Morten Svenson

Immunogenicity of interferon-β in multiple sclerosis patients: Influence of preparation, dosage, dose frequency, and route of administration

A total of 754 consecutive patients with relapsing‐remitting multiple sclerosis were investigated for interferon‐β (IFNβ) antibodies by protein‐G affinity chromatography and antiviral neutralization bioassay during 24 months on 6 MIU (22 μg) of subcutaneous IFNβ‐1a once weekly (n = 143) or three times weekly (n = 160), 6 MIU (30 μg) of intramuscular IFNβ‐1a once weekly (n = 140), or 8 MIU every other day of IFNβ‐1b (n = 311). The proportion of binding antibodies was higher in those receiving IFNβ‐1b compared with 6 MIU of IFNβ‐1a three times weekly (97 vs 89% at 12 months), and fewer became positive if 6 MIU of IFNβ‐1a was administered once weekly (58 vs 89%). Fewer patients on intramuscular than subcutaneous IFNβ‐1a became positive (33 vs 58%). The binding and neutralizing capacities were higher in the IFNβ‐1b group than in the IFNβ‐1a groups; these differences, however, were not significant after 12 months. The number of positive patients varied considerably and depended on the amount of IFN added to the bioassay; adding 10 LU/ml or more masked antibody detection. Antibodies induced by either preparation neutralized both IFNβ species but not IFNα. In conclusion, IFNβ‐induced antibodies are frequently found in multiple sclerosis patients, and IFNβ‐1b is more immunogenic than IFNβ‐1a. The immunogenicity of IFNβ‐1a increases with the frequency of administration and if it is given subcutaneously. Ann Neurol 2000;48:706–712

Autoantibodies to cytokines--friends or foes?

Cytokines form a network of communication signals between cells of the immune system, and between the immune system and other organs. They interact with structurally complex and often dynamically expressed target cell receptors. The recent demonstration of autoantibodies to cytokines, even in sera of normal individuals, suggests further complexities in the way that nature regulates cytokine functions. Based mainly on evidence obtained by investigating autoantibodies to interleukin 1 alpha (IL-1 alpha), Klaus Bendtzen and colleagues discuss the possibility that naturally occurring antibodies may function as specific physiological carriers and regulators of cytokines.

High-avidity autoantibodies to cytokines

Abstract The increased use of recombinant cytokines and cytokine analogues in various disease therapies emphasizes the importance of antibodies to cytokines. Here, Klaus Bendtzen and colleagues discuss how immunological tolerance to cytokines may be broken, and the clinical relevance of cytokine autoantibodies. They also comment on the implications for refined passive immunization and cytokine vaccination strategies.

Binding of cytokines to pharmaceutically prepared human immunoglobulin

Pharmaceutically prepared IgG, pooled from sera of over 2,000 normal individuals, contained both monomeric and dimeric IgG. Each type of IgG bound 125I-labeled interleukin (IL)-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha. Increased binding to IgG was observed if 125I-IL-1 beta was denatured by heating to 39 degrees C. However, the binding of both nondenatured and denatured 125I-IL-1 beta was not inhibited by unlabeled IL-1 beta. In contrast, binding of 125I-IL-1 alpha, 125I-IL-6, and 125I-TNF alpha was inhibited by the corresponding unlabeled cytokine. Papain-digestion of IgG abolished binding of 125I-TNF alpha but failed to influence the displaceable binding of 125I-IL-1 alpha and 125I-IL-6. 125I-TNF alpha was a mixture of trimeric and monomeric forms, the latter being the predominant form at lower concentrations. The apparent saturability of 125I-TNF alpha was explained by a higher nonspecific binding of monomeric than of trimeric 125I-TNF alpha to IgG. The amounts of cytokine antibodies in IgG preparations would contribute approximately 2 micrograms anti-IL-1 alpha IgG and 1 microgram anti-IL-6 IgG per kg body wt during high dose immune globulin therapy. In conclusion, pharmaceutical preparations of human IgG contain specific and neutralizing, high affinity antibodies against IL-1 alpha and IL-6, but not against TNF alpha or IL-1 beta. There are significant methodological pitfalls that hamper detection of IgG autoantibodies against cytokines.

Anti-interleukin-6 antibodies in normal human serum.

High‐avidity IgG antibodies to the cytokine inlerleukin‐6 (IL‐6) were found in sera of apparently healthy adult individuals. These antibodies specifically interfered with an FLISA (enzyme‐linked immunosorbent assay) for IL‐6 in which specific polyclonal rabbit antibodies to human recombinant IL‐6 (rlL‐6) were used. Furthermore. using precipitation of 125‐I‐rIL‐6 with rabbit antibodies to human immunoglobulins (Ig). the sera of 7 out of 6S Danish blood donors were found to contain specific antibodies in substantial amounts. Judged by ELISA interference, gel filtration of sera incubated with 125I‐rIL‐6 and second antibody precipitation of 125I‐rIL‐6. IgG seemed to be the dominant IL‐6 binding protein in these normal sera. Using specific antibodies to human in light chains, it was found that the anti‐lL‐6 antibodies were of polyclonal origin. Moreover, there are at least two epitopes on the IL‐6 molecule, because more than one IgG bound lo some IL‐6 molecules at the same time, The anti‐IL‐6 antibodies did not cross‐react with a number of other human recombinant‐derived and native cytokines. The antibodies recognized native as well as rlL‐6. but preferentially monomeric lL‐6.

High avidity IFN-neutralizing antibodies in pharmaceutically prepared human IgG.

