Peptidoglycan-polysaccharide Polymers Research Articles

Streptococcus species enriched in the oral cavity of patients with RA are a source of peptidoglycan-polysaccharide polymers that can induce arthritis in mice

ObjectivesAnalysis of oral dysbiosis in individuals sharing genetic and environmental risk factors with rheumatoid arthritis (RA) patients may illuminate how microbiota contribute to disease susceptibility. We studied the oral microbiota...

Indocyanine green–enhanced imaging of antigen‐induced arthritis with an integrated optical imaging/radiography system

To evaluate a combined indocyanine green-enhanced optical imaging/radiography system for the detection of arthritic joints in a rat model of antigen-induced arthritis. Arthritis of the knee and ankle joints was induced in 6 Harlan rats, using peptidoglycan-polysaccharide polymers. Three rats served as untreated controls. Optical imaging of the knee and ankle joints was done with an integrated optical imaging/radiography system before and up to 24 hours following intravenous injection of 10 mg/kg indocyanine green. The fluorescence signal intensities of arthritic and normal joints were compared for significant differences, using generalized estimating equation models. Specimens of knee and ankle joints were further processed and evaluated by histology. Immediately after administration, indocyanine green provided a significant increase in the fluorescence signal of arthritic joints compared with baseline values (P < 0.05). The fluorescence signal of arthritic joints was significantly higher compared with that of nonarthritic control joints at 1-720 minutes after intravenous injection (P < 0.05). Fusion of indocyanine green-enhanced optical imaging and radiography allowed for anatomic coregistration of the inflamed tissue with the associated joint. Hematoxylin and eosin staining confirmed marked synovial inflammation of arthritic joints and the absence of inflammation in control joints. Indocyanine green-enhanced optical imaging is a clinically applicable tool for detection of arthritic tissue. Using relatively high doses of indocyanine green, long-term enhanced fluorescence of arthritic joints can be achieved. This may facilitate simultaneous evaluations of multiple joints in a clinical setting. Fusion of indocyanine green-enhanced optical imaging scans with radiography increases anatomic resolution.

Genetic dissection of granulomatous enterocolitis and arthritis in the intramural peptidoglycan-polysaccharide-treated rat model of IBD

Inflammatory arthropathies are common extraintestinal manifestations of inflammatory bowel diseases (IBD). As genetic susceptibility plays an important role in the etiology of IBD, we questioned how granulomatous enterocolitis and arthritis are genetically controlled in an experimental animal model displaying both conditions. Chronic intestinal and systemic inflammation was induced by intramural injection of peptidoglycan-polysaccharide (PG-PS) polymers in the ileocecal region of female F2 progeny derived from susceptible LEW and resistant F344 rats. Animals were followed for 24 days after injection and phenotyped by evaluating gross gut lesions, liver weight and granulomas, hematocrit, white blood cell count, and change in rear ankle joint diameters. Coinheritance of the phenotypic parameters with polymorphic DNA markers was analyzed by genome-wide quantitative trait locus (QTL) analysis. Linkage analysis revealed significant QTLs for enterocolitis and/or related phenotypes (liver granulomas, white blood cell count) on chromosomes 8 and 17. The QTL on chromosome 8 also showed suggestive linkage to arthritis. Significant QTLs for arthritis were detected on chromosomes 10, 13, 15, and 17. Analyses of the modes of inheritance showed arthritogenic contributions by both parental genomes. In addition, several other loci with suggestive evidence for linkage to 1 or several phenotypes were found. Susceptibility to PG-PS-induced chronic intestinal and systemic inflammation in rats is under complex multigenic control in which the genetic loci regulating arthritis are largely different from those controlling enterocolitis. Possible candidate genes within these QTL (including Tnfrsf11a/RANK, Gpc5, Il2ra, and Nfrkb) are also implicated in the respective human diseases.

