Porcine gammadelta T cells: possible roles on the innate and adaptive immune responses following virus infection.

gammadelta T cells recognise different types of antigen in alternative ways to alphabeta T cells, and thus appear to play a complementary role in the immune response. However, unlike alphabeta T cells, the role or function of gammadelta T cells is still unclear. As pigs possess a high proportion of circulating gammadelta T cells, they are suitable large animal model to study gammadelta T cell functions. This as yet has not been fully exploited, leaving porcine gammadelta T cell biology and its role in immunity in its infancy. Foot-and-mouth disease (FMD) high potency "emergency" vaccines are able to induce early protection from challenge and it has been suggested that, in part, there is some involvement of innate immune responses. The antigen component of the vaccine is able to stimulate purified naive pig gammadelta T cells and induce the mRNA of various cytokines and chemokines. This observation suggests that gammadelta T cells probably contribute to the early phase of the immune responses to FMD vaccination, and perhaps infection. A subset of these circulating gammadelta T cells display a phenotype similar to professional antigen presenting cells and are able to take up and present soluble antigen to CD4(+) T cells in a direct cell-cell interaction via MHC class II. This direct interaction between gammadelta T cells and CD4(+) T cells is likely to have a significant influence on the out come of the adaptive immune response.

A sub-population of circulating porcine gammadelta T cells can act as professional antigen presenting cells.

A sub-population of circulating porcine gammadelta T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by gammadelta T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When gammadelta T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with gammadelta T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of gammadelta T cells with chloroquine. These results indicate that a sub-population of circulating porcine gammadelta T cells act as APCs and present antigen via MHC class II.

Mechanism of inactivation of NF-kappa B by a viral homologue of I kappa b alpha. Signal-induced release of i kappa b alpha results in binding of the viral homologue to NF-kappa B.

Activation of the nuclear factor kappa B plays a key role in viral pathogenesis, resulting in inflammation and modulation of the immune response. We have previously shown that A238L, an open reading frame from African swine fever virus (ASFV), encoding a protein with 40% homology to porcine I kappa B alpha exerts a potent anti-inflammatory effect in host macrophages, where it down-regulates NF-kappa B-dependent gene transcription and proinflammatory cytokine production. This paper reveals the mechanism of suppression of NF-kappa B activity by A238Lp. A238Lp is synthesized throughout infection as two molecular mass forms of 28 and 32 kDa, and vaccinia-mediated expression of A238L demonstrated that both proteins are produced from a single gene. Significantly, the higher 32-kDa form of A238L, but not the 28-kDa form, interacts directly with RelA, the 65-kDa subunit of NF-kappa B, indicating that the binding is dependent on a post-translational modification. Immunoprecipitation analysis shows the NF-kappa B p65-A238L p32 heterodimer is a separate complex from NF-kappa B-I kappa B alpha, and it resides in the cytoplasm. Moreover, we show that ASFV infection stimulates the NF kappa B signal transduction pathway, which results in the rapid degradation of endogenous I kappa B alpha, although both forms of A238Lp are resistant to stimulus-induced degradation. Using the proteasome inhibitor MG132, we show that when degradation of I kappa B alpha is inhibited, A238Lp binding to NF-kappa B p65 is reduced. The results suggest that the virus exploits its activation of the NF-kappa B pathway to enable its own I kappa B homologue to bind to NF-kappa B p65. Last, we show that synthesis of I kappa B alpha is increased during ASFV infection, indicating RelA-independent transcription of the I kappa B alpha gene.

The use of permeabilized cells to study the ion requirements of receptor-ligand dissociation in endosomes.

