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Regular Articles |
From the Departments of Periodontology and Oral
Biology*
and Endodontics,
Boston University School of Dental Medicine, Boston University,
Boston, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-1 and TNF play an important role in initiating and coordinating the cellular events that make up the immune system’s response to infection. Many cell types are capable of producing IL-1 and TNF, and almost all are capable of responding to these cytokines.5,6 The biological effects of IL-1 and TNF include activation of leukocytes such as lymphocytes (T and B cells), macrophages, and natural killer cells; fever induction; acute-phase protein release; cytokine and chemokine gene expression; and endothelial cell activation. Under physiological conditions, IL-1 and TNF are induced and released in restricted microenvironments where they have autocrine and paracrine activity. During infection, morbidity can result when the systemic levels are sufficiently high enough to induce shock.
Abu-Amer and colleagues have proposed that TNF activity is critical in mediating bone loss due to the gram-negative bacterial product lipopolysaccharide (LPS).8 They suggest that LPS induces TNF expression, which in turn stimulates osteoclastogenesis and bone loss. This is supported by findings that TNFp55-/- receptor mice have greatly reduced in vivo and ex vivo formation of osteoclasts in response to LPS. Similarly, we have reported that inhibitors of IL-1 and TNF applied in combination significantly reduce osteoclast activity and bone loss in bacteria-induced experimental periodontitis.9
IL-1 activity is conferred by two related proteins, IL-1
and
IL-1ß, both of which bind to IL-1 receptors, termed type I and type
II. The type I IL-1 receptor (IL-1RI) is responsible for specific
signaling, whereas the type II receptor functions as a nonsignaling
decoy receptor.10
Similarly, there are two molecules of
TNF: TNF and TNFß, which have a high degree of structural and
sequence homology and are able to interact with two distinct TNF
receptors, type I (TNFRp55) and type II (TNFRp75).11
Most
of the well-documented inflammatory properties of TNF are mediated by
TNFRp55, while signaling through TNFRp75 appears to reduce
TNF-mediated inflammation.12
IL-1 and TNF share several
biological activities and often have similar effects on inflammatory
processes. In many instances IL-1 and TNF act synergistically in both
in vivo and in vitro studies.13,14
Because IL-1 and TNF have several overlapping functions, it has been difficult to establish the importance of each of these cytokines in the process of inflammation and host resistance to infectious agents. The generation and availability of mice lacking cytokines or cytokine receptors through targeted gene mutation allows for a more exact determination of the role of a particular cytokine in physiological homeostasis and the pathogenesis of disease states. Recently, IL-1 receptor-mutant (IL-1RI-/-) and TNF receptor-mutant (TNFRp55-/- and/or TNFRp75-/-) mice have been generated. These mice do not exhibit gross abnormalities and are capable of developing antibodies to exogenous antigen stimulation.12,15,16 However, TNFRp55-/- mice fail to develop germinal centers in their peripheral lymphoid organs.
To investigate the role of IL-1 and/or TNF in the host response to mixed anaerobic infection in both soft and hard connective tissue, a model that could develop and maintain a conducive growth environment for anaerobic pathogens was used.17,18 Infection of the dental pulp and subsequent osseous lesion formation at the apex of the root fulfills these criteria and was used to examine osteoclastogenesis as well as bacterial penetration, leukocyte recruitment, and soft tissue necrosis. After surgical exposure of the dental pulp and inoculation by six putative pathogens, the infectious sequelae were assessed in control mice and three groups of experimental mice, which lacked IL-1 and/or TNF receptor signaling (IL-1RI-/-, TNFRp55-/--p75-/-, or TNFRp55-/--IL-1RI-/-). The results indicate that neither IL-1 or TNF receptor signaling is essential for bacteria-induced osteoclastogenesis and bone loss. However, both play a critical role in limiting the damage caused by a mixed anaerobic infection, and their activities together afford greater protection than either alone.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For each data point, specimens were obtained from five mice. A total of 150 animals (age 10–14 weeks), including experimental and wild-type mice, were examined. The experimental mice consisted of three different groups with targeted mutations, as have been described12,15,16 : 1) IL-1 receptor type I (IL-1RI-/-); 2) TNF receptors type I and type II (TNFRp55-/--p75-/-); and 3) type I receptors for both IL-1 and TNF (TNFRp55-/--IL-1RI-/-) (generously donated by Immunex R&D Corp., Seattle, WA). The wild-type mice with similar genetic background were C57BL/6 x 129J hybrids (purchased from Jackson Lab, Bar Harbor, ME). Deletion of the targeted genes was verified by polymerase chain reaction (PCR) that could distinguish between homozygous or heterozygous mice with targeted deletions and wild-type mice.
