Dominique Wachsmann

NF-kappaB and the MAP kinases/AP-1 pathways are both involved in interleukin-6 and interleukin-8 expression in fibroblast-like synoviocytes stimulated by protein I/II, a modulin from oral streptococci.

As in rheumatoid arthritis (RA), it was demonstrated recently that bacterial fragments of DNA or rRNA are present in the joint and therefore could play a role in inducing or perpetuating the disease, this work was initiated to define mechanisms that account for the stimulatory activities of the oral streptococcal modulin, protein I/II, on fibroblast‐like synoviocytes (FLSs) from RA patients. FLSs from RA patients were stimulated with protein I/II, and expression of interleukin (IL)‐6 and IL‐8 mRNA was evaluated by reverse transcription–polymerase chain reaction (RT–PCR). Immunoblotting by antibodies specific for activated forms of MAPKs and electrophoretic mobility shift assays (EMSAs) were performed to study downstream signalling, which allowed the synthesis of IL‐6 and IL‐8. We reported that protein I/II interactions with FLSs from RA patients trigger the synthesis and release of IL‐6 and IL‐8. We also demonstrated that protein I/II enhances the phosphorylation of ERK 1/2, p38 and JNKs and that ERK 1/2 and JNK MAPKs seem to play a more important role than p38 in protein I/II‐mediated synthesis of IL‐6 and IL‐8. Our experiments also indicated that stimulation of FLSs with protein I/II induces nuclear translocation of NF‐κB, AP‐1‐binding activity and that NF‐κB plays a major role in IL‐6 and IL‐8 secretion from activated cells.

Design of highly immunogenic liposomal constructs combining structurally independent B cell and T helper cell peptide epitopes

We have designed liposomal diepitope constructs that allow the physical combination, within the same vesicle, of B and Th epitopes as structurally separate entities. The immune response against such constructs was explored using TPEDPTDPTDPQDPSS (TPE), a B cell epitope originating from a Streptococcus mutans surface adhesin and QYIKANSKFIGITEL (QYI), a “universal” Th epitope from tetanus toxin. The two peptides were linked to the outer surface of small (diameter approximately 100u2009nm) unilamellar liposomes by covalent conjugation to two different anchors. To that end we have developed a strategy that allows the controlled chemical coupling of TPE and QYI, functionalized at their N terminus with a thiol, to preformed liposomes containing thiol‐reactive derivatives of phosphatidylethanolamine and the lipopeptide S‐[2,3‐bis (palmitoyloxy)‐(2‐RS)‐propyl]‐N‐palmitoyl‐(R)‐cysteinyl‐alanyl‐glycine (Pam3CAG), respectively. This synthetic construct (administered i.u2009u2009p. to BALB/c mice) induced highly intense (titers >u200920u2009000), anamnestic and long‐lasting (over 2 years) immune responses, indicating that this strategy is successful. Two parameters were of prime importance to elicit this response with our liposomal diepitope constructs: (1) the simultaneous expression of B and Th epitopes on the same vesicle, and (2) the lipopeptide Pam3CAG anchor of the Th epitope QYI could not be replaced by a phosphatidylethanol‐amine anchor (a lesser immune response was observed). Analysis of the antibody response revealed a complex pattern; thus, besides the humoral response (production of IgG1, IgG2a, IgG2b) a superposition of a T‐independent (TI‐2 type) response was also found (IgM and IgG3). These results indicate that liposomal diepitope constructs could be attractive in the development of synthetic peptide‐based vaccines.

Involvement of alpha5beta1 integrins in interleukin 8 production induced by oral viridans streptococcal protein I/IIf in cultured endothelial cells.

Using human endothelial cells, we define a mechanism that accounts for the induction of interleukin 8 (IL‐8) by protein I/IIf, an adhesin from Streptococcus mutans serotype f. We report that protein I/IIf interactions with endothelial cells increased the tyrosine phosphorylation of three cellular components with relative mass of 145u2003000, 125u2003000 and 70u2003000 in endothelial cells. These proteins were identified as phospholipase Cγ (PLCγ), focal adhesion kinase (FAK) and paxillin after immunoprecipitation with monoclonal antibodies (mAbs) and immunoblotting with antiphosphotyrosine mAbs. These results suggested that β1 integrins could be one of the components implicated in the modulin activity of protein I/IIf. By incubating protein I/IIf with either purified α5β1 integrins or with α5β1 integrins overexpressing CHO cells, we demonstrated that α5β1 integrins act as cell receptors for protein I/IIf. We also showed that protein I/IIf interactions with α5β1 integrins lead to IL‐8 secretion. Using specific inhibitors, we demonstrated that protein I/IIf‐induced IL‐8 release involves mitogen‐activated protein kinases (MAPKs), and that PLCγ and PKC also seem to contribute to protein I/IIf stimulation. However, PI‐3K activation is not involved in IL‐8 release. Altogether, these results indicate that, after binding to α5β1 integrins, protein I/IIf induces IL‐8 release by activating the MAPKs signalling pathways.

