IL‐7 and immobilized Kit‐ligand stimulate serum‐ and stromal cell‐free cultures of precursor B‐cell lines and clones

Long‐term proliferating, D_HJ_H‐rearranged mouse precursor B‐cell lines have previously been established in serum‐ and IL‐7‐containing media from fetal liver, but not from bone marrow. Serum and stromal cells expose these pre‐B cells to undefined factors, hampering accurate analyses of ligand‐dependent signaling, which controls pre‐B cell proliferation, survival, residence and migration. Here, we describe a novel serum‐free, stromal cell‐free culture system, which allows us to establish and maintain pre‐B cells not only from fetal liver, but also from bone marrow with practically identical efficiencies in proliferation, cloning and differentiation. Surprisingly, recombinant kit‐ligand, also called stem cell factor, produced as a kit‐ligand‐Fc fusion protein, suffices to replace stromal cells and serum, provided that it is presented to cultured pre‐B cells in an optimal density in plate‐bound, insolubilized, potentially crosslinking form. Additional recombinant CXCL12 and fibronectin have a minor influence on the establishment and maintenance of pre‐B cell lines and clones from fetal liver, but are necessary to establish such cell lines from bone marrow.     

Effects of EGF and TGFβ1 on c‐myc gene expression and DNA synthesis in embryonic hamster palate mesenchymal cells

Previous work has shown that cell proliferation is a major contributor to the early palate morphogenesis in mammals. The present study was undertaken to examine the effect of EGF, TGFβ₁ and their combination on proliferation (measured by DNA synthesis) and on the expression of a growth related proto‐oncogene, c‐myc, in embryonic hamster palate mesenchymal cells (HPMC). Vertically developing hamster palatal shelves were dissected on day 11 of gestation, and trypsinized, and primary cultures were grown in DMEM + 10% serum at 37°C and 5% CO₂. Following appropriate growth factor treatment of HPMC, DNA synthesis was measured by scintillation counting and extracted RNA was subjected to Northern blot analysis. In serum‐starved, pre‐confuent cultures treated with EGF (20 ng/ml), DNA synthesis was stimulated in the presence of 2.5% serum. In contrast, treatment of HPMC with TGFβ₁ (10 ng/ml) in the presence or absence of EGF/serum for 24 hr, or HPMC pre‐treatment with TGFβ₁ (30 min) followed by EGF/serum (24 hr), resulted in an arrest of DNA synthesis. Northern blot analysis of RNA extracted from HPMC showed that as serum‐starved, growth‐arrested cells progressed through G0 to G1 phase of the cell cycle, following EGF treatment, c‐myc was expressed by 1 hr and declined thereafter. In contrast, TGFβ₁ did not support expression of c‐myc. Following pre‐ or co‐treatment with TGFβ₁, the EGF ± serum‐induced expression of c‐myc was seen between 1 and 6 hr. It appears that EGF‐induced expression of c‐myc may be involved in advancing the HPMC in G1, and thus may contribute to the onset of DNA synthesis in HPMC. Since co‐ or pre‐treatment with TGFβ₁ did not inhibit EGF/serum induced expression of c‐myc, it is possible that growth arresting effect of TGFβ₁ may not be exerted directly through inhibition or blockage of c‐myc expression. Anat Rec 254:453–464, 1999. © 1999 Wiley‐Liss, Inc.     

The thioredoxin‐1 and glutathione/glutaredoxin‐1 systems redundantly fuel murine B‐cell development and responses

Antioxidant systems maintain cellular redox homeostasis. The thioredoxin‐1 (Trx1) and the glutathione (GSH)/glutaredoxin‐1 (Grx1) systems are key players in preserving cytosolic redox balance. In fact, T lymphocytes critically rely on reducing equivalents from the Trx1 system for DNA biosynthesis during metabolic reprogramming upon activation. We here show that the Trx1 system is also indispensable for development and functionality of marginal zone (MZ) B cells and B1 cells in mice. In contrast, development of conventional B cells, follicular B‐cell homeostasis, germinal center reactions, and antibody responses are redundantly sustained by both antioxidant pathways. Proliferating B2 cells lacking Txnrd1 have increased glutathione (GSH) levels and upregulated cytosolic Grx1, which is barely detectable in expanding thymocytes. These results suggest that the redox capacity driving proliferation is more robust and flexible in B cells than in T cells, which may have profound implications for the therapy of B and T‐cell neoplasms.     

