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J Dent Res 86(9):878-882, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Hemoglobin and LPS Act in Synergy to Amplify the Inflammatory Response

C. Bodet, F. Chandad, and D. Grenier*

Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Quebec City, Quebec, Canada G1K 7P4

* corresponding author, Daniel.Grenier{at}greb.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular disruption and bleeding during periodontitis likely increase the levels of hemoglobin in gingival crevicular fluid. The aim of this study was to investigate the effect of hemoglobin on the inflammatory responses of human macrophages stimulated with lipopolysaccharides (LPS) isolated from periodontopathogens. The production of interleukin-1 beta (IL-1ß), IL-6, IL-8, and tumor necrosis factor alpha (TNF-{alpha}) by macrophages following challenges with Porphyromonas gingivalis and Fusobacterium nucleatum LPS in the presence or absence of human hemoglobin was analyzed by ELISA. The effect of hemoglobin on LPS-binding to macrophages was evaluated with 3H-LPS. Hemoglobin and LPS from periodontopathogens acted in synergy to stimulate the production of high levels of IL-1ß, IL-6, IL-8, and TNF-{alpha} by macrophages. Hemoglobin also enhanced LPS-binding to macrophages. This study suggests that hemoglobin contributes to increases in the levels of pro-inflammatory mediators in periodontal sites by acting in synergy with LPS from periodontopathogens, thus favoring the progression of periodontitis.

KEY WORDS: cytokine • Fusobacterium nucleatum • periodontitis • Porphyromonas gingivalis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process resulting in the loss of tooth-supporting tissues. Porphyromonas gingivalis and Fusobacterium nucleatum are major periodontopathogens (Moore and Moore, 1994), and their numbers in subgingival plaque increase significantly during the active phase of periodontitis (van Winkelhoff et al., 2002). The host response to these bacterial species and their products, such as lipopolysaccharides (LPS), is a critical determinant in the initiation and progression of periodontitis (Agarwal et al., 1995; Wilson, 1995). Excessive, continuous production of cytokines—including interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor alpha (TNF-{alpha})—in inflamed periodontal tissues is responsible for disease progression and periodontal tissue destruction (Okada and Murakami, 1998).

The periodontal tissue of persons affected with periodontitis is highly infiltrated with macrophages that play an essential role in the host response to periodontopathogens (Okada and Murakami, 1998). A clinical characteristic of periodontitis is vascular disruption and bleeding. Hemoglobin accounts for 95% of the protein content of erythrocytes and is present in both plasma and gingival crevicular fluid. Several studies have revealed a close relationship between bleeding on probing and inflamed gingival tissue (Abrams et al., 1984; Thilo et al., 1986). A correlation between hemoglobin concentration in gingival tissue and the active phase of periodontitis has also been suggested. Indeed, the hemoglobin concentration increases rapidly over the first 7 days following ligature-induced experimental periodontitis in dogs (Hanioka et al., 1989), as well as in human gingiva, with increasing inflammation (Hanioka et al., 1990). Interestingly, the increased hemoglobin concentration in human gingiva is restored to normal levels when the inflammation is resolved (Hanioka et al., 1991). In this study, we hypothesized that hemoglobin acts in synergy with periodontopathogen LPS to amplify the inflammatory responses of human macrophages.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Growth Conditions, and LPS Preparations
P. gingivalis ATCC 33277, Actinobacillus actinomycetemcomitans ATCC 29522, and F. nucleatum subsp. nucleatum ATCC 25586 (hereafter referred to as F. nucleatum) were grown in Todd-Hewitt broth supplemented with hemin (10 µg/mL) and vitamin K (1 µg/mL) at 37°C for 24 hrs under anaerobic conditions. LPS were isolated by the method of Darveau and Hancock (1983).

Monocyte and Macrophage Cultures
We cultivated U937 cells (ATCC CRL-1593.2), a monoblastic leukemia cell line, at 37°C in a 5% CO2 atmosphere in RPMI-1640 medium (HyClone Laboratories, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (RPMI-FBS) and 100 µg/mL of penicillin-streptomycin. Monocytes (2 x 105 cells/mL) were incubated in RPMI-FBS containing 10 ng/mL of phorbol myristic acid (PMA) for 48 hrs, to induce differentiation into adherent macrophage-like cells. Following the PMA treatment, the medium was replaced with fresh medium, and the differentiated cells were incubated for an additional 24 hrs prior to use. Adherent macrophages were washed and suspended in RPMI with 1% heat-inactivated FBS at a density of 1 x 106 cells/mL and incubated in six-well plates (2 x 106 cells/well in 2 mL) at 37°C in a 5% CO2 atmosphere for 2 hrs prior to stimulation.