Effects of the cFMS Kinase Inhibitor 5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) in Normal and Arthritic Rats

The cFMS (cellular homolog of the V-FMS oncogene product of the Susan McDonough strain of feline sarcoma virus) (Proc Natl Acad Sci U S A 83:3331-3335, 1986) kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) inhibits colony-stimulating factor (CSF)-1-induced monocyte growth and bone degradation in vitro and inhibits CSF-1 signaling through cFMS kinase in 4-day models in mice (Proc Natl Acad Sci U S A 102:16078, 2005). In the present study, the kinase selectivity of GW2580 was further characterized, and the effects of chronic treatment were evaluated in normal and arthritic rats. GW2580 selectively inhibited cFMS kinase compared with 186 other kinases in vitro and completely inhibited CSF-1-induced growth of rat monocytes, with an IC(50) value of 0.2 microM. GW2580 dosed orally at 25 and 75 mg/kg 1 and 5 h before the injection of lipopolysaccharide inhibited tumor necrosis factor-alpha production by 60 to 85%, indicating a duration of action of at least 5 h. In a 21-day adjuvant arthritis model, GW2580 dosed twice a day (b.i.d.) from days 0 to 21, 7 to 21, or 14 to 21 inhibited joint connective tissue and bone destruction as assessed by radiology, histology and bone mineral content measurements. In contrast, GW2580 did not affect ankle swelling in the adjuvant model nor did it affect ankle swelling in a model where local arthritis is reactivated by peptidoglycan polysaccharide polymers. GW2580 administered to normal rats for 21 days showed no effects on tissue histology and only modest changes in serum clinical chemistry and blood hematology. In conclusion, GW2580 was effective in preserving joint integrity in the adjuvant arthritis model while showing minimal effects in normal rats.

578. Suppression of Inflammation Following Intramuscular Administration of Pseudotyped AAV-TNFR:Fc Vectors in a Rat Model of Arthritis

TNF-|[alpha]| antagonists such as anti-TNF-|[alpha]| monoclonal antibodies or soluble tumor necrosis factor receptor have proven to be useful therapeutics for the treatment of autoimmune inflammatory diseases including rheumatoid and psoriatic arthritis. In the current study, we employed a monoarticular arthritis model in rats that allowed us to evaluate the long-term effect of TNFR:Fc expression on recurrence of joint inflammation. Monoarticular arthritis was initiated by injection of a small dose of peptidoglycan-polysaccharide polymers (PG-APS) isolated from cell walls of group A streptocci into the hind ankle joint. This resulted in a flare of inflammation that resolved within five days. Intramuscular administration of AAV-TNFR:Fc vectors performed on day seven. To assess the long-term effect of TNFR:Fc expressions on suppression of arthritis in this model, animals were evaluated for four rounds of remission and reactivation of joint inflammation. Reactivation of joint inflammation was induced by intravenous injection of PG-APS. Animals that were treated with vehicle, 109, 5x109 DRP of AAV2/1-TNFR:Fc or 1012 DRP of AAV2-TNFR:Fc developed a significant flare of inflammation in their ankle joints. In these animals, levels of TNFR:Fc protein in the circulation were below 0.75 |[mu]|g/mL. In contrast, when animals were treated with 1010, 1011or 1012 DRP of AAV2/1-TNFR:Fc, levels of TNFR:Fc protein in the circulation were 1.27|[ndash]|8.35 |[mu]|g/mL, 6.8|[ndash]|27.4 |[mu]|g/mL and 19.6|[ndash]|61 |[mu]|g/mL, respectively. The inflammatory response in these animals, as measured by joint volume, was completely suppressed. To increase the transduction efficiency of AAV2/2- TNFR:Fc following administration of 1012 DRP, we employed treatment with proteosome inhibitor that has been shown to enhance AAV transduction. In these animals, levels of TNFR:Fc protein in the circulation increased up to 3.65 |[mu]|g/mL, and led to significant decrease in joint inflammation.

