Aims-This study investigated the presence ofthe cytokine tumour necrosis factor a (TNFa) and the vascular adhesion glyco-

The intracellular Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP-1) is a negative regulator of cell signaling and contributes to the establishment of TCR signaling thresholds in both developing and mature T lymphocytes. Although there is much functional data implicating SHP-1 as a regulator of TCR signaling, the molecular basis for SHP-1 activation in T lymphocytes is poorly defined. A modification of the yeast two-hybrid system was employed to identify in T cells phosphotyrosine-containing proteins capable of binding the SH2 domains of SHP-1. From this yeast tri-hybrid screen, the p85β subunit of phosphatidylinositol 3-kinase and the immunoreceptor tyrosine-based inhibitory motif-containing receptors, leukocyte-associated Ig-like receptor-1 (LAIR-1) and programmed death-1 (PD-1), were identified. Coimmunoprecipitation studies demonstrated that the exclusive phosphotyrosine-containing protein associated with SHP-1 in Jurkat T cells under physiological conditions is LAIR-1. Significantly, this interaction is constitutive and was detected only in the membrane-enriched fraction of cell lysates. Ligand engagement of the SH2 domains of SHP-1 is a prerequisite to activation of the enzyme, and, consistent with an association with LAIR-1, SHP-1 was found to be constitutively active in unstimulated Jurkat T cells. Importantly, a constitutive interaction between LAIR-1 and SHP-1 was also detected in human primary T cells. These results illustrate the sustained recruitment and activation of SHP-1 at the plasma membrane of resting human T cells by an inhibitory receptor. We propose that this mechanism may exert a constitutive negative regulatory role upon T cell signaling.

SUMMARYTo investigate the binding properties of dendritic cells (DC) to vascular endothelium, a comparative analysis was undertaken of DC, monocytes and lymphocytes isolated from the blood of 25 healthy subjects using monolayers of human umbilical vein endothelial cells as the adherence substrate. More blood DC (mean 24% adherence) were adherent to endothelial monolayers than monocytes (mean 18%; P < 0 . 001) and lymphocytes (mean 12%; P < 0 . 001). When the monolayers were pretreated with tumour necrosis factor-alpha (TNF-a) all leucocyte populations exhibited an increased attachment, but there was still greater binding of DC (mean 37% adherence) in comparison with monocytes (mean 23%; P < 0 . 001) and lymphocytes (mean 18%; P < 0 . 001). Flow cytometric analysis revealed that in relation to monocytes and lymphocytes the DC had a higher surface expression of the adhesion molecules CD11a (P < 0 . 05), CD11c (P < 0 . 005) and CD54 (P < 0 . 005) but a lower prevalence of cells bearing CD49d (mean 38%; P < 0 . 05) and the homing receptor CD62L (mean 14%; P < 0 . 001). CD1a was present on 22% of DC and virtually absent from the surface of monocytes and lymphocytes. The intensity of expression of the b 1 -integrins, CD49c, CD49d and CD49e was greater on DC than lymphocytes and monocytes (P < 0 . 05). Antibody blocking studies demonstrated that DC binding to untreated and TNF-a-treated endothelium was dependent upon the expression of CD11a, CD18 and CD49d, and the simultaneous application of anti-CD18 and anti-CD49d antibodies produced an approximate 70% inhibition of adhesion (P < 0 . 001). Thus, the expression of both b 1 -and b 2 -integrins contributes to the adhesive interaction between DC and endothelium.

Extensive evidence has been accumulated to implicate the intracellular protein tyrosine phosphatase, Src homology region 2 domain-containing protein tyrosine phosphatase-1 (SHP-1), as a negative regulator of TCR-signaling thresholds. Specifically, T cells from the SHP-1-deficient mouse, motheaten, exhibit a hyperproliferative phenotype when activated by cognate peptide-pulsed APCs. However, the cellular basis for this phenotype has not been fully explained. Using the intracellular fluorescent dye, CFSE, we show that a greater proportion of motheaten vs control naive CD8+ T cells undergo cell division when activated by peptide-pulsed APCs. Furthermore, there is a greater likelihood of TCRs on SHP-1-deficient vs control T cells binding to peptide/MHC ligands on APCs when using TCR down-regulation as an indirect measure of TCR engagement. In addition, T cell-APC conjugate assays provide direct evidence that a greater proportion of SHP-1-deficient T cells are capable of forming stable conjugates with APCs and this may explain, at least in part, their hyperproliferative response to TCR-triggered stimulation. The physiological relevance of the combined in vitro observations is demonstrated by the significantly enhanced in vivo expansion and CTL capacity generated in mice receiving adoptively transferred SHP-1-deficient naive CD8+ T cells when compared with control T cells.

