Characterization of Inflammatory Cell Infiltrate in Human Dental Pulpitis

doi:10.1111/j.1365-2591.2010.01757.x

Characterization of inﬂammatory cell inﬁltrate in human dental pulpitis

K. F. Bruno1, J. A. Silva1, T. A. Silva2, A. C. Batista3, A. H. G. Alencar1 & C. Estrela1 1Department of Endodontics, Federal University of Goia´s, Goiaˆnia, GO; 2Department of Oral Surgery and Pathology, Dental School, Federal University of Minas Gerais, Belo Horizonte; and 3Department of Stomatology (Oral Pathology), Dental School, Federal University of Goia´s, Goiaˆnia, Brazil

Abstract Results Two microscopic patterns of pulpitis were found: group 1 (G1) (n = 15) had an intense Bruno KF, Silva JA, Silva TA, Batista AC, Alencar AHG, inflammatory infiltrate and mild collagen deposition; Estrela C. Characterization of inflammatory cell infiltrate in conversely, group 2 (G2) (n = 23) had a scarce human dental pulpitis. International Endodontic Journal, 43, inflammatory infiltrate and intense collagen deposition. 1013–1021, 2010. The numbers of CD68+ macrophages (P = 0.004) and Introduction To evaluate the microscopic charac- CD20+ B(P = 0.068) lymphocytes and the density of teristics and densities (per mm2) of tryptase+ mast cells, blood vessels (P = 0.002) were higher in G1 than in CD4+ T helper lymphocytes, CD45RO+ memory T G2. However, a similar number of CD4+ and CD45RO+ lymphocytes, foxp3+ T regulatory lymphocytes, CD20+ T lymphocytes was found in both groups (P > 0.05). B lymphocytes, CD68+ macrophages, and CD31+ blood When present, tryptase+ mast cells were equally vessels in human dental pulpitis (n = 38) and healthy distributed in G1 and G2, whereas foxp3+ T regulatory pulpal tissue (n = 6). lymphocytes were detected in 59% and 14% of the Methodology The pulps of 38 human teeth with a samples of G1 and G2. Controls exhibited lower clinical diagnosis of irreversible pulpitis were removed by numbers of foxp3, tryptase, CD4, CD45RO, CD68 and pulpectomy. The pulp tissue was immersed in 10% CD20 positive cells than G1 and G2. buffered formalin for evaluation using light microscopy. Conclusions Irreversible pulpitis had distinct micro- Tryptase, CD4, CD45RO, foxp3, CD20, CD68, and CD31 scopic features with important quantitative and qual- expressions were analysed using immunohistochemis- itative differences in inflammatory cell infiltration. try; other microscopic features, such as intensity of Keywords: caries, dental pulp, dental pulpitis, inflammatory infiltrate and collagen deposition, were immunological cells, inflammatory cells. evaluated using haematoxylin and eosin stain. Wilcoxon and Mann–Whitney tests were used for statistical Received 10 December 2009; accepted 16 April 2010 analysis. The significance level was set at a = 5%.

1981, Izumi et al. 1995, Okiji et al. 1997, Nanci 2003, Introduction Costa et al. 2009). Bacteria found in dental caries are the most frequent The inflammatory response consists of non-specific aetiological agents of injury to the dental pulp. Inflam- and immediate defense mechanisms, which involve matory and immunologic reactions occur in response vascular-exudative phenomena, such as vasodilatation to microorganisms or their products, which penetrate and increased permeability, as well as infiltration of into the pulp through the dentinal tubules (Bergenholtz inflammatory cells, such as mast cells, neutrophils, and macrophages (Bergenholtz 1990, Izumi et al. 1995, Avery 2002, Abbas & Lichtman 2003). Whilst Correspondence: Professor Carlos Estrela, Centro de Ensino e these cells play an important role in pulp defense, Pesquisa Odontolo´gica do Brasil (CEPOBRAS), Rua C-245, Quadra 546, Lote 9, Jardim Ame´rica, Goiaˆnia, GO, CEP: they also participate in the degradation of the extra- 74.290–200, Brazil (e-mail: [email protected]). cellular matrix by releasing matrix metalloproteinases

