A new role for platelet-endothelial cell adhesion molecule-1 (CD31): inhibition of TCR-mediated signal transduction.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed by endothelial cells, platelets, monocytes, neutrophils, and certain T cell subsets. The PECAM-1 extracellular domain has six Ig-homology domains that share sequence similarity with cellular adhesion molecules. The PECAM-1 cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when appropriately engaged, becomes phosphorylated on tyrosine residues, creating docking sites for nontransmembrane, Src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1 and SHP-2. The purpose of the present study was to determine whether PECAM-1 inhibits protein tyrosine kinase (PTK)-dependent signal transduction mediated by the immunoreceptor tyrosine-based activation motif-containing TCR. Jurkat cells, which coexpress PECAM-1 and the TCR/CD3 complex, were INDO-1AM-labeled and then incubated with anti-CD3epsilon mAbs, anti-PECAM-1 mAbs, or both, and goat anti-mouse IgG was used to cross-link surface-bound mAbs. Calcium mobilization induced by CD3 cross-linking was found to be attenuated by coligation of PECAM-1 in a dose-dependent manner. PECAM-1-mediated inhibition of TCR signaling was attributable, at least in part, to inhibition of release of calcium from intracellular stores. These data provide evidence that PECAM-1 can dampen signals transduced by ITAM-containing receptors and support inclusion of PECAM-1 within the family of ITIM-containing inhibitors of PTK-dependent signal transduction.

Interferon-gamma secretion defects in haemophilia A patients receiving highly purified plasma-derived or recombinant factor VIII.

The outcome of developing immune responses is influenced by interactions among a large and complex network of secreted cytokines. T-cell secretion of interferon-gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha) and TNF-beta, or lymphotoxin contributes to the development of cell-mediated immunity, whereas secretion of interleukin (IL)-4, IL-5 and IL-6 contributes to development of humoral immunity. Humoral immunity to factor VIII (FVIII) develops in approximately 25% of severe haemophilia A patients. The aim of our research was to understand the underlying immune response to FVIII in patients with FVIII inhibitors. We report a defect in IFN-gamma secretion by peripheral blood mononuclear cells (PBMC) derived from haemophilia A patients, which was accompanied by a low level of mitogen-induced proliferation and a significant decrease in the percentage of natural killer (NK) cells. All of the observed defects were found in haemophilia A patients, both with and without FVIII inhibitors, who were free of viral infection and had been treated predominantly or exclusively with monoclonal antibody-purified or recombinant FVIII.

T cell recognition of MHC class II-associated peptides is independent of peptide affinity for MHC and sodium dodecyl sulfate stability of the peptide/MHC complex. Effects of conservative amino acid substitutions at anchor position 1 of influenza matrix protein19-31.

T cells recognize peptide fragments of Ags bound to MHC-encoded molecules. Pockets in the MHC peptide-binding groove accommodate a limited set of amino acid side chains present at anchor positions in peptide; however, the functional significance of accommodation of different side chains at an anchor position in peptide is not clear. A panel of T cell clones was evaluated to test the effect of conservative amino acid substitution at a primary peptide anchor position. Results of T cell stimulation studies were correlated with two well studied characteristics of the peptide/MHC complex, which are the affinity of peptide binding to MHC and the stability of the resulting complex upon PAGE in the presence of SDS. We found that formation of a functional complex required neither high affinity peptide binding nor SDS stability. Furthermore, T cell clones differed in their ability to recognize individual peptide variants, suggesting that some structural aspect of the peptide/MHC complex is influenced by interactions between peptide anchor residues and MHC pockets.

Differential effect of polymorphism at HLA-DR1 beta-chain positions 85 and 86 on binding and recognition of DR1-restricted antigenic peptides.

The valine-glycine dimorphism at position 86 of the DR beta-chain exhibited by most DR alleles has been shown to affect peptide binding. We demonstrate that DR1-restricted antigenic peptides differ in the extent to which binding is affected by amino acid substitution at positions 85 and 86 of the DR1 beta-chain. Binding of peptides derived from influenza hemagglutinin (HA306-320) and tetanus toxin (TT830-843) but not influenza matrix protein (MP19-31) was diminished on cells expressing DR1 beta-chains encoded by DRB1*0102 relative to DRB1*0101. The presence of tyrosine within HA306-320 and TT830-843 vs leucine within MP19-31 at a single DR contact position was revealed by alignment of the peptides according to a DR-binding motif. HA306-320 bearing leucine at this position (HA 306-320L309) bound to DR1 possessing either DRB1*0101- or DRB1*0102-encoded beta-chains suggesting that DR residues may discriminate among peptides based upon amino acid identity at a single position within the peptide. Furthermore, whereas all HA306-320-specific T cell clones recognized HA306-320L309 in the context of DR1 molecules possessing DRB1*0102-encoded beta-chains, some T cell clones failed to recognize HA306-320L309 in the context of DR1 molecules possessing DRB1*0101-encoded beta-chains. These results suggest that peptide conformation may also be affected by amino acid substitution at positions 85 and or 86 of the DR1 beta-chain.

