Partial Biochemical Characterization and Purification of IgG2b Inducing Factor as a New Cytokine from Synovial Fluid of Patients with Rheumatoid Arthritis

Abstract

Rheumatoid arthritis synovial fluid (RA‐SF) contains a novel biological activity, which selectively induces IgG2b antibody production in lipopolysaccharide (LPS)‐activated mouse spleen cells in vitro and in vivo. Our previous studies have shown that this activity is not functionally identical to otherwell‐known cytokines and interleukins. In this study we demonstrate the partial purification and biochemical characterization of the IgG2b inducing activity in RA‐SF. Biochemical characterization revealed that the IgG2b inducing activity in RA‐SF has the following properties: it is a protein, sensitive to pH > 11 and < 4, which is precipitated by 50% of saturated ammonium sulphate and has a molecular weight of 50–70 kDa; it binds to Cibacron‐blue and heparin and its activity is not mediated by immunoglobulins or immune complexes, which are present in RA‐SF. Biochemical characteristics of the IgG2b inducing activity also differ from other cytokines and interleukins. The term IgG2b inducing factor is proposed for this novel activity.

Rheumatoid Inflammatory T‐Cell Clones express mostly Th1 but also Th2 and Mixed (Th0‐Like) Cytokine Patterns

“…This is in agreement with results obtained with T-cell clones isolated from the peripheral blood of normal individuals reacting against mycobacterial antigens and with clones from patients with Lyme arthritis or leprosy [4][5][6][7], This is also consistent with the involvement of Thl cells during delayed type hypersensitivity responses. A similar histological picture is also characteristic of RA synovitis, where the T-cell infiltrate is mainly composed of CD4"*" T cells [9] that are functionally T helper cells, and in addition monocyte/macrophages [8,10], Despite the intense infiltrate and signs of T-cell activation by expression of HLA-DR and VLA-1 antigens [27][28][29][30], detection of T-cell-derived cytokines has been difiicult in RA synovial fiuids [11][12][13][14][15][16]31], They have been shown in low levels that are in sharp contrast with the high production of proinfiammatory monocyte/macrophagederived cytokines. The high levels of IL-1, TNF-a and IL-6 have been directly implicated in joint destruction [32,33], The high amounts of these groups of cytokines have led to the hypothesis that the RA T cells might play a role in the hyperproduction of the proinfiammatory cytokines [32,33], Most of the clones produced high levels of IFN-y, This cytokine has been directly implicated in the initiation and the chronicity of autoimmune diseases and furthermore is a potent inducer of proinfiammatory cytokine production.…”

Synovial Fluid Cytokines in Patients with Rheumatoid Arthritus or Other Arthritic Lesions

“…It is a heparin-binding protein, sensitive to heat and extreme pH values. It has a molecular weight of 50-70 000 [15] and it is not identical to other known cytokines [16,17].…”

IgG2b Inducing Factor from Rheumatoid Arthritis Synovial Fluid Activates Antibody Production in Highly Purified Mouse B Lymphocytes

“…Unbound material containing the IgG2b inducing activity was collected and concentrated to about 1-2 ml by Centriprep-10 (Amicon, Danvers, MA, USA). This fraction was then loaded on a Cibacron-blue sepharose (Blue-Sepharose CL-6B, Pharmacia, Uppsala, Sweden) column, previously shown to bind albumin and interferons [38], and also IgG2bIF [10]. This column was equilibrated with the same buffer as above and bound proteins were eluted with 0.05 M Tris-HCl, 1.5 M NaCl pH 7.1, concentrated to 2.5 ml and desalted with RPMI-1640 by a PD-10 column (Pharmacia, Uppsala, Sweden).…”

“…Semi-purification of IgG2b inducing factor. The IgG2b inducing factor was partially purified from the RA-SF pool mentioned above by using affinity chromatography as described previously [10]. To remove IgG antibodies, 3 ml of pooled RA-SF was layered on a protein G sepharose (Protein G fast flow, Pharmacia, Uppsala, Sweden) column equilibrated with 0.05 M Tris-HCl, 0.1 M NaCl pH 7.1.…”

“…Previously, we have shown that RA-SF can replace T cells for pokeweed mitogen (PWM) stimulation of human B cells into antibody-secreting cells [8] and that RA-SF induces IgG2b antibody production in lipopolysaccharide (LPS) stimulated CBA murine spleen cells [9]. The IgG2b inducing factor (IgG2bIF) in RA-SF is a heparin-binding protein, sensitive to heat and extreme pH values and has a molecular weight of 50-70 kDa [10]. It is not identical to other known cytokines, now found in the joints of RA patients [11,12].…”