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J Dent Res 82(6): 428-432, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Clinical

Transforming Growth Factor-ß and Interleukin 10 in Oral Implant Sites in Humans

G. Schierano1, G. Bellone2, E. Cassarino1, M. Pagano3, G. Preti1,*, and G. Emanuelli2

1 Department of Prosthetic Dentistry,
2 Department of Clinical Physiopathology, and
3 Department of Biomedical Sciences and Human Oncology, University of Turin, Corso Dogliotti 14, 10126 Torino, Italy;

*corresponding author, giulio.preti{at}unito.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cross-talk between cells and cytokines in peri-implant tissue is largely unknown. The immune response in the gingival mucosa appears to favor implant integration over rejection, since titanium-implant-retained overdentures show long-term success. This study evaluates pro-inflammatory (interleukin [IL]-2, interferon [IFN]-{gamma}, IL-12) and anti-inflammatory (IL-4, IL-10, transforming growth factor [TGF]-ß1) cytokine mRNA expression and tissue morphometry in peri-implant soft tissue from patients before and during treatment with Brånemark titanium implants. Immediately after treatment with endosseous implant and overdenture, TGF-ß1 mRNA increased in peri-implant mucosa specimens; transcript accumulation for IL-10 was elevated at 4 months and decreased dramatically thereafter. Transcripts for IL-2, IFN-{gamma}, IL-12, and IL-4 were absent. Healthy osseointegrated implants showed no histological inflammation in most patients. These findings suggest that newly classified TGF-ß and/or IL-10 secreting T regulatory (r)/T helper (h)-3 cells may populate implant insertion sites.

KEY WORDS: Th1 • Th2 • Th3 • cytokines • oral implants


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extensive multicenter studies attest to the long-term success of dental implant therapy (Apse et al., 1991). The mechanisms by which gingival soft tissue tolerates osseointegrated dental titanium implants are unclear. Periodontal stability may depend on the local balance of reactive and suppressor immune cells, their cytokines and mediators. Among cytokines, interleukin (IL)-1ß, IL-6, IL-8, IL-12, tumor necrosis factor (TNF)-{alpha}, and interferon (IFN)-{gamma} serve predominantly pro-inflammatory functions. They are produced by or act on phagocytic and other cells to up-regulate adhesion molecules, nitric oxide synthesis, eicosanoid and metalloproteinase production, and cytokine secretion. They also activate endothelial and connective tissue cells (Dinarello, 2000). Other cytokines—including IL-1 receptor antagonist (IL-1ra), IL-13, transforming growth factor (TGF)-ß, and IL-10—play anti-inflammatory roles (Opal and DePalo, 2000).

Inflammatory cell infiltrates and cytokine production by macrophages/antigen-presenting cells (APC) and CD4+ T-helper (Th) lymphocytes may regulate reaction to gingival injury. Two Th lymphocyte subsets are defined by cytokine secretion pattern (Romagnani, 1996): Th1 cells release IL-2, IFN-{gamma}, and TNF-ß and promote cell-mediated immune-response; Th2 cells secrete IL-4, IL-10, IL-5, IL-13, and induce humoral response. Recently, two novel CD4+ subsets, designated T regulatory (r) and Th3, predominantly producing TGF-ß and/or IL-10, have shown in vitro immunosuppressive activity (Groux et al., 1997).

Although the role of soluble mediators during periodontal tissue injury and disease is established (Seymour and Gemmell, 2001), their importance in host response to oral implants is less clear. Histological and clinical studies show that peri-implant mucosa acts as a normal-periodontal-mucosa-like barrier without signs of inflammation (Preti et al., 1996). This study evaluates pro-/anti-inflammatory cytokine mRNA levels and tissue morphometry in peri-implant soft tissue from patients with Brånemark titanium implants.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Techniques
Eleven patients (six men, five women, mean age 61.4 yrs, range 53-69 yrs, 7-10 yrs edentulous) were rehabilitated with complete dentures. After 6 months’ adaptation, they underwent implantation following the Brånemark mandibular full-denture anchorage protocol, described elsewhere (Schierano et al., 2000). Peri-implant mucosa specimens (diameter, 4-5 mm) were taken at surgical implant insertion (Stage 1), healing abutment connection (Stage 2), and 4, 8, and 12 mos after prosthetic anchorage (Fig. 1Go). Tissue samples were fixed in 10% formalin and paraffin-embedded for histology, or frozen in liquid nitrogen for gene-expression analysis. All patients gave informed, signed consent to participation; the study was approved by the appropriate local institutional review board.



