Levels of ll-37 antimicrobial peptide in the gingival crevicular fluid of young and middle-aged subjects with or without gingivitis.

PURPOSE: LL-37 is an antimicrobial peptide which plays an important role in the innate immunity. The aim of this study was to investigate the LL-37 levels in the gingival crevicular fluid (GCF) of middle-aged and young adults who have either gingivitis or healthy periodontal tissues.
MATERIALS AND METHODS: Forty middle-aged adults (20 healthy controls and 20 with gingivitis) and 41 younger adults (20 healthy controls and 21 with gingivitis) were included in the present study. Probing depth, clinical attachment level, plaque index, and papilla bleeding index were recorded. LL-37 levels in the GCF were analyzed by enzyme-linked immunosorbent assay.
RESULTS: No significant differences were observed in the GCF LL-37 levels between young healthy and middle-aged healthy subjects. Also, there were no significant differences in GCF LL-37 levels between young and middle-aged gingivitis subjects. However, gingivitis groups had significantly higher GCF LL-37 levels than healthy groups (p<0.001). Correlation analysis demonstrated no significant correlation between age and GCF LL-37 levels neither in healthy nor in gingivitis groups.
CONCLUSION: The levels of LL-37 in GCF increase in the presence of gingival inflammation, however, this does not vary according to subjects being young or middle-aged.

Introduction

The reversible inflammation of gingival tissues, gingivitis, is the most common form of periodontal diseases which is caused by the accumulation of dental plaque adjacent to gingival margin (1, 2). 54% of the United States population has been reported to have at least one site with gingivitis (3, 4). Plaque accumulation results in acute inflammatory response dominated by neutrophils. Consequently, collagen homoeostasis changes, and pro-inflammatory cytokines are expressed (1). Neutrophil markers such as leukotriene B4, interleukin-8, and betaglucuronidase increase in the GCF of subjects with gingivitis (5, 6, 7). The severity of gingivitis is affected by the host inflammatory response to bacterial challenge (8, 9).

If the gingivitis is left untreated, it may progress to periodontitis in some patients (10, 11). The tissue damage caused by gingivitis is reversible. The presence of gingivitis is, on the other hand, a risk factor for the development of periodontitis (1, 12). Periodontal diseases are caused by the interactions between the microbial dental plaque and host immune response which plays an important role in the individual susceptibility for periodontitis (1, 13). The presence of dental plaque may trigger innate and adaptive responses. Antimicrobial peptides including LL-37, adrenomedullin and defensins are important contributors of the innate immune response (14).

Neutrophils and epithelial cells express LL-37 antimicrobial peptide (14, 15, 16). It is stored as an inactive precursor in the secondary granules of neutrophils (17, 18).Following neutrophil stimulation, mature LL-37 is released from neutrophils (18). LL-37 modulates the inflammatory and immune responses, accelerates the angiogenesis, promotes wound healing and re-epithelization, and neutralizes the lipopolysaccharides (14, 15, 17). It has been stated that there is an association between LL-37 levels and a number of inflammatory diseases (19, 20, 21). Although blood neutrophils do not have mature LL-37, abundant amount of hCAP18 in patients with Papillon–Lefèvre syndrome has been reported (22).

In an immunohistochemical study, Fransson et al. (23) demonstrated that there was a difference in the inflammatory response to plaque formation between young and old people. However, Tsalikis et al. (24) have shown that the age variable did not have an effect on the cytokine expression in experimental gingivitis. A positive correlation between saliva LL-37 level and age has been reported in childhood (25). Castaneda-Delgado et al. (26) have detected similar amounts of LL-37 in the serum samples of both old and young healthy participants. It has been speculated that the effects age on the immune response and infection may be more severe in older subjects compared to younger people (27). Also, older people present increased susceptibility for infectious diseases (26), which might be caused by the disregulation of immune response (27). The aim of the present study is therefore to investigate the relationship between the age and LL-37 levels in the gingival crevicular fluid (GCF) of middle-aged and young adults who have either gingivitis or healthy periodontal tissues.