This paper demonstrates and characterizes naturally occurring antibodies to interferon (IFN) in human IgG preparations. In vitro neutralization of the antiviral effect of IFN alpha and IFN beta, but not IFN gamma, was observed in 12 of 15 normal IgG preparations. The neutralizing capacity was higher against rIFN alpha 2A and rIFN alpha 2C than against lymphoblastoid IFN alpha and IFN beta. Frühsommer meningoencephalitis hyperimmune IgG and hepatitis-B hyperimmune IgG showed potent neutralization, whereas anti-rhesus D-, anti-rabies-, and anti-tetanus IgG showed weak neutralization. Saturable binding of 125I-rIFN alpha 2A was demonstrated only in those IgG preparations found to neutralize the antiviral effect of IFN. Significant correlation between IFN binding and neutralization capacity was observed. The antibodies bound with Fab to rIFN alpha 2A with an avidity of approximately 30 pM; the majority was of the IgG1 subclass. Maximum binding capacity was 490 pg rIFN alpha 2A/mg IgG. Cross-binding of rIFN alpha 2C, lyIFN alpha N1 and IFN beta occurred with 10 and 100-200 times lower activities than that of rIFN alpha 2A. There was no cross-binding with rIFN gamma or rIL-6. IgG preparations containing anti-IFN antibodies blocked the binding of 125I-rIFN alpha 2A to A549 cells. In conclusion, pharmaceutically prepared human IgG preparations contain variable but significant levels of high-avidity IFN alpha and IFN beta neutralizing antibodies.

Human anti-interleukin 1α antibodies

Abstract Autoantibodies to the cytokine interleukin (IL)-1α are frequently found in sera of apparently healthy humans. We have developed a sensitive radioimmunoassay (RIA) and an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of human serum antibodies to IL-1α at concentrations below 10 pmol/l. The RIA is based on co-precipitation of 125 I-labelled human recombinant IL-1α (rIL-1α) by rabbit antibodies to human immunoglobulins. The ELISA is based on recovery of added rIL-1α to serum samples and takes advantage of the fact that free human autoantibodies to IL-1α in a dose dependent manner reduce recovery of added rIL-1α. The assays correlate exceedingly well ( r = 0.99, P

Distribution and Characterization of Autoantibodies to Interleukin 1α in Normal Human Sera

Antibodies against IL‐1α were detected in sera of apparently healthy individuals. The immunoglobulins belonged to the IgG and class, particularly IgG1. IgG2. and IgG4. [125I]rIL‐1α bound to Tab fragments of IgG. and IgG immune complexes of molecular weights from 160 to 700 kDa were formed in the sera by [125I]rIL‐1α. The occurrence of detectable anti‐IL‐1α IgG in sera of 32 male and 32 female donors was 25 and 22% respectively. As judged by Scatchard analysis of the binding data, the capacity and avidity of binding were greaser in the male than in the female sera (mean capacity to bind [l25I]rIL‐1α 10 [0.7 ‐27] versus 3.3 [0.5‐7.3] ng/ml; and mean Kd:5.5 [5‐7] versus 11 [4 16] PM). The antibodies did not cross‐bind human recombinant IL‐1β, IL‐2, 1L‐6. or tumour necrosis factor alpha (TNF‐α). It is concluded that native IL‐1α seems lo trigger production of specific, high‐avidity IgG antibodies in a relatively large number of normal individuals. These autoantibodies may regulate immunoinflammatory processes involving IL‐1α.

Specific binding of interleukin 1 (IL-1)β and IL-1 receptor antagonist (IL-1ra) to human serum. High-affinity binding of IL-1ra to soluble IL-1 receptor type I

Molecules that bind recombinant interleukin 1 (rIL-1) beta and rIL-1 receptor antagonist (rIL-1ra) with high affinity were detected in sera of healthy individuals. rIL-1 beta bound with dissociation constants in the nanomolar range, and the serum binding capacity was 40-50 ng/ml. rIL-1ra bound with 30 times higher affinity, and the serum binding capacity was 0.7-1 ng/ml. Rabbit antibodies against the recombinant-derived extracellular part of human IL-1 receptor type I (rsIL-1RI) selectively inhibited the binding of 125I-rIL-1ra to the serum factor(s). Almost 70% of the high-affinity IL-1ra-binding capacity was recovered after immunosorption with these antibodies. Binding of 125I-rIL-1ra to rsIL-1RI was blocked by rIL-1 alpha and by rIL-1 beta. In contrast, the purified rIL-1ra-binding factor (IL-1raBF) failed to bind rIL-1 alpha and rIL-1 beta. Gel filtration chromatography indicated a 1:1 binding of rIL-1 beta and rIL-1ra to their respective serum factors. The apparent molecular size of both serum factors was 70-80 kDa. Using SDS-PAGE and autoradiography, IL-1raBF had a molecular size of 60 kDa. We conclude that IL-1raBF, a serum factor which selectively and with high affinity binds IL-1ra (Kd = 70 pM), is related to or identical with a soluble form of IL-1RI. If upregulated during disease, IL-1raBF may constitute yet another level of natural regulation of IL-1 bioactivities.

Cloning and expression of an alternatively spliced mRNA encoding a soluble form of the human interleukin-6 signal transducer gp130.

Both gp130 and alternatively spliced sgp130 were also transcribed by the myeloma cell lines XG‐1, XG‐2, XG‐4, XG‐4CNTF XG‐6, XG‐7, XG‐9, XG‐10, U266 and RPMI 8226. However, XG‐4A cells derived from XG‐4 cells, but growing independently of exogenous IL‐6, did not transcribe sgp130 mRNA. A possible interference with intracrine stimulatory factors by alternatively spliced sgp130 needs to be further investigated.