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  		Figure 1. Phases at which biopsies were taken: surgical stage 1 (baseline) (A); surgical stage 2 (B); mandibular overdenture anchored to 3 implants (4-8-12 mos) (C) via ball-attachment ø 2.25 mm (D); technique of peri-implant soft-tissue biopsies (E) and amount of specimen (F).

 
RNA Extraction and Semi-quantitative RT-PCR
Cytokine mRNA was assessed by semi-quantitative RT-PCR. Briefly, total RNA, isolated from gingival soft tissue by the single-step Trizol method (Life Technologies, Gaithersburg, MD, USA), was reverse-transcribed by Superscript II reverse transcriptase (Life Technologies). Each sample was subjected to initial 20-cycle amplification by human ß-actin specific primers, as described elsewhere (Schierano et al., 2000). Based on the amount of amplified ß-actin PCR product, an equal amount of reverse-transcribed product was amplified by the following cytokine primer pairs in these conditions: (IL-2) 5'-AACTCCTGTCTTGCATTGCA-3' (sense) and 5'-GTGTTGAGATGATGCTTTGAC-3' (antisense) (30 cycles, annealing temperature 56°C; four-second extension at 72°C); (IL-4) 5'-CAACTTTGTCCACGGACAC-3' (sense) and 5'-TCCAACGTACTCTGGTTGG-3' (antisense) (30 cycles, annealing temperature 56°C; 35-second extension at 72°C); (IL-10) 5'-ATGCCCCAAGCTGAGAACCAAGACCCA-3' (sense) and 5'-AAGTCTCAAGGGGCTGGGTCAGCTA-3' (antisense) (30 cycles, annealing temperature 60°C; 30-second extension at 72°C); (IL-12p40) 5'-CCAAGAACTTGCAGCTGAAG-3' (sense) and 5'-TGGGTCTATTCCGTTGTGTC-3' (antisense) (30 cycles, annealing temperature 57°C; 45-second extension at 72°C); (IFN-{gamma}) 5'-ATGAAATATACAAGTTATATCTTGGCTTT-3' (sense) and 5'-GATGCTCTTCGACCTCGAAACAGCAT-3' (antisense) (30 cycles, annealing temperature 54°C; 30-second extension at 72°C); and (TGF-ß1) 5'-GCCCTGGACACCAACTATTGC-3' (sense) and 5'-GCACTTGCAGGAGCGCA-3' (antisense) (30 cycles, annealing temperature 60°C; one-minute extension at 72°C). The PCR reaction mixture contained 5 µL cDNA, 200 µL of each dNTP, 0.4 µmol/L of each upstream- and downstream-specific primer, 1.5 mmol/L MgCl2, 2.5 U of Taq DNA polymerase (Life Technologies), and 1 µCi of [32P]dCTP (3000 Ci/mmol; DuPont-New England Nuclear, Boston, MA, USA) in reaction buffer. PCR products were analyzed by electrophoresis through 6% acrylamide Tris borate-EDTA gel, followed by autoradiography and quantification by Molecular Imager and Molecular Analyst software (Biorad, Hercules, CA, USA). The predicted sizes of PCR products of IL-2, IL-4, IL-10, IL-12p40, IFN-{gamma}, and TGF-ß1 were 368, 450, 345, 352, 355, 494, and 333 bp, respectively. Negative controls omitted RNA from cDNA synthesis and specific PCR amplification. Experiments were repeated three times.

Morphometric Analysis
Formalin-fixed, paraffin-embedded biopsies were stained with hematoxylin-eosin and Giemsa (Carlo Erba, Milan, Italy). Histological findings were analyzed on each slide by cell population. Lymphocytes, plasma cells, and granulocytes were counted at HPF (40x) in 5 randomly chosen fields; results are means ± SE. Fibroblasts were scored: + = few; ++ = moderate; +++ = numerous.

