Interleukin-15 up-regulates interleukin-2 receptor α chain but down-regulates its own high-affinity binding sites on human T and B cells

The cytokines interleukin (IL)-2 and IL-15 share many biological activities as a consequence of their utilization of the β and γ chains of the IL-2 receptor. However, each cytokine binds to a specific receptor α chain; IL-2 with low affinity and IL-15 with high affinity. Here, we demonstrate that IL-15, like IL-2, up-regulates expression of IL-2Rα on human T and B cells, but rapidly down-regulates IL-15 high-affinity binding sites, which represent IL-15Rα. This leads to a decreased responsiveness to IL-15 as measured by induction of Jak3 tyrosine phosphorylation. These results suggest a mechanism by which IL-15, a product of activated macrophages, may cooperate with IL-2 at the initiation of an immune response and enhance subsequent IL-2 responsiveness during T cell expansion.     

Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand‐dependent apoptosis via the alteration of decoy receptor 3

Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis‐inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL–Fas‐dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL‐induced apoptosis. Abnormally high Akt activity suppresses GSK‐3β function, thereby accumulating the nuclear factor of activated T‐cells cytoplasmic 1 (NFATc1) in the nucleus, increasing DcR3 expression in IPF fibroblasts. This alteration protects IPF cells from FasL‐induced apoptosis on collagen. However, the inhibition of Akt or NFATc1 decreases DcR3 mRNA and protein levels, which sensitizes IPF fibroblasts to FasL‐mediated apoptosis. Furthermore, enhanced DcR3 and NFATc1 expression is mainly present in myofibroblasts in the fibroblastic foci of lung tissues derived from IPF patients. Our results showed that when IPF cells interact with collagen matrix, aberrantly activated Akt increases DcR3 expression via GSK‐3β–NFATc1 and protects IPF cells from the FasL‐dependent apoptotic pathway. These findings suggest that the inhibition of DcR3 function may be an effective approach for sensitizing IPF fibroblasts in response to FasL, limiting the progression of lung fibrosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

A quantitative study of lung dysfunction following haemorrhagic shock in rats

Haemorrhagic shock occasionally causes an episode of lung dysfunction, the severity of which appears to correlate with fatal outcome. Our previous study indicated that proinflammatory cytokines, such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β, played a key role in the development of lung dysfunction through recruitment of activated neutrophils by causing pulmonary endothelial cell damage. Here, we examined this issue quantitatively by grading four groups of severity of bleeding in rats. As the amount of bleeding increased, the expression of mRNA for TNF‐α and IL‐1β in the lung tissue and the pulmonary serum levels of both cytokines increased progressively up to 5 h, and the frequency of activated neutrophils increased likewise. The lung dysfunction indices serum lactic dehydrogenase‐3 isozyme (LDH‐3), partial pressure of arterial oxygen (PaO₂) and alveolar‐arterial oxygen tension difference (AaDO₂) in blood deteriorated as the amount of bleeding increased. The frequency of activated neutrophils in the lung correlated well with the LDH‐3 level 5 h after haemorrhagic shock. The present results demonstrate that the increase of proinflammatory cytokines and the recruitment of activated neutrophils into the lung following haemorrhagic shock are quantitatively related to progression of lung dysfunction as the amount of bleeding increases.     

Control of B cell lymphoma recognition via natural killer inhibitory receptors implies a role for human Vγ9/Vδ2 T cells in tumor immunity

The Vγ9/Vδ2 T cell receptor (TCR) is expressed by most human γδ T cells. We show here that cytotoxic T lymphocytes of the Vγ9/Vδ2 subset, but not of the Vδ1 subset of human γδ T cells, express natural killer inhibitory receptors (KIR) with specificity for different HLA class I alleles that down-regulate TCR-mediated signaling in response to HLA class I-expressing B cell lymphomas. Vγ9/Vδ2 T cell clones with a T helper cell phenotype lack KIR and produce lymphokines in response to most human B cell lymphomas, just as they do upon recognition of the HLA class l-deficient human Burkitt's lymphoma Daudi. Thus, human Vγ9/Vδ2 T cells have an innate specificity for nonpolymorphic cell surface structures expressed by many lymphoma cells and their cytotoxic activity is controlled by KIR. These results imply a general role of human Vγ9/Vδ2 T cells in the defense against hematopoietic tumors that is distinct from NK cells.     

