Investigating the structure and function of PEPITEM, a novel inhibitor of T cell transmigration

Peptide inhibitor of trans-endothelial migration (PEPITEM) is a B cell-secreted peptide which inhibits T cell trafficking across cytokine-stimulated endothelium. This homeostatic mechanism is lost in autoimmune and chronic inflammatory diseases, leading to inappropriate T cell trafficking with pathological consequences, e.g. in type-1 diabetes and rheumatoid arthritis. We aimed to investigate the structure and function of PEPITEM in vitro and in vivo to establish its pharmacokinetics, therapeutic potential, and underlying molecular mechanisms. The efficacy of PEPITEM was verified, showing the same regulatory control of T cell trafficking as previously reported. Nuclear magnetic resonance (NMR) studies revealed PEPITEM to be a linear peptide lacking secondary structure. Intravenous administration of radiolabelled native PEPTEIM in wild-type mice showed rapid clearance by the renal circulation, conferring a circulatory half-life of <2 minutes. Common conjugation strategies employed to modify PEPITEM, e.g. PEGylation, did not affect peptide function, demonstrating its potential for therapeutic development. The evolution of PEPITEM from its parent protein, 14-3-3ζ was investigated. PEPITEM is probably cleaved from 14-3-3ζ by matrix metalloproteinase 9 extracellularly after exocytosis from B cells. Interestingly, this generates a 17aa peptide which requires additional proteolytic processing to evolve a smaller biologically active pharmacophore. These observations add to our current knowledge of the PEPITEM paradigm, which appears central to regulated trafficking of T cells. Future work will formulate new PEPITEM versions with suitable pharmacological profiles, and screen them for function in vitro and in vivo to develop suitable therapeutic agents

Platelet P-selectin triggers rapid surface exposure of tissue factor in monocytes

Abstract: Tissue factor (TF) plays a central role in haemostasis and thrombosis. Following vascular damage, vessel wall TF initiates the extrinsic coagulation cascade. TF can also be exposed by monocytes. Inflammatory or infectious stimuli trigger synthesis of new TF protein by monocytes over the course of hours. It has also been suggested that monocytes can expose TF within minutes when stimulated by activated platelets. Here, we have confirmed that monocytes rapidly expose TF in whole blood and further demonstrate that platelet P-selectin exposure is necessary and sufficient. Monocyte TF exposure increased within five minutes in response to platelet activation by PAR1-AP, PAR4-AP or CRP-XL. PAR1-AP did not trigger TF exposure on isolated monocytes unless platelets were also present. In whole blood, PAR1-AP-triggered TF exposure required P-selectin and PGSL-1. In isolated monocytes, although soluble recombinant P-selectin had no effect, P-selectin coupled to 2 µm beads triggered TF exposure. Cycloheximide did not affect rapid TF exposure, indicating that de novo protein synthesis was not required. These data show that P-selectin on activated platelets rapidly triggers TF exposure on monocytes. This may represent a mechanism by which platelets and monocytes rapidly contribute to intravascular coagulation

Cross-reactivity of anti-HMGB1 antibodies for HMGB2.

HMGB1 and HMGB2 are DNA-interacting proteins but can also have extracellular actions during inflammation. Despite their relatively high homology, they may have distinct roles, making it essential to be able to differentiate between the two. Here we examine the specificity of five commercially-available anti-HMGB1 antibodies. By Western blotting of recombinant proteins and HMGB1-/- mouse embryonic fibroblasts, we identified only one HMGB1 antibody that, under our experimental conditions, did not also detect HMGB2. Selecting specific antibodies for HMGB1 and HMGB2 allowed identification of distinct HMGB1 and HMGB2 subcellular pools in primary neutrophils

Monocyte subsets coregulate inflammatory responses by integrated signaling through TNF and IL-6 at the endothelial cell interface.

