Neutralizing activity was only shown in plasma with concentration of IL-1α autoantibodies, the level of which was over1000ng/ml. The affinity of the IL-1α autoantibodies in plasma was between2.1X10-10and1.2X10-9M (mean6.4X10-10M). Our results provide a basis for comparison with IL-1α autoantibodies prevalence between healthy states and disease states, and suggest that IL-1α autoantibodies may play a significant role in modulating the effects of excessive IL-1α at local site or in systemic regions. J. Med. Invest. 44:89-94, 1997

Keywords:autoantibodies, IL-1α, blood donors, aging, IL-1α receptor assay

Interleukin-1 (IL-1), first recognized as an endogenous pyrogen, has subsequently been found to be an important mediator of immune and inflammatory reactions (1-3). It is synthesized in response to infection, injury or antigenic challenge by a variety of cells including monocytes and macrophages (2), and exerts biologic effects on various target cells in normal and pathological conditions (1-3). IL-1exists as two genomic forms, IL-1α and IL-1β, both of which are very similar in function. Although the precussor for IL-1β requires cleavage to reveal its IL-1activity, the proIL-1α is fully active as a precussor and remains intracellulaly (1-3). IL-1α is thought to be involved in cell-mediated responses through cell-to-cell contact (1-3). Despite its host-defense role, IL-1may cause tissue damage and exacerbate chronic inflammation. Accordingly, its potent inflammatory effects are believed to be tightly regulated.
The actions of IL-1are controlled in several ways, such as by regulation of its synthesis and by production of neutralizing factors including a soluble IL-1receptor, and an IL-1receptor antagonist (1, 3, 4, 7). Recently, antibodies against cytokines such as interferons, tumor necrosis factor, IL-6 and IL-10 have been detected in sera of patients with inflammatory and neoplastic diseases and in healthy subjects (8-12). Autoantibodies against IL-1α (IL-1α autoantibodies) have also been found in the sera of patients with inflammatory process and asymptomatic individuals (5, 6, 13-16). It is known that IL-1α autoantibodies neutralize IL-1α specifically (14, 16, 17), while IL-1receptor antagonist blocks the activity of both IL-1α and IL-1β (1, 3). However, the level of plasma IL-1α autoantibodies which neutralizes IL-1α activity was not clear. Thus, the role of IL-1α autoantibodies in blood remains to be defined. However, the reported prevalence of IL-1α autoantibodies in the blood of healthy subjects was inconsistent (6, 13-16). Their frequency of occurrence in normal human blood needs to be established, because fresh plasma is transfused into patients as an element of blood, and also because such data sheds light on the roles of IL-1α autoantibodies in healthy and diseased subjects. We developed sensitive radioimmunoassay (RIA) for IL-1α autoantibodies, and measured IL-1α autoantibodies levels in plasma from a large number of healthy blood donors, considering overall prevalence, sex and age. Furthermore, we characterized the immunoglobulin class of IL-1α autoantibodies as well as elucidating the level of plasma IL-1α autoantibodies to neutralize IL-1α activity.

Study Population
Plasma was obtained from donor blood at the Tokushima Blood Transfusion Center in Japan. Samples were obtained from 6290healthy donors (2313women and 3977men between16yr and 64yr). Samples were separated into donor groups I-VI, respectively aged 16-19, 20-29, 30-39, 40-49, 50-59, and 60-64yr. Age and sex distributions of donors are shown in Table1.