Genotoxic bioactivation of constituents of a diesel exhaust particle extract by the human lung

The ability of the human lung to catalyze genotoxic bioactivation of constituents of diesel exhaust particle (DEP) extract (DEPE) and the identity of the lung enzymes involved in the bioactivation were investigated using human lung tissues obtained from surgical resections. Genotoxicity was determined by lung S9‐catalyzed mutagenicity of DEPE constituents to Salmonella typhimurium TA98NR in the Ames test and by DEPE‐induced pneumocyte DNA damage response as determined by γH2Ax expression in ex vivo tissues. S9 was prepared from lung explants treated ex vivo with either DEPE to induce pulmonary enzymes (DEPE‐S9) or vehicle only (CON‐S9). TA98NR served as the tester strain for the purpose of enhancing and minimizing the contribution of lung S9 and Salmonella, respectively, to DEPE bioactivation. DEPE‐S9 was 2.2–fold more active than CON‐S9 or rat liver S9 in DEPE bioactivation and the bioactivation was inhibited 58, 45, 22, and 16% by α‐naphthoflavone, dicumarol, ketoconazole, and ticlopidine, respectively. Alveolar S9 was less active than bronchioalveolar S9 in DEPE bioactivation. DEPE and diesel exhaust particles (DEP) induced γ‐pH2Ax expression in pulmonary cells. Pulmonary CYP1A1 and NQO1 were induced by DEPE treatment, with the constitutive and induced CYP1A1 distributed throughout all peripheral lung regions, whereas NQO1 was limited in distribution to bronchiolar epithelium. The results show that the human lung is highly active in catalyzing genotoxic bioactivation of diesel emission constituents and that CYP1A and NQO1 play major roles in the reaction. The findings underscore the usefulness of human lung tissues in studies of the pneumotoxicity potential of chemicals to humans. Environ. Mol. Mutagen. 54:158–171, 2013. © 2013 Wiley Periodicals, Inc.     

Parvalbumin-containing interneurons of the human cerebral cortex express nicotinic acetylcholine receptor proteins

Cholinergic fibers from the basal forebrain are known to contact cholinoceptive cortical pyramidal neurons. Recent electrophysiological studies have revealed that nicotinic acetylcholine receptors are also present in human cerebrocortical interneurons. A direct visualization of nicotinic receptor subunits in cortical interneurons has, however, not yet been performed. We have applied double-immunofluorescence using antibodies against parvalbumin --a marker for the Chandelier and basket cell subpopulation of interneurons--and to the alpha4 and alpha7 subunit proteins of the nicotinic acetylcholine receptor. The vast majority of the parvalbuminergic interneurons was immunoreactive for the alpha4 and the alpha7 nicotinic acetylcholine receptor. Provided these receptors would be functional--as suggested by recent electrophysiological findings--the connectivity pattern of cholinergic afferents appears much more complex than thought before. Not only direct cholinergic impact on cortical projection neurons but also the indirect modulation of these by cholinergic corticopetal fibers contacting intrinsic cortical cells would be possible.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Interleukin-2 receptor β and γ chain dysregulation during the inhibition of CD4 T cell activation by human immunodeficiency virus-1 gp120

We have observed that CD4 T lymphocytes from human immunodeficiency virus (HIV)-infected patients marginally express interleukin-2 receptor (IL-2R)β and IL-2Rγ chains which are essential for IL-2 signal transduction. To analyze this observation further, we studied the influence of gp120 on the cell surface expression of IL-2Rβ and IL-2Rγ by purified CD4 lymphocytes in vitro. Cross-linking of the T cell receptors of these lymphocytes initiates entry into the cell cycle as measured by CD69 and CD71 cell surface expression and [³H]thymidine incorporation. It also induces the cell surface expression of IL-2Rβ and IL-2Rγ. We have shown that treatment of the CD4 T lymphocytes with HIV-1 gp120 before anti-CD3 stimulation impedes cell cycle progression as measured by reduced CD71 expression and inhibition of [³H]thymidine incorporation. Furthermore, cell surface expression of IL-2Rβ and IL-2Rγ subunits, which form the functional intermediate-affinity IL-2R, are significantly inhibited. More importantly, addition of exogenous IL-2 does not restore the proliferation of the CD4 T cells treated with gp120, suggesting that cells are anergic and/or that the remaining IL-2R are not functional. This is the first study of IL-2Rβ and IL-2Rγ dysregulation in the context of HIV infection and shows that CD4 is also involved in IL-2R expression.     

