Most B cells in non-lymphoid tissues are naïve

The current view of lymphocyte migration states that na?ve lymphocytes re-circulate between the blood and the lymph via the lymph nodes, but are not able to access non-lymphoid tissues. We examined B lymphocytes in peripheral tissues and found that the majority were phenotypically similar to na?ve B cells in lymphoid tissues and were located within the parenchyma, not associated with blood vessels. The mutation rate within the Vh region of these cells was substantially less than the rate attributed to somatic hypermutation and was identical to that observed in na?ve B cells isolated from the lymph nodes, showing the presence of na?ve B cells in the non-lymphoid organs. Further, using FTY720-treated mice, we showed that na?ve B cells migrate through the peripheral tissues and, using pertussis toxin, that the entry of B cells was not controlled by chemokine-mediated signalling events. Overall, these results show that na?ve B lymphocytes constitute the majority of the total B-cell population in non-lymphoid tissues and suggest that these cells may re-circulate through the periphery as part of their normal migration pathway. This has implications for the current view of the role of na?ve B cells in priming and tolerance.     

Inducible resistance to Fas—mediated apoptosis in B cells

Apoptosis produced in B cells through Fas(APO-1,CD95) triggering is regulated by signals derived from other surface receptors:CD40 engagement produces upregulation of Fas expression and marked susceptibility to Fas-induced cell death,whereas antigen receptor engagement,or IL-4R engagement,inhibits Fas killing and in so doing induces a state of Fas-resistance,even in otherwise sensitive,CD40-stimulated targets.Surface immunoglobulin and IL-4R utilize at least partially distinct path ways to produce Fas-resistance that differentially depend on PKC and STAT6,respectively.Further,surface immunoglobulin signaling for inducible Fas-resistance bypasses Btk,requires NF-κB,and entails new macromolecular synthesis.Terminal effectors of B cell Fas-resistance include the known anti-apoptotic gene products,Bcl-XL and FLIP,and a novel anti-apoptotic gene that encodes FAIM (Fas Apoptosis Inhibitory Molecule).faim was identified by differential display and exists in two alternatively spliced forms;faim-S is broadly expressed,but faim-L expression is tissue-specific.The FAIM sequence is highly evolu tionarily conserved,suggesting an important role for this molecule throughout phylogeny.Inducible resistance to Fas killing is hypothesized to protect foreign antigen-specific B cells during potentially hazardous interactions with FasL-bearing T cells,whereas autoreactive B cells fail to become Fas-resistant and are deleted via Fas-dependent cytotoxicity.Inadvertent or aberrant acquisition of Fas-resistance may permit autoreactive B cells to escape Fas deletion,and malignant lymphocytes to impede anti-tumor immunity.     

Human IgM+CD27+ B cells: memory B cells or "memory" B cells?

Memory B cells are generated in germinal centers (GC) and contribute to serological immunity by rapidly differentiating into plasma cells. Human memory B cells can be identified by the expression of CD27. These cells exhibit more rapid responses than naive (CD27-) B cells following stimulation in vitro, consistent with the heightened kinetics of secondary responses in vivo. CD27+ B cells express mutated Ig V region genes; however a significant proportion continue to express IgM, suggesting the existence of IgM+ memory B cells. The observation that mutated IgM+CD27+ B cells are generated in humans who cannot form GC led to the conclusions that these cells are generated independently of GC and thus are not memory cells and that they mediate responses to T cell-independent Ag. Although some studies support the idea that IgM+CD27+ B cells participate in T cell-independent responses, many others do not. In this review we will provide alternate interpretations of the biology of IgM+CD27+ B cells and propose that they are indeed memory cells.     

Intracellular fate of fibrinogen Bβ chain expressed in COS cells

Full-length fibrinogen Bβ cDNA was subcloned into an expression vector, pBC12BI, and transfected into COS cells. Bβ chain expression was measured by pulse-labelling cells with l-[³⁵S]methionine, immunoprecipitating the Bβ chain with antibody to fibrinogen and separating the nascent radioactive protein by sodium dodecyl sulfate-pholyacrylamine gel electrophoresis (SDS-PAGE). Bβ chain was expressed in transfected COS cells but was not secreted into the medium. Treatment with endoglycosidase H showed that non-secreted Bβ chain contains mannose-rich carbohydrates rather than the complex form of carbohydrate which occurs in plasma fibrinogen and indicates that Bβ chain is not transported to the Golgi apparatus. In transfected COS cells, anitibody to fibrinogen co-immunoprecipitated Bβ chain and 78 kDa immunoglobulin-binding protein (BiP) and antibody to BiP immunoprecipitated BiP and nascent Bβ chains. Non-secreted Bβ chain was degraded intracellularly with a half-life of 5 h by enzymes which were not affected by incubating transfected cells with NH₄Cl, which indicates a non-lysosomal pathway of degradation. These studies indicate that Bβ chain by itself does not contain the signal for fibrinogen secretion and that non-secreted Bβ chain is associated with BiP and degraded in the rough endoplasmic reticulum.     