Statistics
We calculated statistical differences between datasets by generating p values by paired Student’s t tests, at a significance level p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pro- and Anti-inflammatory Cytokine Expression in Gingival Soft Tissue
    Stage 1 (baseline)
To elucidate the influence of implants on pro-/anti-inflammatory cytokine profiles, we determined mRNA expression in baseline gingival soft tissue. As Fig. 2AGo shows, TGF-ß-specific mRNA was identified in most specimens (8/11, 73%); 3/11 (27%) co-expressed IL-10-mRNA. No message was detected for IFN-{gamma}, IL-2, or IL-12p40. When cytokine mRNA expression was normalized to levels of mRNA encoding for ß-actin, TGF-ß predominated among anti-inflammatory cytokines expressed (Fig. 2BGo).



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  		Figure 2. RT-PCR analysis of gingival mucosa for pro-/anti-inflammatory cytokine messages. (A) Lanes 1-11: gingival soft tissue from 11 edentulous patients; Lane 12: peripheral blood mononuclear cells activated with appropriate stimuli (phytohemoagglutin for IFN-{gamma}, IL-2, IL-4, IL-10, and TGF-ß1, and lipopolysaccharide for IL-12p40) as positive control. No bands formed in the absence of cDNA template (Lane 13). Each determination was repeated three times with similar results. PCR for cytokines was amplified for 30 cycles and PCR for ß-actin for 20 cycles. (B) Densitometric values assigned to cytokine bands by Molecular Analyst software were normalized for ß-actin expression, and the ratio of each cytokine to ß-actin was determined. Data are means ± SE of 11 patients (three repeats).

 
    Stage 2 and 4, 8, and 12 Months after Prosthetic Anchorage
Pro-/anti-inflammatory cytokine transcript modulation was monitored in peri-implant gingival soft tissue and compared with pre-surgery mRNA accumulation (Fig. 3Go). IL-2- and IL-4-specific messages were not detected in any RNA sample. In 3/11 patients (27%), Stage 2 gingival soft-tissue specimens simultaneously expressed IL-12p40 and IFN-{gamma} mRNA, both undetectable at other times; concomitantly, TGF-ß1 and IL-10 messages were low or absent (data not shown). The expression pattern of anti-inflammatory cytokine mRNA shows significant TGF-ß1 transcript elevation (8/11 patients, 73%) vs. baseline (p = 0.03). In five patients (45%), IL-10 mRNA became detectable at Stage 2, while in the remaining three patients (27%), no significant change occurred. On average, no statistically significant difference was found vs. baseline IL-10 expression.



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  		Figure 3. Local modulation of pro-/anti-inflammatory cytokine mRNA expression during oral titanium implant rehabilitation. Total RNA from peri-implant gingival soft tissue at Stage 2 and 4, 8, and 12 mos after prosthetic anchorage was subjected to RT-PCR analysis for cytokine and ß-actin mRNA. Densitometric values assigned to cytokine bands by Molecular Analyst software were normalized for ß-actin expression and the ratio of each cytokine to ß-actin determined. Data are means ± SE of 11 patients (three repeats). Significant difference vs. baseline was determined by Student’s t test.

 
From month 4 onward, IL-12p40 and IFN-{gamma} mRNA were absent, except in one patient with chronic inflammation in whom small amounts were detected (data not shown). At 4 mos, anti-inflammatory cytokines increased in all patients, in particular IL-10 (TGF-ß1, p = 0.002 vs. baseline; IL-10, p = 0.0001 vs. baseline). Thereafter, while IL-10 transcript decreased or disappeared, TGF-ß1 mRNA remained expressed, returning to near-baseline levels.

Morphometric Analysis
Biopsies were available from only eight patients. At baseline, subepithelial stroma was loosely woven with abundant fibroblasts and thin connective fibers. Upon mechanical stimulation by prosthetic anchorage, dense collagen fibers accumulated and fibroblast numbers fell. At all stages, lymphocytes were the most common immune cells, mostly at peri-vessel sites or scattered in the stroma or inner epithelial layers, resident rather than infiltrated. Few plasma cells were found, except in the case of chronic inflammation. All specimens were free of infiltrating macrophages, eosinophils, and mastocytes. No significant increase in lymphocyte number or distribution occurred during follow-up (Fig. 4AGo). Representative photomicrographs are in Fig. 4BGo.




