Effect of cytokine and antigen stimulation on peripheral blood lymphocyte syndecan-1 expression

INTRODUCTION: Cytokines are not only produced by activated lymphocytes but also interact with a number of cell-surface molecules on the same cells. Syndecan-1 is one such cell-surface molecule, which has the capacity to bind a variety of growth factors as well as cytokines. The aim of this study was to examine the effects of transforming growth factor beta (TGF-beta), interleukin-1 (IL-1), IL-2, IL-4, lipopolysaccharide (LPS) from Porphyromonas gingivalis and tetanus toxoid on syndecan-1 expression by B and T lymphocytes. METHODS: B and T lymphocytes were obtained from the peripheral blood of healthy donors. Following exposure to the above growth factors, cytokines and antigens, syndecan-1 expression was determined by flow cytometry. RESULTS: Subjects could be categorized as high or low expressors of syndecan-1. In the high-responder group TGF-beta1 alone resulted in a significant increase in syndecan-1 expression by both B and T cells. None of the other cytokines and antigens produced a significant response. When analysed in combination, TGF-beta1 in combination with IL-2, IL-4, P. gingivalis LPS and tetanus toxoid all produced significant increases in syndecan-1 expression by B cells. For T cells, combinations of TGF-beta1 with IL-2 and tetanus toxoid resulted in increased syndecan-1 expression. CONCLUSIONS: Both B and T lymphocytes synthesize the cell-surface proteoglycan syndecan-1 and its expression can be modulated by TGF-beta1, either alone or in combination with IL-2, IL-4 and LPS from P. gingivalis and tetanus toxoid. While these may reflect general responses under inflammatory conditions their biological significance requires further investigation.     

Synergistic effects of lipopolysaccharides from periodontopathic bacteria on pro‐inflammatory cytokine production in an ex vivo whole blood model

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been strongly associated with chronic periodontitis. This disease is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. The secretion of high levels of inflammatory cytokines by those cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of whole blood from periodontitis patients following challenges with whole cells of P. gingivalis, T. denticola, and T. forsythia or their lipopolysaccharides (LPS), individually and in combination. Whole blood collected from seven periodontitis patients was stimulated with whole cells or LPS and the production of interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor alpha (TNF‐α) were quantified by enzyme‐linked immunosorbent assays. The mono and mixed challenges with whole bacterial cells or LPS induced the secretion of high amounts of IL‐1β, IL‐6, IL‐8, and TNF‐α by the mixed leukocyte population from periodontitis patients. In addition, P. gingivalis LPS, T. denticola LPS, and T. forsythia LPS acted in synergy to induce high levels of IL‐1β and TNF‐α. This study suggests that P. gingivalis, T. denticola, and T. forsythia may contribute to the immunodestructive host response characteristic of periodontitis through synergistic effects of their LPS on the inflammatory response induced by a mixed population of leukocytes.     

Whole‐Blood Cultures From Patients With Chronic Periodontitis Respond Differently to Porphyromonas gingivalis but not Escherichia coli Lipopolysaccharide

Background: Porphyromonas gingivalis lipid A heterogeneity modulates cytokine expression in human cells. This study investigates the effects of two lipid A isoforms of P. gingivalis, lipopolysaccharide (LPS)_1435/1449 and LPS₁₆₉₀, on the secretion of proinflammatory and regulatory cytokines in total blood cultures from patients with and without chronic periodontitis (CP). Methods: A cross‐sectional study was conducted in 38 systemically healthy individuals divided in two groups: 1) the CP group (n = 19), in which patients were diagnosed with CP; and 2) the no periodontitis (NP) group (n = 19), which included control patients without CP. Blood samples were collected from all patients, and whole‐blood cell cultures (WBCCs) were stimulated for 48 hours with P. gingivalis LPS_1435/1449 and LPS₁₆₉₀ and Escherichia coli LPS. Unstimulated WBCCs served as negative controls. The secretion of interferon‐γ (IFN‐γ), interleukin‐10 (IL‐10), and transforming growth factor‐β (TGF‐β) was detected in WBCC supernatants by enzyme‐linked immunosorbent assays. Results: E. coli LPS significantly increased the expression of all cytokines in WBCCs from both the NP and CP groups when compared to non‐stimulated cells (control treatment). P. gingivalis LPS preparations increased IFN‐γ levels in the CP group but not in the NP group when compared with controls (P <0.05). P. gingivalis LPS preparations also increased IL‐10 and TGF‐β levels in both CP and NP groups, but P. gingivalis LPS₁₆₉₀ showed a three‐fold increase on IL‐10 production in the NP group (P <0.05) when compared to P. gingivalis LPS_1435/144. Conclusions: These data demonstrate that WBCC cell populations obtained from healthy individuals and patients with CP may differ in the cytokine response to P. gingivalis but not E. coli LPS. This is consistent with the notion that CP alters the systemic WBCC response and that this can be detected by the different P. gingivalis LPS structures.     