Materials and Methods

Study participants

Forty-one participants with gingivitis and 40 subjects with healthy periodontal tissues were included in the present study. Sample was derived from the patients who had been recruited from Ege University, Faculty of Dentistry, Department of Periodontology between 2013 and 2015. Each patient was given detailed information about the study protocol land they signed the consent form. Ethics Committee of Ege University has reviewed and approved the protocol (# 12-2.1/4). Exclusion criteria were as follows: the presence of aggressive or chronic periodontitis, subjects with systemic or infectious diseases, pregnancy, lactation, having periodontal treatment within the past one year, subjects taking oral contraceptive drugs and/or those who have taken antibiotics in the last 3 months, and subjects with a history of smoking. Inclusion criteria were as follows: being a non-smoker who has 16 teeth at least. Subjects, who have healthy periodontal tissues or gingivitis, aged 18 to 30 years, were included in the young adult groups. Subjects aged 40 to 60 years, who have healthy periodontal tissues or gingivitis, were included in middle-aged adult groups. Subjects aged 31 to 39 years were not included in the study. All healthy subjects were free of systemic or periodontal diseases. Healthy patients had no sites with clinical attachment level (CAL)>2 mm or probing depth (PD)>3 mm. Furthermore, they had bleeding on probing (BOP) less than 15% of the sites and no alveolar bone loss radiographically. Subjects with gingivitis had no clinical signs of periodontitis and no radiographic evidence of alveolar bone loss. Patients who had BOP at more than 50% of the examined periodontal areas were diagnosed as having gingivitis.

Twenty middle-aged adults (aged 40 - 59 years) (12 female, 8 male) were included in the healthy middle-aged adult group (MA-Healthy). The mean age of this group was 46.5 ± 5.4 years. Twenty subjects (aged 20-30 years) (13 female, 7 male) were included in the healthy young adult group (Y-Healthy). The mean age of this group was 27 ± 3 years. Twenty subjects (aged 41 - 57 years) (11 female, 9 male) were included in middle-aged adult group with gingivitis (MA-Gingivitis). The mean age of this group was 47.1 ± 5.1 years. Twenty-one subjects (aged 24 - 30 years) (13 female, 8 male) were included in young adult group with gingivitis (Y-Gingivitis). The mean age of this group was 28 ± 4.7 years.

Clinical examination

PD and CAL measurements were performed at six sites per tooth excluding third molars. PD and CAL were measured by William’s periodontal probe (Hu-Friedy, Chicago, IL, USA) in millimeter. Plaque index (PI) (28), BOP (29) and papilla bleeding index (PBI) (30) scores were also determined. Same clinician (O.T) performed all clinical examinations. The intra-examiner reliability was considered to be high since the intra-class correlation coefficient (ICC) was 0.87 and 0.85 for PD and CAL measurements,respectively.

Sample collection and analysis of LL-37 levels

GCF sampling was performed from vestibule sides of the interproximal areas of anterior teeth. In gingivitis patients, sample sites were selected from the teeth with BOP. In healthy group, sample sites were selected from the teeth having PD less than 3 mm without BOP. Samples were collected the day after the periodontal diagnosis. Supragingival plaque was moved away from the sampling sites. Area was isolated and dried, the sample was then obtained using a filter paper (Periopaper, ProFlow, Inc., Amityville, NY, USA) (31). Periotron 8000 device (Oraflow Inc., Helwlett, NY, USA) was used to determine the GCF volume in each strip. Enzyme-Linked ImmunoSorbent Assay (ELISA) kit was used to determine LL-37 levels (Hbt Human LL-37, Hycult Biotechnology,Uden, Netherlands). All procedures were performed according to the manufacturer’s recommendations. The minimum detectable limit of LL-37 was 0.14 ng/ml.

