Possible Role of Calcium in Periodontal Disease*Joseph J. Aleo, Harish Padh, and Appian Subramoniam

Accepted for publication 27 February 1984

The uptake of Ca2+ by endotoxin-challenged 3T6 fibroblasts, in vitro, was studied. In recentyears, the role of calcium in cell injury ultimately leading to cell death has attracted a fairamount of interest. The purpose of the study was to determine whether the direct toxicaction of endotoxin is related to a disturbance in Ca2+ homeostasis. Increased calciumuptake in endotoxin-challenged cells was found to be directly related to the bacterial source

and method of extraction of endotoxin, the cell density of the culture and the pH of themedium. The effect of endotoxin on calcium uptake was completely reversed by polymyxin which is known to neutralize the endotoxicity of lipopolysaccharides. These results implythat the increased calcium uptake may be one of the mechanisms by which endotoxin causesdirect tissue damage. The potential significance of these data to periodontal disease isdiscussed.

It is generally accepted that bacterial endotoxins,potent inflammatory agents, are implicated in some

way(s) in the etiology of inflammatory periodontal dis-ease. Some of the more common indirect deleteriousbiologic effects of endotoxins include activation of thealternate pathway of the complement system,1 promo-tion of polymorphonuclear neutrophil (PMN) Chemo-taxis,2 bone résorption in vitro,3 alteration of the vaso-constrictive properties of catecholamines,4 stimulationof collagenase synthesis by macrophages,5 initiation ofB-lymphocyte blastogenesis6 and many more.

In 1974 Aleo, et al. demonstrated for the first timethat endotoxin and/or endotoxin-like products arefound in the cementum of periodontally involvedteeth.7 The direct cytotoxic effects of these products, invitro, on mouse and human gingival fibroblasts includea limiting of cellular proliferation, a lethal effect whencells are exposed to high concentrations, a preventionor limitation of cellular attachment to root surfaces8and an alteration of cellular organdíes.9

It has long been suspected that these direct cytotoxiceffects may be the result of interaction of endotoxinwith cell membranes. Although the mechanism of thisinteraction is poorly understood and ill-defined,10"'3 a

recent in vitro study14 showed that endotoxins alteredthe surface of several different cell types functionallyand morphologically. These alterations suggest that cel-lular responses to endotoxins, direct or indirect, maydepend almost entirely upon an initial interaction ofendotoxin with cell membranes.

In recent years the role of calcium in cell injury* Supported by National Institute of Dental Research Grant DE

05766 and in part by Hoffmann-La Roche, Inc.

ultimately leading to cell death has attracted consider-able attention stemming principally from the frequentfinding of high levels of calcium leading to calcificationin tissues that have undergone necrosis.15 A number ofstudies appear to support these observations and suggestthat the accumulation of intracellular free calcium maybe common in all injured cells and under certain con-ditions may lead to cell death.16"19

Intracellular concentrations of calcium are approxi-mately -7 m while the extracellular concentrationsare about 10"3 m. If direct toxic effects of endotoxininvolve alterations or injury to the plasma membrane,then one might hypothesize that the homeostasis ofintracellular calcium would be altered by the sheerpresence of the concentration gradients alone even

though, mechanistically, other factors may be involved.Since alterations in calcium homeostasis are capable ofdisrupting cellular functions such as oxidative phospho-rylation pathways,20 structural proteins such asmicrotubules21 and calcium-sensitive enzyme sys-tems,22 one might expect that excessive or prolongedcellular calcium influx not compensated by adequateefflux would produce adverse physiologic changes lead-ing ultimately to cell death.

The purpose of this investigation was to compare thereported direct toxic effects of endotoxin7 with calicumuptake and to correlate any alteration in calcium ho-meostasis with the possible etiology and pathogenesisof periodontal disease.

MATERIALS AND METHODS

Plastic tissue culture flasks having a 25-cm2 growthsurface were used to grow 3T6 fibroblasts in Dulbecco-

642

Volume 55Number 11 Possible Role ofCalcium 643

Vogt modification of Eagles medium with 10% heat-inactivated calf serum and 100 Mg/ml gentamycin inan atmosphere of 7.5% CO2. Depending upon thenature of the experiment to be performed, serial flaskswere seeded with a variety of cell densities and grownfor a series of time intervals. After the growth mediumwas removed, the attached cells were incubated in freshgrowth medium with or without endotoxin for 4 hoursat 37°C, endotoxin from a variety of bacterial sourcesand a variety of extraction procedures was used. Endo-toxin was purchased commercially from Sigma. At theend of the 4-hour incubation period, the medium wasremoved and replaced with fresh medium containing45CaCl2 (0.7-0.9 µ /µ , New England Nuclear), withand without endotoxin, and incubated at 37°C for 2.5minutes (unless otherwise stated). The pH of the me-dium was between 7.3 and 7.5.