Two major monocyte subsets, CD14(+)CD16(−) (classical) and CD14(+/dim)CD16(+) (nonclassical/intermediate), have been described. Each has different functions ascribed in its interactions with vascular endothelial cells (EC), including migration and promoting inflammation. Although monocyte subpopulations have been studied in isolated systems, their influence on EC and on the course of inflammation has been ignored. In this study, using unstimulated or cytokine-activated EC, we observed significant differences in the recruitment, migration, and reverse migration of human monocyte subsets. Associated with this, and based on their patterns of cytokine secretion, there was a difference in their capacity to activate EC and support the secondary recruitment of flowing neutrophils. High levels of TNF were detected in cocultures with nonclassical/intermediate monocytes, the blockade of which significantly reduced neutrophil recruitment. In contrast, classical monocytes secreted high levels of IL-6, the blockade of which resulted in increased neutrophil recruitment. When cocultures contained both monocyte subsets, or when conditioned supernatant from classical monocytes cocultures (IL-6(hi)) was added to nonclassical/intermediate monocyte cocultures (TNF(hi)), the activating effects of TNF were dramatically reduced, implying that when present, the anti-inflammatory activities of IL-6 were dominant over the proinflammatory activities of TNF. These changes in neutrophil recruitment could be explained by regulation of E-selectin on the cocultured EC. This study suggests that recruited human monocyte subsets trigger a regulatory pathway of cytokine-mediated signaling at the EC interface, and we propose that this is a mechanism for limiting the phlogistic activity of newly recruited monocytes

Is CD248 involved in the resolution of inflammation during development of lung sarcoidosis? and the role of adipose tissue-derived stromal cells in the survival of lymphocytes

Recent findings suggest CD248 involvement in inflammation and tissue remodeling/repair. These processes occur during inflammation and fibrogenesis. Therefore we explored the role of CD248 in a mouse model of sarcoidosis. Apolipoprotein-E (ApoE) knock-out, CD248 knock-out, and double knock-out mice were fed a high-fat diet (HFD). Histological analysis was followed with immunofluorescent CD248 staining. ApoE deficiency combined with HFD induced sarcoid-like granulomas. Our results suggest CD248 involvement in the resolution of inflammation. CD248 deficiency influenced disease severity and expression increased in the sarcoid lung. CD248’s influence on resolution is a promising finding. Investigation in other models of inflammation could uncover its immunological role and therapeutic potential. Stromal cells regulate lymphocyte homeostasis in the lymph node and are derived from adipocyte precursors in surrounding fat. Factors regulating lymphocytes in adipose tissue are unknown. We assessed the role of adipose tissue-derived stromal cells in lymphocyte survival. Lymphocyte survival in co-culture with stromal cells was examined using flow cytometry, and expression of key lymphotrophic factors was investigated with real-time PCR. Our findings suggest that pre-adipocytes in the adipose tissue stromal vascular fraction produce IL-7 and CCL19, supporting T lymphocyte survival. Comprehensive understanding of pre-adipocyte modulation of T cells, and how adipose tissue influences immune responses may facilitate development of novel therapies

Platelet P-selectin triggers rapid surface exposure of tissue factor in monocytes

Abstract: Tissue factor (TF) plays a central role in haemostasis and thrombosis. Following vascular damage, vessel wall TF initiates the extrinsic coagulation cascade. TF can also be exposed by monocytes. Inflammatory or infectious stimuli trigger synthesis of new TF protein by monocytes over the course of hours. It has also been suggested that monocytes can expose TF within minutes when stimulated by activated platelets. Here, we have confirmed that monocytes rapidly expose TF in whole blood and further demonstrate that platelet P-selectin exposure is necessary and sufficient. Monocyte TF exposure increased within five minutes in response to platelet activation by PAR1-AP, PAR4-AP or CRP-XL. PAR1-AP did not trigger TF exposure on isolated monocytes unless platelets were also present. In whole blood, PAR1-AP-triggered TF exposure required P-selectin and PGSL-1. In isolated monocytes, although soluble recombinant P-selectin had no effect, P-selectin coupled to 2 µm beads triggered TF exposure. Cycloheximide did not affect rapid TF exposure, indicating that de novo protein synthesis was not required. These data show that P-selectin on activated platelets rapidly triggers TF exposure on monocytes. This may represent a mechanism by which platelets and monocytes rapidly contribute to intravascular coagulation