Maarten P. Hazenberg

Presence of bacterial DNA and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides

OBJECTIVE The continuous presence of bacteria or their degraded antigens in the synovium may be involved in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the presence of bacterial nucleic acids and bacterial cell wall constituents in the joints of patients with RA and other forms of arthritis. METHODS Joint samples were obtained from patients with RA (n = 26), septic arthritis (n = 2), inflammatory osteoarthritis (n = 5), and gout (n = 6), and joint trauma (n = 1). Universal 16S-ribosomal RNA primers were used to detect the presence of bacterial DNA in these samples, using stringent regimens for sample collection and molecular microbiologic analysis. Automated sequencing and comparative data analysis were performed to identify the species. The presence of bacterial peptidoglycan-polysaccharide complexes in synovial tissue was detected by immunohistologic analysis with a specific antibody. RESULTS The bacterial species cultured from the synovium could be identified in both of the patients with septic arthritis. DNA amplicons were also detected in the synovial fluid and/or tissue samples from 5 patients with RA and 2 patients with crystal-induced arthritis; these originated from multiple bacterial species. Staining for peptidoglycan-polysaccharide complexes was positive in the synovial tissue of both patients with septic arthritis, 16 with RA, 4 with inflammatory osteoarthritis, 4 with crystal-induced arthropathy, and 1 with joint trauma. The staining was mainly found in cells in the synovial sublining, including macrophages. CONCLUSION The results indicate that bacterial DNA and bacterial cell wall constituents are retained in the joints of some patients with arthritis, where they might enhance synovial inflammation.

Are intestinal bacteria involved in the etiology of rheumatoid arthritis

Observations in bowel‐related joint diseases give support to this hypothesis. In Crohns disease and ulcerative colitis, the bowel wall inflammation is complicated in about 20% of the patients by joint inflammation. Bowel infection by Salmonella, Shigella and Yersinia can provoke joint inflammation and supports an etiological link between bowel bacteria and arthritis. The arthropathic properties of the most abundant group of intestinal bacteria, i.e. the obligate anaerobic bacteria, were studied in an animal model. Cell wall fragments (CWF), with peptidoglycan as the major component, from some Eubacterium and Bifidobacterium species induced a severe chronic polyarthritis in Lewis rats after a single intraperitoneal injection. Eubacterium was found in numbers of 108‐109 per gram in stools of healthy subjects and rheumatoid arthritis (RA) patients. CWF of isolated strains of E. aerofaciens were arthropathic. Soluble peptidoglycan polysaccharide complexes (PG‐PS) originating from the obligate anaerobic flora were purified from human intestinal contents. PG‐PS from ileostomy fluid that proved to be less processed by intestinal enzymes induced chronic arthritis in rats after a single administration in oil in the base of the tail. It was concluded that the human intestinal bowel contains soluble bacterial cell wall products that are arthropathic in an animal model. Peptidoglycan (PG) or its subunits was reported to be present in mammalian tissues. Immunohistochemical studies from our group showed the presence of intestinal PG‐PS in sections of normal rat spleen. Bacterial cell wall or PG‐induced joint inflammation in rats is proven to be absolutely dependent on functional T cells. T‐cell lines were isolated from the lymph nodes of rats with an E. aerofaciens CWF arthritis. A helper T‐cell line B13 was in vivo arthritogenic in knee or ankle joints upon intravenous injection in rats and proliferated in vitro on syngeneic spleen cells alone, but was additionally stimulated by intestinal PG‐PS and E. aerofaciens CWF. It was postulated that the arthritogenic T cells that seem to be autoreactive are, in fact, recognizing bacterial PG‐PS on antigen‐presenting cells (APC). It is generally accepted that RA is a T‐cell‐dependent process and that therefore the reaction is directed at small peptides bound by the major histocompatibility complex of APC. The only peptides present in arthritis inducing intestinal PG‐PS and in CWF are PG peptides interlinking the sugar chains. We feel that the immunoreaction against PG peptides plays a pivotal role in experimental and human arthritis of an unknown etiology.

The Obligate Anaerobic Faecal Flora of Patients with Crohn's Disease and Their First-Degree Relatives

The obligate anaerobic faecal floras of patients with Crohns disease, their first-degree relatives, and healthy control subjects were compared. The flora of Crohns patients contained more anaerobic gram-positive coccoid rods and gram-negative rods than that of healthy subjects; on this basis patients and healthy subjects formed two clusters with minor overlap. Nine of 26 children of Crohns patients were also included within the Crohns disease cluster. During 5 to 7 years of follow-up study 3 of them presented with remitting abdominal pain, diarrhoea, or weight loss, and in 1 of them Crohns disease was diagnosed; none of the 17 children with a normal flora showed symptoms possibly due to Crohns disease. It is concluded that the abnormal flora may be indigenous to subjects predisposed to Crohns disease, suggesting a direct or indirect relationship between the abnormal faecal flora and Crohns disease.