B7‐1/2, but not PD‐L1/2 molecules,are required on IL‐10‐treated tolerogenic DC and DC‐derived exosomes for in vivo function

Costimulatory molecules, such as B7‐1/2 and PD‐L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7‐1/2 and PD‐L1/2 to the function of IL‐10‐treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL‐10 treatment of DC significantly downregulated surface expression of MHC II, B7‐1, B7‐2, and decreased levels of MHC I and PD‐L2. IL‐10 treatment of DC resulted in a modified costimulatory profile of DC‐secreted exosomes with a reduction in B7‐1, PD‐L1 and PD‐L2. We further demonstrate that absence of B7‐1 or B7‐2 on donor DC results in a loss of ability of IL‐10‐treated DC and their exosomes to suppress the delayed‐type hypersensitivity response, whereas IL‐10‐treated DC deficient in PD‐L1/2 as well as their secreted exosomes retained the ability to suppress delayed‐type hypersensitivity responses. We conclude that B7‐1 and B7‐2, but not PD‐L1 and PD‐L2, on IL‐10‐treated DC and DC‐derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.     

NF‐κB1, NF‐κB2 and c‐Rel differentially regulate susceptibility to colitis‐associated adenoma development in C57BL/6 mice

NF‐κB signalling is an important factor in the development of inflammation‐associated cancers. Mouse models of Helicobacter‐induced gastric cancer and colitis‐associated colorectal cancer have demonstrated that classical NF‐κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF‐κB proteins, including NF‐κB1/p50, NF‐κB2/p52, and c‐Rel, differentially regulate the development of gastric pre‐neoplasia. To investigate the effect of NF‐κB subunit loss on colitis‐associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1^?/?, Nfkb2^?/?, and c‐Rel^?/?mice. Animals lacking the c‐Rel subunit were more susceptible to colitis‐associated cancer than wild‐type mice, developing 3.5 times more colonic polyps per animal than wild‐type mice. Nfkb2^?/? mice were resistant to colitis‐associated cancer, developing fewer polyps per colon than wild‐type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2^?/? mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c‐Rel^?/? mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild‐type counterparts. These observations demonstrate different functions of specific NF‐κB subunits in this model of colitis‐associated carcinogenesis. NF‐κB2/p52 is necessary for the development of colitis, whilst c‐Rel‐mediated signalling regulates colonic epithelial cell turnover following DNA damage. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Chitin induces upregulation of B7‐H1 on macrophages and inhibits T‐cell proliferation

Chitin is a highly abundant glycopolymer, which serves as structural component in fungi, arthropods and crustaceans but is not synthesized by vertebrates. However, vertebrates express chitinases and chitinase‐like proteins, some of which are induced by infection with helminths suggesting that chitinous structures may be targets of the immune system. The chitin‐induced modulations of the innate and adaptive immune responses are not well understood. Here, we demonstrate that intranasal administration of OVA and chitin resulted in diminished T‐cell expansion and Th2 polarization as compared with OVA administration alone. Chitin did not promote nor attenuate Th2 polarization in vitro. Chitin‐exposed macrophages inhibited proliferation of CD4⁺ T cells in a cell–cell contact‐dependent manner. Chitin induced upregulation of the inhibitory ligand B7‐H1 (PD‐L1) on macrophages independently of MyD88, TRIF, TLR2, TLR3, TLR4 and Stat6. Inhibition of T‐cell proliferation was largely dependent on B7‐H1, as the effect was not observed in cocultures with cells from B7‐H1‐deficient mice.     

Mechanism of interleukin‐1β‐induced proliferation in rat hepatic stellate cells from different levels of signal transduction