Immunomodulation of dendritic cells differentiated in the presence of nicotine with lipopolysaccharide from Porphyromonas gingivalis

Tobacco smoking is a significant risk factor for periodontal diseases. Nicotine, one of the most studied constituents in cigarette smoke, is thought to modify immune responses. Dendritic cells (DCs), which are key mediators between innate and adaptive immunity, stimulate naive T cells to differentiate to effector T‐cell subsets that may be actively involved in the immunopathogenesis of periodontal diseases. In this study, we evaluated the effects of nicotine and lipopolysaccharide (LPS) from Porphyromonas gingivalis, alone and in combination, on the functions of human monocyte‐derived DCs to elucidate the mechanism of tissue destruction of smoking‐associated periodontal diseases. P. gingivalis LPS‐stimulated DCs differentiated with nicotine (NiDCs) induced lower T‐cell proliferation and human leukocyte antigen (HLA)‐DR expression, but elevated expression of programmed cell death ligand 1. Additionally, NiDCs impaired interferon‐γ production but maintained interleukin (IL)‐5 and IL‐10 production in co‐cultured T cells. Furthermore, NiDCs produced lower levels of proinflammatory cytokines compared with DCs differentiated in the absence of nicotine. Interestingly, NiDCs preferentially produced the T helper 2 (Th2)‐type chemokines macrophage chemotactic protein‐1 and macrophage‐derived chemokine. These results suggest that the presence of nicotine during differentiation of DCs modulates the immunoregulatory functions of P. gingivalis LPS‐stimulated DCs.     

The Influence of Glycated Hemoglobin on the Cross Susceptibility Between Type 1 Diabetes Mellitus and Periodontal Disease

Background: Periodontal disease is a major complication of type 1 diabetes mellitus (T1DM). The aim of the present study is to investigate the relationship between glycated hemoglobin and circulating levels of interleukin (IL)‐6, IL‐8, and C‐X‐C motif chemokine ligand 5 (CXCL5) in non‐smoking patients suffering from T1DM, with and without periodontitis. In addition, to determine the effect of advanced glycation end products (AGE) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) on IL‐6, IL‐8, and CXCL5 expression by THP‐1 monocytes and OKF6/TERT‐2 cells. Methods: There were 104 participants in the study: 19 healthy volunteers, 23 patients with periodontitis, 28 patients with T1DM, and 34 patients with T1DM and periodontitis. Levels of blood glucose/glycated hemoglobin (International Federation of Clinical Chemistry [IFCC]) were determined by high‐performance liquid chromatography. Levels of IL‐6, IL‐8, and CXCL5 in plasma were determined by enzyme‐linked immunosorbent assay (ELISA). In vitro stimulation of OKF6/TERT‐2 cells and THP‐1 monocytes was performed with combinations of AGE and P. gingivalis LPS. Changes in expression of IL‐6, IL‐8, and CXCL5 were monitored by ELISA and real‐time polymerase chain reaction. Results: Patients with diabetes and periodontitis had higher plasma levels of IL‐8 than patients with periodontitis alone. Plasma levels of IL‐8 correlated significantly with IFCC units, clinical probing depth, and attachment loss. AGE and LPS, alone or in combination, stimulated IL‐6, IL‐8, and CXCL5 expression in both OKF6/TERT‐2 cells and THP‐1 monocytes. Conclusions: Elevated plasma levels of IL‐8 potentially contribute to the cross‐susceptibility between periodontitis and T1DM. P. gingivalis LPS and AGE in combination caused significantly greater expression of IL‐6, IL‐8, and CXCL5 from THP‐1 monocytes and OKF6/TERT‐2 cells than LPS alone.     