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(Tja¨derhane et al. 2001, Gusman et al. 2002, Wahl- cytotoxic T lymphocyte antigen-4 (CTLA-4), and the gren et al. 2002). forkhead transcription factor (Foxp3) (Akbar et al. Mast cells release several biologically active sub- 2003, Chen et al. 2003, Zheng & Rudensky 2007). Of stances responsible for the modulation of inflammatory these, foxp3 expression may be the most specific and immunologic responses, and angiogenesis (Holzer marker of Treg cells. (Chen et al. 2003, Zheng & 1988, Jontell et al. 1998, Rodini et al. 2004, Freitas Rudensky 2007). et al. 2007). The latter is extremely important in Despite the high frequency of dental pulpitis in inflammation and repair processes, in which endothe- endodontic clinics, few studies have described its lial membrane protein CD31 represents a reliable inflammatory and immunologic profiles (Bergenholtz vascular marker (Woodfin et al. 2007). Macrophages 1981, Izumi et al. 1995, Jontell et al. 1998). Therefore, are mononuclear cells responsible for phagocytosis, new studies should investigate the pathogenesis of antigen presentation, and immunomodulation (Jontell pulpal disease so that more efficient therapies may be et al. 1998, Abbas & Lichtman 2003, Hahn & Liewehr developed. 2007a); they also contribute to pulp repair by inducing To understand the pathogenesis and microscopic fibroblast proliferation (Brody et al. 1992) and neovas- characteristics of human dental pulpitis better, this cularization (Polverini 1995). study investigated the presence of several immune The immune response is a more specific defense inflammatory cells (mast cells, T helper lymphocytes, mechanism that becomes active after the inflammatory memory T lymphocytes, regulatory T lymphocytes, B response. Immune reactions are characterized by anti- lymphocytes, and macrophages). genic processing, presentation, and recognition (Avery 2002, Abbas & Lichtman 2003). In the dental pulp, Materials and methods antigen-presenting cells (APCs), which express major histocompatibility complex class II (MHC class II) Dental pulp samples molecules, capture antigenic proteins and present peptide fragments to antigen-specific helper T lympho- This study was approved by the ethics committee of the cytes in lymphonodes. Activated T helper lymphocytes Federal University of Goia´s, Brazil, and informed (CD4+ T helper lymphocytes) rapidly multiply, create consent was obtained from all patients. many clones, differentiate into memory T cells Thirty-eight human teeth were randomly selected (CD45RO+ memory T lymphocytes) (Jontell et al. (19 maxillary incisors and 19 maxillary molars, whose 1998), and, through blood or lymphatic streams pulps were removed from their palatal roots) with a (Pimenta et al. 2003), migrate to the pulp tissue, clinical diagnosis of inflamed pulp from patients (mean where native APCs start phagocytosis and present the age, 29.8 ± 13; 20 men) treated at the Dental antigens directly to these T cells. Once activated, CD4+ Emergency Service of the School of Dentistry, Federal T helper lymphocytes trigger an effective immune University of Goia´s, Brazil. These patients had no response to any new entrance of the aggressor in the history of systemic diseases or had consumed any pulp. According to the cytokine production profile, medicaments in the last 3 months. The clinical diag- CD4+/CD45RO+ T lymphocytes may stimulate the nosis of irreversible pulpitis was based on the follow- migration and activation of macrophages, other CD4+ ing criteria: spontaneous symptomatology; positive T helper lymphocytes, and CD20+ B lymphocytes response to pulp sensibility test (PST; tetrafluoroethane (Jontell et al. 1998, Abbas & Lichtman 2003), which spray); absence of pulp exposure associated with dental produce antibodies against specific antigens (Abbas & caries. The teeth were not associated with periodontal Lichtman 2003). or periapical diseases. Subsets of T lymphocytes, known as T regulatory After the clinical diagnosis of irreversible pulpitis, the (Treg) cells, play an important role in regulating the patients were referred to a specialist for root canal immune and inflammatory responses through secretion treatment. The first step was pulpectomy, according to of anti-inflammatory cytokines such as interleukin-10 the following guidelines: anaesthesia, tooth isolation, and transforming growth factor b (TGF-b) (Abbas & antisepsis of the operative field with 1% sodium Lichtman 2003, Akbar et al. 2003, Chen et al. 2003). hypochlorite, and access cavity preparation on the These cells are characterized by the constitutive occlusal surface of the tooth using a diamond-tipped expression of a transmembrane protein, the alpha spherical bur at high speed and under water-air chain of the receptor for interleukin-2 (CD25), the cooling. The working length was set 1 mm from the