Structural requirements for the interaction of human IgM and IgA with the human Fcα/μ receptor

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fcα/μ receptor (hFcα/μR). Ligand polymerization status was crucial for the interaction, because hFcα/μR binding did not occur with monomeric Ab of either class. hFcα/μR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFcα/μR binding. IgM binding required contributions from both Cμ3 and Cμ4 Fc domains, whereas for dIgA, an exposed loop in the Cα3 domain was crucial. This loop, comprising residues Pro440–Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcαRI and polymeric Ig receptor (pIgR), as well as IgA‐binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440–Phe443 loop resulted in loss of hFcα/μR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA‐binding proteins were shown to inhibit the dIgA–hFcα/μR interaction. Therefore, we have identified a motif in the IgA–Fc inter‐domain region critical for hFcα/μR interaction, and highlighted the multi‐functional nature of a key site for protein–protein interaction at the IgA Fc domain interface.     

Wogonin prevents lipopolysaccharide‐induced acute lung injury and inflammation in mice via peroxisome proliferator‐activated receptor gamma‐mediated attenuation of the nuclear factor‐kappaB pathway

Acute lung injury (ALI) from a variety of clinical disorders, characterized by diffuse inflammation, is a cause of acute respiratory failure that develops in patients of all ages. Previous studies reported that wogonin, a flavonoid‐like chemical compound which was found in Scutellaria baicalensis, has anti‐inflammatory effects in several inflammation models, but not in ALI. Here, the in vivo protective effect of wogonin in the amelioration of lipopolysaccharide (LPS) ‐induced lung injury and inflammation was assessed. In addition, the in vitro effects and mechanisms of wogonin were studied in the mouse macrophage cell lines Ana‐1 and RAW264.7. In vivo results indicated that wogonin attenuated LPS‐induced histological alterations. Peripheral blood leucocytes decreased in the LPS‐induced group, which was ameliorated by wogonin. In addition, wogonin inhibited the production of several inflammatory cytokines, including tumour necrosis factor‐α, interleukin‐1β (IL‐1β) and IL‐6, in the bronchoalveolar lavage fluid and lung tissues after LPS challenge, while the peroxisome proliferator‐activated receptor γ (PPARγ) inhibitor GW9662 reversed these effects. In vitro results indicated that wogonin significantly decreased the secretion of IL‐6, IL‐1β and tumour necrosis factor‐α in Ana‐1 and RAW264.7 cells, which was suppressed by transfection of PPARγ small interfering RNA and GW9662 treatment. Moreover, wogonin activated PPARγ, induced PPARγ‐mediated attenuation of the nuclear translocation and the DNA‐binding activity of nuclear factor‐κB in vivo and in vitro. In conclusion, all of these results showed that wogonin may serve as a promising agent for the attenuation of ALI‐associated inflammation and pathology by regulating the PPARγ‐involved nuclear factor‐κB pathway.     

Secretory immunoglobulin A induces human lung fibroblasts to produce inflammatory cytokines and undergo activation

Immunoglobulin (Ig)A is the most abundant immunoglobulin in humans, and in the airway mucosa secretory IgA (sIgA) plays a pivotal role in first-line defense against invading pathogens and antigens. IgA has been reported to also have pathogenic effects, including possible worsening of the prognosis of idiopathic pulmonary fibrosis (IPF). However, the precise effects of IgA on lung fibroblasts remain unclear, and we aimed to elucidate how IgA activates human lung fibroblasts. We found that sIgA, but not monomeric IgA (mIgA), induced interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1 and granulocyte–macrophage colony-stimulating factor (GM-CSF) production by normal human lung fibroblasts (NHLFs) at both the protein and mRNA levels. sIgA also promoted proliferation of NHLFs and collagen gel contraction comparable to with transforming growth factor (TGF)-β, which is involved in fibrogenesis in IPF. Also, Western blot analysis and real-time quantitative polymerase chain reaction (PCR) revealed that sIgA enhanced production of α-smooth muscle actin (α-SMA) and collagen type I (Col I) by NHLFs. Flow cytometry showed that NHLFs bound sIgA, and among the known IgA receptors, NHLFs significantly expressed CD71 (transferrin receptor). Transfection of siRNA targeting CD71 partially but significantly suppressed cytokine production by NHLFs co-cultured with sIgA. Our findings suggest that sIgA may promote human lung inflammation and fibrosis by enhancing production of inflammatory or fibrogenic cytokines as well as extracellular matrix, inducing fibroblast differentiation into myofibroblasts and promoting human lung fibroblast proliferation. sIgA’s enhancement of cytokine production may be due partially to its binding to CD71 or the secretory component.     