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  		Figure 4. Histological findings. (A) Pattern of immune cell populations analyzed in gingival mucosa specimens from seven patients at Stage 2 and 4, 8, and 12 mos after prosthetic anchorage. Lymphocytes, granulocytes, and plasma cells were counted at HPF (40x) in 5 randomly chosen fields; results are means ± SE. Patients suffering chronic inflammation were excluded (B). Micrograph of gingival soft tissue from a representative patient (Hematoxylin-eosin and Giemsa stain, original magnification x400).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Because titanium-implant-retained overdentures have demonstrated long-term success, gingival mucosa response may be assumed to favor implant integration over rejection. The mechanisms involved may depend on local reactive/suppressor immune cell balance and related cytokines; however, this implant site/cell-cytokine cross-talk has yet to be investigated. RT-PCR plus morphometric analysis enabled us to estimate quantitative and temporal changes of pro-/anti-inflammatory cytokines and the immunocompetent cell composition in peri-implant gingival soft tissue throughout treatment. Gingival mucosa from pre-surgery edentulous patients (Stage 1) preferentially expressed mRNA for TGF-ß1 and, to a lesser extent, IL-10; mRNA for pro-inflammatory cytokines (IL-2, IFN-{gamma}, and IL-12p40) was not detected.

At abutment connection and during 12-month follow-up, TGF-ß1 mRNA levels rose, at Stage 2 and 4 mos, then gradually decreased to baseline levels. By contrast, IL-10 expression increased strongly at 4 mos, decreased at 8 mos, and became almost undetectable at 12 mos, suggesting that these immunosuppressive cytokines combine to regulate immuno-inflammatory balance in peri-implant mucosa. TGF-ß-producing cells are the primary suppressors in animal colitis models, but IL-10 is necessary for these cells to expand, despite Th1 response (Strober et al., 2001).

Specific mRNA for IL-2 and IL-4, two important T-cell growth factors for Th1 and Th2 cells (Mosmann and Coffman, 1989), was not expressed in peri-implant mucosa. These two cytokines, however, may influence gingival-mucosa immune responses to implant insertion, since lack of expression might depend on biopsy timing. Generation of Th1-response depends on IL-12 released by APC. The bioactive heterodimer IL-12, inducing IFN-{gamma} production, leads to macrophage activation and promotes cell-mediated immune responses (Trinchieri, 1998). Th1 cells are generally pro-inflammatory and, if persistent, contribute to autoimmune and chronic inflammatory disease development (Romagnani, 1996). We found limited frequency (3/11, 27%) and duration of Th1-type implant-site reaction.

In agreement with other reports (Apse et al., 1991), our clinical and histomorphometric findings, complementary to cytokine mRNA expression analysis, revealed no long-term inflammatory response in peri-implant tissues (at 12 mos) in most patients (91%). Prominent TGF-ß expression during implantation is linked to successful implant osseointegration, inducing bone formation and affecting orientation, growth, and adhesion-molecule expression in human gingival fibroblasts (Schierano et al., 2000, 2001). TGF-ß1 also inhibits chemokine-receptor expression (Sallusto et al., 1998) and abrogates IL-12 and, in turn, IFN-{gamma} synthesis (Schmitt et al., 1994), possibly explaining the lack of leukocyte infiltration into peri-implant gingival mucosa. The severe, uncontrolled inflammatory reactions observed in TGF-ß1-knockout mice attest to the role of TGF-ß as an anti-inflammatory cytokine (Shull et al., 1992). The mRNA of IL-10, an inhibitor of pro-inflammatory cytokine synthesis (Howard and O’Garra, 1992), is widely expressed in peri-implant tissue. IL-10-knockout mice not only develop intestinal inflammation but also exhibit prolonged, enhanced contact hypersensitivity, possibly leading to severe tissue damage (Berg et al., 1995). IL-10 may also be fundamental in controlling periodontal disease (Gemmell et al., 1997). We therefore contend that TGF-ß1 and IL-10 are likely to affect the gingival microenvironment and local implant-site immune response. Formal assessment is lacking; however, our observations suggest the newly described Th3/Tr-type cytokine profile response.

The functionally unique lineage CD4+, CD25+ Tr cells are thought to be involved in maintaining immune tolerance (Roncarolo and Levings, 2000). Tr cells produce immunoregulatory cytokines, such as IL-10 and TGF-ß, whereby they exert their suppressive functions (Shevach et al., 2001). TGF-ß and IL-10 induce both anergic CD4+ T-cells and Tr cells in experimental autoimmune diseases (Roncarolo and Levings, 2000), tempting speculation that induction of TGF-ß- and IL-10-secreting Tr cells may contribute to poor implant-site response.

