Fenneke G. Joslin

Interleukin-1 inhibitors

DNA sequences that encode Interleukin-1 inhibitors and recombinant-DNA methods for the production of interleukin-1 inhibitors are provided. The DNA sequences encode proteins having interleukin-1 inhibitors activity.

An improved ELISA for anti-native DNA by elimination of interference by anti-histone antibodies

We evaluated an enzyme-linked immunosorbent assay (ELISA) for antibodies to native DNA (nDNA) in which protamine was used to link DNA to polystyrene. Elevated anti-nDNA was largely restricted to patients with systemic lupus erythematosus (SLE), and within this group good correlation between ELISA and the ammonium sulfate assay was obtained. However, substantial background immunoglobulin binding to protamine coated wells was commonly observed, and it was necessary to subtract this activity from each anti-DNA determination. Many of the SLE sera also contained anti-histone antibodies, and this antibody activity showed significant correlation with the binding to protamine. In contrast, methylated bovine serum albumin (mBSA) did not bind anti-histone antibodies and provided a substrate for coupling nDNA to polystyrene. This modified ELISA allowed the quantitation of antibodies to native DNA without the simultaneous binding of anti-histone antibodies.

Reactivity of anti-histone antibodies induced by procainamide and hydralazine☆

Abstract Sera from patients with procainamide (Pr)- or hydralazine (Hy)-induced lupus were examined for anti-histone antibodies by an immunofluorescence assay using histonerecostituted mouse kidney sections and by a solid-phase radioimmunoassay using polystyrene tubes coated with purified histones. Distinct differences were observed between Pr sera and Hy sera. Pr sera were uniformly positive in the immunofluorescence assay on histone-reconstituted tissue sections, but all Hy sera were negative. In solidphase radioimmunoassay, both Pr and Hy sera showed anti-histone activity with Pr sera demonstrating strong reactions with H2A-H2B histone complex but Hy sera demonstrating weak reactions. The difference in the immunofluorescence assay could in most but not all instances be attributed to this difference in anti-H2A-H2B activity. Antibodies were not detected to DNA or to nonhistone nuclear antigens Sm, nuclear ribonucleoprotein, and SS-B La . The results indicate that both Pr and Hy induce antibodies to histones and that anti-histone antibodies induced by Pr differ both quantitatively and qualitatively from those induced by Hy.

IL-1ra ELISA: reduction and alkylation of synovial fluid eliminates interference by IgM rheumatoid factors

IL-1 and a specific receptor antagonist of IL-1, IL-1ra, may play important roles in the pathophysiology of rheumatoid arthritis and in other types of inflammatory synovitis. Measurement of IL-1ra in synovial fluids and in other body fluids may lead to a greater understanding of its possible activity as a modulator of the immune and inflammatory systems in vivo. Therefore, a modified sandwich ELISA was developed to measure IL-1ra protein concentration in synovial fluids. The antibodies used in this ELISA were polyclonal and derived from rabbits hyperimmunized with human recombinant IL-1ra. IgM rheumatoid factors within synovial fluid resulted in false elevation of determined IL-1ra by the sandwich ELISA through binding of the primary and secondary antibodies. Reduction and alkylation of synovial fluid samples before application to the ELISA plate eliminated the interference caused by greater than or equal to 2000 micrograms/ml IgM rheumatoid factor (latex agglutination titer of 1/5.120). This ELISA was specific for IL-1ra; there was no detection of IL-1 alpha, IL-1 beta, or lysozyme. The sensitivity of this ELISA was less than 200 pg/ml, making it a useful assay for the accurate measurement of synovial fluid IL-1ra protein concentration.

Characteristics of chondrocyte responses to a human interleukin 1-like factor.

The objective of these studies was to characterize some aspects of collagenase production by rabbit articular chondrocytes cultured with stimulated monocyte supernatants. Supernatants from human monocytes stimulated with 20 ng/ml bacterial lipopolysaccharide (LPS) induced the synthesis and secretion of latent collagenase by the chondrocytes beginning at 6 hr. The time course and dose response of collagenase production by the chondrocytes were identical using crude monocyte supernatants or semipurified interleukin 1 (IL-1). Recombinant or purified human interleukin 2 failed to induce collagenase production in the cultured chondrocytes. The response of the chondrocytes was inhibited by actinomycin D or cycloheximide and not by corticosteroids. Phorbol myristate acetate (PMA) alone failed to directly stimulate the chondrocytes. However, PMA led to enhanced collagenase production by chondrocytes when incubated with submaximal amounts of LPS-stimulated monocyte supernatant or semipurified IL-1. LPS alone in amounts between 0.1 and 10.0 micrograms/ml directly stimulated collagenase production in chondrocytes between 4 and 11 days in culture. These data confirm those of other laboratories that IL-1 may be the active factor in monocyte supernatants responsible for inducing collagenase production in cultured chondrocytes. Further characterization of this response indicates that the collagenase is not preformed in the cells and stimulation of its production is not inhibited by corticosteroids. Cell supernatants or IL-1 preparations containing PMA as low as 1.0 ng/ml or LPS as low as 1.0 microgram/ml may give falsely high values for IL-1 activity when assayed by stimulation of collagenase production in cultured chondrocytes.