Antigen-presenting cells containing bacterial peptidoglycan in synovial tissues of rheumatoid arthritis patients coexpress costimulatory molecules and cytokines

OBJECTIVE Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by intimal lining hyperplasia and massive infiltration of the synovial sublining by antigen-presenting cells (APCs), lymphocytes, and plasma cells. Peptidoglycan (PG), a major cell wall component of gram-positive bacteria, which is abundantly expressed in all mucosa, is believed to be involved in the pathogenesis of RA because of its ability to induce the production of proinflammatory cytokines as well as to induce arthritis in rodents. While PG has been detected in APCs in RA joints, little is known about the role of these cells in RA. In this study, the presence and immune competence of PG-containing cells in synovial tissues from 14 RA and 14 osteoarthritis (OA) patients were analyzed in situ. METHODS Using immunohistochemistry, we examined the coexpression of phenotypic markers, costimulatory molecules, and cytokines by PG-containing cells. RESULTS PG was present in higher numbers in RA than in OA synovial tissues, although the difference was not significant. PG-containing cells were mainly macrophages, but some mature dendritic cells also contained PG. A high percentage of PG-containing cells in both RA and OA synovial tissues coexpressed HLA-DR. CD40, CD80, and CD86 expression by PG-containing cells was higher in RA than in OA tissues. Furthermore, PG-containing cells coexpressed cytokines, which modulate inflammatory reactions, in particular, tumor necrosis factor alpha and interleukins 6 and 10. CONCLUSION The results suggest that PG-containing cells may contribute to inflammation within the microenvironment of the joint in RA patients.

Gut flora induces and maintains resistance against streptococcal cell wall-induced arthritis in F344 rats

Streptococcal cell wall (SCW)‐induced arthritis is a chronic, erosive polyarthritis that can be induced in susceptible Lewis rats by one i.p. injection of an aqueous, sterile suspension of SCW. F344 rats are resistant to chronic joint inflammation. Our previous studies showed a correlation between susceptibility to SCW‐induced arthritis and the ability to mount SCW‐specific T cell responses, suggesting tolerance to SCW as a putative mechanism. Here we prevented the induction of tolerance to bacterial epitopes in F344 rats by using them germ‐free and analysed susceptibility to arthritis subsequently. In addition, we conventionalized germ‐free F344 rats at different times before induction of arthritis. Our results show that germ‐free F344 rats are susceptible to SCW‐induced arthritis with a similar severity, chronicity, incidence and onset as Lewis rats. Moreover, T cells isolated from germ‐free F344 rats were able to respond to SCW. Conventionalization dramatically moderates arthritis and makes T cells unresponsive to SCW again. Thus, in normal rats (F344) a state of tolerance to arthritogenic epitopes is induced (neonatally) and maintained through life by the bacterial flora, resulting in resistance to bacterium‐induced artritides. In arthritis‐prone (Lewis) rats, this tolerance is deficient and/or easily broken.

Humoral immune response against Campylobacter jejuni lipopolysaccharides in Guillain-Barre and Miller Fisher syndrome

In this study we characterized the IgG antibodies against lipopolysaccharides (LPS) of Campylobacter jejuni in serum from patients with Guillain-Barré syndrome (GBS), Miller Fisher syndrome (MFS), C. jejuni enteritis and normal controls. In patients with GBS and MFS long-lasting titers of IgG1 and IgG3 antibodies against LPS from GBS and MFS associated C. jejuni were found. The subclass and course of these antibodies were highly associated with those of antibodies against GM1 and GQ1b in GBS and MFS patients. However, in C. jejuni enteritis and normal controls anti-LPS antibodies were predominantly IgG2. Antibody binding with LPS was reduced after treatment with choleratoxin and sialidases, suggesting that the ganglioside-like epitopes in LPS are immunodominant. These results further indicate that antecedent C. jejuni infections determine the specificity and isotype of anti-ganglioside antibodies in GBS and MFS patients.

