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Studies of the mechanisms of pathogenesis of periapical lesions were undertaken using a rat model of surgical pulp exposure. In this model, periapical lesions develop rapidly between days 0 and 15 (active phase) and more slowly thereafter (chronic phase). A Gram-negative anaerobic flora, similar to that seen in human beings, are quickly established. Lesions contain a mixed inflammatory cell infiltrate consisting of T cells, neutrophils, B cells, macrophages, and plasma cells. Helper T cells predominate during the active phase, whereas suppressor T cells are more frequent in the chronic phase. Extracts of periapical lesions contain bone-resorbing activity, the highest levels of which are present when lesions are actively expanding. Most bone-resorbing activity is mediated by the cytokine interleukin-1 alpha, as determined by biochemical criteria and antibody neutralization studies. Prostaglandin2 accounts for 10% to 15% of resorptive activity. Cells that express interleukin-1 alpha were identified in pulp beginning on day 2 after exposure and in periapical tissue beginning on day 7, as determined by in situ hybridization and immunostaining. Macrophages, fibroblasts, neutrophils, and osteoclasts were positive for interleukin-1 alpha mRNA and protein. Cells that express tumor necrosis factor alpha were also detected, whereas cells expressing interleukin-1 beta or tumor necrosis factor beta were absent. Finally, periapical bone destruction was inhibited by 60% by treatment with interleukin-1 receptor antagonist. These studies establish a key role for interleukin-1 alpha in the pathogenesis of periapical lesions in the rat model.

Extracts of human periapical granulomas were tested for the presence of bone-resorbing activity. All granulomas (10 of 10) contained low but significant levels of bone-resorbing activity, ranging from 2.1 to 4.9% treatment-% control/mg specific 45Ca release, as determined by the fetal rat long bone assay. Healthy periodontal ligament and dental pulp had no significant resorbing activity. In characterization studies, the resorbing activity in an extract pool was unaffected by the presence of polymyxin B, indicating an active moiety distinct from lipopolysaccharide. Resorbing activity was also unaffected by heating to 56 degrees C for 30 min, but was completely abolished by proteinase K treatment or heating to 70 degrees C, indicating that activity was largely protein mediated. Fast performance liquid chromatography gel filtration studies demonstrated that activity could be resolved to two major peaks, of M(r) 30,000 to 60,000 (I), and 15,000 to 20,000 (II), with a minor peak present at < 1,000 (III). Peak III was identified as prostaglandin E2 by radioimmunoassay. In inhibition studies, virtually all of the resorbing activity present was inhibited by anti-interleukin 1 beta (69%) and anti-tumor necrosis factor beta (66%) antisera, whereas anti-interleukin 1 alpha and antitumor necrosis factor alpha had no effect. Treatment with the cyclooxygenase inhibitor indomethacin also reduced activity by 74%. Taken together, these data demonstrate that most bone-resorbing activity present in chronic human periapical lesions is attributable to the action of resorptive cytokines interleukin 1 beta and tumor necrosis factor beta, acting via both indomethacin-dependent and independent pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

To identify the mediators that stimulate periapical bone resorption following infection, a rat model system was used in which active (rapid) and chronic (slow) phases of bone destruction can be distinguished. Extracts of inflammatory tissues from active lesions contained high levels of bone-resorbing activity, which was destroyed by proteinase K and heat (70 degrees C), but was unaffected by polymyxin B, indicating the presence of protein mediator(s) rather than lipopolysaccharide. Fast-performance liquid chromatography gel filtration of extracts of active lesions demonstrated that most activity was associated with macromolecules of MW 30-60 kDa and 15-20 kDa, consistent with bone resorptive cytokines, including interleukin 1 (IL-1) and tumor necrosis factor (TNF). Inhibition with cytokine-specific antisera demonstrated that resorbing activity in active lesions was significantly neutralized by anti-IL-1 alpha, whereas anti-IL-1 beta, anti-TNF alpha and anti-TNF beta had only slight effect. A lower amount of resorbing activity was present in extracts of chronic lesions, which was also neutralized only by anti-IL-1 alpha. Inflammatory tissue explants produced more IL-1 alpha than IL-1 beta in vitro, confirming findings with extracts, and high levels of IL-1 alpha were present in active lesions by radioimmunoassay. These data indicate that bone resorption stimulated by bacterial infection is primarily mediated by IL-1 alpha in this model. The similarity of cytokines in active and chronic lesions suggests that quantitative rather than qualitative differences in these mediators may account for lesion progression.

