George Grigoriadis

The Rel subunit of NF-kappaB-like transcription factors is a positive and negative regulator of macrophage gene expression: distinct roles for Rel in different macrophage populations.

The role of Rel in the monocyte/macrophage lineage was examined in mice with an inactivated c‐rel gene. Although the frequency of monocytic cells was normal in Rel−/− mice, we show that Rel serves distinct roles in regulating gene expression and immune effector function in different mature macrophage populations. Stimulated Rel−/− resident peritoneal macrophages produced higher than normal levels of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), granulocyte colony‐stimulating factor (G‐CSF) and interleukin‐6 (IL‐6), but tumour necrosis factor‐alpha (TNF‐alpha) production was not induced. Diminished cytotoxic activity exhibited by resident Rel−/− macrophages was consistent with reduced nitric oxide production resulting from impaired up‐regulation of inducible nitric oxide synthase expression. While a similar altered pattern of IL‐6 and TNF‐alpha expression was observed in stimulated Rel−/− peritoneal effusion macrophages, cytotoxic activity, nitric oxide, GM‐CSF and G‐CSF production by these cells was normal. The alternate regulation of certain genes in the two macrophage populations coincided with different patterns of nuclear Rel/NF‐kappaB complexes expressed in normal resident and elicited cells. Collectively, these results establish that Rel is a positive or negative regulator of transcription in macrophages and that Rel has distinct roles in different macrophage populations.

The transcription factors c-rel and RelA control epidermal development and homeostasis in embryonic and adult skin via distinct mechanisms.

ABSTRACT Determining the roles of Rel/NF-κB transcription factors in mouse skin development with loss-of-function mutants has been limited by redundancy among these proteins and by embryonic lethality associated with the absence of RelA. Using mice lacking RelA and c-rel, which survive throughout embryogenesis on a tumor necrosis factor alpha (TNF-α)-deficient background (rela −/− c-rel −/− tnfα−/−), we show that c-rel and RelA are required for normal epidermal development. Although mutant fetuses fail to form tylotrich hair and have a thinner epidermis, mutant keratinocyte progenitors undergo terminal differentiation to form an outer cornified layer. Mutant basal keratinocytes are abnormally small, exhibit a delay in G1 progression, and fail to form keratinocyte colonies in culture. In contrast to the reduced proliferation of mutant keratinocytes during embryogenesis, skin grafting experiments revealed that the mutant epidermis develops a TNF-α-dependent hyperproliferative condition. Collectively, our findings indicate that RelA and c-rel control the development of the epidermis and associated appendages during embryogenesis and regulate epidermal homeostasis in a postnatal environment through the suppression of innate immune-mediated inflammation.

Mitogenic actions of endothelin-1 and epidermal growth factor in cultured airway smooth muscle.

1. Hyperplasia of airway smooth muscle contributes to the increase in bronchomotor responsiveness that characterizes asthma. We have investigated the mitogenic potential of endothelin‐1 (ET‐1) and epidermal growth factor (EGF) in guinea‐pig cultured airway smooth muscle and the relationship of these actions to tyrosine phosphorylation and increases in intracellular calcium (Ca2+i).

Detection of BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders.

Hairy cell leukemia has been shown to be strongly associated with the BRAF V600E mutation. We screened 59 unenriched archived bone marrow aspirate and peripheral blood samples from 51 patients with hairy cell leukemia using high resolution melting analysis and confirmatory Sanger sequencing. The BRAF V600E mutation was detected in 38 samples (from 36 patients). The BRAF V600E mutation was detected in all samples with disease involvement above the limit of sensitivity of the techniques used. Thirty-three of 34 samples from other hematologic malignancies were negative for BRAF mutations. A BRAF K601E mutation was detected in a patient with splenic marginal zone lymphoma. Our data support the recent finding of a disease defining point mutation in hairy cell leukemia. Furthermore, high resolution melting with confirmatory Sanger sequencing are useful methods that can be employed in routine diagnostic laboratories to detect BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders.

Coagulation-induced shedding of platelet glycoprotein VI mediated by factor Xa

This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinase-mediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n = 36, P < .0001), and 14.6 ng/mL (95% confidence interval, 7.9-27.1 ng/mL) in healthy subjects (n = 25, P = .002). In conclusion, coagulation-induced GPVI shedding via FXa down-regulates GPVI under procoagulant conditions. FXa inhibitors have an unexpected role in preventing GPVI down-regulation.