A Sialidase‐Deficient Porphyromonas gingivalis Mutant Strain Induces Less Interleukin‐1β and Tumor Necrosis Factor‐α in Epi4 Cells Than W83 Strain Through Regulation of c‐Jun N‐Terminal Kinase Pathway

Background: Porphyromonas gingivalis is one of the major periodontal pathogens. In a previous study, a mouse abscess model showed that sialidase deficiency of P. gingivalis weakened its virulence, but the mechanism behind this observation remains unknown. Methods: A sialidase‐deficient mutant strain (△PG0352) and a complemented strain (com△PG0352) were constructed. Epi4 cells were stimulated by wild‐type strain P. gingivalis W83, △PG0352, or com△PG0352. Real‐time polymerase chain reaction was carried out to detect expression of virulent genes in P. gingivalis and interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α in epi4 cells. Activities of sialidase, gingipains, and lipopolysaccharide (LPS) were compared among the different P. gingivalis strains. Levels of IL‐1β and TNF‐α in the epi4 cells supernatant were detected by enzyme‐linked immunosorbent assay and levels of p38, extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase (JNK), and phospho‐c‐Jun were detected by western blotting. Results: Compared with P. gingivalis W83 and com△PG0352, activities of Kgp and Rgp gingipains and amount of LPS decreased in △PG0352, whereas there were no differences in LPS activity among these three strains. Level of phospho‐JNK was lower in epi4 cells stimulated by △PG0352. △pG0352 induced less IL‐1β and TNF‐α and more IL‐8 in epi4 cells; differences in IL‐1β and TNF‐α could not be detected after JNK blocking. Conclusion: A sialidase‐deficient P. gingivalis mutant strain induces less IL‐1β and TNF‐α in epi4 cells than W83 strain through regulation of JNK pathway.     

Induction of interleukin (IL)-1β, IL-10, IL-8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans

Trindade SC, Olczak T, Gomes‐Filho IS, Moura‐Costa LF, Cerqueira EMM, Galdino‐Neto M, Alves H, Carvalho‐Filho PC, Xavier MT, Meyer R. Induction of interleukin (IL)‐1β, IL‐10, IL‐8 and immunoglobulin G by Porphyromonas gingivalis HmuY in humans. J Periodont Res 2012; 47: 27–32. © 2011 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis, an anaerobic gram‐negative bacterium, is associated with chronic periodontitis. This study was undertaken to evaluate the production of interleukin (IL)‐1β, IL‐8 and IL‐10 by human peripheral blood mononuclear cells (PBMC) stimulated with P. gingivalis antigens and to assess the levels of serum immunoglobulin (Ig)G, IgA and IgG subclasses raised against P. gingivalis HmuY protein. Material and Methods: PBMC from patients with chronic periodontitis (CP) and from nonperiodontitis (NP) control subjects were stimulated with P. gingivalis antigens, and the cytokine levels in the culture supernatants were determined by ELISA. The specificity of serum antibodies raised against HmuY was analyzed by Western blotting and by ELISA. Results: Compared with the NP controls, the CP patients produced higher levels of total serum IgG and IgG1 specific for P. gingivalis HmuY. No differences were found between CP and NP groups in the production of IL‐1β and IL‐8 by PBMC stimulated with total P. gingivalis antigens. Only P. gingivalis lipopolysaccharide (LPS) induced higher levels of IL‐10 in the CP group. Higher levels of IL‐1β and IL‐10 were induced by HmuY than by other antigens derived from the wild‐type P. gingivalis strains. In contrast, total antigens derived from the hmuY‐deletion mutant strain induced the production of significantly higher levels of IL‐8 and significantly lower levels of IL‐1β. Conclusion: Our data suggest that P. gingivalis HmuY may be considered an immunogenic protein associated with host–pathogen interactions.     

Blocking Proinflammatory Cytokine Release Modulates Peripheral Blood Mononuclear Cell Response to Porphyromonas gingivalis

