Darrell Pilling

Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation

Fibroblasts are important sentinel cells in the immune system and, here, it is proposed that these cells play a critical role in the switch from acute inflammation to adaptive immunity and tissue repair. It is suggested that chronic inflammation occurs because of disordered fibroblast behaviour in which failure to switch off their inflammatory programme leads to the inappropriate survival and retention of leukocytes within inflamed tissue.

RGD peptides induce apoptosis by direct caspase-3 activation

Synthetic peptides containing the arginine–glycine–aspartate (RGD) motif have been used extensively as inhibitors of integrin–ligand interactions in studies of cell adhesion, migration, growth and differentiation,,, because the RGD motif is an integrin-recognition motif found in many ligands. Here we report that RGD-containing peptides are able to directly induce apoptosis without any requirement for integrin-mediated cell clustering or signals. We show that RGD-containing peptides enter cells and directly induce autoprocessing and enzymatic activity of pro-caspase-3, a pro-apoptotic protein. Using the breast carcinoma cell line MCF-7, which has a functional deletion of the caspase-3 gene, we confirm that caspase-3 is required for RGD-mediated cell death. In addition to an RGD motif, pro-caspase-3 also contains a potential RGD-binding motif, aspartate–aspartate–methionine (DDM), near the site of processing to produce the p12 and p17 subunits. On the basis of the ability of RGD–DDX interactions to trigger integrin activation, we suggest that RGD peptides induce apoptosis by triggering conformational changes that promote pro-caspase-3 autoprocessing and activation. These findings provide an alternative molecular explanation for the potent pro-apoptotic properties of RGD peptides in models of angiogenesis, inflammation and cancer metastasis,,.

Inhibition of T cell apoptosis in the rheumatoid synovium.

Synovial T cells in rheumatoid arthritis are highly differentiated and express a phenotype suggesting susceptibility to apoptosis (CD45RB dull, CD45RO bright, Bcl-2 low, Bax high, Fas high). However, no evidence of T cell apoptosis was found in synovial fluid from any of 28 patients studied. In contrast, synovial fluid from 10 patients with crystal arthritis showed substantial levels of T cell apoptosis. The failre of apoptosis was not an intrinsic property of rheumatoid synovial T cells, as they showed rapid spontaneous apoptosis on removal from the joint. Synovial T cells from rheumatoid arthritis and gout patients could be rescued from spontaneous apoptosis in vitro either by IL-2R gamma chain signaling cytokines (which upregulate Bcl-2 and Bcl-XL) or by interaction with synovial fibroblasts (which upregulates Bcl-xL but not Bcl-2). The phenotype of rheumatoid synovial T cells ex vivo (Bcl-2 low, Bcl-xL high) suggested a fibroblast-mediated mechanism in vivo. This was confirmed by in vitro culture of synovial T cells with fibroblasts which maintained the Bcl-xL high Bcl-2 low phenotype. Synovial T cells from gout patients were Bcl-2 low Bcl-xL low and showed clear evidence of apoptosis in vivo. Inhibition experiments suggested that an integrin-ligand interaction incorporating the Arg-Gly-Asp motif is involved in fibroblast-mediated synovial T cell survival. We propose that environmental blockade of cell death resulting from interaction with stromal cells is a major factor in the persistent T cell infiltration of chronically inflamed rheumatoid synovium.

Purification of human blood eosinophils by negative selection using immunomagnetic beads.

A simple method for isolating highly purified eosinophils from human blood is described. Buffy coats from normal individuals (eosinophil counts less than 0.4 x 10(9)/litre) were centrifuged through a two layer Percoll density gradient, to produce a granulocyte fraction containing neutrophils and eosinophils. Neutrophils were extracted from this fraction using a monoclonal antibody (CLB FcR gran 1) against CD 16 (Fc gamma R III) in a direct or indirect selection procedure using immunomagnetic beads (Dynabeads). This negative immunoselection produced eosinophils of greater purity and with a superior capacity to mount a respiratory burst than eosinophils isolated by a method employing metrizamide.