MMP-3 expression and release by rheumatoid arthritis fibroblast-like synoviocytes induced with a bacterial ligand of integrin α5β1

Fibroblast-like synoviocytes (FLSs) play a major role in the pathogenesis of rheumatoid arthritis (RA) by secreting effector molecules that promote inflammation and joint destruction. How these cells become and remain activated is still elusive. Both genetic and environmental factors probably play a role in transforming FLSs into inflammatory matrix-degrading cells. As bacterial products have been detected in the joint and shown to trigger joint inflammation, this study was undertaken to investigate whether a bacterial ligand of integrin α5β1, protein I/II, could contribute to the aggressive behavior of RA FLSs. Protein I/II is a pathogen-associated molecular pattern (PAMP) isolated from oral streptococci that have been identified in the joints of RA patients. The response of RA and osteoarthritis FLSs to protein I/II was analyzed using human cancer cDNA expression arrays. RT-PCR and pro-MMP-3 (pro-matrix metalloproteinase) assays were then performed to confirm the up-regulation of gene expression. Protein I/II modulated about 6% of all profiled genes. Three of these, those encoding IL-6, leukemia inhibitory factor, and MMP-3, showed a high expression level in all RA FLSs tested, whereas the expression of genes encoding other members of the cytokine or MMP-family was not affected. Furthermore, the up-regulation of MMP-3 gene expression was followed by an increase of pro-MMP-3 release. The expression of interferon regulatory factor 1 and fibroblast growth factor-5 was also up-regulated, although the expression levels were lower. Only one gene, that for insulin-like growth factor binding protein-4, was down-regulated in all RA FLSs. In contrast, in osteoarthritis FLSs only one gene, that for IL-6, was modulated. These results suggest that a bacterial ligand of integrin α5β1 may contribute to the aggressive behavior of RA FLSs by inducing the release of pro-inflammatory cytokines and a cartilage-degrading enzyme, such as IL-6 and MMP-3, respectively.

Local response in rat to liposome-associated Streptococcus mutans polysaccharide-protein conjugate

The effect of gastric intubation with soluble or liposome-associated Streptococcus mutans serovar polysaccharide, 74-kDa saliva receptor (74K SR protein) or polysaccharide-74K SR protein conjugate on the locally induced salivary IgA response and memory in rats was investigated. Animals immunized on four successive days with soluble antigens showed a weak salivary anti-74K SR protein or anti-polysaccharide IgA response. Rats primed and boosted by a single injection of liposome-associated 74K SR protein or polysaccharide-74K SR protein conjugate developed a salivary anti-74K SR protein IgA and IgG primary and secondary response. A primary anti-polysaccharide response was only observed in saliva of animals immunized with either high concentration of liposome-associated polysaccharide or liposome-associated polysaccharide-74K SR protein conjugate. However, a secondary local anti-polysaccharide IgA response was detected in animals boosted with liposome-polysaccharide-74K SR protein conjugate. No such anamnestic response was seen when high dose of liposome-associated polysaccharide was used to boost the animals. Furthermore, the salivary anti-polysaccharide IgA response paralleled the anti-74K SR protein IgA response. These studies showed that intragastric immunization of rats with liposome-associated polysaccharide-74K SR protein conjugate produced a local anti-polysaccharide IgA memory.

Impaired release of IL‐18 from fibroblast‐like synoviocytes activated with protein I/II, a pathogen‐associated molecular pattern from oral streptococci, results from defective translation of IL‐18 mRNA in pro‐IL‐18

Proinflammatory cytokines such as tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β and IL‐18 are key mediators of joint inflammation during rheumatoid arthritis (RA). This chronic inflammation may result from a non‐specific innate immune response that could be triggered by a wide variety of microorganisms, because numerous bacterial fragments have been identified in the joints of RA patients. As we have demonstrated previously that protein I/II, a pathogen‐associated molecular pattern (PAMP) from oral streptococci, triggers IL‐6 and IL‐8 gene expression and release from either THP‐1 cells or fibroblast‐like synoviocytes (FLSs), we next explored the capacity of protein I/II to induce the synthesis and release of IL‐18 in THP‐1 cells and in FLSs isolated from either RA or osteoarthritis (OA) patients. We demonstrate that protein I/II induced IL‐18 mRNA in both THP‐1 cells and FLSs but, in contrast to THP‐1 cells, gene expression was not associated with the synthesis of the corresponding protein in FLSs. Furthermore, our studies revealed that FLSs did not express the biologically inactive precursor, pro‐IL‐18, in response to protein I/II. Using actinomycin D, we also showed that IL‐18 mRNA is unstable in FLSs. Taken together, these data indicate that lack of IL‐18 release from activated FLSs results from a defect in translation of IL‐18 mRNA into pro‐IL‐18 because of rapid degradation of IL‐18 mRNA.