Measurement of IL-1α autoantibodies
Human recombinant IL-1α (rIL-1α) was prepared as described previously (18). IL-1α autoantibodies in plasma were measured by a radioimmunoassay (RIA). Briefly, duplicate50μl samples of plasma or standard dilutions of monoclonal antibodies to IL-1α (ANOC301, purity >95%),ranging from5to640ng/ml, were mixed with 125I-rIL-1α. Each sample was mixed with 0.1%bovine serum albumin/phosphate buffer saline (BSA/PBS) containing 5mM ethylenediaminetetraacetic acid (EDTA)/0.05%NaN3, and each standard dilution of rIL-1α was mixed with 0.1% BSA/PBS containing5mM EDTA/0.05%NaN3 and carrier buffer (2% bovine γ globulin). After antigen-antibody binding had reached equilibrium by overnight incubation at room temperature, each sample was mixed with 25%polyethylene glycol (MW6000) and incubated for1hr. Then, the tubes were centrifuged at3000rpm for15min, and the radioactivity of the precipitates were counted for1min in an automated γ-spectrometer. The IL-1α autoantibodies titer was determined from the standard dilution curve using IL-1α monoclonal antibody (Fig.1).Plasma was considered IL-1α autoantibodies-positive at levels over 5ng/ml. Autoantibodies for IL-1β (IL-1β autoantibodies) in the plasma were measured by the same method as described for IL-1α autoantibodies, substituting IL-1α by IL-1β. The sensitivity of this assay for IL-1β autoantibodies was5ng/ml.

Characterization of IL-1α autoantibodies in plasma
To characterized IL-1α autoantibodies, we used plasma containing a high IL-1α autoantibodies level and investigated immunoglobulin class of IL-1α autoantibodies. The plasma was applied to a protein A agarose column. After washing with the binding buffer (50mM sodium borate with 3M NaCl, pH9.0), the bound material was eluted with elution buffer (100mM glycine, pH2.5) and collected in2ml fraction. The IL-1α autoantibodies level in each fraction was measured by RIA. The plasma IL-1α autoantibodies was also fractionated by gel filtration using Sepharose 6B column. Plasma was applied to a column preequilibrated with50mM Tris-HCl containing 0.5M NaCl and 5mM EDTA (pH8.0) and eluted with the same buffer. The elute was collected in3ml fractions and the IL-1α autoantibodies concentration of each fraction was measured by RIA.

IL-1α receptor assay
Neutralizing activity of IL-1α autoantibodies in plasma was examined by IL-1receptor assay as described previously (19). 125I-labeled IL-1α was prepared by the same way as described in the method of RIA. The specific activity of 125I-labeled IL-1α was130-150μCi/g. BALB/3T3fibroblasts (the American Type Culture Collection) were maintained in a Dulbecco's minimum essential medium (Nissui Pharm. Co. Ltd., Tokyo, Japan) containing10%fetal calf serum (Hyclone Inc., Utah). 3T3 fibroblasts (1X106) were incubated with500μl of culture medium containing 125I-labeled IL-1α (105cpm) and10%sera from the patients in12-well plates (Corning Glass Works, NY) for2hr at4°C. Nonspecific binding was determined in the presence of 1μg/ml of unlabeled IL-1α. The cell monolayers were then rinsed 3times with 1ml of PBS then solublized with1ml of a mixture of1%SDS and0.2N NaOH. The radioactivity was counted in a γ-counter.

Equilibrium binding assay
To determine the affinity of IL-1α autoantibodies in plasma for human IL-1α, the dissociation constant (Kd) of IL-1α autoantibodies/IL-1α complexes was measured. The plasma IL-1α was incubated with increasing amount of 125I-IL-1α in the final volume of 200μl in RPMI1640, 1%BSA. After4hr incubation at 4°C, IL-1α autoantibodies/IL-1α complexes were precipitated with protein G-sephadex and the radioactivity of the precipitate was measured by a γ counter. Ratio (B/F) of specific bound 125I-IL-1α (B) vs free 125I-IL-1α (F) were calculated, then plotted vs specific bound 125I-IL-1α amounts calculated according to the IL-1α specific activity. According to Scatchard, the Kd value was determined by the slope of the obtained straight line (Kd=-1/slope) and expressed in concentration according to the reaction volume.

Statistical Analysis
Statistical analysis was performed by the χ2test, and values were considered significantly different at p<0.05.