Inhibition of T cell apoptosis by IFN-β rapidly reverses nuclear translocation of protein kinase C-δ

Type I interferons rescue activated human T cells from cytokine deprivation‐induced apoptosis. Our data now show that IFN‐β also rapidly inhibits apoptotic signals induced through the Fas receptor (CD95) in human T cells. To identify upstream signaling elements that could be targets of IFN‐β, we have studied protein kinase C (PKC). PKC‐δ is actively involved in the regulation of apoptosis and immunofluorescence staining revealed that early in apoptosis PKC‐δ accumulated in the nucleus. Addition of IFN‐β to T cells already deprived of survival factors or treated with anti‐Fas antibody caused a rapid retranslocation of PKC‐δ away from the nucleus. Furthermore, the generation of a constitutively active catalytic fragment by cleavage of PKC‐δ by caspase 3 occurred only after translocation of full‐length PKC‐δ to the nucleus. IFN‐β also inhibited caspase 3 and the proteolytic activation of PKC‐δ. We conclude from these studies that nuclear translocation of PKC‐δ is an early event in T cell apoptosis and that IFN‐β rapidly reverses this process.

Inhibition of T Cell Apoptosis in the Aqueous Humor of Patients with Uveitis by IL-6/Soluble IL-6 Receptor trans-Signaling

A fundamental mechanism of immune privilege in the eye is the induction of T lymphocyte apoptosis. Intraocular inflammation in uveitis implies compromise of immune privilege. This study sought to determine whether apoptosis of T cells is actively inhibited in patients with uveitis and by what pathways this may occur. Apoptotic lymphocytes were found to be absent from aqueous humor (AqH) of virtually all patients with recent-onset uveitis. However, T cells removed from the eye were highly susceptible to both spontaneous and Fas ligand-induced apoptosis in vitro. AqH from patients with uveitis had no modulatory effect on Fas ligand-induced apoptosis, but strongly suppressed survival factor deprivation-induced apoptosis. In contrast, noninflammatory AqH from patients undergoing cataract surgery had no modulatory effects on apoptosis at all. These data suggest that triggering of the Fas pathway is diminished in uveitis, and also that homeostatic resolution through survival factor deprivation-induced apoptosis is inhibited by factors present in AqH. The most widely recognized pathways, common γ-chain cytokines and type I IFNs, did not contribute to AqH-mediated T cell survival. High levels of both IL-6 and soluble IL-6R were found in AqH. IL-6 alone did not induce T cell survival, because IL-6R expression on T cells in AqH was too low to facilitate signaling. However, combinations of IL-6 and soluble IL-6R were highly effective inhibitors of T cell apoptosis, suggesting that the trans-signaling pathway is likely to be a key mediator of T cell apoptosis inhibition mediated by uveitis AqH.

Type 1 IFN Maintains the Survival of Anergic CD4+ T Cells

Anergic T cells have immunoregulatory activity and can survive for extended periods in vivo. It is unclear how anergic T cells escape from deletion, because both anergy and apoptosis can occur after TCR ligation. Stimulation of human CD4+ T cell clones reactive to influenza hemagglutinin peptides can occur in the absence of APCs when MHC class II-expressing, activated T cells present peptide to each other. This T:T peptide presentation can induce CD95-mediated apoptosis, while the cells that do not die are anergic. We found that the death after peptide or anti-CD3 treatment of a panel of CD4+ T cell clones is blocked by IFN-β secreted by fibroblasts and also by IFN-α. This increases cell recovery after stimulation, which is not due to T cell proliferation. This mechanism for apoptosis inhibition rapidly stops protein kinase C-δ translocation from the cytoplasm to the nucleus, which is an early event in the death process. A central observation was that CD4+ T cells that are rescued from apoptosis after T:T presentation of peptide by IFN-αβ remain profoundly anergic to rechallenge with Ag-pulsed APCs. However, anergized cells retain the ability to respond to IL-2, showing that they are nonresponsive but functional. The prevention of peptide-induced apoptosis in activated T cells by IFN-αβ is a novel mechanism that may enable the survival and maintenance of anergic T cell populations after TCR engagement. This has important implications for the persistence of anergic T cells with the potential for immunoregulatory function in vivo.