Purification and partial characterization of rat macrophage Fc receptor and binding factor for IgA

By using a biotinylated ligand and Western blotting techniques, a receptor (RFc alpha) and a binding factor (BF) for IgA were detected, respectively, on membrane and in the cell-free culture supernatant of rat peritoneal macrophages. Extraction of the RFc alpha was obtained by solubilization of macrophages with Nonidet P-40, and purification was performed by HPLC affinity chromatography on a column derivatized with IgA. RFc alpha is formed of two subunits, with molecular masses of 56 and 70 kDa, which are both involved in the IgA binding ability of rat peritoneal macrophages. IgABF was recovered from the cell-free supernatant of a short-term culture of rat macrophages and was affinity-purified in the same manner as RFc alpha. Like RFc alpha, IgABF retained its IgA binding activity in its native, as well as denatured form. Since the molecular masses of RFc alpha and IgABF are similar, and IgABF competes with RFc alpha for IgA binding, one can assume that IgABF probably represents a shed RFc alpha.

33 - Post-Infectious Arthritis: Reactive Arthritis or Slow Infectious Arthritis?

The discovery that a large number of inflammatory disorders are the consequence of an interaction between microbial and human immunogenetic factors has been one of the major conceptual revolutions. This concept also applies with a great deal of pertinence to arthritis, where many forms which are useful to explain inflammatory phenomena are induced by microbial compounds (adjuvant arthritis, streptococcal wall arthritis). In man, the best examples are the various types of post-infectious arthritis, which include not only reactive and post-streptococcal arthritis, but also post-infectious forms occurring as the result of viral or parasitic infections. The mechanisms underlying these post-infectious disorders are most certainly of interest for ones understanding of the common forms of inflammatory rheumatism such as rheumatoid arthritis. In this chapter, different mechanisms are discussed which allow one to explain the occurrence of post-infectious arthritis of bacterial origin. The abundant literature relating to post-infectious arthritis, in particular reactive arthritis, serves as a guideline for the chapter.

Protein I/II from Oral Viridans Streptococci Modulates Expression of E-Selectin, ICAM-1 and VCAM-1, and Promotes Transendothelial Migration of Neutrophils in Vitro

Oral viridans streptococci which are major commensal bacteria of the oropharyngeal cavity have been associated with chronic as well as acute inflammatory diseases such as subacute endocarditis, subacute glomerulonephritis, rheumatic arthritis, sepsis and toxic shock. These diseases are probably the consequence of a local infection which has gain access to the bloodstream. Despite the fact that mechanisms of pathogenesis remained undefined, several reports have suggested that production in excess of proinflammatory cytokines may be involved in the development of the former diseases and we reported recently that oral viridans streptococci induce the release of TNF-α, IL-1β, IL-6, IL-8 from monocytes, of IL-6 and IL-8 from endothelial cells and of IL-6 from epithelial cells. We provide also evidence that oral viridans streptococci exert these effects probably by engaging two cell surface adhesins which belong to the recently defined class of modulins (1): protein I/II and a cell-wall polysaccharide (RGP). If RGP’s dependent signalling pathway appears to be related to binding to the CD14 receptor, protein I/II stimulates monocytes, epithelial and endothelial cells through lectin interactions. Accumulation of leucocytes in perivascular tissues is also a key step in inflammatory disorders and is correlated to a sequential upregulation of E-selectin, VCAM-1 and ICAM-1 expression on endothelium. Protein I/II, insofar as it is able to exert immunomodulatory effects on monocytes and endothelial cells, could contribute in an indirect or direct manner to the up-regulation, on endothelial cells of these adhesion molecules that allow binding and then penetration of leukocytes into tissues. This effect could contribute to the pathology of the diseases associated to oral viridans streptococci. To gain a broader insight into these mechanisms, we studied the direct effect of protein I/II on human saphenous vein endothelial cells (HSVEC) E-selectin, ICAM-1 and VCAM-1 expression and on the subsequent neutrophil migration.