NF-κB subunit specificity in hemopoiesis

Summary:  Although the diverse functions served by the nuclear factor‐κB (NF‐κB) pathway in virtually all cell types are typically employed to deal with stress responses, NF‐κB transcription factors also play key roles in the development of hemopoietic cells. This review focuses on how NF‐κB transcription factors control various aspects of thymic T‐cell and myeloid cell differentiation that include its roles in hemopoietic precursors, conventional αβ T cells, CD4+ regulatory T cells, natural killer T cells, γδ T cells, macrophages, and dendritic cells.

c-Rel Controls Multiple Discrete Steps in the Thymic Development of Foxp3+ CD4 Regulatory T Cells

The development of natural Foxp3+ CD4 regulatory T cells (nTregs) proceeds via two steps that involve the initial antigen dependent generation of CD25+GITRhiFoxp3−CD4+ nTreg precursors followed by the cytokine induction of Foxp3. Using mutant mouse models that lack c-Rel, the critical NF-κB transcription factor required for nTreg differentiation, we establish that c-Rel regulates both of these developmental steps. c-Rel controls the generation of nTreg precursors via a haplo-insufficient mechanism, indicating that this step is highly sensitive to c-Rel levels. However, maintenance of c-Rel in an inactive state in nTreg precursors demonstrates that it is not required for a constitutive function in these cells. While the subsequent IL-2 induction of Foxp3 in nTreg precursors requires c-Rel, this developmental transition does not coincide with the nuclear expression of c-Rel. Collectively, our results support a model of nTreg differentiation in which c-Rel generates a permissive state for foxp3 transcription during the development of nTreg precursors that influences the subsequent IL-2 dependent induction of Foxp3 without a need for c-Rel reactivation.

Modulating T regulatory cells in cancer: how close are we?

Regulatory T cells (Tregs) are a specialized subset of CD4 T cells that have an indispensable role in maintaining immune homeostasis and tolerance. Although studies in mice and humans have clearly highlighted that the absence of these cells results in severe autoimmunity and inflammation, increased Treg numbers and/or function is not always beneficial. This is best exemplified in certain cancers where increased Tregs promote cancer progression by interfering with immune surveillance. Conversely, in other types of cancers that have an inflammatory component, Tregs can inhibit cancer progression by dampening inflammation. In this review article, we provide a historical perspective of the discovery of Tregs, followed by a summary of the existing literature on the role of Tregs in malignancy.

Tumor progression locus 2 (Tpl2) deficiency does not protect against obesity-induced metabolic disease.

Obesity is associated with a state of chronic low grade inflammation that plays an important role in the development of insulin resistance. Tumor progression locus 2 (Tpl2) is a serine/threonine mitogen activated protein kinase kinase kinase (MAP3K) involved in regulating responses to specific inflammatory stimuli. Here we have used mice lacking Tpl2 to examine its role in obesity-associated insulin resistance. Wild type (wt) and tpl2−/− mice accumulated comparable amounts of fat and lean mass when fed either a standard chow diet or two different high fat (HF) diets containing either 42% or 59% of energy content derived from fat. No differences in glucose tolerance were observed between wt and tpl2−/− mice on any of these diets. Insulin tolerance was similar on both standard chow and 42% HF diets, but was slightly impaired in tpl2−/− mice fed the 59% HFD. While gene expression markers of macrophage recruitment and inflammation were increased in the white adipose tissue of HF fed mice compared with standard chow fed mice, no differences were observed between wt and tpl2−/− mice. Finally, a HF diet did not increase Tpl2 expression nor did it activate Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), the MAPK downstream of Tpl2. These findings argue that Tpl2 does not play a non-redundant role in obesity-associated metabolic dysfunction.

The NF-κB1 transcription factor prevents the intrathymic development of CD8 T cells with memory properties

The role of specific members of the NF‐κB family of transcription factors in CD8 T‐cell selection and development is largely unknown. Here, we show that mice lacking NF‐κB1 develop a unique population of conventional CD8 single‐positive (SP) thymocytes with memory T cell‐like properties that populate peripheral immune organs. Development of this memory‐like population is not due to PLZF+ thymocytes and instead coincides with changes in CD8 T‐cell selection. These include a reduction in the efficiency of negative selection and a dependence on MHC class Ia or Ib expressed by haematopoietic cells. These findings indicate that NF‐κB1 regulates multiple events in the thymus that collectively inhibit the excess development of CD8+ thymocytes with memory cell characteristics.