A new cell enzyme-linked immunosorbent assay demonstrates gamma interferon suppression by beta interferon in multiple sclerosis

Abstract

Multiple sclerosis (MS) is a demyelinating disorder of the central nervous system of unknown etiology. Immune mechanisms involving the proinflammatory cytokine gamma interferon (IFN-gamma) are believed to play an important role in the pathogenesis of MS. IFN-beta-1b has been introduced as a treatment for MS and was found to reduce the number and severity of clinical exacerbations. To examine the influence of IFN-beta-1b on myelin basic protein (MBP)-specific and phytohemagglutinin-induced IFN-gamma production, we developed a cell-released capturing enzyme-linked immunosorbent assay (CRC-ELISA), which rapidly measures spontaneous and antigen- or mitogen-induced cellular IFN-gamma production. CRC-ELISA documented a significant MBP-specific T-cell response in the blood of untreated MS patients, as assessed by IFN-gamma production. This response was suppressed in MS patients treated with IFN-beta-1b. The present work confirms in vivo the in vitro suppressive effects of IFN-beta-1b on IFN-gamma production in MS. Moreover, it provides a powerful new technique for detection of cytokines.

FIG. 1

Scheme of CRC-ELISA for detection of cell-released cytokines (for example, IFN-γ, as in the present study). The method can detect cell-released cytokines produced in response to specific antigens or mitogen or produced spontaneously. ABC, avidin-biotin alkaline phosphatase-complex.

FIG. 2

rIFN-γ standard curve. The curve was designed from the absorbencies, obtained after capturing and detecting different known concentrations of rIFN-γ (see Materials and Methods), to high-binding microtiter plate wells precoated with anti-IFN-γ MAb. The absorbencies of the known IFN-γ concentrations were entered into a data set of a graphical computer software program as y-axis data with a linear scale. The IFN-γ units were entered into the same data set as x-axis data with a logarithmic scale. Into another data set, the absorbencies of the specimens were entered as y-axis data. Units corresponding to these absorbencies were measured from the standard curve and are shown as x-axis values. Dashed lines indicate the minimum and maximum units that the computer can detect from the assay’s standard curve. During the study more than 10 standard curves were made, and there was no significant variation among the curves.

FIG. 3

Comparison of the CRC-ELISA with a conventional ELISA of supernatants of mononuclear cell suspensions and with the ELISPOT assay. Suspensions of PBL from 10 MS patients were plated at 2 × 10⁵ cells per well and cultured for 48 h. Triplicate cultures were exposed to the optimal dilution of MBP or PHA. Control cultures received no stimulation. Data are the results of five different experiments. Error bars indicate standard deviations. MNC, mononuclear cells; SC, secreting cells.

FIG. 4

Effects of IFN-β-1b treatment on MBP-specific and PHA-induced IFN-γ production. Suspensions of PBL from 15 IFN-β-1b-treated and 29 untreated MS patients, 17 patients with OND, and 15 healthy controls (HC) were plated at a cell number of 2 × 10⁵ per well and cultured for 48 h. Triplicate cultures were exposed to the optimal dilution of MBP or PHA or left without stimulation. Note the lower IFN-γ production in response to MBP and PHA in IFN-β-1b-treated MS patients compared to production in untreated MS patients. Error bars indicate standard deviations.