Multi-component immune knockout: A strategy for studying the effective components of traditional Chinese medicine

Rheumatoid arthritis is an autoimmune disease characterized by the production of two known antibodies - rheumatoid factor and anti-citrullinated peptide antibody (ACPA) - against common autoantigens that are widely expressed within and outside the joints. The interactions between genes and environment are crucial in all stages of the disease, involving namely genes from major histocompatibility complex locus, and antigens such as tobacco or microbes (e.g. Porphyromonas gingivalis). T and B cells are activated as soon as the earliest phases of the disease, rheumatoid arthritis appearing as a Th1 and Th17 disease. Inflammatory cytokines have a considerable importance in the hierarchy of the processes involved in RA. The joint destruction seen in RA is caused not only by cytokine imbalances, but also by specific effects of the Wnt system and osteoprotegerin on osteoclasts and by matrix production dysregulation responsible for cartilage damage. Both innate and adaptative immunity demonstrated their respective cornerstone position in rheumatoid arthritis, since targeted treatments has been efficiently developed against TNF-α, IL-6 receptor, IL-1β, CD20 B cells and T-cell/Dendritic cell interactions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters. Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure lymphocyte proliferation by flow cytometry has become one of the most widely utilised assays for assessing lymphocyte responses. The properties of CFSE make it ideal for such a task, covalently labelling cells with a long-lived fluorescence of high intensity and low variance with minimal cell toxicity. No dye in the last 20 years has been capable of replicating CFSE in these respects. However, currently CFSE is limited to following a maximum of 7 cell divisions and is not compatible for use with ubiquitously available fluorescein conjugates or other fluorescent molecules with spectral properties similar to fluorescein, such as EGFP. Here we characterise two new fluorescent dyes for measuring lymphocyte proliferation, Cell Trace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD), which have different excitation and emission spectra to CFSE and, consequently, are compatible with fluorescein conjugates. We found that while both CTV and CPD can label cells to a high fluorescence intensity, which is long-lived and has low variability and low toxicity and makes them ideal for long-term tracking of non-dividing lymphocytes in vivo, CTV offers possibly the best available alternative to CFSE in the analysis of cell divisions. We also describe how intercellular dye transfer and cell autofluorescence can affect division resolution with the three different dyes and describe labelling conditions for the three dyes that produce ultra-bright lymphocytes for in vivo tracking studies and allow up to 11 cell divisions to be detected when using CFSE and CTV as the fluorescent dyes. Copyright © 2012 Elsevier B.V. All rights reserved.