Infection of the dental pulp stimulates a host immune response in the periapical region (the periapical "lesion") with the concomitant resorption of bone. The cell composition of human or rat periapical lesions is mixed, consisting of T, B, and "null" lymphocytes, plasma cells, macrophages, polymorphonuclear leukocytes, and mast cells. Cells are thus present which mediate a broad spectrum of immunological phenomena. In order to determine which of these mechanisms contribute to periapical bone resorption, a rat model system has been used in which periapical lesions are induced by pulp exposure and infection from the oral environment. In this model, a rapid period of lesion expansion and bone destruction occurs between days 1 and 15 after exposure (active phase), with a chronic phase characterized by lesion stabilization commencing thereafter. Phenotypic analysis of the cellular infiltrate has shown that T helper cells predominate during the active phase, whereas increased numbers of T suppressor cells are associated with chronicity. Extracts of periapical lesions contain bone-resorbing activity, as determined by the release of 45Ca from prelabeled a rat long bones in organ culture. Higher levels of bone-resorbing activity are present during the active than in the chronic phase. Characterization of bone-resorbing activity indicates the presence of resorptive mediators which are heat labile and protease sensitive and which are distinct from lipopolysaccharide. Preliminary biochemical fractionation indicates that resorptive mediators fractionate in the 15,000- to 60,000-dalton molecular mass range, presumably cytokines such as interleukin 1 and tumor necrosis factor. These studies demonstrate the utility of the rat model for studying mechanisms of periapical lesion pathogenesis and implicate T helper cell-mediated activities, in particular those leading to macrophage activation and cytokine production, in periapical lesion expansion.

The mechanism of bone destruction in periapical lesions was studied using a rat model system. Periapical lesions were induced by pulp exposure and infection from the oral environment. Lesions expanded most rapidly between induction on day 0 and day 15 ("active phase"), with enlargement occurring at a slower rate thereafter (days 20 and 30, "chronic phase"), as assessed by radiography and automated image analysis. Pooled extracts of day 15 periapical tissues contained significant levels of bone resorbing activity (BRA), as determined by 45Ca release from fetal rat long bones. Normal rat dental pulp and periodontal ligament contained no activity. In kinetic experiments, highest levels of BRA were detected in active phase tissues on days 10 and 15, with BRA declining thereafter in chronic phase tissues to near baseline levels by day 30. In characterization studies, BRA in pooled day 15 tissue extracts was unaffected by treatment with polymyxin B, but was completely abolished by proteinase K treatment or heating to 70 degrees C, indicating an active moiety distinct from bacterial LPS, probably a protein(s). FPLC gel filtration chromatography revealed that BRA could be resolved into four major peaks, of MW 30-60 kD (Peak I); 15-20 kD (II); 1-2 kD (III); less than 1kD (IV), consistent with the presence of the following bone resorptive mediators: (I) cytokines TNF alpha, TNF beta and/or unprocessed IL-1 alpha; (II) processed IL-1 alpha and/or IL-1 beta; (IV) PGE2. These findings demonstrate that bone resorbing activity is temporally related to bone destruction, and that the activity present during the rapid phase of periapical lesion expansion is primarily attributable to bone resorptive cytokines.

Interleukin 1 beta is a potent bone resorptive cytokine which also mediates soft tissue destruction through the stimulation of prostaglandin production, and the induction of collagenase and other proteases. This constellation of activities suggests a role for IL-1 beta in the pathogenesis of human periodontitis. Levels of IL-1 beta were therefore determined in tissue obtained from (1) diseased, active (2) diseased, inactive and (3) healthy sites from 12 patients with destructive adult periodontitis. Disease activity was defined as attachment loss of greater than or equal to 2.5 mm, as determined by sequential probing and the tolerance method. IL-1 beta was extracted from homogenates of tissue biopsies taken at surgery, and levels were quantified by ELISA. IL-1 beta was found to be present in most patient tissue samples, with levels ranging from 0-82 ng/ml. Disease active sites had higher IL-1 beta levels (p less than 0.05) than inactive and healthy sites. Diseased inactive sites were divided into 2 groups, those losing small amounts of attachment (0.5-2.0 mm, worsening sites) and those which showed no change or attachment gain (stable sites). Stable diseased sites had IL-1 beta levels which were comparable to those found in healthy sites, and which were significantly different from active sites (p less than 0.02). Worsening sites had IL-1 beta levels intermediate between the levels in stable and active sites. Detection of disease activity occurred more frequently at sites with IL-1 beta levels greater than 25 ng/ml (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 beta (IL-1 beta) is the predominant form of IL-1 produced by macrophages. IL-1 beta possesses numerous and diverse biological activities. Several of these activities, including fibroblast proliferation, potentiation of the immune response, and stimulation of bone resorption may be of relevance to the pathogenesis of periodontal disease. This study was designed to examine the presence of IL-1 beta in human periodontal tissue. An antiserum directed against the N-terminal segment (117-131) of human IL-1 beta was used to detect IL-1 beta using immunofluorescent staining techniques. IL-1 beta positive staining cells were observed in both normal and diseased tissue and were limited to the lamina propria. Brightly staining cells were increased by almost 3-fold in periodontally diseased tissue when compared to normal tissue. Low intensity staining cells were equally distributed in the normal and diseased specimens. We propose that IL-1 beta and IL-1 beta produced by cells in periodontal tissues may be related to the pathological processes associated with periodontal disease.