1014 International Endodontic Journal, 43, 1013–1021, 2010 ª 2010 International Endodontic Journal Bruno et al. Immunological aspects of dental pulpitis apex according to periapical radiographs. A small Collagen deposition was characterized by eosinophilic diameter K-file was introduced between the root canal area with reduced cellularity and blood vessels density wall and the pulp tissue to create space and release the or even acellular eosinophilic region lacking blood tissue, and then the tissue was carefully excised with a vessels. The collagen deposition was classified as mild, Hedstro¨m file. After pulpectomy, root canal prepara- when most fields (>7) had <35% of the graticule tion and filling were performed at the same appoint- space filled by collagen and intense, when most fields ment. (>7) had more than 35% of the graticule space filled In addition, healthy dental pulps (control) (n =6) by collagen. were collected from human permanent erupted teeth The calcification was scored as absent, mild, and (without clinical dentine caries) extracted for ortho- intense, and the necrotic areas were considered as dontic reasons. present or absent. The pulp tissues were immersed in 10% buffered formalin for further evaluation using light microscopy Immunohistochemistry in the Oral Pathology Laboratory of the Federal University of Goia´s, Brazil. Sections of 3 lm were deparaffinized and dehydrated. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide. The sections were submitted to Light microscopy antigen retrieval (Table 1). Sections were then blocked All the pulp tissue specimens were fixed in 10% by incubation with 3% normal goat serum for 20 min. buffered formalin (pH 7.4) and embedded in paraffin Mast cells, T helper lymphocytes, memory T lympho- blocks. Microscopic features, including intensity of the cytes, regulatory T lymphocytes, B lymphocytes, inflammatory infiltrate, collagen deposition, calcifica- macrophages, and blood vessels were detected using tion, and necrotic areas, were evaluated using a 5-lm anti-tryptase, anti-CD4, anti-CD45RO, anti-foxp3, anti- section of each sample stained with haematoxylin and CD20, anti-CD68, and anti-CD31 antibodies, respec- eosin (HE). tively (Table 1). The slides were then incubated with the The intensity of the associated inflammatory infil- primary antibodies for 18 h at 4 C. After washing in trate (absent, mild or intense) was determined for TBS, the sections were treated with the Novolink System each dental pulp specimen. After analysing 10 repre- (Novocastra, Newcastle, UK) or the LSAB+system, HRP sentative microscopic high-power fields (400· magni- Peroxidase Kit (Dako, Carpinteria, CA, USA) (Table 1), fication) using a graticule (Carl Zeiss, Go¨ttingen, and then incubated in 3,3¢-diaminobenzidine (DAB) Germany), specimens were classified according to (Dako) for 2–5 min. Finally, the sections were stained inflammation: a) no inflammation, when most fields with Mayer’s haematoxylin and covered. Samples of (>7) had no inflammatory cells; b) mild inflammation, tonsil tissue were used as positive controls for all when most fields (>7) had <35% of the graticule markers. Negative controls were obtained by omitting space filled by inflammatory cells; c) intense inflam- primary antibodies and using 1% PBS-BSA and non- mation, when most fields (>7) had more than 35% of immune rabbit (X0902, Dako) or mouse (X501-1, Dako) the graticule space filled by inflammatory cells. serum.

