Role of Citrullinated Collagen in Autoimmune Arthritis.

Citrullination of proteins plays an important role in protein function and it has recently become clear that citrullinated proteins play a role in immune responses. In this study we examined how citrullinated collagen, an extracellular matrix protein, affects T-cell function during the development of autoimmune arthritis. Using an HLA-DR1 transgenic mouse model of rheumatoid arthritis, mice were treated intraperitoneally with either native type I collagen (CI), citrullinated CI (cit-CI), or phosphate buffered saline (PBS) prior to induction of autoimmune arthritis. While the mice given native CI had significantly less severe arthritis than controls administered PBS, mice receiving cit-CI had no decrease in the severity of autoimmune arthritis. Using Jurkat cells expressing the inhibitory receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), Western blot analysis indicated that while CI and cit-CI bound to LAIR-1 with similar affinity, only CI induced phosphorylation of the LAIR ITIM tyrosines; cit-CI was ineffective. These data suggest that cit-CI acts as an antagonist of LAIR-1 signaling, and that the severity of autoimmune arthritis can effectively be altered by targeting T cells with citrullinated collagen.

Pathways involved in the synergistic activation of macrophages by lipoteichoic acid and hemoglobin.

Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-ß mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response.

Directed evaluation of enterotoxigenic Escherichia coli autotransporter proteins as putative vaccine candidates.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies. METHODOLOGY: Available ETEC genomes were examined to identify AT coding sequences present in pathogenic isolates, but not in the commensal E. coli HS strain. Passenger domains of the corresponding autotransporters were cloned and expressed as recombinant antigens, and the immune response to these proteins was then examined using convalescent sera from patients and experimentally infected mice. PRINCIPAL FINDINGS: Potential AT genes shared by ETEC strains, but absent in the E. coli commensal HS strain were identified. Recombinant passenger domains derived from autotransporters, including Ag43 and an AT designated pAT, were recognized by antibodies from mice following intestinal challenge with H10407, and both Ag43 and pAT were identified on the surface of ETEC by flow cytometry. Likewise, convalescent sera from patients with ETEC diarrhea recognized Ag43 and pAT, suggesting that these proteins are expressed during both experimental and naturally occurring ETEC infections and that they are immunogenic. Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC. CONCLUSIONS: Passenger domains of conserved autotransporter proteins could contribute to protective immune responses that develop following infection with ETEC, and these antigens consequently represent potential targets to explore in vaccine development.

Peptides derived from platelet non-integrin collagen-receptors or types I and III collagen inhibit collagen-platelet interaction.

Platelet-collagen interaction plays an important role in hemostasis and pathological thrombosis. Upon an injury to the subendothelium of a blood vessel wall, platelets adhere to the denuded substrate, aggregate, and release biological substances. Many investigators have explored the use of blocking agents to interrupt the final step of binding fibrinogen on glycoprotein (GP) IIb/IIIa of activated platelets. A potent peptide is Arg-Gly-Asp-Ser (RGDS) and its derivatives in various forms. Results from many clinical trials show that the efficacy of these antagonists does not lie in blocking the adhesion of platelets to the distal site(s) of the injury as expected. Because type I and type III collagens are predominant components of blood vessel walls, other laboratories and ours have defined various active peptides from either collagen molecules or platelets as useful for blocking collagen-platelet interaction. An active hybrid peptide derived from both platelet types I and type III collagen receptors that abolishes type I and type III collagen-induced platelet aggregation has been obtained. The hybrid peptide inhibits the binding of type I and type III collagens to washed human platelets, platelet aggregation, and the adhesion of washed platelets to rabbit aortic segments. However, the usefulness of the defined hybrid peptide in preventing thrombi formation in vivo requires further investigation.

Type I and type III collagen-platelet interaction: inhibition by type specific receptor peptides.

We have previously cloned and characterized a platelet receptor for type III collagen (47 kDa) from a human bone marrow cDNA phage library and defined two active peptides. We also cloned and characterized a platelet receptor for type I collagen (65 kDa) and defined an active peptide. Our objective was to study whether there is type specificity of these active peptides. We have engineered a mutant receptor clone by replacing one of the two active peptides of the platelet receptor for type III collagen with the active peptide of the platelet receptor for type I collagen. The replacement of an active peptide at the amino terminal end (rMIII) of the platelet receptor for type III collagen with the type I collagen active peptide was done without altering the hydrophilicity of the protein. This purified recombinant protein reacts with polyclonal anti-47-kDa and anti-65-kDa active peptide antibodies. The purified recombinant protein inhibits both types I and III collagen-induced platelet aggregation. This rMIII also inhibits the adhesion of washed platelets to rabbit aortic segments (natural matrix) in a dose-dependent manner. The chemically synthesized hybrid peptide of each active peptide of platelet type I and type III collagen receptors inhibits types I and III collagen-induced platelet aggregation in a dose-dependent manner. These results suggest that there is a type specific reactive site on platelets for type I and type III collagens.

Role of platelet endothelial form of nitric oxide synthase in collagen-platelet interaction: regulation by phosphorylation.

Different pathways have been reported to be involved in platelet-collagen interaction. We have reported that the platelet endothelial form of nitric oxide synthase (eNOS) and the platelet receptor for type I collagen, p65, are closely associated. But the controlling mechanism underlying the generation of nitric oxide (NO) by the eNOS has not been fully explored. In this investigation, Western blot analyses of time course samples with anti-phosphorylated tyrosine, and anti-serine/threonine showed a marked increase in serine/threonine phosphorylation of eNOS during type I collagen-induced platelet aggregation. Meanwhile, the eNOS activity measured by the conversion of [3H]-arginine to [3H]-citrulline is significantly decreased. Correlation of type I collagen-induced platelet aggregation and the activity of eNOS in the presence of the serine/threonine phosphatase inhibitor, okadiac acid and the tyrosine phosphatase inhibitor, vanadate were performed with PRP. Results show the decrease in eNOS activity by adding okadiac acid correlated with the inhibitory effect on platelet aggregation in a dose-dependent manner. On the other hand, vanadate significantly inhibits platelet aggregation and also inhibits eNOS activity when the concentration of vanadate is greater than 2 mM. These results suggest that phosphorylation of serine/threonine and tyrosine residues control the activity of eNOS through different mechanisms to affect collagen-induced platelet aggregation.

Cloning, characterization, and functional studies of a 47-kDa platelet receptor for type III collagen.

A 1.2-kb cDNA fragment encoding a platelet 47-kDa protein has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide of the sequenced amino terminus of the purified platelet protein with a poly(dT)(12).(dG) by polymerase chain reaction. A computer search revealed that the cDNA represents the coding sequence of a protein with a fragmentary homology to several proteins. Using a prokaryotic expression system, pBad TOPO-47 cDNA, a 47-kDa recombinant protein was obtained and purified to apparent homogeneity by nickel-nitrilotriacetic acid resin and collagen affinity column. The recombinant protein binds to type III but not type I collagen-Sepharose 2B affinity columns. Anti-47-kDa but not anti-65-kDa antibody inhibits the binding of the recombinant protein to the type III collagen-coated micro titer wells in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type III collagen-induced platelet aggregation also in a dose-dependent manner. We have defined two active peptides from the cloned deduced amino acid sequence. Both peptides inhibit type III but not type I collagen-induced platelet aggregation in a dose-dependent fashion. These results suggest that the active binding site of the platelet receptor to type III collagen resides in these portions of the protein.