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Transforming Growth Factor-β and Interleukin 10 in Oral Implant Sites in Humans
1 Department of Prosthetic Dentistry, Correspondence: *corresponding author, giulio.preti{at}unito.it
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]-  , IL-12) and anti-inflammatory (IL-4, IL-10, transforming growth factor [TGF]-β1) 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-β1 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-, IL-12, and IL-4 were absent. Healthy osseointegrated implants showed no histological inflammation in most patients. These findings suggest that newly classified TGF-β 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
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)-1β, IL-6, IL-8, IL-12, tumor necrosis factor (TNF)-  , and interferon (IFN)- 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)-β, 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- 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.
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. 1  ). 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.
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 β-actin specific primers, as described elsewhere (Schierano et al., 2000). Based on the amount of amplified β-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- ) 5'-ATGAAATATACAAGTTATATCTTGGCTTT-3' (sense) and 5'-GATGCTCTTCGACCTCGAAACAGCAT-3' (antisense) (30 cycles, annealing temperature 54°C; 30-second extension at 72°C); and (TGF-β1) 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-, and TGF-β1 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
Statistics
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. 2A shows, TGF-β-specific mRNA was identified in most specimens (8/11, 73%); 3/11 (27%) co-expressed IL-10-mRNA. No message was detected for IFN-, IL-2, or IL-12p40. When cytokine mRNA expression was normalized to levels of mRNA encoding for β-actin, TGF-β predominated among anti-inflammatory cytokines expressed (Fig. 2B).
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. 3 ). 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- mRNA, both undetectable at other times; concomitantly, TGF-β1 and IL-10 messages were low or absent (data not shown). The expression pattern of anti-inflammatory cytokine mRNA shows significant TGF-β1 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.
From month 4 onward, IL-12p40 and IFN- 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-β1, p = 0.002 vs. baseline; IL-10, p = 0.0001 vs. baseline). Thereafter, while IL-10 transcript decreased or disappeared, TGF-β1 mRNA remained expressed, returning to near-baseline levels.
Morphometric Analysis
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-β1 and, to a lesser extent, IL-10; mRNA for pro-inflammatory cytokines (IL-2, IFN- , and IL-12p40) was not detected. At abutment connection and during 12-month follow-up, TGF-β1 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-β-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-
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-β 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-β1 also inhibits chemokine-receptor expression (Sallusto et al., 1998) and abrogates IL-12 and, in turn, IFN- 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-β, whereby they exert their suppressive functions (Shevach et al., 2001). TGF-β 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-β- 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-β1 production (Mosmann and Sad, 1996). Th3-type cells appear to be distinct from Th2-type cells, since CD4+ TGF-β-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.
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 for publication March 4, 2002. Revision received January 28, 2003. Accepted for publication February 17, 2003.
Journal of Dental Research, Vol. 82, No. 6,
428-432 (2003)
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