Regulatory B cells are reduced in the blood in patients with granulomatosis with polyangiitis, and fail to regulate T-cell IFN-γ production.

Regulatory B (Breg) cells can dampen inflammation, autoreactivity, and transplant rejection. We investigated the frequencies, phenotypes, and function of Breg cells in granulomatosis with polyangiitis (GPA) to gain further knowledge as to whether there are numerical alterations or limitations of their ability to regulate T-cell function. Frequencies and phenotypes of CD24hiCD27+ and CD24hiCD38hi B-cells in the blood were determined with flow cytometry in 37 GPA patients (22 in remission and 15 with active disease) and 31 healthy controls (HC). A co-culture model was used to study the capacity of Breg cells to regulate T-cell activation and proliferation in cells from 10 GPA patients in remission and 12 HC. T-cell cytokine production in vitro and levels in plasma were determined with enzyme-linked immunosorbent assay. Frequencies of CD24hiCD27+ B-cells were reduced both during active disease and remission compared with HC (P = 0.005 and P = 0.010, respectively), whereas CD24hiCD38hi B-cells did not differ. Patient CD24hiCD27+ B-cells exhibited decreased expression of CD25 but increased expression of PD-L1 and PD-L2 during remission. B-cells from GPA patients regulated T-cell proliferation but failed to regulate interferon (IFN)-γ production (median T-cells alone 222 ng/ml vs. T-cells + B-cells 207 ng/ml, P = 0.426). IFN-γ was also elevated in patient plasma samples (P = 0.016). In conclusion, GPA patients exhibit altered numbers and phenotypes of CD24hiCD27+ B-cells. This is accompanied by a disability to control T-cell production of Th1-type cytokines during remission, which might be of fundamental importance for the granulomatous inflammation that characterizes the chronic phase of this disease.

Sizing Up Extracellular DNA: Instant Chromatin Discharge From Cells When Placed in Serum-Free Conditions.

How do you wash cells? Three out of four of our colleagues use experimental procedures during everyday lab-bench work that can severely impair data interpretation depending on how cells are handled. We show here that a subpopulation (2-3%) of human leukocytes immediately induce a yet unclassified lytic cell death, concomitant with discharge of chromatin entities and cell elimination, when placed in protein-free solutions (i.e., PBS and HBSS). DNA release was not restricted to hematopoietic cells but occurred also in HEK293T cells. Albumin, fetal bovine serum, polyethylene glycol, and Pluronic F-68 supplements prevented chromatin discharge. Expelled chromatin was devoid of surrounding membranes but maintained its original nuclear shape, although ∼10 times enlarged. These structures differed from DNA appearance after osmotic or detergent-induced cell lysis. Besides sounding a cautionary note to the neutrophil extracellular trap (NET) research community, in which ∼50% of all published studies used protein-free media for NET-formation, our study also provides a rapid tool for analysis of chromatin organization.

Altered IL-32 Signaling in Abdominal Aortic Aneurysm.

INTRODUCTION AND OBJECTIVE: Interleukin (IL)-32 is a pro-inflammatory cytokine not previously studied in relation to abdominal aortic aneurysm (AAA). The aim of this study was to elucidate the expression and localization of IL-32 in AAA. METHODS: Expression and localization of IL-32 in human aortic tissue was studied with immunohistochemical analysis and Western blot (AAA: n = 5; controls: n = 4). ELISA was used to measure IL-32 in human plasma samples (AAA: n = 140; controls: n = 37) and in media from cultured peripheral blood mononuclear cells (PBMCs) from 3 healthy donors. IL-32 mRNA in PBMCs, endothelial cells, aortic smooth muscle cells (SMCs), and aortic tissue samples of AAA (n = 16) and control aortas (n = 9) was measured with qPCR. RESULTS: IL-32 was predominantly expressed in SMCs and T-cell-rich areas. Highest mRNA expression was observed in the intima/media layer of the AAA. A weaker protein expression was detected in non-aneurysmal aortas. Expression of IL-32 was confirmed in isolated T cells, macrophages, endothelial cells, and SMCs, where expression was also inducible by cytokines such as interferon-γ. There was no difference in IL-32 expression in plasma between patients and controls. CONCLUSION: IL-32 signaling is altered locally in AAA and could potentially play an important role in aneurysm development. Further studies using animal models would be helpful to study its potential role in AAA disease.

Marginal-Zone B-Cells Are Main Producers of IgM in Humans, and Are Reduced in Patients With Autoimmune Vasculitis.

In mice, B1 and marginal zone (MZ) B-cells play an important role in prevention of autoimmunity through production of regulatory cytokines and natural antibodies. There is limited knowledge about the human counterparts of these cells. We therefore investigated functions of MZ-like B-cells and the frequency of circulating MZ-like and B1-like B-cells in healthy controls (HC), as well as in patients with autoimmune vasculitis to learn more about the role of these cells in autoimmune disease. After stimulation with CpG oligodeoxynucleotides (ODN) of class B in vitro, MZ-like B-cells were the main producers of IgM whereas switched memory B-cells primarily produced IgG and IgA. TNF and IL-10 were produced by both MZ-like and switched memory B-cells. Neither antibody nor TNF/IL-10 production by the B-cell subsets differed between patients and HC. Patients with autoimmune vasculitis, irrespective of disease activity, had lower percentage and absolute numbers of circulating MZ-like B-cells, and lower absolute numbers of B1-like B-cells. The percentage of B1-like B-cells was reduced during active disease. These findings remained significant when the analysis was confined to active treatment-naïve patients (disease onset).Our results suggest that human innate-like B-cells might have a physiological role in prevention of autoimmunity.