BST2/Tetherin is constitutively expressed on human thymocytes with the phenotype and function of Treg cells

In contrast to peripheral plasmacytoid DCs (pDCs), thymic pDCs constitutively express low levels of IFN‐α. This leads to induction of interferon secondary genes (ISGs) in medullary thymocytes, raising the question whether IFN‐α may play a role in T‐cell development. When characterizing further differences between peripheral and thymic pDCs, we found that thymic pDCs have a phenotype consistent with an “activated signature” including expression of TNF‐α and bone marrow stromal cell antigen 2 (BST2), but no expression of ILT7. Given that BST2 is induced by IFN‐α, and IFN‐α secretion is controlled by interaction between ILT7 and BST2, this regulatory pathway is apparently lost in thymic pDCs. Further, we also show that BST2 is constitutively expressed on a subset of medullary thymocytes at the mRNA and protein level reflecting a history of IFN‐α transduced signals. The majority of BST2⁺ thymocytes express CCR5 rendering them prevalent targets for R5‐tropic HIV infection. Moreover, BST2⁺ thymocytes express Foxp3 and CD25, consistent with the phenotype of natural Treg cells, and exert suppressive activity as they impair the proliferation of autologous CD3⁺ thymocytes. Collectively, our results suggest that low levels of IFN‐α secreted by thymic pDCs play an important role in the development of natural Treg cells.     

Effects of a-adrenoceptor subtype-selective antagonists on the human saphenous vein in vivo

The effects of the α-adrenoceptor subtype-selective antagonists prazosin (α¹) and yohimbine (α₂) on the saphenous vein of six healthy male subjects were investigated in vivo. The drugs were infused locally into the congested (40 mmHg), long saphenous vein constricted by simultaneous local infusion of noradrenaline (NA). Prazosin 10^-9 M (concentration in the infusion solution, infusion rate 0.3 ml min^_I) did not reduce the NA-induced venoconstriction, but at a concentration of 10^-8 M there was a significant reduction; in two subjects no response to NA could be elicited in the presence of 10^-8 M prazosin. Prazosin 10^-7 M caused no further reduction of the NA effect compared to that produced by 10^-8 M in three of the subjects, whereas in one, prazosin 10^-9, 10^-8 and 10^-7 M caused a dose-dependent blockade. Yohimbine, 10^-9, 10^-8 and 10^-7 M caused a dose-dependent reduction of the NA-induced venoconstriction in all subjects. The results suggest that the human saphenous vein is endowed with functionally important populations of both α₂- and a₁-adrenoceptors.     

Postjunctional α1- and α2-adrenoceptors mediating contraction in isolated human groin arteries and veins

By means of selective agonists and antagonists for α₁- and α₂-receptors, the α-receptor subtypes in human groin arteries and veins were characterized and compared. In the arteries the α₁-receptor blocker prazosin caused a concentration-dependent parallel displacement of the noradrenaline (NA) concentration-response (cr) curve without reduction of maximum (pA₂=9.86); the selective α₂-receptor antagonist rauwolscine in the concentration 10^-8 M caused a right-ward shift of the NA cr-curve without reduction of E_max, but 10^-7 M and 10^-6 M caused little or no further shift. In the veins, the two antagonists had the opposite effects. Rauwolscine caused a concentration-dependent right-ward shift of the NA cr-curve without depression of maximum (pA₂=9.03); prazosin 10^-9 M significantly displaced the NA cr-curve, whereas 10^-8 M and 10^-7 M caused little or no further shift. The responses to the α₂-receptor agonist clonidine in the arteries were too small to allow calculations of pEC50 values; in the veins contractions were elicited in all vessel segments investigated (pEC₅₀=6.24). Phenylephrine, selective for α₁-receptors, was significantly more potent in arteries than in veins. NA was significantly more potent in veins than in arteries. It is concluded that in human groin vessels, there is a functional predominance of arreceptors in the arteries and of a2-receptors in the veins.