The mannose receptor mediates the transport of high-mannose glycoproteins from the cell surface to lysosomes in macrophages. The binding of ligand to the receptor is dependent on both pH and Ca2+. Upon internalization, ligands enter an acidic pre-lysosomal compartment where receptor-ligand dissociation takes place. Acidification is driven by an endosomal proton pump and anion transport is coupled to this acidification step. A permeabilized-cell assay has been designed to characterize the ionic requirements for receptor-ligand dissociation in endosomes. The plasma membrane of macrophages has been permeabilized selectively with digitonin without affecting endosomal membranes. Receptor-ligand dissociation in permeabilized cells required ATP and was blocked by proton ionophores. Di-isothiocyanostilbene-disulphonic acid and N-ethylmaleimide also blocked dissociation, but mitochondrial ATPase inhibitors and vanadate were ineffective. To explore the nature of the anion requirement for acidification, the ability of different anions to compensate for Cl- was tested. For the halide series, Br- was as equally effective as Cl- in supporting receptor-ligand dissociation, but I- was inhibitory. Citrate and gluconate were only partially effective, while SO4(2-), NO3- and PO4(2-) blocked dissociation. Addition of Ca2+ to permeabilized-cell preparations impaired ATP-dependent dissociation without affecting endosome acidification. These results suggest that the endosomal membrane has a Ca2+ conductance that would permit the rapid efflux of Ca2+ from endosomes during acidification, and this would appear to be a necessary step for efficient sorting of Ca2+-dependent receptors from their ligands.

Human CD3-epsilon gene contains three miniexons and is transcribed from a non-TATA promoter.

The antigen receptor of the T lymphocyte consists of two variable T-cell receptor chains (either TCR-alpha, TCR-beta or TCR-gamma, TCR-delta) noncovalently linked to four different invariant membrane proteins (CD3-gamma, CD3-delta, CD3-epsilon, and the CD3-zeta homodimer). The CD3 genes are expressed early in thymocyte development, preceding the rearrangement and expression of the T-cell receptor genes. Here we report the isolation and structural analysis of the human CD3-epsilon gene. The gene consisted of nine exons. Three exons, encoding the junction of leader peptide and mature protein, were extremely small (21, 15, and 18 base pairs, respectively). The murine gene contained only two such miniexons, the sequences of which were not homologous to those of the three human miniexons. But from comparisons of intron sequences the regions surrounding the human miniexons III and IV appeared to be closely related to those surrounding the murine miniexons III and IV. The most-3' miniexon in the human gene (IVa) had no murine counterpart and appeared not to duplicate any of the other miniexons. Sequence analysis of CD3-epsilon cDNA clones isolated from four independent libraries gave no evidence for alternative use of these miniexons. Like CD3-delta, the CD3-epsilon gene was transcribed from a weak, nontissue-specific, TATA-less promoter. Pulsed-field electrophoresis showed that the human CD3-epsilon gene was separated from the CD3-gamma, CD3-delta gene pair by at least 30 kilobases, but by no more than 300 kilobases.

Isolation and characterization of a mannose-specific endocytosis receptor from rabbit alveolar macrophages.

Rabbit alveolar macrophages express a plasma-membrane receptor that recognizes glycoprotein ligands bearing terminal mannose, fucose or N-acetylglucosamine residues. Macrophage membranes were washed extensively with buffers containing high salt and mannose or EDTA to remove endogenously bound ligand, before Triton X-100 extraction. The extracts were chromatographed on mannose-Sepharose. Elution with mannose, followed by dialysis and a second mannose-Sepharose step with EDTA elution, produced a preparation that migrated as single protein band of Mr 175,000 on SDS/polyacrylamide-gel electrophoresis. The purified protein binds mannose-BSA (bovine serum albumin) with a dissociation constant of 1.9 X 10(-8) M. Ligand binding is Ca2+ and pH-dependent, with maximal binding at neutral pH and low binding below pH 6.0. The binding of 125I-mannose-BSA is inhibited by ligands bearing high-mannose oligosaccharides, such as mannan or beta-glucuronidase, as well as the monosaccharides mannose, fucose and N-acetylglucosamine. Galactose, galactosylated BSA, glucose and mannose 6-phosphate are non-inhibitory. Amino acid compositional analyses indicate that the receptor contains high concentrations of aspartate/asparagine and glutamate/glutamine, and low amounts of methionine. The carbohydrate composition was studied by lectin overlays of electrophoretically transferred receptor, and the results indicate the presence of N-linked complex and O-linked sialylated oligosaccharides. A protein of Mr 175,000 was immunoprecipitated from radio-iodinated macrophage membranes with an antibody generated against purified rabbit lung mannose receptor.

Isolation and characterization of a mannose-specific endocytosis receptor from human placenta.