Bacterial Strains and Growth Conditions
Surgically exposed dental pulps were inoculated with six putative oral pathogens, a facultative anaerobe (Streptococcus mutans) and five obligate anaerobes: Streptococcus mutans (American Type Culture Collection (ATCC) 25175), Streptococcus intermedius (ATCC 27335), Peptostreptococcus micros (ATCC 33270), Porphyromonas gingivalis (ATCC 33277), Prevotella intermedius (ATCC 25611), and Fusobacterium nucleatum (ATCC 49256). The bacteria were grown under anaerobic conditions. During the inoculation procedures, anaerobic conditions were maintained with an N2 environment.
Induction of Osteolytic Lesions
Animals were anesthetized with a combination of Xylazine-20 (Ben Venue Laboratory, Bedford, OH) and Ketaset (Fort Dodge Laboratory, Fort Dodge, IA) and placed on a retraction board, and the mesial cusps of the first mandibular molars were surgically removed to expose the dental pulp. One hundred microliters of a viscous bacterial mixture (containing 108 of each of the six bacteria strains in 2% methylcellulose) were placed on the tooth surface. Five mice from each group were sacrificed by CO2 asphyxiation at 0, 3, 7, 14, 21, and 38 days after pulp exposure. The mandibles were dissected, immersed in 4% paraformaldehyde at 4°C for 4 hours, decalcified in EDTA, and prepared for cryostat sectioning as described previously.19 Some sections were stained with hematoxylin and eosin (H&E), and others were assessed for tartrate-resistant acid phosphatase (TRAP) activity, using a kit from Sigma (St. Louis, MO). In addition, immunostained (F4/80) cells were identified using a biotinylated secondary antibody and a detection kit from Vector Laboratories (Burlington, MA) as previously described.19 This antibody recognizes peripheral monocytic cells, including monocytes, macrophages, and dendritic cells, but not osteoclast precursors. TRAP staining and F4/80 immunostaining were performed on frozen serial sections in coordination with the H&E-stained slides. Subsequent analysis was performed by "blinded" observers.
Image Analysis and Measurements
Analysis of osseous lesions was restricted to histological sections representing the maximum lesion size for a given specimen. Measurements were made on serial histological sections, using computer-assisted image analysis. All images of the slides were coded by one person and analyzed by another person, making the measurements double blind. The results were verified by a second examiner. Interexaminer and intraexaminer variation was generally less than 5%.
Degree of Tissue Necrosis
Necrosis in the pulpal tissue of the distal root of the mandibular first molar was assessed at a magnification of x400. The tissues within the root canal were divided into three equal parts: coronal third, middle third, and apical third. The following scale was used to assess the spread of necrosis from the coronal to the apical third: 0, no necrosis; 1, partial necrosis of coronal third; 2, total necrosis of coronal third; 3, partial necrosis of middle third; 4, total necrosis of middle third; 5, partial necrosis of apical third; and 6, total necrosis of apical third. The highest score represented the necrosis status of that canal and was used in statistical analysis.
Osteoclastogenesis and Osteoclastic Activity
The number of osteoclasts was determined by counting multinucleated TRAP-positive cells in direct contact with bone at a magnification of x200 and expressed as the number per millimeter of bone length. In addition, the relative depth of Howship’s lacunae was evaluated for each osteoclast at a magnification of x400 as an indicator of osteoclast activity. The following scale was used for assessment of osteoclast activity: 1, shallow lacunae; 2, moderate lacunae (less than a half the diameter of the osteoclast); and 3, deep lacunae (more than a half the osteoclast diameter). The numbers of osteoclasts in shallow, moderate, or deep Howship’s lacunae were calculated separately for a given specimen. The osteoclast activity score, which was used in statistical analysis, was determined by calculating the sum of the number of osteoclasts in each major group multiplied by the value assigned to that group (1, shallow; 2, moderate; 3, deep lacunae).