DOES SMALL INTESTINE-LIVER AXIS PLAY A ROLE IN PEDIATRIC OBESITY RELATED LIVER DAMAGE ? A PILOT STUDY

Background: Studies in an obese animal model and in some obese adults have recently suggested that a derangement of the small intestine-liver axis may play a role in the development of obesity related liver abnormalities. Aim & Methods: To examine small intestinal data [basal & lactulose H2 breath test (BT), permeability to mannitol/cellobiose (spectrophotometric method), serum levels of circulating bound endotoxins (LAL chromogenous method) and of Abs to bacterial wall products (peptidoglycan-polysaccharide (PG-PS) polymers; ELISA test) which reach the liver through the portal flow] in obese children (age 6-13 y) with and without hypertransaminasemia and/or ultrasonographic (US) bright liver not due to other causes of hepatopathy. Results: Up to now we have enrolled 27 obese children with [group 1, n = 16, mean age 10.8 y; BMI 30.6; ALT 52 ± 25 U/L) and without (group 2, n = 11, mean age 10.5 y (p = NS); BMI 27.5 (p = NS), ALT 21 ± 9 U/L (P = 0.001)] liver disease. 100% pts had familial obesity; 75% vs. 36% had familial history of liver disease (p = 0.06). Obesity duration, food habits, skinfold tickness and body circumferences, bioimpedance analysis of % lean and fat mass, visceral and subcutaneous US fat, serum lipids, IL6, TNFa and iron status were comparable in the two groups. As expected, fasting insulin to glucose ratios (FGIR) and n. insulin-resistant (FGIR <7) pts were different between groups [(5.5 ± 2.9 vs. 8.2 ± 4.1 (p = 0.05) and 12/16 vs. 4/11 (p = 0.06), respectively]. As for intestinal data, there were no differences in prevalence of small intestine bacterial overgrowth evaluated by H2 BT and abnormalities of intestinal permeability (1 case/group) & serum levels of bound endotoxins (9.2 ± 4.0 vs 12.0 ± 6.5 pg/ml). Serum IgA (but not IgG and IgM) Abs to PG-PS were significantly higher in group 1 (1.0 ± 0.4 vs. 0.7 ± 0.2 pg/ml; p < 0.05). We are now evaluating changes of the aforementioned data during a double blind treament with probiotics vs. placebo. Summary & Conclusions: Apart from Abs to PG-PS, so far we have not found relevant evidences suggesting that intestinal abnormalities may greatly contribute towards liver damage in pediatric obese children.(Acknowledgements: Italian MIUR 2003 & ELFID).

880. Suppression of Inflammation in a Rat Model of Arthritis Following Intramuscular Administration of AAV-TNFR:Fc Vector Pseudotyped with AAV Type 1 Capsid

TNF-alpha antagonists such as anti-TNF-alpha monoclonal antibodies or soluble tumor necrosis factor receptors have proven to be useful therapeutics for the treatment of autoimmune inflammatory diseases including rheumatoid and psoriatic arthritis. Recently, we have demonstrated that local (intra-articular) or systemic (intramuscular) administration of an AAV2-ratTNFR:Fc vector, encoding the rat tumor necrosis factor-a receptor: immunoglobulin (IgG1) Fc fusion gene, to rats with experimental arthritis led to suppression of disease as reflected in decreased inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and mRNA expression of joint proinflammatory cytokines. In the current study we employed a monoarticular arthritis model in rats that allow us to evaluate the long-term effect of TNFR:Fc expression on recurrence of joint inflammation. Monoarticular arthritis was initiated by injection of a small dose of peptidoglycan-polysaccharide polymers (PG-APS) isolated from cell walls of group A streptocci into the hind ankle joints. This resulted in a flare of inflammation that resolved within 5 to 7 days. Thirty-two days later, reactivation of joint inflammation was induced only in the joints previously injured by intravenous injection of subarthropathic dose of PG-APS. Twenty-six days later, rats were administered intramuscularly with either vehicle or with AAV-TNFR:Fc vector pseudotyped with AAV type 1 capsid at a dose of 1E12 DNase-resistant particles (DRP). Four weeks post-vector administration, the mean levels of circulating TNFR:Fc protein was 21.5 μg/mL. PG-APS was then administered intravenously to induce a recurring flare of joint inflammation. As expected, animals that were treated with vehicle developed a significant flare of inflammation in their ankle joints. In contrast, the inflammatory response, measured by joint volume, was completely suppressed in AAV2/1-TNFR:Fc-treated animals. To assess the long-term effect of TNFR:Fc expression on suppression of arthritis in this model, animals are currently being evaluated for another round of remission and reactivation of joint inflammation.