Macrophage Stimulation
The macrophages were stimulated with P. gingivalis LPS or F. nucleatum LPS (0.1, 1, and 10 µg/mL) in the presence or absence of human erythrocyte hemoglobin (Sigma, St. Louis, MO, USA; 10, 50, and 100 µg/mL). After a 24-hour incubation, the supernatants were collected and stored at –20°C until used. Cells incubated in culture medium with or without hemoglobin, but not stimulated with LPS, were used as controls. Macrophage viability was evaluated by an MTT (3-[4,5-diethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany).

Determination of Cytokine Production
We used commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) to quantify IL-1ß, IL-6, IL-8, TNF-{alpha}, and Regulated on Activation Normal T-cell Expressed and Secreted (RANTES) concentrations in the cell-free culture supernatants, according to the manufacturer’s protocols.

Radiolabeling of LPS and 3H-LPS Binding
F. nucleatum and P. gingivalis 3H-LPS were prepared by a modification of a procedure described previously (Rokita and Menzel, 1997). The binding of 3H-LPS to hemoglobin-, BSA-, or gelatin-coated microplate wells, and the effect of hemoglobin on 3H-LPS binding to macrophages were investigated (see APPENDIX).

Statistical Analyses
Statistical analyses were performed by the Student’s t test for paired values. To determine the synergistic effect of LPS-hemoglobin on cytokine production, we added the cytokine levels induced by hemoglobin alone to the levels induced by LPS alone and compared them with the levels induced by LPS in the presence of hemoglobin. The data were considered significant at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To evaluate the effect of hemoglobin on LPS-induced pro-inflammatory cytokine production, we stimulated macrophages with P. gingivalis and F. nucleatum LPS (0.1, 1, and 10 µg/mL) in the presence or absence of hemoglobin (10, 50, and 100 µg/mL). A hemoglobin concentration of 100 µg/mL caused a 23% reduction in macrophage viability, whereas concentrations of 50 µg/mL and 10 µg/mL resulted in 16% and 7% reductions in macrophage viability, respectively. In addition, the treatment with both hemoglobin and LPS did not result in a further decrease in cell viability compared with the treatment with hemoglobin alone. Stimulation of macrophages with hemoglobin alone induced the secretion of IL-1ß, TNF-{alpha}, IL-6, and IL-8 (Figs. 1Go, 2Go), as well as RANTES (data not shown). This effect was concentration-dependent, and IL-8 was the most strongly induced.


Figure 1
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  		Figure 1. Secretion of IL-1ß (A), TNF-{alpha} (B), IL-6 (C), and IL-8 (D) by macrophages stimulated with LPS from P. gingivalis in the presence or absence of hemoglobin (10, 50, 100 µg/mL) for 24 hrs. Cytokine secretion was assessed by ELISA. The data are the means ± standard deviations of triplicate assays. *p < 0.05.

 

Figure 2
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  		Figure 2. Secretion of IL-1ß (A), TNF-{alpha} (B), IL-6 (C), and IL-8 (D) by macrophages stimulated with LPS from F. nucleatum in the presence or absence of hemoglobin (10, 50, 100 µg/mL) for 24 hrs. Cytokine secretion was assessed by ELISA. The data are the means ± standard deviations of triplicate assays. *p < 0.05.

 
A synergistic effect of both P. gingivalis and F. nucleatum LPS and hemoglobin on IL-1ß secretion was observed with hemoglobin at concentrations of 50 and 100 µg/mL (Figs. 1AGo, 2AGo). At a hemoglobin concentration of 100 µg/mL, the synergistic effect was observed at all concentrations of both LPS. In contrast, while a synergistic effect was observed at all F. nucleatum LPS concentrations in combination with a hemoglobin concentration of 50 µg/mL, a significant (p < 0.05) enhancement of LPS-induced IL-1µ secretion was observed only at a high concentration of P. gingivalis LPS (10 µg/mL). Hemoglobin (50 and 100 µg/mL) and LPS at all concentrations tested acted in synergy to enhance TNF-{alpha} production by macrophages (Figs. 1BGo, 2BGo). A synergistic effect between hemoglobin at all concentrations tested and LPS was also observed for IL-6 secretion (Figs. 1CGo, 2CGo). More specifically, a significant effect was observed at a high concentration (10 µg/mL) of P. gingivalis LPS and at a lower concentration of F. nucleatum LPS (1 and 10 µg/mL). Hemoglobin also enhanced LPS-induced IL-8 secretion by macrophages (Figs. 1DGo, 2DGo). At P. gingivalis LPS concentrations of 0.1 and 1 µg/mL, the synergistic effect on IL-8 secretion was observed only at a high hemoglobin concentration (100 µg/mL) (Fig. 1DGo). At a higher P. gingivalis LPS concentration (10 µg/mL), a significant effect was observed at all the hemoglobin concentrations tested. A synergistic effect on IL-8 production by macrophages was observed at a low concentration of F. nucleatum LPS (0.1 µg/mL) at all the hemoglobin concentrations tested (Fig. 2DGo). At a high concentration of F. nucleatum LPS (10 µg/mL), a significant synergistic effect was observed only with a hemoglobin concentration of 10 µg/mL. Similar effects of hemoglobin on LPS-induced cytokine secretion by macrophages were also observed with A. actinomycetemcomitans LPS (Appendix Fig.). While hemoglobin alone enhanced RANTES secretion, it had no significant synergistic effect in the presence of LPS (data not shown). In addition, the effect of hemoglobin on cytokine production by macrophages following stimulation with whole P. gingivalis cells was also investigated, and no significant effect was observed (Appendix Table).