Table 1 Antibodies and protocols of immunohistochemical reactions

Antibodiesa (clone) Dilution Antigen retrieval Secondary antibody

Anti-Tryptaseb (AA1) 1 : 2000 No exposure Kit-LSABb Anti-CD4c (4B12) 1 : 100 EDTA buffer (pH = 9.0 for 30 min at 95 C) Kit-Novolinkc Anti-CD45ROc (UCHL1) 1 : 300 Citrate buffer (pH = 6.0 for 30 min at 95 C) Kit-Novolinkc Anti-foxp3d (236A/E7) 1 : 400 Citrate buffer (pH = 6.0 for 30 min at 95 C) Kit-LSABb Anti-CD20b (L26) 1 : 2000 Citrate buffer (pH = 6.0 for 45 min at 95 C) Kit-LSABb Anti-CD68c (KP1) 1 : 1000 Citrate buffer (pH = 6.0 for 45 min at 95 C) Kit-LSABb Anti-CD31c (1A10) 1 : 200 Citrate buffer (pH = 6.0 for 30 min at 95 C) Kit-Novolinkc aAll antibodies were mouse monoclonal. bDako (Carpinteria, CA, USA). cNovocastra (Newcastle, UK). dSanta Cruz Biotechnology (Santa Cruz, CA, USA).

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5–10 representative and consecutive microscopic high- Histochemistry power fields (400· magnification); at this magnifica- Mast cells density was also evaluated using toluidine tion, each field of the graticule had an area of blue (TB) (Heaney et al. 1997). Sections of 5 lm were 0.0961 mm2. The location and distribution of cells deparaffinized and hydrated with water. TB staining and blood vessels were also analysed. For all analysis, was performed with a 1% TB solution diluted in one slide with two representative sections for each phosphate buffer (pH 4–6) for 45 s. After rising in sample was evaluated. Descriptive analyses were phosphate buffer for 1 min, sections were quickly expressed as mean ± standard deviation (SD) of n dehydrated through 70%, 96% ethanol and acetone observations per mm2. The Wilcoxon test was used to p.a. to xylene and mounted in synthetic resin. compare all cell populations, and the non-parametric Mann–Whitney test for comparisons between experimental groups. A P-value of <0.05 was considered to Quantitative and qualitative analysis be statistically significant. The numbers of tryptase+ mast cells, CD4+ T helper lymphocytes, CD45RO+ memory T lymphocytes, Results foxp3+ T regulatory lymphocytes, CD20+ B lymphocytes, and CD68+ macrophages in the human dental The microscopic features of the samples analysed pulps were calculated using the graticule. All CD31+ revealed two distinct patterns of pulpitis: dental pulp blood vessels with a vascular lumen were counted, with intense inflammatory infiltrate and mild collagen even if they were very small (microvessels). All immune deposition (n = 15) (Group 1 – G1) (Fig. 1a–c), and inflammatory cells and blood vessels were counted in dental pulp with scarce inflammatory infiltrate and

(a) (b)

(e) (f) Figure 1 Dental pulp with intense inflammatory infiltrate and mild collagen deposition (a–c) and dental pulp with scarce inflammatory infiltrate and intense collagen deposition (d–f). Preserved, dilated and congested blood vessels (b and e – arrow), and calcifications (c and f – arrow). HE, Original Magnification: a,d, ·100; b,c,e,f, ·400.