NF-κB activation in myeloid cells mediates ventilator-induced lung injury

Background

Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated.

Methods

To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKβ^△mye), IL-6^-/- to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI.

Results

Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1β, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKβ^△mye mice as well as in IL6^-/- to WT chimeric mice.

Conclusion

Given that IKKβ^△mye mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB–IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.     

Soluble HLA-G induces NF–кB activation in natural killer cells

Human leukocyte antigen (HLA)-G is an immunomodulatory molecule discovered for the first time in the maternal–fetal interface. In cancer context, where high number of natural killer (NK) cells is described, the presence of HLA-G in its soluble form is thought to be essential for NK cells signaling. To evaluate intracellular signaling in NK cells upon HLA-G soluble forms stimulation, we investigate the role of soluble HLA-G (HLA-G5- and HLA-G1 shedding form) stimulation on classical nuclear factor (NF)–κB pathway activation. We reported that these two forms of soluble HLA-G could activate NF–κB in NK cells. NF–κB activation in NK cells does implicate neither phosphatidylinositol 3-kinase (PI3K) nor MEK (MAP kinase kinase) as demonstrated after specific inhibition experiments. We demonstrated elsewhere that NF–κB activation in NK cells is not implicated in cytotoxicity inhibition by HLA-G. Our findings may suggest the important role played by NF–κB activation after soluble HLA-G stimulation in other NK cells function.     

B cells biology in systemic lupus erythematosus—from bench to bedside

Systemic lupus erythematosus(SLE) is a debilitating autoimmune disease that can involve multi-organs. B cells play a central role in the immunopathogenesis via antibody-dependent and antibody-independent ways. Excessive autoantibodies production, hyperresponsiveness and prolonged survival of autoreactive B cells are characteristics of SLE. In this article, mechanisms of self-tolerance loss of B cells and promising B cell-targeting therapies in lupus are reviewed and discussed.     

1,25-dihydroxyvitamin D3 impairs NF-κB activation in human naïve B cells

1α,25-dihydroxyvitamin D(3) (calcitriol), the bioactive metabolite of vitamin D, modulates the activation and inhibits IgE production of anti-CD40 and IL-4 stimulated human peripheral B cells. Engagement of CD40 results in NF-κB p50 activation, which is essential for the class switch to IgE. Herein, we investigated by which mechanism calcitriol modulates NF-κB mediated activation of human na?ve B cells. Na?ve B cells were predominantly targeted by calcitriol in comparison with memory B cells as shown by pronounced induction of the VDR target gene cyp24a1. Vitamin D receptor activation resulted in a strongly reduced p105/p50 protein and mRNA expression in human na?ve B cells. This effect is mediated by impaired nuclear translocation of p65 and consequently reduced binding of p65 to its binding site in the p105 promoter. Our data indicate that the vitamin D receptor reduces NF-κB activation by interference with NF-κB p65 and p105. Thus, the vitamin D receptor inhibits costimulatory signal transduction in na?ve B cells, namely by reducing CD40 signaling.     

Retigeric acid B exhibits antitumor activity through suppression of nuclear factor-κB signaling in prostate cancer cells in vitro and in vivo

