C. Bird

A simple sensitive bioassay for interleukin-1 which is unresponsive to 103 U/ml of interleukin-2

A subclone, NOB-1, of the mouse EL-4 line constitutively produces very little interleukin-2 but in response to interleukin-1 produces high concentrations of interleukin-2. Co-stimulation with mitogen, phorbol esters or calcium ionophores was not required. NOB-1 is not responsive to tumour necrosis factor alpha, tumour necrosis factor beta, interferon gamma and lipopolysaccharide. The NOB-1 line was used in conjunction with a CTLL line to detect less than 1 pg/ml interleukin-1. Rapid assay was performed by co-culturing the EL-4 cells with CTLL cells. By incorporating a pre-incubation step, followed by thorough washing of the EL-4 cells, responses to interleukin-1 were maintained, but interleukin-2 had no effect. The assay was used to detect interleukin-1 in serum samples and to evaluate neutralizing antisera to interleukin-1.

Anti-cytokine autoantibodies in autoimmunity: preponderance of neutralizing autoantibodies against interferon-alpha, interferon-omega and interleukin-12 in patients with thymoma and/or myasthenia gravis

We have screened for spontaneous anticytokine autoantibodies in patients with infections, neoplasms and autoimmune diseases, because of their increasingly reported co‐occurrence. We tested for both binding and neutralizing autoantibodies to a range of human cytokines, including interleukin‐1alpha (IL‐1α), IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, IL‐18, interferon‐alpha2 (IFN‐α2), IFN‐ω, IFN‐β, IFN‐γ, tumour necrosis factor alpha (TNF‐α), transforming growth factor beta‐1 (TGF‐β1) and granulocyte‐macrophage colony stimulating factor (GM‐CSF), in plasmas or sera. With two notable exceptions described below, we found only occasional, mostly low‐titre, non‐neutralizing antibodies, mainly to GM‐CSF; also to IL‐10 in pemphigoid. Strikingly, however, high‐titre, mainly IgG, autoantibodies to IFN‐α2, IFN‐ω and IL‐12 were common at diagnosis in patients with late‐onset myasthenia gravis (LOMG+), thymoma (T) but no MG (TMG–) and especially with both thymoma and MG together (TMG+). The antibodies recognized other closely related type I IFN‐α subtypes, but rarely the distantly related type I IFN‐β, and never (detectably) the unrelated type II IFN‐γ. Antibodies to IL‐12 showed a similar distribution to those against IFN‐α2, although prevalences were slightly lower; correlations between individual titres against each were so modest that they appear to be entirely different specificities. Neither showed any obvious correlations with clinical parameters including thymoma histology and HLA type, but they did increase sharply if the tumours recurred. These antibodies neutralized their respective cytokine in bioassays in vitro; although they persisted for years severe infections were surprisingly uncommon, despite the immunosuppressive therapy also used in most cases. These findings must hold valuable clues to autoimmunizing mechanisms in paraneoplastic autoimmunity.

Cytokines in skin lesions of psoriasis

Cytokine levels were compared in aqueous extracts of stratum corneum from psoriatic lesions and normal heel. Samples from heel contained high levels of interleukin-1 alpha (IL-1 alpha) and beta measured in immunoassays, although only the IL-1 alpha was biologically active. No other cytokines could be detected in heel samples. Interleukin-1 (IL-1) levels were dramatically reduced in lesional samples. A neutrophil chemoattractant was found in all lesional extracts, and was demonstrated to be mainly interleukin-8 (IL-8) using a specific neutralizing antiserum. Tumor necrosis factor alpha (TNF-alpha) and beta (TNF-beta), and interferon alpha (IFN-alpha) and gamma (IFN-gamma) were detected in lesional extracts using immunoassays, however, no equivalent biological activities could be detected. Interleukins 2 (IL-2), 4 (IL-4), and 6 (IL-6), granulocyte and granulocyte/macrophage colony stimulating factor (GM-CSF), could not be detected in any samples. IL-8 is therefore the only biologically active cytokine shown in this study to be elevated in psoriatic lesional extracts, and may therefore play a role in the pathogenesis of the disease.