Alternatively, our findings may be indicative of involvement of the newly classified Th3 subset with mucosal T-helper function and suppressive activity via predominant TGF-ß1 production (Mosmann and Sad, 1996). Th3-type cells appear to be distinct from Th2-type cells, since CD4+ TGF-ß-secreting cells with suppressive properties have been generated from IL-4-deficient animals (Weiner, 2001) which do not develop Th2 responses (Noben-Trauth et al., 1996). Current studies focus on characterization of the cell-surface profile and suppressive function of these putative gingival Th3/Tr cells.


   ACKNOWLEDGMENTS
 
This work was supported by MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca, Roma, Italy) and by Nobel Biocare Italiana, Agrate Brianza, Milan, Italy . We are indebted to Frances Cooper as English-language editor.

Received March 4, 2002; Last revision January 28, 2003; Accepted February 17, 2003


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apse P, Zarb GA, Schmitt A, Lewis DW (1991). The longitudinal effectiveness of osseointegrated dental implants. The Toronto Study: peri-implant mucosal response. Int J Periodont Rest Dent 11:94–111.

Berg DJ, Kuhn R, Rajewsky K, Muller W, Menon S, Davidson N, et al. (1995). Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J Clin Invest 96:2339–2347.

Dinarello CA (2000). Proinflammatory cytokines. Chest 118:503–508.[Abstract/Free Full Text]

Gemmell E, Marshall RI, Seymour GJ (1997). Cytokines and prostaglandins in immune homeostasis and tissue destruction in periodontal disease. Periodontol 2000 14:112–143.

Groux H, O’Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, et al. (1997). A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737–742.[Medline]

Howard M, O’Garra A (1992). Biological properties of interleukin 10. Immunol Today 13:198–200.[ISI][Medline]

Mosmann TR, Coffman RL (1989). Heterogeneity of cytokine secretion patterns and functions of helper T cells. Adv Immunol 46:111–147.[ISI][Medline]

Mosmann TR, Sad S (1996). The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 17:138–146.[ISI][Medline]

Noben-Trauth N, Kropf P, Müller I (1996). Susceptibility to Leishmania major infection in interleukin-4-deficient mice. Science 271:987–990.[Abstract]

Opal SM, DePalo VA (2000). Anti-inflammatory cytokines. Chest 117:1162–1172.[Abstract/Free Full Text]

Preti G, Bassi F, Barbero P, Lorenzetti M, Valente G (1996). Histological changes in edentulous oral mucosa under implant-supported overdentures. J Oral Rehabil 23:651–654.[ISI][Medline]

Romagnani S (1996). Th1 and Th2 in human diseases. Clin Immunol Immunopathol 80:225–235.[ISI][Medline]

Roncarolo MG, Levings MK (2000). The role of different subsets of T-regulatory cells in controlling autoimmunity. Curr Opin Immunol 12:676–683.[ISI][Medline]

Sallusto F, Lanzavecchia A, Mackay CR (1998). Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol Today 19:568–574.[ISI][Medline]

Schierano G, Bassi F, Gassino G, Mareschi K, Bellone G, Preti G (2000). Cytokine production and bone remodeling in patients wearing overdentures on oral implants. J Dent Res 79:1675–1682.[Abstract/Free Full Text]

Schierano G, Bellone G, Manzella C, Preti G, Emanuelli G (2001). In vitro effect of transforming growth factor-beta on adhesion molecule expression by human gingival fibroblasts cultured in the presence of a titanium abutment. J Periodontol 72:1658–1665.[ISI][Medline]

Schmitt E, Hoehn P, Huels C, Goedert S, Palm N, Rude E, et al. (1994). T helper type 1 development of naive CD4+ T cells requires the coordinate action of interleukin-12 and interferon-gamma and is inhibited by transforming growth factor-beta. Eur J Immunol 24:793–798.[ISI][Medline]

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Shevach EM, McHugh RS, Piccirillo CA, Thornton AM (2001). Control of T-cell activation by CD4+ CD25+ suppressor T-cells. Immunol Rev 182:58–67.[ISI][Medline]

Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, et al. (1992). Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 359:693–699.[Medline]

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Trinchieri G (1998). Interleukin-12: a cytokine at the interface of inflammation and immunity. Adv Immunol 70:83–243.[ISI][Medline]

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This Article
Right arrow Abstract Freely available
Right arrow Figures Only
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Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schierano, G.
Right arrow Articles by Emanuelli, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schierano, G.
Right arrow Articles by Emanuelli, G.

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