Inflammatory Cell Recruitment
The number of polymorphonuclear leukocytes was counted manually from an image projected onto a color monitor at a magnification of x1000. A counting template was centered at a fixed distance from the apical foramina of distal roots throughout the selected section of each different specimen of the H&E-stained slides. Within these designated areas, the number of polymorphonuclear leukocytes was counted using their identifying characteristics, such as darkly stained cells with multilobed, horseshoe-shaped nuclei. The number of F4/80 immunopositive monocytic cells, which had a distinctly round and darkly stained nucleus, was similarly counted. However, F4/80 immunopositive histiocytic cells with a dendritic appearance were excluded from these counts because of difficulty in obtaining accurate measurements and because they were found in relatively high numbers in noninflamed specimens.
Bacterial Penetration of Tissue
Inoculation of the dental pulp with six putative oral pathogens was carried out as described above. Six groups of mice were tested—three groups of mice with targeted deletions (IL-1RI-/-, TNFRp55-/--p75-/-, TNFRp55-/--IL-1RI-/-), matched wild-type control mice, and two groups of normal CD-1 mice, which served as negative controls for sterile technique. Eight days after inoculation, the mice were carefully disinfected with Povidone iodine (Baxter Health Care Corp., Deerfield, IL) followed by 70% ethanol, and the lower jaw was microdissected and immediately placed in a sterile petri dish. The apical third of the distal root of the experimental tooth was microdissected in a sterile lamina flow hood and then crushed while submerged in a liquid dental transport medium (Anaerobe Systems, San Jose, CA). Serial dilutions were performed, bacteria were grown under anaerobic conditions, and the number of bacterial colonies formed was determined 7 days later by "blinded" examiners.
Statistical Analysis
The degree of tissue necrosis, size of osteolytic lesions, osteoclastogenesis and osteoclast activity scores, number of polymorphonuclear and mononuclear phagocytes, and the number of bacterial colonies recovered from the apical third of each root were analyzed using one-way analysis of variance with Tukey-Kramer’s post hoc test to determine significance at a given time point. Significance was established at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The model used is useful for following the spread of
bacteria-induced tissue damage, which occurs reproducibly and
predictably involves necrosis of soft tissue followed by formation of
an osteolytic lesion. After inoculation with one facultative and five
obligate anaerobic bacteria, little or no tissue necrosis was observed
at day 7 in wild-type mice (Figure 1)
.
However, in mice lacking responses to IL-1 and TNF, the tissue within
the root was completely necrotic at this time point. Quantitative
assessment of the progression of tissue necrosis is shown in Figure 2
. There were no statistical differences
between the experimental groups or the experimental and control groups
on day 3. However, significant differences were noted between the three
experimental groups of IL-1 or TNF receptor-deficient mice and
the wild-type group at day 7 and later time points. By day
14, a maximum or nearly maximum necrosis score was observed for mice in
each of the experimental groups, whereas the control group score was
significantly less. In fact, the wild-type mice did not have a maximum
necrosis score even at day 38.
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Bacteria that colonize the upper portion of the dental pulp
eventually penetrate to the end of the tooth root. Because this
progression is predictable and reproducible, it serves as an excellent
model for the study of bacterial penetration of connective tissue.
Figure 3
indicates that bacterial
penetration is dramatically greater in mice lacking IL-1 and/or TNF
responses. The mean number of bacterial colonies in receptor-deficient
mice was significantly higher than that in wild-type mice; 4,700-fold
for TNFRp55-/--p75-/- mice, 7,600-fold for
IL-1RI-/- mice, and 18,500-fold for
TNFRp55-/--IL-1RI-/- mice. Furthermore,
bacterial penetration of the tissue was significantly greater in mice
lacking both IL-1 and TNF responses compared to those lacking either
IL-1 or TNF responses alone. There were no significant differences in
the bacterial colonies formed between IL-1RI-/- and
TNFRp55-/--p75-/- mice. To establish that
bacteria were not introduced into the specimens during dissection and
bacterial sampling, bacterial colony counts were made for mice that had
been treated in the same way as the experimental groups but did not
undergo surgical exposure and bacterial inoculation of the dental pulp.