P. gingivalis and F. nucleatum 3H-LPS were tested for their capacity to bind to hemoglobin-coated microplate wells. No significant differences were noted in the binding to hemoglobin-coated wells and to control wells coated with BSA or gelatin (data not shown). The effect of hemoglobin on 3H-LPS-binding to macrophages was then investigated (Fig. 3Go). The binding of P. gingivalis and F. nucleatum LPS was significantly enhanced in the presence of hemoglobin at 10, 50, and 100 µg/mL, but not in the presence of BSA (100 µg/mL; data not shown). For instance, at 50 µg/mL, hemoglobin enhanced the binding of P. gingivalis and F. nucleatum LPS by approximately 25% and 100%, respectively. This effect was observed in the presence or absence of serum, which contains proteins that participate in the interaction of LPS with cells, including LPS-binding protein (LBP) and soluble CD14 (sCD14) (data not shown).


Figure 3
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  		Figure 3. Effect of hemoglobin on LPS-binding to macrophages. 3H-LPS from P. gingivalis and F. nucleatum were added to macrophages at a final concentration of 1 µg/mL in the presence or absence of hemoglobin (10, 50, 100 µg/mL). After 24 hrs, the quantity of 3H-LPS bound to macrophages was determined with the use of a multi-purpose scintillation counter. A value of 100% was assigned to the amount of LPS bound in the absence of hemoglobin. *p < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Periodontal tissue destruction is accompanied by gingival vascular disruption and bleeding. Several studies have supported a close relationship between bleeding on probing and inflammatory reactions of the gingiva (Abrams et al., 1984; Thilo et al., 1986), as well as between hemoglobin concentrations in gingival tissues and gingival inflammation (Hanioka et al., 1989, 1990, 1991). We investigated here the effect of hemoglobin on periodontopathogen LPS-induced cytokine secretion by macrophages.

Few groups have reported the capacity of hemoglobin to stimulate the production of pro-inflammatory cytokines (McFaul et al., 2000; Carrillo et al., 2002). Our study confirmed this property of hemoglobin and showed, for the first time, the induction of IL-1ß and RANTES production by macrophages. This suggests that the increased hemoglobin concentration observed in inflamed gingiva (Hanioka et al., 1990) may participate in the inflammatory process by stimulating the secretion of pro-inflammatory cytokines by leukocytes.

Hemoglobin increased periodontopathogen LPS immunostimulatory activity. Indeed, the secretion of pro-inflammatory cytokines (IL-1ß, TNF-{alpha}, and IL-6) and a chemokine (IL-8) by macrophages following stimulation with P. gingivalis, F. nucleatum, and A. actinomycetemcomitans LPS was strongly enhanced in the presence of hemoglobin. This suggests that this synergistic effect is not specific to one type of periodontopathogen LPS. In addition, the fact that no significant effect was observed with P. gingivalis whole cells (ATCC 33277 and gingipain null mutant of ATCC 33277) suggests that the synergistic effect occurs only with detached LPS. This can be related to the availability of lipid A in detached LPS, a moiety anchored into the membranes of bacterial cells in attached LPS. Indeed, hemoglobin can induce conformational changes in the lipid A moiety of LPS, a phenomenon associated with an increase in LPS-induced cytokine induction (Jurgens et al., 2001). The synergistic effect between hemoglobin and LPS, leading to enhanced production of inflammatory mediators associated with periodontitis progression, may play an important role in the development of this disease. On the one hand, gingival crevicular fluid levels of endotoxins have been correlated with gingival inflammation (Simon et al., 1971) and are higher in periodontitis sites (Fine et al., 1992). In contrast, the concentration of hemoglobin has been reported to increase in human gingiva with increasing inflammation (Hanioka et al., 1990). This suggests that increasing levels of these factors in combination may play a role in the continuous, high expression of inflammatory mediators by host cells in inflamed gingiva, thus contributing to the progression of periodontitis.