Standard curve of IL-1α autoantibodies in RIA
The standard curve of IL-1α autoantibodies, as measured by RIA, is shown in Fig.1. From this standard curve, the IL-1α autoantibodies concentration in plasma of normal healthy subjects was calculated, and ranged from5to640ng/ml by this assay.

Prevalence of IL-1α and β autoantibodies in normal human plasma
Prevalence of IL-1α autoantibodies in plasma are shown in Table2. IL-1α autoantibodies was detected in 917 (14.6%) of samples. Its prevalence was higher in males than in females in general (16.6%vs11.2%, p<0.01) and in groups II and IV (p<0.01). As shown in Table2, the prevalence of IL-1α autoantibodies increased with age:in groups I and II, it was less than those in groups III, IV, V and VI (p<0.01), but there were no significant differences in the groups over 40yr. In a parallel study, we measured IL-1β autoantibodies in plasma from about500 normal subjects. We could not detect IL-1β autoantibodies in plasma from any subjects (data not shown).

Distribution of subjects with high titers of IL-1α autoantibodies in the blood
The proportions of subjects with an IL-1α autoantibodies level exceeding 1000ng/ml among the antibody-positive individuals are shown in Figure2. The percentage of subjects with a high titer of IL-1α autoantibodies increased steadily with age, being2 (2.9%)in group I, 8 (6.1%) in group II, 27 (10.9%) in group III, 41(13.2%) in group IV, 27 (19.7%) in group V and7 (29.2%) in group VI.

Characterization of plasma IL-1α autoantibodies
To characterize IL-1α autoantibodies, we used plasma containing a high IL-1α autoantibodies level. The sample was applied to a protein A agarose column. After washing with the binding buffer, we obtained the IgG fraction using an elution buffer. RIA showed that the high titer of IL-1α autoantibodies was presented only in the IgG fraction (Fig.3). Furthermore, the plasma was fractionated by gel filtration resulting in its elution with an apparent molecular weight of100,000-200,000, IL-1α autoantibodies corresponded to that of IgG (Fig.4). The affinity of the IL-1α autoantibodies in several samples, examined by the Scatchard analysis, was between2.1X10-10 and1.2X10-9 M (Table3).

Neutralizing activity of IL-1α autoantibodies
First, we examined whether neutralizing activity of IL-1α autoantibodies was estimated by IL-1α receptor assay using BALB/3T3 fibroblasts. Standard of IL-1 receptor assay was performed using monoclonal antibody to IL-1α (ANOC301). As shown in Fig.5, monoclonal antibody (ANOC301) showed neutralizing activity in a dose dependent manner. Next, to examine the neutralizing activity of IL-1α autoantibodies in plasma, we performed an IL-1α receptor assay. Plasma from subjects with a concentration of IL-1α auto-antibodies, whose level was over1000ng/ml, blocked binding of 125I-labeled IL-1α to the IL-1receptor, showing neutralizing activity against IL-1α (Fig.6). Plasma levels of IL-1α autoantibodies much less than1000ng/ml or without detectable titers showed no detectable neutral-izing activity.