Previously, we reported that retigeric acid B (RB), a natural pentacyclic triterpenic acid isolated from lichen, inhibited cell growth and induced apoptosis in androgen-independent prostate cancer (PCa) cells. However, the mechanism of action of RB remains unclear. In this study, we found that using PC3 and DU145 cells as models, RB inhibited phosphorylation levels of IκBα and p65 subunit of NF-κB in a time- and dosage-dependent manner. Detailed study revealed that RB blocked the nuclear translocation of p65 and its DNA binding activity, which correlated with suppression of NF-κB-regulated proteins including Bcl-2, Bcl-x(L), cyclin D1 and survivin. NF-κB reporter assay suggested that RB was able to inhibit both constitutive activated-NF-κB and LPS (lipopolysaccharide)-induced activation of NF-κB. Overexpression of RelA/p65 rescued RB-induced cell death, while knockdown of RelA/p65 significantly promoted RB-mediated inhibitory effect on cell proliferation, suggesting the crucial involvement of NF-κB pathway in this event. We further analyzed antitumor activity of RB in in vivo study. In C57BL/6 mice carrying RM-1 homografts, RB inhibited tumor growth and triggered apoptosis mainly through suppressing NF-κB activity in tumor tissues. Additionally, DNA microarray data revealed global changes in the gene expression associated with cell proliferation, apoptosis, invasion and metastasis in response to RB treatment. Therefore, our findings suggested that RB exerted its anti-tumor effect by targeting the NF-κB pathway in PCa cells, and this could be a general mechanism for the anti-tumor effect of RB in other types of cancers as well.     

Co-operative effects of angiotensin II and caerulein in NFκB activation in pancreatic acinar cells in vitro

Angiotensin II is a vasoactive peptide that controls blood pressure and homeostasis. Emerging evidence shows that locally generated angiotensin II plays a crucial role in normal physiology, as well as pathophysiological conditions such as pancreatitis. We recently reported that angiotensin II activates pancreatic NFκB in obstructive pancreatitis. However, the specific cell type responsible for this activation remains unclear. In this study, we investigated whether pancreatic acinar cells respond to angiotensin II. These cells are the most abundant pancreatic cells and the most vulnerable to pancreatitis. Pancreatic acinar AR42J cells were used as an in vitro model of pancreatic inflammation. Our results demonstrated that treatment with caerulein, a cholecystokinin receptor agonist, induced hypersecretion and NFκB activation, as demonstrated by elevated amylase secretion and degradation of inhibitor of NFκB (IκBβ). Angiotensin II, either alone or in combination with caerulein, augmented IκBβ degradation. Pre-treatment with losartan, an antagonist of the angiotensin type I (AT₁) receptor, abolished NFκB activation by angiotensin II and caerulein in a dose-dependent manner. Treatment with PD123319, a blocker of the angiotensin type II (AT₂) receptor, enhanced the activation of NFκB by angiotensin II and caerulein. Preliminary data further demonstrated that angiotensin II could extend caerulein-induced ERK1/2 activation in acinar cells. These results indicated that inflammation triggered by hyperstimulation of pancreatic acinar cells is enhanced by angiotensin II, via the AT₁ receptor. In contrast, stimulation of the AT₂ receptor protects against caerulein-induced NFκB activation. The differential roles of the AT₁ and AT₂ receptors might be useful in developing potential therapies for pancreatic inflammation.     

Can resting B cells present antigen to T cells?

Antigen stimulation of T lymphocytes can occur only in the presence of an antigen-presenting cell (APC). An ever-increasing number of cell types have been found to act as APCs; these include macrophages, splenic and lymph node dendritic cells, and Langerhans' cells of the skin. Although activated B lymphocytes and B cell lymphomas are known to serve as APCs, it has been generally believed that resting B cells cannot perform this function. However, in recent studies we have found that resting B cells can indeed present soluble antigen to T cell clones as well as to antigen-primed T cells. The previous difficulty in demonstrating this activity can be explained by the finding that, in contrast to macrophages and dendritic cells, the antigen-presenting ability of resting B cells is very radiosensitive. Macrophages are usually irradiated with 2000-3300 rads to prevent them from incorporating [3H]thymidine in the T cell proliferation assay. Resting B cells, however, begin to lose presenting function at 1500 rads and have completely lost this activity at 3300 rads. It was also possible to distinguish two distinct T cell clonal phenotypes when resting B cells were used as APCs on the basis of two different assays (T cell proliferation, and B cell proliferation resulting from T cell activation). The majority of T cell clones tested were capable of both proliferating themselves and inducing the proliferation of B cells. Some T cells clones, however, could not proliferate in the presence of antigen and B cell APCs, although they were very good at inducing the proliferation of B cells. This suggests that there are two distinct pathways of T cell activation, one leading to T cell proliferation and the other leading only to the release of lymphokines (as measured by the polyclonal activation of B cells).     