Statistical analysis

Statistical Package for Social Sciences (SPSS) software (SPSS Inc., ver. 17.0, Chicago, IL, USA) was used in this study. Post hoc power was 0.86 for LL-37 total amount. Chi square test was used for gender comparisons among the study groups. Periodontal variables calculated from the samples taken from whole-mouth and sampling sites were compared either with parametric or non-parametric tests. Mann-Whitney U test was used to compare GCF LL-37 levels between the study groups. Pearson correlation coefficient was used to analyze the relationship between the age and LL-37 levels in GCF.

Results

The distribution of the male and female participants was similar in the study groups.

Periodontal variables

Whole mouth PD, CAL, BOP, PBI, and PI scores in Y-Healthy group were similar to those found in MA-Healthy group. No significant differences were observed in whole mouth PD, CAL, BOP, PBI, and PI scores between Y-Gingivitis and MA-Gingivitis groups. Both gingivitis groups had significantly higher PI, BOP, and PBI scores of whole mouth compared to their healthy counterparts (p<0.001) (Table 1).

Table 1.

Mean and standard deviations (SD) of gingival index (GI), plaque index (PI) and the DMFS (Decayed, Missing, Filled, Surfaces) scores stratified by previous visits to the dentist, having dental fear, receiving oral health care information previously, using dental floss and mouthrinse, and having dental scaling previously.* indicates p<0.05 in Student t Test.

Clinical variables	Y-Healthy	Y-Gingivitis	MA-Healthy	MA-Gingivitis
	(n=20)	(n=21)	(n=20)	(n=20)
PD	2.1±0.5	2.5±0.5	2.1±0.4	2.4±0.5
CAL	0.13±0.1	0.12±0.1	0.14±0.1	0.11±0.1
PI	1.6 (0.6-2.8)	3.2(2.3-4.5)*	1.5 (0.9-3)	3 (2.2-4.5)*
BOP	11±5	78±12*	13±3	80±14*
PBI	0.1 (0.1-0.1)	2.1 (1-3.1)*	0.1 (0.1-0.2)	2 (1- 3)*

Y-Healthy and MA-Healthy groups had similar PD, PI scores and GCF volumes of sampling sites. PD, PI, PBI, and GCF volumes of sampling sites were found to be similar in Y-Gingivitis and MA-Gingivitis groups. There were significant differences in PD, PI, PBI values, and GCF volumes of sampling sites between Y-Gingivitis and Y-Healthy groups (p<0.001). Similarly, significant differences were observed in PD, PI, PBI values, and GCF volumes of sampling sites between MA-Healthy and MA-Gingivitis groups (p<0.001) (Table 2).

Table 2.

Periodontal parameters of the sampling sites in the study groups (PD: probing depth, CAL: clinical attachment level, PI: plaque index, PBI: papilla bleeding index, GCF:gingival crevicular fluid, Y: young, MA: middle aged, *indicates significant difference from healthy counterpart).

Clinical variables	Y-Healthy	Y-Gingivitis	MA-Healthy	MA-Gingivitis
	(n=20)	(n=21)	(n=20)	(n=20)
PD	2 (1-2)	3 (2-4)*	2 (1-2)	3 (2-5)*
CAL	-	-	-	-
PI	2 (1-3)	3 (2-4)*	2 (1-3)	3 (2-5)*
PBI	-	2 (1-4)*	-	2 (1-4)*
GCF volume (µl)	0.2±0.1	0.38±0.14*	0.2±0.1	0.42±0.12*

LL-37 levels in gingival crevicular fluid

Table 3 presents total amount and concentration LL-37 in GCF. No significant differences were observed in the total amount and concentration of LL-37 between Y-Healthy and MA-Healthy subjects. Also, there were no significant differences in total amount and concentration of LL-37 between Y-Gingivitis and MA-Gingivitis subjects. MA-Gingivitis group had significantly higher total amount of GCF LL-37 compared to that of MA-Healthy group (p<0.001). Similarly, Y-Gingivitis group had significantly higher GCF LL-37 total amount than Y-Healthy group (p<0.001). A significant difference was observed in GCF LL-37 concentration between Y-Healthy and Y-Gingivitis groups (p<0.05), while there was no significant difference in the LL-37 concentration between MA-Healthy and MA-Gingivitis groups. Correlation analysis demonstrated no significant correlation between age and GCF LL-37 levelsin healthy and gingivitis groups.

