Site-Specific Neutrophil Migration and CXCL2 Expression in Periodontal Tissue

Abstract

The oral microbial community is the best-characterized bacterial ecosystem in the human host. It has been shown in the mouse that oral commensal bacteria significantly contribute to clinically healthy periodontal homeostasis by influencing the number of neutrophils that migrate from the vasculature to the junctional epithelium. Furthermore, in clinically healthy tissue, the neutrophil response to oral commensal bacteria is associated with the select expression of the neutrophil chemokine CXCL2 but not CXCL1. This preliminary study examined the contribution of commensal bacteria on neutrophil location across the tooth/gingival interface. Tissue sections from the root associated mesial (anterior) of the second molar to the root associated distal (posterior) of the second molar were examined for neutrophils and the expression of the neutrophil chemokine ligands CXCL1 and CXCL2. It was found that both the number of neutrophils as well as the expression of CXCL2 but not CXCL1 was significantly increased in tissue sections close to the interdental region, consistent with the notion of select tissue expression patterns for neutrophil chemokine expression and subsequent neutrophil location. Furthermore, mice gavaged with either oral Streptococcus or Lactobacillus sp. bacteria induced a location pattern of neutrophils and CXCL2 expression similar to the normal oral flora. These data indicate for the first time select neutrophil location and chemokine expression patterns associated with clinically healthy tissue. The results reveal an increased inflammatory load upon approaching the interproximal region, which is consistent with the observation that the interproximal region often reveals early clinical signs of periodontal disease.

Keywords: bacteria; cell signaling; chemokines; epithelia; gingiva; histochemistry.

Figure 1.

Diagram of the sectioning and sample selection (not to scale), as well as an example of the area analyzed for data collection. JE, junctional epithelium; OE, oral epithelium.

Figure 2.

Examples of immunohistochemistry staining. (A) Junctional epithelium (JE) of a negative control (no secondary antibody) specific pathogen-free (SPF) mouse also represents a stain intensity grade of 0. (B) JE of a neutrophil-stained SPF mouse. (C) JE of a neutrophil-stained germ-free mouse. (D) JE (mesial, anterior) of a black 6 SPF mouse stained with CXCL2, with a stain intensity of 2. (E) JE (midtooth) of a black 6 SPF mouse stained with CXCL2, stain intensity of 1. (F) JE (distal, posterior) of a black 6 SPF mouse stained with CXCL2, with a stain intensity of 2. Magnification, ×20.

Figure 3.

Examination of neutrophil migration across the tooth. (A) The average number of neutrophils, with 95% confidence intervals, for each slide of germ-free mice (germ-free), specific pathogen-free mice, germ-free mice gavaged with Lactobacillus sp., and germ-free mice gavaged with Streptococcus sp. (1–12), from the root associated mesial to the root associated distal of the second molar. (B) Quadratic trend analysis of neutrophil per mouse. (C) Mean neutrophil count per mouse. All groups were statistically significantly different from each other. Groups separated by shaded areas are statistically different at P = 0.05. 95% CI, 95% confidence interval; GF, germ-free.

Figure 4.

Examination of CXCL2 expression levels across the tooth. (A) CXCL2 stain intensity (0 indicates no stain and 3 indicates heavy stain) across the tooth with 95% confidence intervals for each slide of germ-free mice, specific pathogen-free mice, germ-free mice gavaged with Lactobacillus sp., and germ-free mice gavaged with Streptococcus sp. (1–12) from the root associated mesial to the root associated distal of the second molar. (B) Quadratic trend analysis of CXCL2 stain intensity per mouse. (C) Mean CXCL2 stain intensity averaged per mouse (all were statistically significantly different from each other). Groups separated by shaded areas are statistically different at P = 0.05). 95% CI, 95% confidence interval; GF, germ-free.

Figure 5.

Examination of CXCL1 expression levels across the tooth. (A) CXCL1 stain intensity (0 indicates no stain and 3 indicates heavy stain) across the tooth with 95% confidence intervals for each slide of germ-free mice (germ-free), specific pathogen-free mice, germ-free mice gavaged with Lactobacillus sp., and germ-free mice gavaged with Streptococcus sp. (1–12) from the root associated mesial to the root associated distal of the second molar. No pattern difference is observed. (B) Quadratic trend analysis of CXCL1 stain intensity per mouse shows no signficant difference between the groups of mice. (C) Mean CXCL1 stain intensity averaged per mouse shows no significant differnce between the groups of mice. 95% CI, 95% confidence interval; GF, germ-free.