The effect of interleukin-1 beta, the major component of osteoclast-activating factor (OAF), on bone formation by fetal rat osteoblast-rich cells was investigated. An in vitro culture system developed by Ecarot-Charrier et al. (1983) and Bellows et al. (1986) was utilized in which osteoblasts form mineralized nodules which closely resemble woven bone. Continuous exposure of cultures to homogenous IL-1 beta resulted in potent inhibition of mineralized nodule formation, which was half maximal at 0.1 U/ml (7.5 X 10(-13) M). Bone formation may thus be considerably more sensitive to IL-1 beta than is bone resorption (half maximal at 3.8 X 10(-11) M). Inhibition of bone formation occurred when cultures were exposed to IL-1 beta at both early and late time periods and was unaffected by the presence of indomethacin or by the osteoclast inhibitors calcitonin and gamma-interferon. Instead, IL-1 beta exerted multiple inhibitory effects on osteoblast functions, including inhibition of collagen and noncollagen protein synthesis, alkaline phosphatase expression, and cell replication. On a dose response basis, the inhibition of protein synthesis correlated most closely with inhibition of bone formation. IL-1 beta is clearly inhibitory rather than stimulatory for bone formation as assessed in this system and is therefore unlikely to function as a coupling factor linking the processes of bone resorption and bone formation.

Strain 2 guinea pigs, immunized with Lys10-Lys(Dnp) in CFA and repeatedly injected intraperitoneally with adjuvant, developed ascites. The fluid was harvested over 8 months in total amounts up to 2 liters per animal and contained substantial amounts of cells and antibody which reacted with the immunizing antigen and related peptides. The antibody was of the IgG and IgA classes and showed restricted heterogeneity. Among synthetic Dnp-oligopeptides, both the cells, studied by antigen-stimulated thymidine incorporation, and the purified antibody, studied by fluorescence quenching, demonstrated the same specificity for the immunizing antigen as has previously been noted in lymph node cells and in serum antibody. The technique offers a means for studying more cells and more antibody than has previously been possible from individual guinea pigs.

A preliminary serotherapeutic trial was undertaken with a monoclonal antibody designated antibody 89 (Ab 89) directed against a lymphoma-associated antigen. In vitro studies demonstrated that Ab 89 could mediate complement-dependent lysis and macrophage adherence but not antibody-dependent cell-mediated cytotoxicity. To evaluate toxicity and therapeutic efficacy, two courses of Ab 89 were administered to a patient with an Ab 89-reactive tumor. Transient decreases in the number of circulating tumor cells and the appearance of circulating dead cells were noted with the infusion of Ab 89. Following administration of 150 mg or more of Ab 89, small amounts of antibody could be demonstrated on circulating tumor cells at a time when no free antibody was found in the serum. The inability to deliver a significant amount of Ab 89 to tumor cells in vivo is thought to be secondary to a circulating tumor antigen. Following each infusion, the amount of this blocking antigen decreased but could not be entirely cleared from the serum. This study provides preliminary evidence for the lack of clinical toxicity of a monoclonal antibody and identifies circulating blocking antigens as a significant obstacle to serotherapy.

Four monoclonal antisera-identifying antigens with the identical tissue distribution and molecular weight of previously described Ia-like antigens were characterized. Two of these antisera, I-1 and I-2, identified antigens expressed on the HLA-D/DR positive cells from all HLA heterozygous individuals. Further characterization on homozygous typing cells (HTC's) demonstrated that I-2 was not reactive with most Dw7 and Dw11 HTC's. Monoclonal antisera, termed I-LR1 and I-LR2, defined polymorphic Ia-like antigens that demonstrated restricted expression on cells from HLA heterozygous individuals. Antigen I-LR1 was expressed on cells from 60% of HLA heterozygotes and its reactivity with HTC's did not conform to any previously described monotypic or supertypic HLA-D/DR pattern. In contrast, I-LR2 was expressed on 40% of HLA heterozygotes and identified only HLA-DR3, 5 and 6 HTC's. Studies of families with HLA recombinants permitted the demonstration that the I-LR1 and I-LR2 antigens are tightly linked to the HLA-D/DR locus. These experiments permit the direct demonstration by immunoprecipitation, linkage studies, and MHC recombinant families that the p29,34 complex in man is closely linked to or is within the HLA-D/DR locus. These studies suggest that the human Ia-like antigens are more heterogeneous than previously demonstrated and that monoclonal antisera will be useful in further defining the structural, genetic, and functional characteristics of these molecules.