Hepatic stellate cells (HSCs) are the major producers of collagen in the liver. Their conversion from resting cells to proliferative, contractile, and activated cells is a critical step leading to liver fibrosis that is characterized by the deposition of excessive extracellular matrix. Interleukin‐1 (IL‐1) may play a role in maintaining HSC in a proliferative state that is responsible for hepatic fibrogenesis. The aim of this study was to study the roles of the IL‐1 type I receptor (IL‐1R1), c‐Jun N‐terminal kinase (JNK), and activation protein‐1 (AP‐1) in IL‐1β–mediated proliferation in rat HSCs. We showed that IL‐1β can upregulate proliferation in rat HSCs; however, inhibition of the JNK pathway could inhibit HSCs proliferation. Furthermore, IL‐1β activated IL‐1R1 expression, the JNK signaling pathway, and AP‐1 activity in a time‐dependent manner in rat HSCs. These data demonstrate that IL‐1β could promote the proliferation of rat HSCs and that the IL‐1R1, JNK, and AP‐1 pathways were involved in this process. In summary, IL‐1β‐induced proliferation is possibly mediated by the IL‐1R1, JNK, and AP‐1 pathways in rat HSCs. Therefore, drugs that block these pathways may inhibit the proliferation of HSCs and suppress liver fibrosis.     

Hypoxia‐inducible factor‐1α perpetuates synovial fibroblast interactions with T cells and B cells in rheumatoid arthritis

Synovial fibroblast hyperplasia, T‐cell hyperactivity, B‐cell overactivation, and the self‐perpetuating interactions among these cell types are major characteristics of rheumatoid arthritis (RA). The inflamed joints of RA patients are hypoxic, with upregulated expression of hypoxia‐inducible factor‐1α (HIF‐1α) in RA synovial fibroblasts (RASFs). It remains unknown whether HIF‐1α regulates interactions between RASFs and T cells and B cells. We report here that HIF‐1α promotes the expression of inflammatory cytokines IL‐6, IL‐8, TNF‐α, and IL‐1β, and cell–cell contact mediators IL‐15, vascular cell adhesion molecule (VCAM)‐1, thrombospondin (TSP)‐1, and stromal cell‐derived factor (SDF)‐1 in RASFs. Furthermore, HIF‐1α perpetuates RASF‐mediated inflammatory Th1‐ and Th17‐cell expansion while differentially inhibiting regulatory B10 and innate‐like B cells, leading to increased IFN‐γ, IL‐17, and IgG production and decreased protective natural IgM secretion. Our findings suggest that HIF‐1α perpetuates the interactions between RASFs and T cells and B cells to induce inflammatory cytokine and autoantibody production, thus exacerbating the severity of RA. Targeting HIF‐1α may provide new therapeutic strategies for overcoming this persistent disease.     

Markers aiding the diagnosis of chondroid tumors: an immunohistochemical study including osteonectin,bcl‐2, cox‐2, actin,calponin, D2‐40 (podoplanin), mdm‐2, CD117 (c‐kit), and YKL‐40

Chondroid tumors comprise a heterogenous group of benign to overt malignant neoplasms, which may be difficult to differentiate from one another by histological examination. A group of 43 such tumors was stained with nine relevant antibodies in an attempt to find consistent marker profile(s) for the different subgroups. Archival material from three extraskeletal myxoid chondrosarcomas, five chordomas, five chondromyxoid fibromas, five chondroblastomas and 25 chondrosarcomas was stained with antibodies against osteonectin, bcl‐2, cox‐2, actin, calponin, D2‐40 (podoplanin), mdm‐2, CD117 (c‐kit) and YKL‐40. All 25 chondrosarcomas showed a positive staining reaction for D2‐40, none for actin and CD117, and a partial reactivity for bcl‐2 (36%). Chondroblastomas (5/5) and chondromyxoid fibromas (2/5) were the only tumors with a positive reaction for actin, and all chondroblastomas (n=5) and extraskeletal myxoid chondrosarcomas (n=3) were positive for bcl‐2. In contrast to all other tumors, two of three extraskeletal myxoid chondrosarcomas were also positive for CD17 and negative for osteonectin, cox‐2, mdm‐2 and actin. All five chordomas were negative for D2‐40 and positive for mdm‐2 and YKL‐40. The diagnosis of chondrosarcoma may be aided by its positivity for D2‐40 and YKL‐40 and its lack of reactivity for actin and CD117. This should be seen in the light of no reaction for D2‐40 in chordomas and a corresponding lack of reaction for osteonectin, cox‐2, mdm‐2 and actin in extraskeletal myxoid chondrosarcomas. A convincing immunoreactivity for calponin and/or actin in chondromyxoid fibromas and chondroblastomas may also be helpful in differentiating these tumors from chondrosarcomas.     