As expected, no bacterial colonies were recovered from the dental pulp
tissue of this negative control group (data not shown).
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The photomicrographs shown in Figure 4
demonstrate the development of large
osteolytic lesions in each of the experimental groups 38 days after
bacterial inoculation. In the wild-type mice, there was a small
osteolytic lesion with a light to moderate infiltration by inflammatory
cells. Large osteolytic lesions in the IL/TNF receptor ablated groups
were characterized by a severe inflammatory cell infiltrate with
evidence of abscess formation.
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represents a quantitative
assessment of the role of IL-1/TNF receptor signaling in the formation
of osteolytic lesions at various time points. On day 7 the size of the
osteolytic lesions was significantly greater in the
TNFRp55-/--IL-1RI-/- mice compared to
wild-type mice, whereas on days 14, 21, and 38, all three experimental
groups had osteolytic lesions that were significantly larger than those
of the control group. Moreover, on day 38, the size of lesions in
TNFRp55-/--IL-1RI-/- mice was significantly
greater than that observed in
TNFRp55-/--p75-/- or
IL-1RI-/- mice, indicating the importance of both IL-1
and TNF in combination in limiting bacteria-induced osteolysis. At no
time was there a statistical difference in the size of lesions between
the mice with deleted responses to either IL-1 or TNF alone.
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Microscopic examination of TRAP-stained sections demonstrated that
osteoclastogenesis proceeds rapidly in mice lacking IL-1/TNF receptor
signaling, whereas it increases slowly and consistently in wild-type
mice (Figure 6)
. For the experimental
groups after day 7, osteoclastogenesis reached a plateau and then
gradually decreased, whereas it continued to increase for the wild-type
mice. By day 38, osteoclastogenesis in the wild-type mice exceeded that
found in the IL-1 and/or TNF receptor-deficient groups. A decline in
osteoclast number at later time points in the experimental groups may
be due to changes in osseous architecture as the lesions become very
large. No significant differences in osteoclastogenesis were found
between TNFRp55-/--p75-/- and
IL-1RI-/- mice at any time points.
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. Day 7 was chosen for
this analysis because the osseous architecture was similar for all
groups at that time point. In the wild-type mice, most of the
Howship’s lacunae were shallow, whereas in the
TNFRp55-/--IL-1RI-/- group, osteoclasts
were found in deep as well as moderate and shallow lacunae. The
TNFRp55-/--p75-/- and
IL-1RI-/- mice had intermediate values between these two
groups. Thus a greater proportion of osteoclasts were in moderate or
deep lacunae in the mice lacking IL-1 or TNF responses compared to
normals, and the greatest proportion in moderate or deep lacunae were
observed in mice lacking both IL-1R1 and TNF-Rp55. The "osteoclast
activity score," which takes into account the activity per osteoclast
and the number of osteoclasts, was significantly larger for
TNFRp55-/--IL-1RI-/- mice than for the two
other experimental groups and wild-type mice. No significant
differences were found between the scores of
TNFRp55-/--p75-/- or
IL-1RI-/- mice compared to wild-type controls.