Hemoglobin increased the toxicity or lethality of LPS in animal models, an effect attributed in part to the capacity of hemoglobin to increase the TNF-{alpha} response (Bloom et al., 1998; Su et al., 1999). Other factors, such as the migration inhibitory factor and glutathione, have been reported to enhance the TNF-{alpha} induction by a gamma globin chain of fetal hemoglobin and LPS in mouse and human cells (Gorczynski et al., 2006). We also observed that the TNF-{alpha} response of macrophages to periodontopathogen LPS was strongly enhanced by hemoglobin. Yang et al.(2002) reported that LPS binding to globin (hemoglobin minus heme) antagonized the action of LPS on cultured macrophages and in mice. They speculated that the heme moiety of hemoglobin is likely involved in the effects of hemoglobin-LPS interactions. In our study, we found that P. gingivalis and F. nucleatum LPS bound to hemoglobin, although it also attached to gelatin and BSA. The binding of hemoglobin to Escherichia coli and Proteus mirabilis LPS increases the biological activity of LPS by promoting the disaggregation of LPS complexes (Kaca et al., 1994), which can result in better accessibility of the binding and recognition groups of LPS to target structures such as serum and membrane proteins. Another study reported that hemoglobin induced conformational changes in the lipid A moiety of LPS, which resulted in an increase in LPS-induced cytokine induction (Jurgens et al., 2001). In addition, it has been suggested that the intercalation of a hemoglobin-LPS complex in the cell membrane may act as a cell activator at the sites of signaling proteins, such as Toll-like receptors (Jurgens et al., 2001; Brandenburg et al., 2003). It is likely that many of these proposed mechanisms contribute to the synergistic effect between hemoglobin and LPS on the inflammatory response, but further studies are needed to clarify this issue.

Hemoglobin also enhances LPS binding to human endothelial cells (Roth, 1996). In the present study, we showed that hemoglobin enhanced the binding of periodontopathogen LPS to macrophages. This capacity of hemoglobin is likely involved in the enhanced cytokine response to LPS observed in the presence of hemoglobin. Gorbenko (1999) reported that hemoglobin penetrates the phospholipid layer of model membranes. An intercalation of hemoglobin-LPS complexes in phospholipid liposomes corresponding to the composition of human macrophages has also been reported (Brandenburg et al., 2003). This may also lead to the increased binding of LPS to macrophages observed in the presence of hemoglobin. Since hemoglobin does not exhibit specific binding properties to periodontopathogen LPS, other phenomena, such as the disaggregation of the LPS complex by hemoglobin, are likely involved in the enhanced binding of LPS to macrophages.

The average concentration of hemoglobin in plasma is between 10 and 40 µg/mL (Dacie and Lewis, 1991). The protein composition and concentration of gingival crevicular fluid bathing the periodontal pocket are derived from gingival capillary beds, and are similar to those of serum (Cimasoni, 1983). The vascular disruption and bleeding that occur during periodontitis likely increase the levels of free hemoglobin in gingival crevicular fluid. In this study, we observed a synergistic effect, in a limited number of conditions, with a hemoglobin concentration of 10 µg/mL, whereas we observed the effect in the majority of the conditions tested with a higher hemoglobin concentration (50 and 100 µg/mL). This provides support for the hypothesis that a synergistic effect between LPS and hemoglobin occurs, most especially in pathological conditions such as inflamed periodontal tissues, where there are higher hemoglobin concentrations. In addition, P. gingivalis and F. nucleatum possess both hemagglutinating and hemolytic activities, conferring on them the ability to agglutinate and lyse red blood cells (Gaetti-Jardim Junior and Avila-Campos, 1999; Olczak et al., 2005). This suggests that periodontopathogens may contribute to enhancing the release of hemoglobin into gingival crevicular fluid and periodontal tissue during the frequent bleeding stages characterizing periodontitis. Higher levels of hemoglobin, which is a source of iron for periodontopathogens, may thus contribute to promoting bacterial growth and the subsequent release of LPS, as well as to increasing the host inflammatory response.

Our study provides clear evidence that hemoglobin contributes to enhancing the levels of pro-inflammatory mediators in periodontal sites by acting in synergy with LPS from periodontopathogens, a phenomenon that may contribute significantly to the progression and severity of periodontitis.


   ACKNOWLEDGMENTS
 
This study was supported by the Canadian Institutes of Health Research. A preliminary report was presented at the 35th Annual Meeting of the AADR in Orlando (2006).


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received November 30, 2006; Last revision April 25, 2007; Accepted May 8, 2007


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Brandenburg K, Garidel P, Andra J, Jurgens G, Muller M, Blume A, et al. (2003). Cross-linked hemoglobin converts endotoxically inactive pentaacyl endotoxins into a physiologically active conformation. J Biol Chem 278:47660–47669.[Abstract/Free Full Text]

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