The current prevalence of AgP in children and young adults in the US is around 1 to 2% and is estimated to be up to 3 times more prevalent in African-Americans (Albandar and Tinoco, 2002
). However, early stages of this disease can go undiagnosed until clear signs of bone destruction are present. If early stages of disease are detected in a young population, treatment can be applied in a more appropriate timeframe, potentially preventing the rapid progression of AgP. More importantly, an understanding of AgP disease mechanisms will also allow for a more appropriate treatment regimen for this disease.
Detection and identification of mediators in the gingival crevicular fluid (GCF) of both healthy and diseased periodontal sites could be important adjuncts to early disease diagnosis as well as provide insight into disease mechanisms. In the present study, we demonstrated elevated levels of at least 9 inflammatory mediators in the GCF of LAP participants’ diseased sites compared with their healthy sites, as well as with healthy sites in healthy participants. These results are in agreement with those of other studies reporting higher GCF levels of IL-1β and GM-CSF in the GCF of individuals with generalized aggressive periodontitis (GAP) when compared with that of periodontally healthy participants (Toker et al., 2008
; Teles et al., 2010
). In particular, IL1β increases monocyte cytotoxicity as well as the adhesion of neutrophils, monocytes, T-cells, and B-cells at the site of infection. IL1β also assists the humoral and adaptive immune response by stimulating B-cell proliferation and T-cell production of IL2, which was also found to be elevated in LAP diseased sites. Although IL10 is often considered an anti-inflammatory cytokine, elevated levels of IL10 in diseased GCF are also responsible for differentiation of cytotoxic T-cells and act as a CD8+ T-cell chemoattractant, promoting an adaptive response capable of local tissue destruction observed in LAP. Collectively, these inflammatory mediators represent a pro-inflammatory response in the local tissues which has the potential to serve as a marker for LAP disease progression. Our methodology for GCF collection differed from those of previous studies in the time of collection. We used 10 sec of collection time (instead of 30 sec as reported by some studies) to avoid saturating the strip, especially in diseased sites. Analysis of our preliminary data has shown that 10 sec would give us enough protein content to be detected by the Luminex analysis without need for dilutions (data not shown).
This study also reports lower levels of 3 inflammatory mediators [MCP1, IL4, and IL8] in the GCF of LAP participants’ diseased sites when compared with healthy sites. Interestingly, MCP1 and IL8 also showed negative correlations with plasma levels of LPS. While MCP1 is responsible for monocyte chemotaxis and has been proposed to have a role in osteoclast differentiation, its expression is suppressed by GMCSF (Kim et al., 2005
). Therefore, the elevated levels of GMCSF in diseased sites found in the present study could explain the reciprocal lower levels of MCP1 in these sites. Similar to findings by Ozmeric et al. (1998)
, the present study showed IL8 levels to be no different or slightly lower in individuals with LAP than in healthy control individuals. IL8 is mainly responsible for neutrophil chemotaxis as well as inducing neutrophil degranulation. Therefore, it is usually expressed at early stages of insult and possibly not during the chronic inflammatory stage seen upon clinical presentation of LAP. IL8 also inhibits the adhesion of leukocytes to activated endothelial cells and therefore possesses some anti-inflammatory activities. Similarly, IL8 plays an important role in the process of wound healing which seems to be absent or dysfunctional in the case of LAP. While Ozmeric et al. (1998)
suggested that less active IL-8 production in spite of a dense bacterial stimulation in LAP could indicate impaired protection against periodontal infections, it is also possible that the stage of disease during which evaluation was performed and/or wound-healing defects occurred explains the lower levels observed.
Interestingly, GCF from a healthy site of one of the siblings who was initially diagnosed as periodontally healthy presented with mediator levels similar to those of diseased LAP sites. This specific site presented disease breakdown at a later time-point (data not shown). Some of the mediators associated with LAP diseased sites (TNFα, GMCSF, IL2, IL12p40, IL1β, IFNγ, IL6, and IL10) as well as some of those associated with healthy sites (IL8 and MCP1) were elevated. This may suggest that different soluble mediators are associated with different stages of periodontal disease and thus may have the potential to be used as adjuncts to clinical measurements to allow for possible early diagnosis. Longitudinal follow-up of our healthy siblings and unrelated control individuals, as well as healthy sites of LAP participants, will enable us to evaluate the role of GCF mediators in disease initiation and other stages of disease progression.
Periodontal disease has been previously correlated positively with plasma LPS, C-reactive protein concentrations, as well as macrophage cytokine production (Pussinen et al., 2004
). Although the disease in the present study is present in a younger population and is localized to specific oral sites, elevated systemic levels of endotoxin were observed in LAP, and those levels were strongly correlated with clinical parameters and most elevated cyto/chemokines. Overgrowth of Gram-negative bacteria and periodontal pocket ulceration and inflammation may result in increased release of lipopolysaccharide (LPS) to circulation, which in turn activates host immune cells in the production of inflammatory markers, perpetuating an inflammatory cycle.
Some studies have reported AgP to be associated with polymorphonuclear cell (PMN) abnormalities (Van Dyke et al., 1982
; Genco et al., 1986
) or depressed neutrophil chemotaxis (Van Dyke et al., 1985
). Other authors have reported elevated presence of pro-inflammatory cytokines, such as TNFα and IL1, in the plasma of individuals with AgP (Shapira et al., 1994
), where this elevation is the biological basis for altered neutrophil function. We have reported a systemic hyper-inflammatory response, an elevated release of inflammatory mediators in response to TLR2 and TLR4 stimulation, in our LAP cohort, as well as an attenuated systemic inflammatory response of the healthy siblings (Shaddox et al., 2010
). Similarly, we reported a localized high prevalence of MMPs (Alfant et al., 2008
) in the same cohort. The present study reports a pro-inflammatory cytokine milieu also in the GCF, which correlates with elevated plasma LPS levels and LAP defining clinical parameters. Analysis of these data, taken together, provides a model in which a localized inflammatory response can lead to local tissue destruction, resulting in systemic release of immune activating agents such as LPS. In populations prone to immune hyper-responsiveness, this systemic release may lead to a perpetuating inflammatory cycle where local inflammation recruits systemically activated immune cells, leading to excessive and rapid local tissue destruction.
Possible limitations of the present study were: (1) collection of 1 GCF strip from a diseased and a healthy site, which means that there could be variations in the degree of inflammation and disease activity in different oral sites, so it would be interesting to evaluate inflammatory patterns in different sites in future studies; and (2) the limited quantity of LAP in primary dentition, which means that as we add more primary dentition disease to this cohort, we will be able to evaluate possible differences in inflammatory patterns and systemic response in primary vs. permanent dentition LAP.