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Table 2
demonstrates that IL-1 or
TNF receptor signaling is not required for rapid recruitment of
polymorphonuclear and mononuclear phagocytes. In fact, at day 3,
recruitment of phagocytes was observed for all groups, with
TNFRp55-/--IL-1RI-/- mice exhibiting a
higher degree of mononuclear phagocyte recruitment compared to the
others. By day 7 in the
TNFRp55-/--IL-1RI-/- mice and by day 14 in
IL-1RI-/- or
TNFRp55-/--p75-/- mice, leukocyte counts
could not be assessed because of the large degree of tissue necrosis,
demonstrating the importance of IL-1/TNF receptor signaling in
protecting the host from bacteria-mediated soft tissue necrosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF.20
We used this
model to investigate the role of IL-1 and TNF activity in the
protecting the host from a mixed, largely gram-negative, anaerobic
infection. Furthermore, the experimental groups allowed us to directly
assess whether IL-1 or TNF receptor signaling was essential for the
induction of bacteria-induced osteoclastogenesis. IL-1 and TNF are produced in response to infectious organisms, both in vitro and in vivo. Once produced, they may exert a beneficial or deleterious effect, depending on the quantity in which they are produced and the time period over which production is sustained. High systemic levels of IL-1 or TNF may create morbidity by the induction of shock.5,6 We found that IL-1 and TNF individually represent critical cytokines in protecting the host from mixed anaerobic infections. Without IL-1 or TNF receptor signaling, bacteria were able to penetrate the loose connective tissue with much greater ease, as determined by bacterial colony counts from infected tissue and from observing the rate of necrosis caused by infection. Interestingly, IL-1 receptor signaling and TNF receptor signaling were both important, with little difference noted between them. However, in mice deficient in both IL-1 and TNF receptor signaling (IL-1R-/--TNFR-p55-/-), the consequence of bacterial infection was more severe and more rapid. It should also be noted that the IL-1R-/--TNFR-p55-/- mice possessed functional TNFR-p75, which has been shown to reduce inflammation.12 A direct comparison of TNFR-p55-/- and TNFR-p75-/- mice, which was not undertaken here, would be required to establish the impact of TNFR-p75 on the progression of an anaerobic bacterial infection, the host response, osteoclastogenesis, and bone loss in this model.
IL-1 and TNF are prototypic cytokines that stimulate osteoclast formation and activity. In animals lacking functional IL-1 or TNF receptors there was a dramatic increase in bone destruction, as evidenced by enhanced osteoclastogenesis, a shift toward deeper Howship’s lacunae, and loss of bone mass. From these results it is clear that bone resorption can occur independently of IL-1R-1 and TNF-Rp55/p75 receptor signaling. The finding was surprising given that IL-1 and TNF have been shown to play prominent roles in bone loss associated with estrogen deficiency in mice21 and that TNF has been suggested to be an essential mediator of LPS-induced bone loss.8 Furthermore, we have recently reported that IL-1 and/or TNF have been shown to play central roles in bacteria-induced experimental periodontitis.9 Thus results presented here indicate that a mixed anaerobic infection can induce the production of mediators other than IL-1 or TNF that are capable of initiating severe destruction of bone. Thus it is likely that IL-1 or TNF participates in osteoclastogenesis in many inflammatory processes, but under certain conditions such as anaerobic infection, other mediators are more important. The process of osteoclast differentiation is not fully understood, but numerous factors have the potential to induce osteoclast differentiation and bone resorption, including arachidonic acid metabolites, IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor and osteoclast differentiation factor.3,4
Polymorphonuclear leukocytes are particularly important in protecting the host from bacterial infection. The area over which polymorphonuclear leukocytes accumulate typically reflects the degree to which bacterial contamination has spread.22 We noted that polymorphonuclear leukocyte recruitment in normal mice was concentrated in an area close to the apex of the tooth root, whereas in IL-1 receptor- and/or TNF receptor-deficient mice, a dense polymorphonuclear leukocyte infiltrate was spread over a relatively large area. This would suggest that IL-1 and/or TNF are important in localizing an infection and restricting its spread. We also found that there was rapid and equivalent accumulation of both mononuclear and polymorphonuclear leukocytes in normal and experimental mice. Thus formation of an inflammatory cell infiltrate occurs efficiently without IL-1 or TNF activity and suggests that endothelial changes necessary for leukocyte recruitment occur without activation by these cytokines. However, in their absence, there was greater tissue damage and bacterial penetration, indicating a diminished capacity to clear a mixed anaerobic infection. In vitro studies suggest that monocyte products such as IL-1 or TNF may represent important components in stimulating the antimicrobial activity of neutrophils during infection.23,24 Future studies may determine whether there is a diminished antimicrobial function of neutrophils in IL-1 receptor- or TNF receptor-deficient mice in vivo, and whether this plays a role in their enhanced susceptibility to mixed anaerobic infection.
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Acknowledgements |
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Footnotes |
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Supported by a grant from the National Institute of Dental and Craniofacial Research (DE07559).
Dr. Chen’s present address is Dental Department, Tri-Service General Hospital and School of Dentistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Accepted for publication August 24, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated neutrophils. J Immunol 1993, 151:4821-4828[Abstract]
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