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(a) (b)

Figure 2 CD31+ blood vessels (brown stain) distributed in dental pulp with intense inflammatory infiltrate and mild collagen deposition (a) and in dental pulp with scarce inflammatory infiltrate and intense collagen deposition (b). Immunohistochemical staining. Original magnification: a and b, ·400. intense collagen deposition (n = 23) (Group 2 – G2) (mean = 6.6 cells mm)2). We did not identify mast (Fig. 1d–f). No samples had necrotic areas, 90% had cells in these samples using TB staining. Similarly, areas of calcification, and all had preserved blood foxp3+ T regulatory lymphocytes (Fig. 3b) were found vessels (Figs 1 and 2). The main microscopic features of in 34% of the samples at densities ranging from 1.1– the samples are summarized in Table 2. Both pulpitis 27.1 cells mm)2 (mean = 10.49 cells mm)2). The groups: Group 1 (dental pulp with intense inflamma- tryptase+ mast cells and foxp3+ T regulatory lympho- tory infiltrate and mild collagen deposition) and Group cytes were absent in control samples. 2 (dental pulp with scarce inflammatory infiltrate and Results also showed that, in all samples, there were intense collagen deposition) had a similar mean age G1 lower densities of CD4+T helper lymphocytes (Fig. 3c) (31.2 ± 12.8 years) and G2 (28.8 ± 15.1 years). and CD45RO+ memory T lymphocytes (Fig. 3d) than of Although two distinct patterns of pulpitis were found CD68+ macrophages (Fig. 3e) and CD20+ B lympho- using microscopy, both G1 and G2 specimens had the cytes (Fig. 3f) (Wilcoxon, P < 0.05 for all compari- same clinical characteristics (spontaneous symptom- sons). atology; positive response to pulp sensibility test; The number of CD68+ macrophages per mm2 was absence of pulp exposure associated with dental caries). significantly higher in G1 than in G2 (Mann–Whitney, The immune inflammatory cells evaluated were P = 0.004) (Fig. 4). Similarly, the density of CD20+ B diffusely distributed throughout the pulp tissue in all lymphocytes was higher in G1 than in G2, although samples (n = 38). Tryptase+ mast cells (Fig. 3a) were this difference was not statistically significant (Mann– found in 24% of the samples at a low density Whitney, P = 0.068). A similar number of CD4+ and CD45RO+ T lymphocytes was found in both G1 and G2 (Mann–Whitney, P > 0.05) (Fig. 4). The control group Table 2 Microscopic characterization of human dental + pulpitis (n = 38) showed significantly lower numbers of CD68 macrophages, CD20+ B lymphocytes, CD4+ T helper lympho- Number cytes, and CD45RO+ memory T lymphocytes when Microscopic feature of cases % compared to the G1 and G2 groups. Inflammatory infiltrate Absent 0 – When present, mast cells were equally distributed in Mild 23 60.5 G1 and G2. However, foxp3+ cells were found in 59% Intense 15 39.5 Collagen deposition Absent 0 – and 14% of the samples of G1 (n = 10) and G2 (n = 3). + Mild 15 39.5 Findings also revealed a higher number of foxp3 T Intense 23 60.5 regularory lymphocytes in G1 (mean = 13.7 - Calcification Absent 4 10.5 cells mm)2) than in G2 (mean = 6.2 cells mm)2). Mild 16 42.1 The number of CD31+ blood vessels was significantly Intense 18 47.4 Necrotic areas Absent 38 100.0 higher in G1 than in G2 (Mann–Whitney, P = 0.002) Present 0 – (Fig. 2). There were no significant differences between Density of CD31+ Low (£144) 23 60.5 the pulpitis (G1 mean = 190.00 blood vessel mm)2 blood vessels (mean: High (>144) 15 39.5 and G2 mean = 120.2 blood vessel mm)2) and control 144 blood vessels mm)2) groups (132.5 blood vessel mm)2).