Surface expression of Hsp70B’ in response to proteasome inhibition in human colon cells

Hsp70B’ was expressed on the surface of HT-29 and CRL-1809 but not SW-480 human colon cell lines in response to proteasome inhibition as detected using flow cytometry. Surface expression was not detected under non-stress conditions nor was heat shock an inducer of surface expression in the three cell lines tested. Phylogenetic analysis indicated that the Hsp70B’ protein sequence was most closely related to another major inducible human Hsp70, Hsp72. Hsp70B’ appeared to be recently diverged, as homologs for Hsp70B’ have not been found in rodents. Hsp72 and Hsp70B’ shared 100% amino acid sequence identity in their predicted peptide-binding regions suggesting that they bind the same peptide substrates, perhaps in extracellular antigen presentation. Amino acid sequence differences were concentrated in the lid regions and the C-terminal domains raising the possibility that Hsp72 and Hsp70B’ bind different co-chaperones or cell surface receptors.     

B cells as a therapeutic target for IFN-β in relapsing-remitting multiple sclerosis

IFN-β-1b is a first-line immunomodulatory therapy for relapsing-remitting multiple sclerosis (RR MS). However, its effects on B cells have not been characterized. In vitro studies of B cells derived from RR MS patients revealed that IFN-β-1b decreases B cells' stimulatory capacity, as detected by inhibition of the Ag-specific T cell proliferative response upon Ag presentation by IFN-β-1b-treated B cells. Our study has identified that IFN-β-1b inhibited B cells' stimulatory capacity in RR MS patients and healthy controls through the suppression of CD40 and CD80 expression, whereas the MHC class I and II expression was not changed. IFN-β-1b in vitro treatment inhibited B cell secretion of IL-1β and IL-23 and induced IL-12 and IL-27. Supernatants transferred from IFN-β-1b-treated B cells inhibited Th17 cell differentiation, as they suppressed gene expression of the retinoic acid-related orphan nuclear hormone receptor C and IL-17A and secretion of IL-17A. In addition, IFN-β-1b induced B cells' IL-10 secretion, which may mediate their regulatory effect. Studies of B cells derived from RR MS patients treated with recombinant s.c. injected IFN-β-1b revealed that they induced a significantly lower proliferative response in allogenic MLR than the B cells from untreated patients. Further confirming the IFN-β-1b in vitro-induced changes in B cell cytokine secretion, B cells derived from the IFN-β-1b-treated patients secreted significantly lower levels of IL-1β and IL-23 and higher levels of IL-12 and IL-27 in comparison with the B cells derived from untreated patients. We conclude that IFN-β-1b exerts its therapeutic effects in part by targeting B cells' functions that contribute to the autoimmune pathogenesis of RR MS.     

Differential expression of FCRLA in naïve and activated mouse B cells

FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.     

Opposing roles for TRAF1 in the alternative versus classical NF-κB pathway in T cells

T cells lacking TRAF1 hyperproliferate in response to T cell receptor signaling but have impaired signaling downstream of specific TNFR family members such as 4-1BB. Here we resolve this paradox by showing that while TRAF1 is required for maximal activation of the classical NF-κB pathway downstream of 4-1BB in primary T cells, TRAF1 also restricts the constitutive activation of NIK in anti-CD3-activated T cells. Activation of the alternative NF-κB pathway is restricted in unstimulated cells by a cIAP1/2:TRAF2:TRAF3:NIK complex. Using knockdown of NIK by siRNA we show that in activated CD8 T cells TRAF1 is also involved in this process and that constitutive activation of the alternative NF-κB pathway is responsible for costimulation independent hyperproliferation and excess cytokine production in TRAF1-deficient CD8 T cells compared with WT CD8 T cells. The T cell costimulatory molecule 4-1BB critically regulates the survival of activated and memory CD8 T cells. We demonstrate that stimulation through 4-1BB induces cIAP1-dependent TRAF3 degradation and activation of the alternative NF-κB pathway. We also show that while both TRAF1 and cIAP1 have non-redundant roles in suppressing the alternative NF-κB pathway in T cells activated in the absence of costimulation, activation of the classical NF-κB pathway downstream of 4-1BB requires TRAF1, whereas cIAP1 plays a redundant role with cIAP2. Collectively these results demonstrate that TRAF1 plays a critical role in regulating T cell activation both through restricting the costimulation independent activation of NIK in activated T cells and by promoting the 4-1BB-induced classical NF-κB pathway.