Cytokine-mediated inhibition of apoptosis in non-transformed T cells and neutrophils can be dissociated from protein kinase B activation

In the absence of survival‐inducing cytokines activated T cells and neutrophils enter apoptosis spontaneously. Phosphatidylinositol 3‐kinase (PI3 K) activation and signaling through PKB/AKT have been widely linked to the inhibition of apoptosis by cytokines. Here we have investigated the role of PKB in the inhibition of spontaneous apoptosis of activated human CD4+ T cells and neutrophils. We used a range of cytokines known to induce survival and/or activation of PKB. We found activation of PKB in T cells treated with IL‐2 and insulin, and neutrophils cultured with N‐formyl‐Met‐Leu‐Phe (fMLP), insulin or granulocyte‐macrophage colony‐stimulating factor. Insulin did not inhibit apoptosis in neutrophils or T cells and fMLP did not delay neutrophil apoptosis. Intriguingly, IFN‐β induced PI3 K‐dependent survival in both cell types, but did not activate PKB. IL‐2 mediated rescue of T cells from apoptosis but no induction of proliferation occurred in thepresence of LY294002, an inhibitor of PI3 K, which also blocked subsequent PKB activation. The main role of PI3 K in IL‐2‐mediated signaling may therefore be in the regulation of proliferation. These findings suggest that activation of PKB and inhibition of apoptosis can be dissociated in cytokine‐mediated rescue of non‐transformed CD4+ T cells and neutrophils.

MHC restriction of synovial fluid lymphocyte responses to the triggering organism in reactive arthritis. Absence of a class I‐restricted response

Synovial fluid mononuclear cells (SFMC) from patients with reactive arthritis (ReA) show marked proliferative responses to preparations of the organism triggering the arthritis. Initial studies with MHC‐specific MoAbs have indicated that a significant element of these proliferative responses is mediated by class II MHC‐restricted CD4+ T cells. It is imperative to establish the presence or absence of a class I‐restricted response, for two reasons. Firstly, the association of ReA with the MHC class I molecule. HLA B27, raises the possibility of there being a B27‐restricted response to the triggering organism. Secondly, a number of the organisms associated with ReA are intracellular pathogens, whose antigens might be expected to be presented by class I MHC molecules. In an effort to identify a class I MHC‐restricted pathogen‐specific response in the SFMC of ReA patients, we have assessed the proliferative responses of SFMC depleted of CD4+ T cells. Responses were grossly diminished by CD4+ T cell depletion. We also investigated Chlamydia‐specific cytotoxicity in the SFMC of patients with sexually acquired ReA in a system using productive chlamydial infection to produce both targets and effectors. Significant antigen specific cytotoxicity was not seen. These experiments do not provide evidence to support the existence of pathogen‐specific responses by CD8+, class 1‐restricted synovial fluid T cells in ReA.

The kinetics of interaction between lymphocytes and magnetic polymer particles

Magnetic polymer-coated particles linked to antibodies are considered to be an efficient rosetting matrix for immunoselection. We have shown that a 20:1 bead:target cell ratio and a 90 min incubation period are the optimal conditions for specific binding of monoclonal antibody-labelled cells to goat anti-mouse IgG-coated beads. Higher ratios or longer incubation periods resulted in considerable non-specific binding. Characterisation of the optimal conditions for specific depletion of lymphocyte subpopulations showed that (a) a range of bead:target cell ratios and incubation periods can be used, with resulting high efficiency and specificity; (b) multiple monoclonal antibodies can be used simultaneously for the depletion of diverse lymphocyte subpopulations; (c) non-specific bead-to-cell binding does not affect the specificity and efficiency of magnetic depletion; (d) specific binding of one bead only was adequate for effective magnetic separation. These findings define the most economical, specific and efficient conditions of use of beads for negative immunoselection but preclude the use of beads as an analytical rosetting medium.