Kupffer-cell depletion attenuates colonic and extracolonic granulomatous inflammation in chronic colitis

Intramural injection of peptidoglycan-polysaccharide polymers into the distal colon in rats induces granulomatous colitis associated with extracolonic manifestations. We sought to clarify the effects of Kupffer-cell depletion induced by the intravenous administration of gadolinium on colonic and extracolonic inflammation in this model. The effects of Kupffer-cell depletion on acute and chronic inflammation were evaluated 3 days and 3 weeks after injection of peptidoglycan-polysaccharide, respectively. We assessed the effects of gadolinium on colonic cytokine levels in vivo and the viability of elicited peritoneal macrophages and peptidoglycan-polysaccharide-induced production of nitrite, an indirect index of nitric oxide production, by these cells in vitro. A single injection of gadolinium caused a marked decrease in the number of Kupffer cells stained with antibodies within 3 days. Gadolinium treatment did not alter acute inflammation at 3 days. Repeated treatment with gadolinium dramatically attenuated grossly observed chronic inflammation, including thickening of colon wall, hepatic and splenic nodules, and swelling of foot joints; and significantly reduced the proportional areas occupied by granulomas in the colon, liver, and spleen at 3 weeks. These protective effects were reflected in significant reduction in colon and liver weights; gross scores; colonic myeloperoxidase activity, an indirect quantitative index of granulocyte infiltration; colonic interleukin-1β levels; plasma nitrite and nitrate levels; and decreased tendency toward arthritis. Although gadolinium did not cause injury in elicited peritoneal macrophages in vitro, the compound dose-dependently attenuated peptidoglycan-polysaccharide–induced production of nitrite by these cells. Chronic Kupffer-cell depletion attenuates peptidoglycan-polysaccharide–induced granulomatous inflammation in the colon, liver, and spleen and reduces the incidence of arthritis, possibly by suppressing the production of interleukin-1β and nitric oxide.

Novel erythropoiesis stimulating protein (darbepoetin alfa) alleviates anemia associated with chronic inflammatory disease in a rodent model.

We developed a rodent model of noninfectious systemic inflammation to examine the pathogenesis of the associated anemia of chronic disorders (ACD), to evaluate the similarity of this ACD model to human ACD, and to evaluate the potential efficacy of novel erythropoiesis stimulating protein (darbepoetin alfa) as an ACD therapy. Lewis rats were immunized with peptidoglycan-polysaccharide polymers (PG-APS), the chronic inflammation and associated ACD were characterized, and the effects of darbepoetin alfa treatment on complete blood counts (CBC), red blood cell (RBC) indices, and iron metabolism were analyzed weekly. Acutely inflamed rats had reduced peripheral blood (PB) RBC counts and hemoglobin (Hb) concentrations and increased reticulocyte counts. PB RBC numbers normalized during chronic inflammation, but RBC remained hypochromic and microcytic. Consequently, the rats remained chronically anemic. Anemic rats had fluctuating serum erythropoietin (EPO) concentrations, but mean EPO concentrations never varied significantly from baseline control levels. Histology of anemic rat spleen sections revealed reticuloendothelial siderosis. Total serum iron concentrations were chronically low. Peritoneal exudate cells (PEC) isolated from anemic rats and stimulated with PG-APS in vitro produced more interleukin (IL)-1alpha and interferon (IFN)-gamma, and significantly more tumor necrosis factor (TNF)-alpha and IL-10 than control cultures. Darbepoetin alfa restored Hb concentrations to baseline levels within 2 to 7 weeks, depending on dosage. A refined treatment strategy restored Hb to baseline and maintained those levels with reduced dosing. ACD in this rodent model closely replicates human ACD. Darbepoetin alfa treatment reversed ACD in this model by increasing RBC production and RBC hemoglobinization while reducing siderosis and hypoferremia.

Bacterial cell wall polymers promote intestinal fibrosis by direct stimulation of myofibroblasts.

Normal luminal bacteria and bacterial cell wall polymers are implicated in the pathogenesis of chronic intestinal inflammation. To determine the direct involvement of bacteria and their products on intestinal fibrogenesis, the effects of purified bacterial cell wall polymers on collagen and cytokine synthesis were evaluated in intestinal myofibroblast cultures established from normal fetal and chronically inflamed cecal tissues. In this study, the intestines of Lewis rats were intramurally injected with peptidoglycan-polysaccharide polymers. Collagen and transforming growth factor (TGF)-beta1 mRNA levels were measured and correlated with mesenchymal cell accumulation by immunohistochemistry. The direct effects of cell wall polymers on fibrogenic cytokine and collagen alpha1 (type I) expression were evaluated in intestinal myofibroblast cultures. We found that intramural injections of bacterial cell wall polymers induced chronic granulomatous enterocolitis with markedly increased collagen synthesis and concomitant increased TGF-beta1 and interleukin (IL)-6 expression. Intestinal myofibroblast cultures were established, which both phenotypically and functionally resemble the mesenchymal cells that are involved in fibrosis in vivo. Bacterial cell wall polymers directly stimulated collagen alpha1 (I), TGF-beta1, IL-1beta, and IL-6 mRNA expression in the intestinal myofibroblasts derived from both normal and inflamed cecum. Neutralization of endogenous TGF-beta1 inhibited in vitro collagen gene expression. From our results, we conclude that increased exposure to luminal bacterial products can directly activate intestinal mesenchymal cells, which accumulate in areas of chronic intestinal inflammation, thus stimulating intestinal fibrosis in genetically susceptible hosts.

