The immune system contains natural regulatory cells important in the maintenance of tolerance. Although this suppressive function is usually attributed to CD4 regulatory T cells, recent reports have revealed an immunoregulatory role for IL-10-producing B cells in the context of several autoimmune diseases including collagen-induced arthritis. In the present study, we attribute this suppressive function to a B cell subset expressing high levels of CD21, CD23, and IgM, previously identified as transitional 2-marginal zone precursor (T2-MZP) B cells. T2-MZP B cells are present in the spleens of naive mice and increase during the remission phase of arthritis. Following adoptive transfer to immunized DBA/1 mice, T2-MZP B cells significantly prevented new disease and ameliorated established disease. The suppressive effect on arthritis was paralleled by an inhibition of Ag-specific T cell activation and a reduction in cells exhibiting Th1-type functional responses. We also provide evidence that this regulatory subset mediates its suppression through the secretion of suppressive cytokines and not by cell-to-cell contact. The ability to regulate an established immune response by T2-MZP B cells endows this subset of B cells with a striking and previously unrecognized immunoregulatory potential.

We have previously reported that IL10+ regulatory B cells, known to play an important role in controlling autoimmunity and inflammatory disorders, are contained within the Transitional-2 immature (T2) B cell pool (T2Bregs). Therapeutic strategies facilitating their enrichment or enhancing their suppressive activity are highly attractive. Here we report that agonistic anti-CD40 specifically targets T2 B cells and enriches B regs upon short term in vitro culture. Whilst transfer of unmanipulated T2 B cells, isolated from mice with established lupus, failed to confer protection to diseased mice, transfer of in vitro anti-CD40-generated T2 B cells (T2-like-Bregs) significantly improved renal disease and survival by an IL-10-dependent mechanism. T2-like-Bregs readily accumulated in the spleen after transfer, suppressed Th1 responses, induced the differentiation of IL-10+CD4+T cells and conveyed regulatory effect to CD4+T cells. In addition, in vivo administration of agonistic anti-CD40, currently on trial for the treatment of cancer, halted and reversed established lupus. Taken together our results suggest a novel cellular approach for the amelioration of experimental lupus.

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Atherosclerosis is considered a chronic inflammatory disease in which monocytes and macrophages are critical. These cells express CD14, toll-like receptor (TLR) 2, and TLR4 on their surfaces, are activated by minimally modified low-density lipoprotein (mmLDL) and are capable of secreting pro-inflammatory cytokines. The aim of this research was thus to demonstrate that the activation of CD14, TLR2, and TLR4 by mmLDL induces the secretion of cytokines.MethodsHuman monocytes and macrophages were incubated with monoclonal antibodies specific for CD14, TLR4, and TLR2 prior to stimulation with mmLDL. Cytokine secretion was then compared to that observed upon mmLDL stimulation in untreated cells.ResultsStimulation with mmLDL induced the secretion of pro-inflammatory cytokines. Blocking CD14 in monocytes inhibited secretion of interleukin (IL)-1β (72%), IL-6 (58%) and IL-10 (63%), and blocking TLR4 inhibited secretion of IL-1β by 67%, IL-6 by 63% and IL-10 by 60%. Blocking both receptors inhibited secretion of IL-1β by 73%, IL-6 by 69% and IL-10 by 63%. Furthermore, blocking TLR2 inhibited secretion of IL-1β by 65%, IL-6 by 62% and IL-10 by 75%. In macrophages, we found similar results: blocking CD14 inhibited secretion of IL-1β by 59%, IL-6 by 52% and IL-10 by 65%; blocking TLR4 inhibited secretion of IL-1β by 53%, IL-6 by 63% and IL-10 by 61%; and blocking both receptors inhibited secretion of IL-1β by 69%, IL-6 by 67% and IL-10 by 65%. Blocking TLR2 in macrophages inhibited secretion of IL-1β by 57%, IL-6 by 40% and IL-10 by 72%.ConclusionOur study demonstrates that CD14, TLR4, and TLR2 participate in the immune response against mmLDL by inducing the production of pro-inflammatory cytokines in both monocytes and macrophages. These findings suggest that the activation of these receptors by mmLDL contributes to the inflammatory process of atherosclerosis.

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The objective of this study was to determine the diagnostic performance of the percentage of serum prolactin (PRL) precipitated with polyethylene glycol (PEG) for the detection of macroprolactinemia in systemic lupus erythematosus (SLE) patients with hyperprolactinemia. Serum samples from SLE patients were examined. Serum PRL was measured by immunoradiometric assay (IRMA) and samples with hyperprolactinemia (> 20 ng/ml) were submitted to PEG precipitation, gel filtration chromatography and affinity chromatography with protein-G sepharose. A comparative survey was used. Among 259 consecutive serum samples from SLE patients, PRL was > 20.1 ng/ml in 43 samples (16.6%). Gel filtration showed a predominant pattern of macroprolactinemia (> 100 kDa) in 14 (32.6%), a predominant pattern of monomeric PRL (23 kDa) in 27 (62.7%), and a variable pattern in two (4.7%). All sera with a predominant pattern of macroprolactinemia displayed anti-PRL autoantibodies by affinity chromatography for IgG. The best cut-off point for percentage of serum PRL precipitated with PEG for detection of macroprolactinemia was > or = 58.4%. Sensitivity and specificity were 100 and 96.6%, respectively. We can conclude that PEG precipitation is a convenient and simple procedure to screen for the presence of macroprolactinemia in sera from SLE patients. Precipitations > or = 58.4% are indicative of the presence of, and those < 50% the absence of, macroprolactinemia. However, samples with precipitations between 50 and 58.3% require gel filtration chromatography to characterize the predominant molecular form of PRL. Therefore, it is important to take these findings into account in future studies that aim to establish a relationship between PRL and disease activity in SLE.