Background: Chronic periodontitis (CP) is an inflammatory disease in which cytokines play a major role in the progression of disease. Anti‐inflammatory cytokines (interleukin 4 [IL‐4] and IL‐10) were reported to be absent or reduced in diseased periodontal tissues, suggesting an imbalance between the proinflammatory and anti‐inflammatory mediators. This study tests the hypothesis that there is cellular crosstalk mediated by proinflammatory and anti‐inflammatory cytokines and that blocking proinflammatory cytokine (tumor necrosis factor‐α [TNF‐α] and IL‐1) production will enhance anti‐inflammatory cytokine (IL‐4 and IL‐10) production from peripheral blood mononuclear cells (PBMCs) in response to Porphyromonas gingivalis. Methods: PBMCs were isolated from individuals diagnosed with CP or healthy individuals and cultured for 24 hours. Concanavalin A (ConA) was used as an activator of lymphocyte function. Live and heat‐killed P. gingivalis or lipopolysaccharide from P. gingivalis were used as the bacterial stimulants. TNF‐α and IL‐1 production was neutralized by specific antibodies against TNF‐α and IL‐1α or IL‐β. Culture supernatants were evaluated by enzyme‐linked immunosorbent assay for TNF‐α, IL‐1β, IL‐4, and IL‐10 production. Results: Live P. gingivalis did not result in any significant IL‐10 or IL‐4 release, whereas heat‐killed P. gingivalis led to a significant increase in IL‐10 levels compared with unstimulated or live P. gingivalis–stimulated cells from both healthy individuals or those with CP. Overall, PBMCs from patients with CP produced significantly lower IL‐10 in response to ConA and P. gingivalis, suggesting chronic suppression of the anti‐inflammatory cytokine production. Blocking the proinflammatory cytokine response did not result in any substantial change in IL‐10 or IL‐4 response to live P. gingivalis. Blocking the proinflammatory cytokine response restored IL‐10 production by cells from CP in response to P. gingivalis lipopolysaccharide. Conclusions: These findings suggest that PBMCs from patients with CP have suppressed anti‐inflammatory cytokine production that can, in part, be restored by neutralizing proinflammatory cytokines. Monocytes are an important source of IL‐10 production, and monocyte‐derived IL‐10 might play a regulatory role in the pathogenesis of CP.     

Interleukin‐1 and interleukin‐8 in nicotine‐ and lipopolysaccharide‐exposed gingival keratinocyte cultures

Johnson GK, Guthmiller JM, Joly S, Organ CC, Dawson DV. Interleukin‐1 and interleukin‐8 in nicotine‐ and lipopolysaccharide‐exposed gingival keratinocyte cultures. J Periodont Res 2010; 45: 583–588. © 2010 John Wiley & Sons A/S Background and Objective: Tobacco use is associated with increased periodontal destruction in both cigarette smokers and smokeless tobacco users. Gingival keratinocytes are the first cells in contact with microbial and tobacco components and play a key role in the innate immune response to these agents. The objective of this study was to evaluate the effect of nicotine and bacterial lipopolysaccharide (LPS) alone and in combination on gingival keratinocyte production of interleukin‐1α (IL‐1α) and interleukin‐8 (IL‐8). Material and Methods: Gingival keratinocyte cultures were established from 10 healthy, non‐tobacco‐using subjects. The cells were stimulated for 24 h with 1 μm or 1 mm nicotine and/or 10 μg/mL Escherichia coli or Porphyromonas gingivalis LPS. Interleukin‐1α and IL‐8 proteins were quantified using ELISAs. Results: Compared with untreated cultures, 1 mm nicotine stimulated production of IL‐1α (p < 0.001); E. coli and P. gingivalis LPS increased IL‐8 production (p = 0.0014 and p = 0.0232, respectively). A combination of nicotine and LPS produced the highest cytokine quantities. Amounts of IL‐1α and IL‐8 following 1 mm nicotine and LPS exposure were significantly greater than in untreated cultures (p < 0.001). Interleukin‐8 was also responsive to 0.1 μm nicotine combined with E. coli or P. gingivalis LPS compared with control cultures (p < 0.0001 and p = 0.0029, respectively). Both cytokines tended to be elevated following the combined treatment relative to nicotine or LPS treatment alone. Conclusion: These results demonstrate that nicotine and LPS differentially regulate IL‐1 and IL‐8 production by gingival keratinocytes. Combined treatment tended to elevate cytokine production further, which may have implications for the progression of periodontitis in tobacco users.     

Lipopolysaccharide from Escherichia coli but not from Porphyromonas gingivalis induce pro-inflammatory cytokines and alkaline phosphatase in dental follicle cells

Objectives

Dental follicle cells (DFCs) as periodontal precursor cells are the natural source for cellular therapies of periodontitis. Periodontitis is initiated after the infection of the periodontium with oral pathogens such as the Gram-negative bacteria Porphyromonas gingivalis. Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative bacteria. Previous studies have shown that especially P. gingivalis LPS induces the expression of pro-inflammatory cytokines in PDL cells and disturbs the differentiation of dental stem cells. Our study investigated the administration of LPS to DFCs for the first time.