Subtherapeutic corticosteroids potentiate the ability of interleukin 10 to prevent chronic inflammation in rats

Background & Aims: Interleukin (IL)-10, which inhibits macrophages and T-helper lymphocyte type 1 (TH 1) lymphocytes, attenuates chronic granulomatous inflammation induced by bacterial cell wall polymers. This study determines whether corticosteroids enhance the protective effects of IL-10 in cultured peripheral blood mononuclear cells (PBMNCs) and in vivo when started before or after the onset of experimental chronic granulomatous inflammation. Methods: Intestines of Lewis rats were injected intramurally with streptococcal peptidoglycan-polysaccharide (PG-APS) polymers. Daily murine recombinant IL-10 and/or dexamethasone (DEX) therapy was started 12 hours before or at several intervals after PG-APS injection. Results: IL-10 plus corticosteroids additively inhibited IL-1β secretion in human PBMNCs but preserved the beneficial IL-1RA/IL-1β ratio induced by IL-10. IL-10 started before PG-APS injection significantly attenuated intestinal and extraintestinal inflammation, with even more pronounced effects in combination with subtherapeutic doses of DEX. The combination of DEX decreased the effective dose of IL-10 by at least one half. After onset of systemic inflammation using doses effective for prevention, IL-10 monotherapy had nearly no benefit and DEX plus IL-10 was similar to the mild therapeutic effect of DEX alone. Conclusions: The combination of IL-10 and corticosteroids allows lower doses of both agents in preventing chronic intestinal and systemic inflammation. However, timing of IL-10 administration is a critical variable in regulating inflammation. GASTROENTEROLOGY 1998;115:856-865

Streptococcal cell wall arthritis.

Streptococcal cell wall (SCW) arthritis in rats is an experimentally-induced inflammatory model with many features that resemble rheumatoid arthritis (RA) in humans. In this unit, Lewis rats are injected with an aqueous suspension of Group A SCW streptococcal cell wall peptidoglycan-polysaccharide polymers (SCW PG-PS) and observed for the development of arthritis. The resulting arthritis is biphasic. An acute phase typically develops within 48 hr, followed 10 to 21 days later by a chronic phase which persists for months. Support protocols are included for preparing Group A PG-PS and measuring PG-PS concentration.

Interleukin 10 suppresses experimental chronic, granulomatous inflammation induced by bacterial cell wall polymers.

Interleukin 10 (IL10) inhibits monocyte/macrophage and T lymphocyte effector functions. This study examined the effect of systemically administered IL10 on acute and chronic granulomatous enterocolitis, hepatitis, and arthritis in a rat model. Lewis rats were injected intramurally with streptococcal peptidoglycan-polysaccharide (PG-APS) polymers. Beginning 12 hours before PG-APS injection, rats were treated daily with subcutaneous murine recombinant IL10 or vehicle for three or 17 days. IL10 attenuated acute enterocolitis in a dose dependent fashion (p < 0.01). Protective effects were more profound in the chronic granulomatous phase with decreased enterocolitis and markedly inhibited leucocytosis, hepatic granulomas, and chronic erosive arthritis (p < 0.001). IL10 downregulated tissue IL1, IL6, tumour necrosis factor alpha, and interferon gamma gene expression, consistent with the in vitro effects of IL10 on PG-APS-stimulated splenocytes. Caecal IL1 protein concentrations and IL2 and interferon gamma secretion by in vitro stimulated mesenteric lymph nodes were downregulated in IL10 treated animals. These results indicate that exogenous IL10 can inhibit experimental granulomatous inflammatory responses and suggest that IL10 treatment could be an effective new therapeutic approach in human disorders such as Crohn's disease, rheumatoid arthritis, and sarcoidosis.

Local production of corticotropin releasing hormone is increased in experimental intestinal inflammation in rats.