Bacterial peptidoglycan polysaccharides in sterile human spleen induce proinflammatory cytokine production by human blood cells

Peptidoglycan (PG) is the major component of the cell wall of gram-positive bacteria. In vitro, PG isolated from conventional bacterial cultures can induce secretion of proinflammatory cytokines by human monocytes, indicating that PG may be involved in immune responses against infections by gram-positive bacteria. To investigate the biologic activity of PG in human tissues, an improved method was developed to isolate significant amounts of PG from sterile human spleen tissue. Biochemical analysis demonstrated that PG isolated from human spleen is largely intact. Human whole blood cell cultures were able to produce the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 and -6 after stimulation with PG isolated from human spleen. Cytokine induction was not sensitive to inhibition by polymyxin B, in contrast to lipopolysaccharide. Collectively, the data show that intact PG in sterile human tissue is biologically active and may induce local proinflammatory cytokine production.

Purification and characterization of N-acetylmuramyl-L-alanine amidase from human plasma using monoclonal antibodies

N-Acetylmuramyl-L-alanine amidase (EC 3.5.1.28) cleaves the amide bond between N-acetyl muramic acid and L-alanine in the peptide side chain of different peptidoglycan products. The enzyme was purified from human plasma using a three-step column chromatography procedure. Monoclonal antibodies were produced against the purified human enzyme. By coupling of a high affinity monoclonal antibody to sepharose beads an immunoadsorbent column was prepared. Using this second purification method it was possible to purify large amounts of the amidase from human plasma in a single step. SDS-PAGE showed one single band of 70 kDa and two-dimensional electrophoresis showed the presence of multiple isomeric forms of the protein with pI between 6.5 and 7.9. Two different methods were used for determination of substrate specificity, a HPLC method separating peptidoglycan monomers from the reaction products after incubation with amidase and a colorimetric method when high molecular weight peptidoglycan was used as a substrate for amidase. It is shown that the disaccharide tetra peptide, disaccharide penta peptide and the anhydro disaccharide tetrapeptide are good substrates for the amidase and that muramyl dipeptide and disaccharide dipeptide are not a substrate for the amidase. Using one of the monoclonal antibodies against the amidase it was shown in FACScan analysis that N-acetylmuramyl-L-alanine amidase is present in granulocytes but not in monocytes from unstimulated peripheral blood of a healthy donor. The presence of N-acetylmuramyl-L-alanine amidase in granulocytes is a novel finding and perhaps important for the inactivation of biologically active peptidoglycan products still present after hydrolysis by lysozyme.

Induction of chronic arthritis in rats by cell wall fragments of anaerobic coccoid rods isolated from the faecal flora of patients with Crohn's disease

Crohns disease and ulcerative colitis are accompanied by seronegative arthritis in about one fifth of the cases. In the present study, cell wall fragments from major residents such as Eubacterium, Coprococcus and Peptostreptococcus species, isolated from the faecal flora of patients with Crohns disease, were tested for properties to induce chronic arthritis in Lewis rats. Cell wall fragments from Eubacterium contortum strains Me44 and Me47 were found to induce chronic arthritis; Peptostreptococcus productus strain C 18 cell wall fragments induced acute self-limiting arthritis. Coprococcus comes strain Me46 cell walls, on the other hand, were found to be lethal to the majority of rats inoculated, whereas those which survived did not develop acute or chronic arthritis. The results indicate that intraperitoneal injection of a single dose of cell wall fragments from bacteria that are major residents of the human anaerobic faecal flora can induce chronic inflammatory joint disease in the rat.

Binding to faeces and influence on human anaerobes of antimicrobial agents used for selective decontamination

The degree of binding of ampicillin, cephradine, co-trimoxazole, gentamicin, nalidixic acid, neomycin, polymyxin B and tobramycin by faecal substance as well as the influence of these antibiotics on human intestinal obligate anaerobes was investigated. In contrast to ampicillin, cephradine, co-trimoxazole and nalidixic acid, the nonabsorbable antibiotics polymyxin B and neomycin were bound to a considerable degree by human faeces. The binding of tobramycin and gentamicin to the solid part of faeces was less effective. The inhibitory effect of co-trimoxazole, gentamicin, nalidixic acid, neomycin, polymyxin B and to-bramycin on the human obligate anaerobes was weak as compared with ampicillin and cephradine.Drugs which effectively eliminate Enterobacteriaceae from the gastrointestinal tract and which have a moderate effect on obligate anaerobes, like polymyxin B, are particularly suitable for selective decontamination of the gastrointestinal tract. The strong inactivating binding of aminoglycosides and polymyxin B to faeces accounts for the relatively high oral dose needed for a suitable faecal concentration.