Table 3.

Means and ranges of the LL-37 amount and concentration values in the gingival crevicular fluid (GCF) of the study groups (Y: young, MA: middle aged, * indicates significant difference from healthy counterpart).

GCF LL-37 level	Y-Healthy	Y-Gingivitis	MA-Healthy	MA-Gingivitis
	(n=20)	(n=21)	(n=20)	(n=20)
Total amount (ng/site)	0.21 (0.01-1.63)	0.93 (0.2-25) *	0.23(0.01-1.42)	0.97 (0.1-2.4)*
Concentration (ng/µl)	1.37 (0.01-8.35)	2.78 (0.43-8.21)*	2.35 (0.01-8.35)	3.13 (0.44-10.83)

Discussion

There are several studies investigating LL-37 antimicrobial peptide in periodontal diseases. However, to the best of out knowledge, there is no study evaluating the possible difference between age groups in the presence or absence of gingival inflammation. Findings of the present study showed no difference in LL-37 in GCF levels in samples taken from healthy or inflammatory gingiva. Furthermore, no significant correlation was observed between age and GCF LL-37 levels. Nevertheless, our results also suggest that the inflammation of the gingiva has an increasing effect on the GCF LL-37 levels, which is independent from the participants’ age.

It has been suggested that the inflammatory response to plaque formation in young and old people might be different (23, 27). However, Tsalikis et al. (24) showed that age did not affect cytokine expression in experimental gingivitis. Similarly, Castaneda-Delgado et al. (26) have found similar amounts of LL-37 in the serums obtained from healthy elder and healthy young subjects. Davidopoulou et al. (25) have investigated the relation of salivary concentration of LL-37 levels with age in young children, and they have found a positive correlation between LL-37 concentration and age. In the present study, young and middle-aged participants had similar amount of GCF LL-37 not only in the healthy group, but also in the gingivitis group. Therefore, our data suggest that being a young or middle-aged participant as defined in this study does not affect the levels of GCF LL-37 levels in healthy, as well as inflammatory conditions of gingiva. However, in the present study, we hypothesized that age might be a factor affecting GCF LL-37 levels because of the disregulation of immune response in older people. Davidopoulou et al. (25) stated that there was a positive correlation between salivary LL-37 concentration and age. These conflicting results might be explained by the age difference of the samples. Davidopoulou et al. (25)’s sample included children aged from 2 to 18 years old which is much younger than the mean age of our study population. In addition, Davidopoulou et al. (25) investigated LL-37 levels in saliva but not in GCF.

Although there are several studies investigating the effect of periodontitis on LL-37 levels in GCF and saliva, a very limited number of articles have evaluated LL-37 levels in the presence of only gingival inflammation. Our findings demonstrated that gingival inflammation has an effect on GCF LL-37 levels regardless of age. Both young and middle aged subjects with gingivitis had significantly higher levels of GCF LL-37 compared to their healthy counterparts. This result is in accordance with those of Hosokawa et al. (32) in which the authors showed that neutrophil LL-37 expression was more prominent in inflammatory lesions than healthy gingiva by immunohistochemistry.

In the present study, there were two age groups which were categorized as 18 to 30 and 40 to 60 years. The main limitation of this design is to exclude the subjects younger than eighteen and older than sixty. Therefore, studies including younger, as well as older, subjects than those of the present study are needed to reveal the true effect of age on GCF LL-37. Additionally, further studies investigating the effect of age on GCF LL-37 levels in the presence of periodontitis would provide more information about this issue.

Conclusion

To the best of our knowledge, this is the first study evaluating the effect of age on GCF LL-37 levels during the gingival inflammation or in the healthy state of the gingiva. Our findings indicate that the presence of gingival inflammation increased the LL-37 levels in GCF; however, this does not vary according to subjects being young or middle-aged.