Corticotropin releasing hormone (CRH) suppresses immunological functions via stimulation of the pituitary-adrenal axis, but is also found in peripheral tissues. Peripheral proinflammatory activity of CRH is suggested by increased tissue concentrations in arthritis and in vitro immunostimulatory effects. This study evaluated intestinal CRH concentrations, immunolocalisation, and synthesis in chronic enterocolitis and investigated in vitro responsiveness of lamina propria mononuclear cells to CRH. Chronic granulomatous enterocolitis was induced by intramural injection of peptidoglycan-polysaccharide polymers in the ileocaecal region of Lewis rats. CRH protein was measured in caecal specimens by immunohistochemistry and radioimmunoassay and caecal CRH mRNA expression was analysed by reverse transcriptase polymerase chain reaction. In the chronically inflamed caecum abundant immunoreactive CRH was found in inflammatory cells, mesenchymal cells, as well as in myenteric plexi. In contrast, only a few CRH containing cells were detected in normal and HSA injected control caecums. Moreover, caecal CRH protein levels were increased during chronic enterocolitis. Local CRH synthesis as indicated by mRNA expression was considerably increased in chronic enterocolitis whereas it was undetectable or low in uninflamed caecum. In addition, CRH stimulated in vitro proliferation of lamina propria mononuclear cells and inhibited mitogen induced proliferation. Increased CRH protein and mRNA expression in chronic enterocolitis and responsiveness of intestinal mononuclear cells to CRH indicate an immunomodulatory role for locally produced CRH in intestinal inflammation.

Bacterial Cell Wall Polymer-Induced Granulomatous Inflammation

Local or systemic injection of peptidoglycan-polysaccharide polymers, which are the primary structural components of cell walls of nearly all bacteria, leads to acute inflammation, which can develop into chronic, spontaneously relapsing, granulomatous inflammation in a number of organs. Evolution into chronic granulomatous inflammation is dependent upon persistence of poorly biodegradable cell wall polymers within tissues, genetically determined host susceptibility, and generation of a T-lymphocyte-mediated immune response. Intraperitoneal injection of peptidoglycan-polysaccharide fragments from group A streptococci or selected intestinal bacteria into susceptible Lewis rats leads to chronic, spontaneously reactivating erosive arthritis and hepatic granulomas. Subserosal (intramural) injection of poorly biodegradable cell wall fragments into the distal intestine of Lewis rats induces chronic, spontaneously relapsing granulomatous enterocolitis with associated arthritis, hepatic granulomas, anemia, and leukocytosis. Chronic inflammation does not occur in T-lymphocyte-deficient rats and is prevented by cyclosporin-A therapy and degradation of peptidoglycan by the muralytic enzyme, mutanolysin. Moreover, resistant Buffalo and Fischer F344rats, the latter sharing identical MHC antigens with Lewis rats, develop only acute inflammation with no chronic granulomatous response. Peptidoglycan-polysaccharide polymers activate almost every limb of the inflammatory response. Blockade of specific pathways suggests that interleukin-1, transforming growth factor-β, plasma kallikrein, and T lymphocytes are dominant mediators of peptidoglycan-polysaccharide-induced arthritis, hepatic granulomas, and enterocolitis. Because of the similarity of immune mechanisms of these rat models to human disease, bacterial cell wall-induced inflammation provides unique opportunities to study pathogenic mechanisms of granuloma formation in response to ubiquitous microbial agents and to test novel therapeutic agents.

Patterns of prostaglandin E 2 and leukotriene B 4 production in inflammation induced by peptidoglycan-polysaccharide polymers of bacterial cell walls

The changing levels of prostaglandin E 2 (PGE 2) and leukotriene B 4 (LTB 4) were compared over a period of 1 h to 15 days in two kinds of inflammatory responses induced in the rat air pouch by chemically similar polymers of peptidoglycan-polysaccharide (PG-PS) isolated from cell walls of group A streptococci. The smaller polymers (100 s, average mol. wt 5.3 × 10 6) induced a more severe acute response over the first 24 h, whereas, from 3 to 15 days the larger polymers (10 p, average mol. wt 5 × 10 8) induced greater chronic inflammation, as measured by pouch fluid volume, number of infiltrating cells and weight of granulation tissue. The most prominent difference between the two kinds of responses in the pattern of PGE 2 and LTB 4 were: (a) A shift between 1 to 6 h to much higher production of PGE 2 in the exudate induced by large polymers. During this time the level of LTB 4 also increased, but continued to be higher in the exudate induced by small polymers. (b) The sustained production of relatively high levels of LTB 4 and PGE 2 in the large polymer exudate at 15 days. Changes such as these indicate that this model is useful for analyzing mediators which regulate the evolution of acute into chronic inflammation.