Plasmacytoid dendritic cells and type 1 interferon promote peripheral expansion of forkhead box protein 3+ regulatory T cells specific for the ubiquitous RNA‐binding nuclear antigen La/Sjögren's syndrome (SS)‐B

RNA‐binding nuclear antigens are a major class of self‐antigen to which immune tolerance is lost in rheumatic diseases. Serological tolerance to one such antigen, La/Sjögren's syndrome (SS)‐B (La), is controlled by CD4⁺ T cells. This study investigated peripheral tolerance to human La (hLa) by tracking the fate of hLa‐specific CD4⁺ T cells expressing the transgenic (Tg) 3B5.8 T cell receptor (TCR) after adoptive transfer into lymphocyte‐replete recipient mice expressing hLa as a neo‐self‐antigen. After initial antigen‐specific cell division, hLa‐specific donor CD4⁺ T cells expressed forkhead box protein 3 (FoxP3). Donor cells retrieved from hLa Tg recipients displayed impaired proliferation and secreted interleukin (IL)?10 in vitro in response to antigenic stimulation. Transfer of highly purified FoxP3‐negative donor cells demonstrated that accumulation of hLa‐specific regulatory T cells (T_reg) was due primarily to expansion of small numbers of donor T_reg. Depletion of recipient plasmacytoid dendritic cells (pDC), but not B cells, severely hampered the accumulation of FoxP3⁺ donor T_reg in hLa Tg recipients. Recipient pDC expressed tolerogenic markers and higher levels of co‐stimulatory and co‐inhibitory molecules than B cells. Adoptive transfer of hLa peptide‐loaded pDC into mice lacking expression of hLa recapitulated the accumulation of hLa‐specific T_reg. Blockade of the type 1 interferon (IFN) receptor in hLa Tg recipients of hLa‐specific T cells impaired FoxP3⁺ donor T cell accumulation. Therefore, peripheral expansion of T_reg specific for an RNA‐binding nuclear antigen is mediated by antigen‐presenting pDC in a type 1 IFN‐dependent manner. These results reveal a regulatory function of pDC in controlling autoreactivity to RNA‐binding nuclear antigens.     

Dimorphic HLA‐B signal peptides differentially influence HLA‐E‐ and natural killer cell‐mediated cytolysis of HIV‐1‐infected target cells

As a mechanism of self‐protection, signal peptides cleaved from human leukocyte antigen (HLA) class I products bind to HLA‐E before the complex interacts with the natural killer (NK) cell receptor CD94/NKG2A to inhibit NK‐mediated cell lysis. Two types of the signal peptides differ in their position 2 (P2) anchor residue, with P2‐methionine (P2‐M) having higher HLA‐E binding affinity than P2‐threonine (P2‐T). All HLA‐A and HLA‐C molecules carry P2‐M, whereas HLA‐B products have either P2‐M or P2‐T. Epidemiological evidence suggests that P2‐M is unfavourable in the context of HIV‐1 infection, being associated with accelerated acquisition of HIV‐1 infection in two African cohorts. To begin elucidating the functional mechanism, we studied NK‐mediated killing of CD4⁺ T cells and monocyte‐derived macrophages infected with two laboratory‐adapted HIV‐1 strains and two transmitted/founder (T/F) viruses. In the presence of target cells derived from individuals with the three HLA‐B P2 genotypes (M/M, M/T and T/T), NK‐mediated cytolysis was elevated consistently for P2‐T in a dose‐dependent manner for all cell and virus combinations tested (P = 0·008–0·03). Treatment of target cells with an anti‐HLA‐E monoclonal antibody restored NK‐mediated cytolysis of cells expressing P2‐M. Observations on cell lysis were also substantiated by measurements of HIV‐1 p24 antigen in the culture supernatants. Overall, our experiments indicate that the anti‐HIV‐1 function mediated by NK cells is compromised by P2‐M, corroborating the association of HLA‐B genotype encoding P2‐M with accelerated HIV‐1 acquisition.     