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(a) (b)

(e) (f)

Figure 3 Representative immunostain- ing of tryptase+ mast cells (a), foxp3+ T regulatory cells (b), CD4+ cells (c), CD45RO+ cells (d), CD68+ cells (e), and CD20+ cells (f) distributed in inﬂamed dental pulp. Immunohistochemical staining. Original magniﬁcation: a–f, ·400.

et al. 2009), age-related changes (Tranasi et al. 2009), Discussion and others. The microscopic evaluation of pulpitis revealed differ- The presence of mast cells in the inflamed pulp, ent patterns of response: pulps with intense inflam- especially during the chronic phase of inflammation, matory infiltrate and mild collagen deposition, and has been reported (Farnoush 1984, Walsh et al. 1995). pulps with scarce inflammatory infiltrate and intense Mast cells do not seem to contribute only to the early collagen deposition. However, these two distinct vascular or specific immune responses in the initial response patterns seen microscopically were not clin- dental pulp pathosis, but are also involved in chronic ically confirmed, an indication of the difficulties of phases of pulp inflammation, such as in pulp polyps establishing associations between histopathological (Freitas et al. 2007). and clinical events, a finding corroborated by other In relation to the staining techniques for mast cells, studies (Seltzer et al. 1963, Na¨rhi 2003). The corre- both toluidine blue (TB) and immunohistochemistry lation between symptoms and histopathological identification techniques reliably identify mast cells changes in pulpitis seems to be poor and the deter- granules; although recent studies (Batista et al. 2005, mination of the type and extent of the inflammatory Oliveira-Neto et al. 2007) showed that the immuno- changes on the basis of symptoms is inaccurate histochemistry method is more specific than metachro- (Seltzer et al. 1963). It is important to consider that matic staining by TB. In accordance, TB was not useful inflammatory response and subsequent repair process for identification of mast cells in this study. in dental pulps may be influenced by several factors Most inflamed pulps had no mast cells, and when such as type and duration of infection (Hahn & present, their number was small, which is in agreement Liewehr 2007a,b), traumatic dental injury (Bruno with previous studies that reported small populations or