Effects of localized HLA class II beta chain polymorphism on binding of antigenic peptide and stimulation of T cells.

The relationship between HLA-DR1 polymorphism and recognition of antigen by T cells was investigated. Two allelic variants of HLA-DR1, which differ by amino acid substitution at positions 85 and 86 of the beta chain, were characterized for the effect of substitution on recognition of foreign antigen by DR1-restricted T cells. Substitution of alanine and valine for valine and glycine residues at positions 85 and 86 of the DR1 beta chain resulted in deficient T-cell stimulation as demonstrated by the requirement for higher concentrations of antigen to induce maximal levels of T-cell proliferation, induction of lower levels of proliferation at optimal antigen concentrations, and slower kinetics of formation of stimulatory peptide-DR1 complexes. Direct binding studies employing both biotinylated and radioiodinated forms of antigenic peptide demonstrated quantitatively lower levels of peptide bound to substituted DR1 molecules and low levels of site-specific binding as assessed by competitive inhibition analyses. The effect of MHC class II polymorphism on peptide-binding affinity as opposed to induction of appropriate peptide conformation and the impact of polymorphism at DR1 beta chain positions 85 and 86 on allorecognition of HLA-DR1 are discussed.

Assessment of chemokinetic behavior of inflammatory lung macrophages in a linear under-agarose assay.

Migration of cells in response to a chemoattractant gradient is influenced by directed migration (chemotaxis) and stimulated random motility (chemokinesis). The present study quantitated the chemokinetic motility of normal and inflammatory lung macrophages by performing the linear under-agarose assay in the presence of uniform concentrations of chemoattractant. Under these conditions, cell motility can be likened to a molecular diffusion process. Mathematical analyses which describe molecular diffusion were then applied, allowing the quantitation of the parameter, mu, the cellular equivalent to the molecular diffusivity constant. Determination of changes in mu as a function of chemoattractant concentration revealed that the chemokinetic motility of alveolar macrophages recovered during the early stages of acute pulmonary inflammation was greater than that of normal alveolar macrophages and macrophages recovered later in the inflammatory response. The correlation of differences in macrophage chemokinesis with macrophage maturation and the relevance of these differences to macrophage accumulation during inflammation are discussed.

Pathogenesis of acute pulmonary inflammation in strain 2 and strain 13 guinea pigs.

An acute inflammatory response was elicited in the lungs of strain 2 and 13 guinea pigs following immunization and aerosol challenge with ovalbumin. The pulmonary inflammatory response, characterized by hemorrhage and influx of inflammatory cells, progressed from initiation at 12-hours postchallenge through resolution at 96-hours postchallenge. Inflammatory and immunoregulatory cells, recovered by bronchoalveolar lavage, showed quantitative changes in their relative contribution to the bronchoalveolar cell infiltrate over the course of inflammation. Changes in concentrations of macrophages and T cells, in particular, are discussed in terms of their possible contributions to initiation and resolution of acute pulmonary inflammation.

Cellular immunoregulation of acute pulmonary inflammation in strain 2 guinea pigs.

Subclasses of lung immunoregulatory T cells were analyzed during acute pulmonary inflammation in strain 2 guinea pigs and compared with T cell subpopulations in the peripheral circulation. Immunized animals were aerosol-challenged with specific antigen and sacrificed at 12-, 24-, 48-, 72-, and 96-hours postchallenge. Mononuclear cells, isolated from peripheral blood and bronchoalveolar lavage, were enriched for T cells. The percentage of helper T cells, as well as antigen-specific blastogenesis, in recovered pulmonary T cells exhibited maximal values at 12-, 24-, and 96-hours postchallenge. In contrast, the presence of suppressor T cells correlated with decreased blastogenesis and antigen-specific suppression in isolated lung cells at 72-hours postchallenge. Since changes in pulmonary cells did not correlate with those found in the peripheral circulation, immunoregulatory events in these two compartments may be distinct. These results indicate that the proportions of lung T cell subclasses, as well as their in vitro functional activity, are altered over the course of pulmonary disease. Such changes in immunoregulatory cell populations may be important in the mediation of disease pathogenesis.