S1P3 confers differential S1P‐induced migration by autoreactive and non‐autoreactive immature B cells and is required for normal B‐cell development

During B‐cell development, immature B‐cell fate is determined by whether the BCR is engaged in the bone marrow. Immature B cells that are non‐autoreactive continue maturation and emigrate from the marrow, whereas autoreactive immature B cells remain and are tolerized. However, the microenvironment where these events occur and the chemoattractants responsible for immature B‐cell trafficking within and out of the bone marrow remain largely undefined. Sphingosine 1‐phosphate (S1P) is a chemoattractant that directs lymphocyte trafficking and thymocyte egress and in this study we investigated whether S1P contributes to B‐cell development, egress and positioning within the bone marrow. Our findings show that immature B cells are chemotactic toward S1P but that this response is dependent on Ag receptor specificity: non‐autoreactive, but not autoreactive, immature B cells migrate toward S1P and are shown to require S1P3 receptor for this response. Despite this response, S1P3 is shown not to facilitate immature B‐cell egress but is required for normal B‐cell development including the positioning of transitional B cells within bone marrow sinusoids. These data indicate that S1P3 signaling directs immature B cells to a bone marrow microenvironment important for both tolerance induction and maturation.     

Tim‐1+ B cells suppress T cell interferon‐gamma production and promote Foxp3 expression,but have impaired regulatory function in coronary artery disease

Atherosclerosis and its associated coronary artery disease (CAD) represent another chronic low‐grade inflammatory disorder. Regulatory B cells (Bregs) possess essential functions in maintaining peripheral tolerance and inhibiting pathogenic inflammation through IL‐10. Here, we investigated one subset of Bregs, Tim‐1⁺ B cell, and its role in atherosclerosis and CAD patients. In healthy individuals, IL‐10‐producing B cells were predominantly found in the Tim‐1⁺ B cells. Upon stimulation of the B cell receptor (BCR) and Toll‐like receptor 9 (TLR‐9) by anti‐BCR antibodies and CpG, respectively, the Tim‐1⁺ B cells could further upregulate IL‐10 expression. In contrast, the Tim‐1⁺ B cells were present at normal frequency in CAD patients, but showed impaired capacity to upregulate IL‐10 with or without BCR + CpG stimulation. The stimulated Tim‐1⁺ B cells from healthy individuals also suppressed expression of interferon gamma (IFN‐γ), an atherogenic cytokine in T cells, in an IL‐10‐dependent fashion, and strongly promoted the expression of Foxp3 in naive CD4⁺CD45RO^? T cells. In contrast, the Tim‐1⁺ B cells from CAD patients were unable to suppress IFN‐γ secretion, and only minimally increased the expression of Foxp3 in naive CD4⁺CD45RO^? T cells. Despite this, the frequency of Tim‐1⁺ B cells in the atherosclerotic lesions from CAD patients was inversely correlated with the frequency of IFN‐γ‐expressing T cells. Together, these results demonstrated that CAD patients presented an inflammatory disorder in regulatory B cells, which could be used as a therapeutic target.     

Islets of Langerhans from prohormone convertase‐2 knockout mice show α‐cell hyperplasia and tumorigenesis with elevated α‐cell neogenesis

Antagonism of the effects of glucagon as an adjunct therapy with other glucose‐lowering drugs in the chronic treatment of diabetes has been suggested to aggressively control blood glucose levels. Antagonism of glucagon effects, by targeting glucagon secretion or disabling the glucagon receptor, is associated with α‐cell hyperplasia. We evaluated the influence of total glucagon withdrawal on islets of Langerhans using prohormone convertase‐2 knockout mice (PC2‐ko), in which α‐cell hyperplasia is present from a young age and persists throughout life, in order to understand whether or not sustained glucagon deficit would lead to islet tumorigenesis. PC2‐ko and wild‐type (WT) mice were maintained drug‐free, and cohorts of these groups sampled at 3, 12 and 18 months for plasma biochemical and morphological (histological, immunohistochemical, electron microscopical and image analytical) assessments. WT mice showed no islet tumours up to termination of the study, but PC2‐ko animals displayed marked changes in islet morphology from α‐cell hypertrophy/hyperplasia/atypical hyperplasia, to adenomas and carcinomas, these latter being first encountered at 6–8 months. Islet hyperplasias and tumours primarily consisted of α‐cells associated to varying degrees with other islet endocrine cell types. In addition to substantial increases in islet neoplasia, increased α‐cell neogenesis associated primarily with pancreatic duct(ule)s was present. We conclude that absolute blockade of the glucagon signal results in tumorigenesis and that the PC2‐ko mouse represents a valuable model for investigation of islet tumours and pancreatic ductal neogenesis.