Strategies for detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals

An important aspect of evaluating the safety of therapeutic biologicals is the assessment of the unwanted immunogenicity of such biologicals in recipients. Properly planned immunogenicity studies with appropriately devised strategies are critical if valid conclusions concerning the unwanted immunogenicity are to be derived. Such studies need to be conducted using carefully selected and validated procedures. Several techniques are available for detection and measurement of immunogenicity including immunoassays, radioimmunoprecipitation assays (RIPAs), surface plasmon resonance (SPR) and bioassays. A combination of methods for characterization of the induced antibodies is usually necessary for a detailed understanding of the type(s) of antibodies generated against a therapeutic product. This review considers the benefits and limitations of the various techniques available for antibody detection and outlines a strategy for the assessment of unwanted immunogenicity of therapeutic products.

Detection and measurement of cytokines.

Accurate and sensitive methods for the measurement and detection of cytokines are an obvious pre-requisite for the study of cytokine biology, biochemistry and the possible involvement of these molecules in pathology. In this review, the various methods available for cytokine measurement and detection (bioassays, immunoassays and other procedures) are described and compared. A critical appraisal of the potential advantages and limitations of the techniques is included.

Cytokines in sera from insulin-dependent diabetic patients at diagnosis.

Cytokines are known to play an important role in autoimmunity and have been suggested to be involved in the pathogenesis of insulin‐dependent diabetes (IDDM). In the present study we have measured IL‐1, IL‐2, IL‐4, IL‐6, interferon‐gamma(IFN‐γ)and tumour necrosis factor (TNF) (using both immunoassays and bioassays) in sera from 50 patients affected by IDDM at the time of clinical diagnosis and 51 age and sex matched controls. Detectable levels of IL‐I, IL‐2, IL‐6 and IFN‐μgK were found in the serum of a small percentage of subjects and were not significantly different between patients and controls. IL‐4 was detectable in a higher number of both patients and controls and circulating TNF‐α (> I U/ml) was found in a percentage of patients (24%) significantly higher than controls (P<0.01). Raised levels of TNF‐α were detectable using an immunoenzymatic assay whereas TNF bioactivity in these samples was negligible. We conclude that the presence of immunoreactive TNF‐α in the patients sera may reflect an increased localized production of this cytokine at pancreatic level. However, the measurement in serum of other cytokines does not add information on the role that they may play in the pathogenesis of IDDM.

Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy

In this study, the development of neutralizing and non‐neutralizing GM‐CSF antibodies and the clinical consequences related to the induction of these antibodies were analysed in 20 patients with metastatic colorectal carcinoma receiving a combination therapy of Escherichia coli‐derived GM‐CSF and a colon carcinoma‐reactive MoAb in the absence of any concomitant chemotherapy. The recombinant human GM‐CSF was administered subcutaneously for 10 days every month for 4 months. Following the first cycle of treatment, no GM‐CSF antibodies were detected, but during subsequent therapy, 19 of the 20 patients studied developed GM‐CSF binding antibodies. However, only a proportion (40%) of the 19 antibody‐positive patients developed antibodies that neutralized the biological activity of GM‐CSF in an in vitro bioassay. The presence of GM‐CSF neutralizing antibodies was associated with a significant reduction in GM‐CSF‐induced expansion of leucocytes, neutrophils and eosinophils. Such clinical effects were not apparent in patients with non‐neutralizing antibodies. Further characterization of sera from patients with neutralizing antibodies showed that, in most cases, the antibodies neutralized the biological activity of GM‐CSF preparations derived using different expression systems (chinese hamster ovary cells and yeast), suggesting that these antibodies may have the potential to cross‐react with endogenously produced GM‐CSF. These effects should be considered before therapeutic use of cytokines, particularly in patients who are not immunosuppressed, and therefore capable of mounting an effective immune response. Our results indicate that assessment of production of neutralizing antibodies induced during cytokine therapy can be used to predict diminished clinical response to further therapy.

DEMONSTRATION OF CYTOKINES IN BIOLOGICAL MEDICINES PRODUCED IN MAMMALIAN CELL LINES

Commercially produced biological medicines may contain cytokines secreted by mammalian cell lines. Several such cell lines were found to produce interleukin 6 and, after stimulation, to secrete interleukin 1, tumour necrosis factor, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. High levels of interleukin 6 were detected in several vaccines and rDNA-derived proteins, and certain vaccines contained interleukin 1, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Some preparations of human monoclonal antibodies were also found to contain interleukin 1 and tumour necrosis factor. Cytokines may contribute to certain types of adverse reactions to these products.