The present findings indicate that cellular activation may occur during the development of PVR, and suggest that these cytokines may be locally produced by cells infiltrating epiretinal membranes. The presence of IL-1 beta, IL-6 and TNF alpha mRNA-positive cells within retinal membranes provides further evidence of a pathogenic role of these cytokines in proliferative vitreoretinopathy.

The intracellular Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase (SHP-1) has been characterized as a negative regulator of T cell function, contributing to the definition of T cell receptor signaling thresholds in developing and peripheral mouse T lymphocytes. The activation of SHP-1 is achieved through the engagement of its tandem SH2 domains by tyrosine-phosphorylated proteins; however, the identity of the activating ligand(s) for SHP-1, within mouse primary T cells, is presently unresolved. The identification of SHP-1 ligand(s) in primary T cells would provide crucial insight into the molecular mechanisms by which SHP-1 contributes to in vivo thresholds for T cell activation. Here we present a combination of biochemical and yeast genetic analyses indicating CD22 to be a T cell ligand for the SHP-1 SH2 domains. Based on these observations we have confirmed that CD22 is indeed expressed on mouse primary T cells and capable of associating with SHP-1. Significantly, CD22-deficient T cells demonstrate enhanced proliferation in response to anti-CD3 or allogeneic stimulation. Furthermore, the co-engagement of CD3 and CD22 results in a raising of TCR signaling thresholds hence demonstrating a previously unsuspected functional role for CD22 in primary T cells. SHP-1,1 an intracellular protein-tyrosine phosphatase, has been demonstrated to be a negative regulator of TCR signaling thresholds (1). SHP-1 is normally maintained in a catalytically inactive state whereby activation minimally requires the engagement of the amino-terminal SH2 domain of SHP-1 by phosphotyrosine (PY)-containing ligand (2, 3). It is predicted that SHP-1-activating ligand(s) exists on mouse naïve T cells based on substantial functional evidence indicating SHP-1 to be catalytically active in naïve T cells (1, 4 -8). It is currently assumed that SHP-1 is activated by one or more components of the TCR signaling pathway. Indeed, the intracellular proteintyrosine kinase, ZAP-70, has been proposed to bind SHP-1 (9).However, the best evidence of SHP-1-associating molecules in other hemopoietic cells relates to the family of immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors (10). In particular, ITIM receptors Ly49 and CD66a associate with SHP-1 in subpopulations of primary T cells, but to date there has been no definition of the ITIM receptors that activate SHP-1 in the majority of mouse primary T cells (11).In the first instance, we have exploited SHP-1-deficient moth-eaten T cells to assist in the definition of genuine associations between SHP-1 and TCR signaling components in CD3/TCR-stimulated mouse primary T cells. Our results reveal no binding of the CD3 invariant chains or ZAP-70 to SHP-1 in mouse primary T cells following TCR/CD3 ligation. However, by employing pervanadate (PV) to induce a robust tyrosine phosphorylation of cellular proteins in primary T cells, we demonstrated that a glycosylated tyrosyl phosphoprotein of 150 kDa, (pp150) associates with SHP-1 in mouse peripheral T cells. We have ident...

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This study provides biochemical and functional evidence pertaining to the role of the intracellular protein tyrosine phosphatase, SHP‐1, in influencing thresholds for TCR activation. Although the loss of SHP‐1 in thymocytes from motheaten mice had minimal effects on the initial rise of cytosolic Ca2+ concentration following TCR triggering, the post‐stimulation equilibrium levelsof Ca2+ were consistently elevated. In keeping with a SHP‐1 effect on PLCγ function, IP3 generation was increased in SHP‐1 deficient thymocytes. Importantly, we demonstrate that loss of SHP‐1 results in a relaxation of the normally stringent co‐stimulatory requirements for IL‐2 production. SHP‐1 deficient single‐positive CD4+ thymocytes revealed a significantly enhanced capacity to produce IL‐2 in response to anti‐CD3 stimulation alone. In contrast, the simultaneous triggering of CD3 and CD28 was required for equivalent IL‐2 production in control single‐positive CD4+ thymocytes. Furthermore, SHP‐1 deficient thymocytes generated an increased and prolonged proliferative response to anti‐CD3 stimulation alone. In addition, the simultaneous triggering of CD28 and CD3 resulted in equivalent proliferative responses in SHP‐1‐deficient and control thymocytes, suggesting that a strong co‐stimulatory signal is able to override the effect of SHP‐1 loss on TCR hyperresponsiveness. Collectively, these results suggest that SHP‐1, rather than acting directly on TCR signaling, may indirectly raise thresholds for TCR triggering by modulating co‐stimulatory signals.