Materials and methods

We evaluated cell proliferation (WST1 assay), expression of cytokines IL1β, IL8 and IL6 (real-time RT-PCR) and the osteogenic differentiation of DFCs (ALP-activity and Alizarin red staining) in the presence of P. gingivalis LPS and Escherichia coli LPS.

Results

All tested pro-inflammatory cytokines were highly increased after E. coli LPS treatment. P. gingivalis LPS induces only the expression of IL8, but this expression was significantly lower than that after E. coli LPS administration. The ALP activity was significantly higher in DFCs after the administration of E. coli LPS than after administration of P. gingivalis LPS or under normal cell differentiation conditions. However, the mineralization was inhibited with LPS from both bacterial species.

Conclusion

LPS disturbs osteogenic differentiation in DFCs. Moreover, the failure of pro-inflammatory cytokines induction in DFCs after the administration of P. gingivalis LPS differs greatly from that of PDL fibroblasts. These immunological properties of DFCs have to be considered for cellular therapies of periodontitis with DFCs.     

Lethal outcome caused by Porphyromonas gingivalis A7436 in a mouse chamber model is associated with elevated titers of host serum interferon‐γ

Background/aims: Septic shock caused by gram‐negative bacteria has been associated with cytokines produced by hosts. Porphyromonas gingivalis A7436, a disseminating strain, caused septic shock‐like symptoms and even animal death in a mouse chamber model. However, P. gingivalis exhibits lower endotoxin activities in its lipopolysaccharide than other typical gram‐negative bacteria. In this study, we examined the effects of P. gingivalis lethal infection on host pro‐inflammatory cytokines production. Methods: Nude and normal BALB/c mice were infected with a lethal dose of P. gingivalis A7436 using a mouse chamber model. Serum levels of tumor necrosis factor, interleukin (IL)‐1β, IL‐12 and interferon‐γ were evaluated. The effects of tumor necrosis factor inhibitor (thalidomide) and anti‐interferon‐γ antibody on infection outcomes were examined. Results: All nude mice survived infectious challenge, whereas 100% of normal mice died with abdominal lesions. Bacterial cultures indicated P. gingivalis dissemination to the circulation. Serum levels of tumor necrosis factor, IL‐1β and IL‐12 showed no significant differences between nude and normal mice. Thalidomide treatment did not protect normal mice from death but decreased remote lesion occurrence, with concurrent reduced bacterial counts recoverable from blood. There was a 3.5‐fold elevation in normal mice serum interferon‐γ titers compared to those of nude mice and anti‐interferon‐γ antibody treatment resulted in 100% protection from lethal outcome. Conclusion: Lethal outcome following P. gingivalis A7436 infection is T‐lymphocyte dependent and involves an increase in systemic interferon‐γ levels. The data further indicate that P. gingivalis transvascular dissemination (bacteremia) alone is not sufficient for lethal outcome.     

Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell‐surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram‐negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), and tumor necrosis factor‐α (TNF‐α) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer‐membrane proteins from P. gingivalis ATCC 53977. Outer‐membrane protein from P. gingivalis enhanced the production of IL‐6 and IL‐8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL‐8 production activity of polysaccharide from P. gingivalis was higher than that of other cell‐surface components. The levels of IL‐6 and IL‐8 released from the P. gingivalis lipopolysaccharide‐treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer‐membrane protein or lipopolysaccharide inhibited the IL‐6 and IL‐8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer‐membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

Inflammatory response of uric acid produced by Porphyromonas gingivalis gingipains

Uric acid is a potential metabolite that serves as a danger‐associated molecular pattern (DAMP) and induces inflammatory responses in sterile environments. Porphyromonas gingivalis is a keystone periodontopathogen, and its gingipain proteases play a critical role in the pathogenesis of periodontitis. In this study, we demonstrate that P. gingivalis gingipains play a role in THP‐1 macrophage uric acid production by increasing the expression and activity of xanthine oxidoreductase (XOR). Uric acid sodium salt induces caspase‐1 activation, cell death, and the expression of proinflammatory cytokines, including IL‐1α, IL‐6, and IL‐8, in the human keratinocyte HOK‐16B cell line. Our results suggest that gingipain‐induced uric acid can mediate inflammation in periodontal tissue cells.