A receptor which recognizes glycoproteins bearing terminal mannose residues has been isolated from human placental membranes. Washed membranes were solubilized with buffer containing 1% Triton X-100 and applied to a mannose-Sepharose affinity column. The column was eluted with buffer containing 200 mM mannose and 1% cholate. The major protein eluted exhibited a molecular weight of 175 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein binds 125I-labeled mannosylated bovine serum albumin in a saturable fashion with a dissociation constant of 4 nM. Ligand binding is pH-dependent with maximal binding above pH 6.5. This binding can be inhibited with EDTA, mannose, fucose, mannan, beta-glucuronidase, and bovine serum albumin conjugated to fucose. Polyclonal antibodies generated against the mannose binding protein immunoprecipitate a single 175-kDa protein species from both surface-iodinated and biosynthetically labeled human monocyte-derived macrophages.

Identification of the macrophage mannose receptor as a 175-kDa membrane protein.

Mannose-lactoperoxidase, a neoglycoprotein prepared by reaction of lactoperoxidase with cyanomethyl 1-thiomannoside, bound to alveolar macrophages at 4 degrees C (Kd = 5.8 X 10(-8) M) and was rapidly internalized at 37 degrees C (K uptake = 2 X 10(-8) M). Mannose-lactoperoxidase binding and uptake were blocked by yeast mannan, and mannose-lactoperoxidase inhibited uptake of 125I-labeled mannose-BSA (bovine serum albumin). Radioiodination of cells with surface-bound mannose-lactoperoxidase was carried out in the presence of glucose and glucose oxidase. A major polypeptide (175 kDa) was radioiodinated by this procedure. Iodination of the 175-kDa polypeptide appeared to be receptor-mediated, since it was blocked by the presence of yeast mannan. Specific iodination was absent from receptor-negative cells. To demonstrate that the 175-kDa species is a ligand-binding protein, cells were iodinated by the standard lactoperoxidase method. Washed cells were then allowed to bind mannose-BSA. Receptor-ligand complexes, prepared by detergent extraction, were passed over anti-BSA IgG affinity columns. Mannose, but not mannose 6-phosphate or galactose, eluted a radioactive protein from the column that migrated with an apparent molecular mass of 175 kDa on NaDodSO4/PAGE. Detergent extracts of crude membranes prepared from macrophage-enriched whole rabbit lung were adsorbed to mannose-Sepharose; the fraction obtained by elution with mannose contained two protein components of 175 and 55 kDa. Subsequent chromatography on N-acetylglucosamine-agarose yielded a single protein of 175 kDa. The 175-kDa polypeptide was shown to bind 125I-labeled mannose-BSA in a precipitation assay. This binding could be blocked with mannan or mannose-BSA. The results indicate that the cell-surface mannose receptor is a 175-kDa protein.

Soluble asparaginase-dextran conjugates show increased circulatory persistence and lowered antigen reactivity.

Oxidized dextrans of increasing molecular weight were bound covalently to Erwinia carotovora asparaginase. The resulting conjugates retained 50% of their enzyme activity and showed marked resistance to proteolysis by trypsin and chymotrypsin and inactivation by asparaginase-specific antibody. When tested in-vivo, the larger molecular weight conjugates showed prolonged circulatory survival in both immune and non-immune animals and failed to elicit full type III hypersensitivity or anaphylactic reactions when injected into sensitized guinea-pigs. Rabbits could tolerate multiple doses of the asparaginase conjugate without developing an immunity to the enzyme. A conjugate showing increased circulatory half-life and lowered antigen reactivity should have therapeutic potential.

Mannose-specific oligosaccharide recognition by mononuclear phagocytes.

Glycoproteins terminating in mannose are recognized by receptors on macrophages. The mannose receptor is expressed by a variety of macrophages but expression is closely regulated. Activated macrophages, for example, express little mannose receptor activity. Kinetic and fractionation experiments suggest that cell surface mannose receptors recycle to and from an acidic, pre-lysosomal compartment. Preliminary evidence suggests that the mannose receptor is a large polypeptide and that it is structurally related to the mannose binding protein found in serum. The mannose receptor may, among other possibilities, regulate the extracellular levels of lysosomal hydrolases.