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foxp3+ T regulatory lymphocytes may suppress T cells (Lehner 2008) and, consequently, reduce their number, which was suggested by the lower numbers of both CD4+ and CD45RO+ T lymphocytes than of CD68+ macrophages and CD20+ B lymphocytes in all the samples analysed. Nevertheless, the role that T regulatory lymphocytes play in the pathogenesis of pulpitis should be explored further. The greater number of B than of T lymphocytes in pulpitis suggests a humoral response mediated by antibodies. After the activation of T cells by APCs, T cells secrete cytokines and differentiate into several effectors: CD4+ T helper lymphocytes, CD45RO+ mem- + Figure 4 Density of CD4+ T helper lymphocytes, CD45RO+ ory T lymphocytes, foxp3 T regulatory lymphocytes, + memory T lymphocytes, CD68+ macrophages, CD20+ B and CD8 cytotoxic lymphocytes. The nature of the lymphocytes per mm2 in G1 (dental pulp with intense responses of these effector cells depends on the dose, inflammatory infiltrate and mild collagen deposition) and G2 affinity, and nature of the antigen, as well as on the (dental pulp with scarce inflammatory infiltrate and intense type and concentration of cytokines in the tissue. collagen deposition). Two representative sections for each According to current knowledge, an increase in the sample were analysed. Results are expressed as mean of dose of antigenic peptide leads to changes in the CD4+ positive cells per mm2 ± SD. The Mann–Whitney test was T helper lymphocytes response pattern of cytokine used to compare groups at a level of significance set at 0.05. production (Hahn & Liewehr 2007b). CD4+ Th1 cells (#) Significant difference in CD68+ macrophages density in comparison of G1 with G2; (*) Control group showed produce interleukin 2 (IL-2) and interferon-gamma significantly lower numbers of CD4+ T helper lymphocytes, (IFN-c) predominantly, which play a role in the CD45RO+ memory T lymphocytes, CD68+ macrophages, and recruitment and activation of macrophages, whereas CD20+ B lymphocytes when compared to the groups 1 and 2. Th2 cells produce IL-4, IL-5, and IL-6, which stimulate B lymphocytes proliferation, differentiation, and activation (Jontell et al. 1998, Abbas & Lichtman 2003). no mast cells in non-inflamed pulps, as well as in early In teeth with deep caries, there is a predominance of pulpitis (Farnoush 1984, Freitas et al. 2007). This Th2-type response pattern, which leads to an increase reduced population of mast cells in pulpitis may interfere in the number of B lymphocytes in the pulp (Jontell with the pulpal defense mechanism, because they are et al. 1998, Hahn & Liewehr 2007b). immunoregulatory cells that release vasoactive amines, There are large numbers of CD68+ macrophages in enzymes, and cytokines, such as tumoral necrosis factor pulpitis; in fact, they are the most prevalent immune a (TNF-a). Conversely, the absence of mast cells might be cells in the tissue (Jontell et al. 1998) and their number interpreted as a form of modulation of the inflammatory is likely to increase with the progression of caries (Hahn response of the pulp, which minimizes increases in tissue & Liewehr 2007b). The presence of these cells in deep pressure and prevents tissue necrosis. Massive mast cell caries may be associated with the expression of degranulation in pulps would result in a significant chemokine ligands, such as monocyte chemotactic increase in inflammatory cell recruitment and vasodila- protein-1 (CCL2/MCP-1), and macrophage inflamma- tation, and, consequently, high intrapulpal pressure and tory protein 3-alpha (CCL20/MIP-3a) (Nakanishi et al. extensive tissue destruction (Walsh 2003). 2005, Hahn & Liewehr 2007b). Also, macrophages Small numbers of foxp3+ T regulatory lymphocytes play an important role in tissue homeostasis and repair were found in most samples, a highly important finding after inflammation, as they stimulate fibroblast prolif- because this appears to be the first study to detect these eration by releasing basic fibroblast growth factor cells in inflamed pulp tissue. Interestingly, high (FGFb) (Brody et al. 1992, Hahn & Liewehr 2007b), amounts of foxp3+ T regulatory lymphocytes were as well as in revascularization, because they release found in pulps with intense inflammatory infiltrate vascular endothelial growth factor (VEGF) (Polverini (G1), which may indicate that they affect immunity 1995, Hahn & Liewehr 2007b, Mattuella et al. control (Lehner 2008) and may prevent exacerbated 2007a,b). FGFb leads to an increase in collagen fibre inflammation and rapid necrosis of pulpal tissue. Also, production, which was also confirmed in this study

