Interferon-α induces altered transitional B cell signaling and function in Systemic Lupus Erythematosus.

Previous studies suggest that the B cells of patients with Systemic Lupus Erythematosus (SLE) are hyper-responsive to BCR crosslinking; however, it has been unclear whether this is the result of altered B cell signaling or differences in various B cell subpopulations in SLE patients as compared to healthy controls. Here we have developed a novel Phosflow technique that permits examination of cell signaling in distinct B cell subpopulations stratified based upon developmental stage and cell surface IgM levels, which we use to show that the naïve B cells of SLE patients are hyper-responsive to IgM receptor crosslinking, resulting in increased SYK phosphorylation. We further demonstrate that this hyper-responsiveness is most marked in the transitional B cell subset and that it is associated with altered function, resulting in decreased apoptosis and increased proliferation of these cells. Examination of repeated samples from the same patients revealed that the hyper-responsiveness fluctuated over time, suggesting that it may be mediated by pro-inflammatory factors rather than genetic variations between patients. In support of this concept, incubation of healthy control B cells with IFN-α or SLE plasma induced the hyper-responsive phenotype, which was blocked by anti-IFN-α antibody. Furthermore, no obvious correlation was seen between genetic variants that are proposed to alter BCR signaling and the increased SYK phosphorylation. The findings suggest that pro-inflammatory factors, in particular Type I IFNs, modulate B cell function in SLE in a way that could contribute to the breach of tolerance in this condition.

Epistatic suppression of fatal autoimmunity in New Zealand black bicongenic mice.

Numerous mapping studies have implicated genetic intervals from lupus-prone New Zealand Black (NZB) chromosomes 1 and 4 as contributing to lupus pathogenesis. By introgressing NZB chromosomal intervals onto a non-lupus-prone B6 background, we determined that: NZB chromosome 1 congenic mice (denoted B6.NZBc1) developed fatal autoimmune-mediated kidney disease, and NZB chromosome 4 congenic mice (denoted B6.NZBc4) exhibited a marked expansion of B1a and NKT cells in the surprising absence of autoimmunity. In this study, we sought to examine whether epistatic interactions between these two loci would affect lupus autoimmunity by generating bicongenic mice that carry both NZB chromosomal intervals. Compared with B6.NZBc1 mice, bicongenic mice demonstrated significantly decreased mortality, kidney disease, Th1-biased IgG autoantibody isotypes, and differentiation of IFN-γ-producing T cells. Furthermore, a subset of bicongenic mice exhibited a paucity of CD21(+)CD1d(+) B cells and an altered NKT cell activation profile that correlated with greater disease inhibition. Thus, NZBc4 contains suppressive epistatic modifiers that appear to inhibit the development of fatal NZBc1 autoimmunity by promoting a shift away from a proinflammatory cytokine profile, which in some mice may involve NKT cells.

Anti-dsDNA and Antichromatin Antibody Isotypes in Serologically Active Clinically Quiescent Systemic Lupus Erythematosus.

OBJECTIVE: Serologically active clinically quiescent (SACQ) patients with systemic lupus erythematosus (SLE) are clinically quiescent despite serologic activity. Since studies suggest that antichromatin antibodies are more sensitive than anti-dsDNA antibodies in detecting active SLE, and that immunoglobulin (Ig) G, in particular complement-fixing subclasses, may be more pathogenic than IgM, we investigated the levels of anti-dsDNA and antichromatin isotypes in SACQ patients as compared to non-SACQ patients with SLE. METHODS: Levels of IgM, IgA, IgG, and IgG1-4 antichromatin and anti-dsDNA were measured by ELISA. SACQ was defined as ≥ 2 years with the SLE Disease Activity Index 2000 (SLEDAI-2K) at 2 or 4 from serologic activity, during which patients could be taking antimalarials, but not corticosteroids or immunosuppressives. Unselected non-SACQ patients with SLE were used as comparators. SACQ patient serum samples were further stratified based on subsequent development of flare, defined as clinical SLEDAI-2K ≥ 1 and/or treatment initiation. Nonparametric statistics were used, and generalized estimating equations were applied to account for multiple samples in the same patient. RESULTS: SACQ patients' complement-fixing antichromatin and anti-dsDNA IgG subclasses were significantly higher than those of non-SACQ patients. When the sample drawn latest in a SACQ period was analyzed, there was no difference between antichromatin or anti-dsDNA isotype or IgG subclass levels between patients who flared and those who remained SACQ, nor were consistent trends seen when samples were examined during SACQ and flare in the same patient. CONCLUSION: The SACQ phenotype does not arise from a lack of pathogenic anti-dsDNA and/or antichromatin autoantibodies. Neither increases in antichromatin nor anti-dsDNA isotype or IgG subclass levels were predictive of or coincident with flare in SACQ patients.

Dual regulation of lysophosphatidic acid (LPA1) receptor signalling by Ral and GRK.

Lysophosphatidic acid (LPA) is a major constituent of blood and is involved in a variety of physiological and pathophysiological processes. LPA signals via the ubiquitously expressed G protein-coupled receptors (GPCRs), LPA(1) and LPA(2) that are specific for LPA. However, in large, the molecular mechanisms that regulate the signalling of these receptors are unknown. We show that the small GTPase RalA associates with both LPA(1) and LPA(2) in human embryonic kidney (HEK 293) cells and that stimulation of LPA(1) receptors with LPA triggers the activation of RalA. While RalA was not found to play a role in the endocytosis of LPA receptors, we reveal that LPA(1) receptor stimulation promoted Ral-dependent phospholipase C activity. Furthermore, we found that GRK2 is required for the desensitization of LPA(1) and LPA(2) and have identified a novel interaction between RalA and GRK2, which is promoted by LPA(1) receptor activity. Taken together, these results establish RalA and GRK2 as key regulators of LPA receptor signalling and demonstrate for the first time that LPA(1) activity facilitates the formation of a novel protein complex between these two proteins.

Beta-arrestin/Ral signaling regulates lysophosphatidic acid-mediated migration and invasion of human breast tumor cells.

The lipid mediator lysophosphatidic acid (LPA) plays a role in cancer progression and signals via specific G protein-coupled receptors, LPA(1-3). LPA has been shown to enhance the metastasis of breast carcinoma cells to bone. However, the mechanisms by which LPA receptors regulate breast cancer cell migration and invasion remain unclear. Breast cancer cell proliferation has been shown to be stimulated by Ral GTPases, a member of the Ras superfamily. Ral activity can be regulated by the multifunctional protein beta-arrestin. We now show that HS578T and MDA-MB-231 breast cancer cells and MDA-MB-435 melanoma cells have higher expression of beta-arrestin 1 mRNA compared with the nontumorigenic mammary MCF-10A cells. Moreover, we found that the mRNA levels of LPA1, LPA2, beta-arrestin 2, and Ral GTPases are elevated in the advanced stages of breast cancer. LPA stimulates the migration and invasion of MDA-MB-231 cells, but not of MCF-10A cells, and this is mediated by pertussis toxin-sensitive G proteins and LPA1. However, ectopic expression of LPA1 in MCF-10A cells caused these cells to acquire an invasive phenotype. Gene knockdown of either beta-arrestin or Ral proteins significantly impaired LPA-stimulated migration and invasion. Thus, our data show a novel role for beta-arrestin/Ral signaling in mediating LPA-induced breast cancer cell migration and invasion, two important processes in metastasis.