Oral Peptidoglycan-Polysaccharide Stimulates Systemic Immunocompetency in Germ-free Mice

Germ-free and conventional mice were fed sterile peptidoglycan-polysaccharide polymers to determine whether intestinal bacterial cell wall products influence systemic immunity. Germ-free mice fed saline had significantly less footpad swelling after challenge with sheep erythrocytes than conventional mice. Feeding peptidoglycan-polysaccharide polymers to germ-free mice restored their deficient cellular immune response to conventional levels. Feeding peptidoglycan-polysaccharide did not alter anti-erythrocyte antibody production in germ-free mice. Spontaneous in vitro proliferation of unfractionated splenocytes taken from germ-free mice fed peptidoglycan-polysaccharide polymers was enhanced compared with proliferation of splenocytes from germ-free mice fed human serum albumin. In vitro conconavalin A stimulation of unfractionated splenocytes obtained from germ-free mice fed cell wall polymers resulted in a significant decrease in proliferation relative to that seen for germ-free mice fed human serum albumin. Respective proliferation results were abrogated when splenocytes were fractionated by removal of plastic-adherent cells. These results suggest that luminal peptidoglycan-polysaccharide polymers are important in the functional development of systemic cellular immunity, and that plastic-adherent splenocytes regulate this response.

Phlogistic properties of peptidoglycan-polysaccharide polymers from cell walls of pathogenic and normal-flora bacteria which colonize humans.

PG-PS polymers which can induce experimental chronic inflammation in joints and other tissues can be isolated from the cell walls of human pathogens, such as group A streptococci, as well as from certain indigenous bacterial species which colonize the human intestinal tract. The structural and biological properties that are required for cell wall fragments to express this remarkable activity are still not well defined, but polymer size, resistance to tissue enzymes, and capacity to sustain activation of complement, macrophages, neutrophils, and T cells are properties associated with the most active preparations. There is increasing evidence that PG-PS structures with arthropathogenic activity occur in the human intestinal lumen and that these polymers can be translocated systemically. These observations support the concept that PG-PS, derived from a variety of bacterial species, can be part of the etiology of rheumatoid arthritis and other chronic inflammatory diseases. Since the PG component provides a common element to which all individuals are exposed, it follows that susceptibility is related to efficiency of disposal of bacterial cell wall debris, as well as to cytokine networks and immune cell function (51).

Superantigen can reactivate bacterial cell wall-induced arthritis.

Intravenous injection of toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus, can reactivate arthritis in a rat ankle joint that has been previously inflamed by injection of peptidoglycanpolysaccharide polymers isolated from the cell walls of group A streptococci. The severity and chronicity of this renewed arthritis is dose dependent and at higher doses (125 micrograms/kg) a prolonged joint inflammation with pannus formation and marginal erosion of cartilage and bone is induced after a single injection of TSST-1. Only modest synovial hyperplasia is induced in control ankle joints by systemic injection of TSST-1. Another superantigen, streptococcal pyrogenic exotoxin induces a much weaker, acute reactivation of arthritis that resolves by 2 days. Repeated injections of TSST-1 at 7-day intervals give the same undiminished pattern of joint response, but the joint swelling persists at a higher level with each succeeding injection. Cyclosporin A suppresses all phases of the recurrent arthritis, indicating that TSST-1 could be functioning through its property of a superantigen activating T lymphocytes. II-1 receptor antagonist and anti-TNF-alpha neutralizing antibody, which reduce reactivation of arthritis by peptidoglycan-polysaccharide polymers, have no effect on reactivation by TSST-1. This experimental model provides a means to examine in vivo the possible role of superantigens in rheumatoid arthritis and related diseases, and to analyze the cellular and molecular pathways induced by this family of microbial products.

Phlogistic properties of peptidoglycan-polysaccharide polymers from cell walls of pathogenic and normal-flora bacteria which colonize humans.

Phlogistic properties of peptidoglycan-polysaccharide polymers from cell walls of pathogenic and normal-flora bacteria which colonize humans.