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because collagen deposition was found at varying Batista AC, Rodini CO, Lara VS (2005) Quantification of mast degrees in both groups. cells in different stages of human periodontal disease. Oral The blood vessels of all the samples examined were Diseases 4, 249–54. preserved. The highest density of CD31+ blood vessels Bergenholtz G (1981) Inflammatory response of the dental was seen in pulps with large numbers of inflammatory pulp to bacterial irritation. Journal of Endodontics 7, 100–4. Bergenholtz G (1990) Pathogenic mechanisms in pulpal cells (G1), as well as increased numbers of macrophag- disease. Journal of Endodontics 16, 98–101. es, cells that are responsible for the production of VEGF, Brody AR, Bonner JC, Overby LH (1992) Interstitial pulmo- a potent factor that induces angiogenesis and vascular nary macrophages produce platelet-derived growth factor permeability (Hahn & Liewehr 2007b). The increased that stimulates rat lung fibroblast proliferation in vitro. number of vessels may contribute to the reparative Journal of Leukocyte Biology 51, 640–8. response of the pulp–dentine complex because of the Bruno KF, Alencar AH, Estrela C, Batista AC, Pimenta FC augmented transport of inflammatory cells, nutrients, (2009) Microbiological and microscopic analysis of the pulp and oxygen to the site of inflammation (Polverini 1995, of non-vital traumatized teeth with intact crowns. Journal of Woodfin et al. 2007). Applied Oral Science 17, 508–14. Recent advances in immunology have disclosed the Chen W, Jin W, Hardegen N (2003) Conversion of peripheral + + + + enormous complexity of the immune regulatory sys- CD4 CD25 naive T cells to CD4 CD25 regulatory T cells by TGF-b induction of transcription factor Foxp3. The Journal tem. The dental pulp is equipped to mount an innate of Experimental Medicine 198, 1875–86. and adaptive immune response to caries, which Costa CAS, Figueiredo JAP, Hebling J, Estrela C (2009) Pulpal involves several types of cells and inflammatory medi- biology. In: Estrela C, ed. Endodontic Science, 2nd edn. Saõ ators (Hahn & Liewehr 2007a,b). Knowledge of the Paulo: Artes Me´dicas, pp. 1–22. pathogenesis of pulp disease may help to develop more Farnoush A (1984) Mast cells in human dental pulp. Journal of efficient therapies. Endodontics 10, 250–2. Freitas P, Novaretti CP, Rodini CO, Batista AC, Lara VS (2007) Mast cells and lymphocyte subsets in pulps from healthy Conclusion and carious human teeth. Oral Surgery Oral Medicine Oral Irreversible dental pulpitis revealed distinct microscopic Pathology Oral Radiology and Endodontics 103, 95–102. features with important quantitative and qualitative Gusman H, Santana RB, Zehnder M (2002) Matrix metalloproteinase levels and gelatinolytic activity in clinically differences in inflammatory cell infiltration. Further healthy and inflamed human dental pulps. European Journal experimental studies should be conducted to determine of Oral Sciences 110, 353–7. the clinical and microbiological factors involved and Hahn CL, Liewehr FR (2007a) Innate immune responses of the how they affect the inflammatory response outcome dental pulp to caries. Journal of Endodontics 33, 643–51. and consequently the pulpal prognosis. Hahn CL, Liewehr FR (2007b) Update on the adaptive immune responses of the dental pulp. Journal of Endodontics 33, 773–81. Acknowledgements Heaney LG, Leggett P, Maxwell P, Bharucha H, Ennis M This work was supported by grants from the National (1997) A comparison of three standard methods of identi- Council for Scientific and Technological Development fying mast cells in endobronchial biopsies in normal and (CNPQ grants 401305/2005-8 to T.A.S and 471878/ asthmatic subjects. Allergy 52, 836–43. Holzer P (1988) Local effector function of capsaicin-sensitive 2006-5 to A.C.B.). We thank Erildo Ribeiro da Silva sensory nerve endings: involvement of tachykinins, calcito- (Laboratory of Oral Pathology, Federal University of nin gene-related peptide and other neuropeptides. Neurosci- Goia´s, Brazil) for his excellent technical support. ence 24, 739–68. Izumi T, Kobayashi I, Okamura K, Sakai H (1995) Immuno- References histochemical study on the immunocompetent cells of the pulp in human non-carious and carious teeth. Archives of Abbas AK, Lichtman AH (2003) Cellular and Molecular Oral Biology 40, 609–14. Immunology, 5th edn. Philadelphia: Saunders. Jontell M, Okiji T, Dahlgren U, Bergenholtz G (1998) Immune Akbar AN, Taams LS, Salmon M, Vukmanovic-Stejic M (2003) defense mechanisms of the dental pulp. Critical Reviews in The peripheral generation of CD4+ CD25+ regulatory T cells. Oral Biology & Medicine 9, 179–200. Immunology 109, 319–25. Lehner T (2008) Special regulatory T cell review: the Avery JK (2002) Oral Development and Histology, 3rd edn. New resurgence of the concept of contrasuppression in immuno- York: Thieme. regulation. Immunology 123, 40–4.

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