The lymphokine osteoclast-activating factor (OAF) was purified to homogeneity. OAF was produced by human peripheral blood mononuclear cells stimulated with concanavalin A and phorbol myristate acetate under serum-free culture conditions. OAF was purified by sequential gel filtration, ion-exchange, and reverse-phase HPLC by following bone resorptive activity. Homogeneity was indicated by the criteria of a single 17,800-dalton band on silver-stained polyacrylamide gels, a single pI 6.8 band on isoelectric focusing, and a single aminoterminal sequence. Purified OAF stimulated half-maximal release of calcium from fetal rat long bones at a concentration of approximately 0.66 ng/ml. The amino-terminal sequence of OAF was determined and found to be identical to that of interleukin 1 beta. Homogeneous OAF possessed an activity of 8.2 X 10(6) U/mg in the thymocyte proliferation assay. Because the m.w., isoelectric point, amino-terminal sequence, and specific activity in the thymocyte proliferation assay are the same for homogeneous OAF and interleukin 1 beta, we conclude that they are the same molecule, and that interleukin 1 beta is the major protein with OAF activity produced by lectin-stimulated peripheral blood mononuclear cells.

Guinea pig lymph node lymphocytes were separated into T and B cell fractions on immunoabsorbent columns. Separated cells were functionally distinct: T cells proliferated in response to ConA, PHA, soluble and alloantigen, whereas anti-Ig reagents only stimulated B cells. The in vitro proliferative response of guinea pig lymph node T lymphocytes was then shown to be highly discriminating when elicited by a series of structurally similar synthetic DNP-oligolysine antigens. Proliferation was always most extensive in response to the homologous, immunizing antigen, and less intense to cross-reacting DNP-oligolysines. Specificity of proliferation was maintained in the absence of both B lymphocytes and antibody secreting cells, suggesting that T cell recognition is not "acquired" from B cells or secreted antibody, but is a property inherent to the T cell.

The interaction of interleukin 1 (IL-1), a locally produced factor, and parathyroid hormone (PTH), a systemic factor, in stimulating bone resorption was examined using fetal rat long bone organ culture. Concentrations of IL-1 and PTH, which stimulated little bone resorption when present singly, produced marked resorption when present simultaneously. This synergistic interaction of IL-1 and PTH was not affected by the presence of the prostaglandin synthetase inhibitor indomethacin. Both interleukin 1 alpha and interleukin 1 beta were capable of producing synergy. Synergy was not produced by sequential exposure of bone to IL-1 and PTH, but required the simultaneous presence of both mediators. The leftward shift in the dose response curve of PTH produced by IL-1 may be an important mechanism controlling localized bone resorption. A role for IL-1 in stimulating bone resorption in pathologic conditions, such as arthritis and periodontal disease, is strengthened by the finding that even low concentrations of IL-1 can produce resorptive effects by synergistic interaction with PTH.

T helper (TH) and T suppressor (TS) cells were enumerated in developing rat periapical lesions in order to identify the cell type associated with the active phase of lesion formation. Experimental pulp exposures were made in all molar teeth of 29 Sprague-Dawley rats, and teeth were left open to the oral environment. On days 15, 20, 30, and 90 after induction, inflammatory cells were isolated from periapical lesions, and T helper (W3/25+), T suppressor (OX8+), and total T cells (W3/13+) were enumerated by reactivity with monoclonal antibodies. During the early, most active phase of lesion development (day 15), TH cells outnumbered TS cells (TH/TS = 1.7). However, by day 20, when lesion expansion had slowed, the TH/TS ratio was reversed (0.9) and remained so for up to 90 days after induction (TH/TS = 0.7). Total T cell numbers did not change over the period of observation. TH cells thus predominated during the active phase of lesion development, whereas TS cells were associated with the chronic phase. This dynamic TH/TS reversal indicates that immune responses which occur within periapical lesions are highly regulated. In addition, the predominance of TH during the active phase of lesion development suggests that TH-mediated activities may be involved in the bone destruction and immune cell activation within periapical lesions.