High-affinity specific autoantibodies to various cytokines, such as IL-1α, IL-6, IL-10 and interferon, are known to be present in normal individuals (5, 6, 8-16). Recently, pharmaceutical preparations of human IgG were reported to contain antibodies against these cytokines (20, 21),indicating that blood products containing these antibodies are likely to be transfused into patients and making it necessary to clarify the prevalence of these antibodies in donor blood.
Despite several reports on the presence of IL-1α autoantibodies in normal subjects, its prevalence has been discrepant (6, 13-16). Furthermore, age-associated alterations in humoral immunity have been recognized widely, with aging associated with a decline in antibodies against pathogens and an increase in autoantibodies (22, 23).Although Hansen et al. reported that by a second-antibody precipitation assay, the prevalence of IgG antibodies against IL-1α depended on both sex and age (24), the number of subjects was apparently not enough to conclude the relation between the prevalence of IL-1α autoantibodies and age or sex. Therefore, to clarify the prevalence of IL-1α autoantibodies, we measured the level of IL-1α autoantibodies in plasma from a large number of healthy blood donors by RIA.
We found that about 15%of plasma samples from healthy donors contained detectable IL-1α autoantibodies at a level of over5ng/ml. Similar to other autoantibodies, their prevalence increased with age. We found their prevalence to be higher in males than in females, an unexpected difference. Therefore, the contradiction in the earlier reports concerning the prevalence of IL-1α autoantibodies may be due to the differences in the age and sex of the subjects studied. Samples with a high level of IL-1α autoantibodies (>1000ng/ml) increased with age. However, we could not detect any IL-1β autoantibodies in plasma. With column chromatography, IL-1α autoantibodies eluted with an apparent molecular weight of100,000-200,000and corresponding to a subclass of the IgG fraction. Moreover, a substantial amount of this material bound to protein A which binds with high specificity to the Fc regions of IgG1, IgG2 and IgG4 (25). These results suggested that IL-1α autoantibodies were of the IgG class and our results were similar to previous reports (13, 17, 26).
The role of IL-1α autoantibodies is still unknown, but reported effects include inhibition of the biologic activity of IL-1α but not IL-1β (5, 14, 17). It has been shown that Fab fragments of the antibodies bind with high affinity to human recombinant IL-1α (13, 27), and that they interfere with its T-cell stimulatory function (26). We confirmed that the plasma containing high titers of this antibodies blocked binding of 125I-labeled IL-1α to IL-1receptors on the surface of BALB/3T3fibroblasts. Our results showed that IL-1α autoantibodies neutralize IL-1α which is consistent with other reported results (14, 16, 17). We showed that a level of IL-1α autoantibodies over1000ng/ml of plasma was needed to neutralize IL-1α. The affinity of the IL-1α autoantibodies in plasma examined by Scatchard analysis showed high affinity, which suggested that this antibody may play a physiological role against overproduced IL-1α .
Although the production of antibodies against IL-1α in healthy subjects can not be adequately explained, there are some interesting reports. IL-1α has been demonstrated in various body fluids and tissues, and is produced by keratinocytes and alveolar macrophages (2). IL-1α is thought to function through cell-to-cell interaction and to be involved in local immune reactions (1-3). It is suggested that IL-1α autoantibodies in some healthy subjects, produced in response to IL-1α presented in normal tissues, helps to hold the immunoinflammatory processes involving IL-1α in check. Furthermore, IL-1α autoantibodies may inhibit the systemic reaction of excess proIL-1α which has a biological function and increase in these processes. It was not clear why the prevalence of IL-1α autoantibodies in plasma was increased with age. Recently, the relation between cytokine and aging was reported (28, 29). Catania et al. showed the plasma concentration of IL-1receptor antagonist and soluble TNF receptor were greater in healthy aged subjects than younger controls (29). We suppose that unapparent inflammation in aged subjects may cause cell activation and release of cytokine antagonist including IL-1receptor antagonist, soluble TNF receptor and IL-1α autoantibodies.
In this study, we demonstrated the prevalence of IL-1α autoantibodies in plasma from a large number of healthy blood donors in general and by age and sex. These results would suggest that monitoring the blood donor pools for level of IL-1α autoantibodies would be useful in disease states. Recently, cytokine inhibitor included the IL-1receptor antagonist and the soluble IL-1receptor was administrated to patients with immunoinflammatory diseases as a new therapeutic approach (3, 30, 31). In this regard, high concentrations of IL-1α autoantibodies which were prepared from normal subjects may be useful as a new type of IL-1α antagonist.

This study was supported by a Grant-in-aid for Scientific Research (C) from the Ministry of Education, Science and Culture of Japan.

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Received for publication August 1, 1997;accepted August 15, 1997.

1 Address correspondence and reprint requests to Fumitaka Ogushi, M.D., Ph.D., Third Department of Internal Medicine, The University of Tokushima School of Medicine, 3-18-15, Kuramoto-cho, Tokushima, Japan.