Incubations were stopped by removing the radioac-tive medium and washing the cell sheet 4 times with 5ml of ice cold buffer. The washing buffer was composedof 155 mM NaCl, 7 min KCl, 0.8 ihm MgCl2, 1.8 mMCaCl2 and 25 mM HEPES. The pH was adjusted to 7with NaOH at 4°C. The washed cell sheet was digestedby adding 2.5 ml 0.4 NaOH. After standing overnight,an aliquot (2 ml) of the dissolved cells was mixed with10 ml of Aquasol (New England Nuclear) in a scintil-lation vial. Radioactivity was determined by liquidscintillation spectrometry using a Beckman 150 scintil-lation counter.

In studying the influence of pH on 45Ca2+ uptake,

incubations were carried out in the HEPES mediumdescribed above with the following modifications using145 mM NaCl and adding 20 mM glucose. The pH ofthe medium was adjusted with NaOH to the desiredpH. The cells were incubated in this HEPES mediumfor 1 hour after being grown in regular growth medium.Following this pre-incubation in HEPES medium,endotoxin in 0.2 ml of saline was added to the experi-mental flasks and an equal volume of saline was addedto the control flasks, with both being incubated for 4hours at 37°C in air. The medium was then changedwith fresh 45CaCl2 HEPES medium and incubated for2.5 minutes. The incubation was terminated and 45Ca2+influx was determined as described above. Total proteinin all experiments was determined by the method ofBradford23 using bovine serum albumin as a standard.

RESULTS

Time Course of Calcium UptakeThe uptake of 45Ca2+ by 3T6 fibroblasts in the culture

system described was rapid for the first 30 minutes ofincubation, reaching a steady state beyond this point(Fig. 1). The steady state denotes a balance betweeninflux and efflux. To minimize the influence of effluxon the uptake studies, all experiments were performedusing a standardized 2.5-minute exposure of the cellsto the radioactive medium. This proved to be the mosteffective and shortest time interval necessary to performall the technical aspects related to the procedure.

o Time Course of ^Ca Uptake

O 4 10 20 30Time (minutes)

60

Figure 1. The culture medium described in the Material and Methods section was usedfor the i5CaCl2 incubation. The approximate cell densitywas 6 to 7' x 10* cells/flask and the pH of the medium was 7.1 to 7.4. Each point represents the mean ± standard deviation.

644 Aleo, Padh, Subramoniam J. Periodontol.November, 1984

Calcium Uptake: Effect of EndotoxinWhen the cultured cells were exposed to a range of

endotoxin concentrations, the increased uptake of45Ca2+, under the conditions described, was directlyproportional to the concentration of endotoxin in themedium (Fig. 2). Concentrations larger than 200 /xg/ml of endotoxin were not used because they wouldseriously affect cell viability.7 To maximize any effectof endotoxin on cellular 45Ca2+ uptake, 200 ßg of endo-toxin per ml of culture medium was used in all exper-iments unless otherwise indicated.Calcium Uptake: Effect of Various Endotoxins

Previously unpublished data from this laboratoryhave shown that the effect of endotoxin will vary withthe bacterial source of the endotoxin and the methodof extraction. It is also conceivable that commerciallyobtained endotoxin may differ from lot to lot and anylong-range study should consider this possible differ-ence. These factors no doubt contribute substantiallyto the many differences in data in similar studies re-ported in the literature.

Of the four endotoxins purchased commercially(Sigma), Escherichia coli 0127:P>8, butanol extract,proved to be the most effective and was used in allexperiments. Differences attributable to methods ofextraction can be seen when E coli 055:B5 TCA extractis compared with the phenol extract (Fig. 3).Calcium Uptake-Endotoxin Challenged Cells: Effect ofCell Density

Although some differences were expected in 45Ca2+

uptake in different cell densities, the magnitude of thedifferences was not anticipated (Fig. 4). Cell densitywas the only factor involved in this series ofexperimentssince all other conditions such as endotoxin, pH, etc.were consistent with previous experiments. The possi-ble role of physical factors and/or cellular metabolicchanges at different cell densities would suggest thatthere are population-dependent requirements for cul-tured cells which must be considered in any in vitrostudy.Calcium Uptake-Endotoxin Challenged Cells: Effect ofPolymyxin

It has long been recognized that polymyxin sulfatecan protect against some of the toxic manifestations ofendotoxin.24 An unexpected finding was that poly-myxin itself caused an increase in 45Ca2+ influx whichwas dose-responsive (Fig. 5). The combination of poly-myxin and endotoxin, however, caused a markedreduction in 45Ca2+ uptake, approaching control levels.Polymyxin is known to inactivate endotoxin by form-ing a stable complex with the toxic lipid A region ofendotoxin25 and it appears from these data that thecomplex also inactivates the action of polymyxin B.Calcium Uptake-Endotoxin Challenged Cells: Influenceof pH

The pH of the culture medium appears to be a criticalfactor in 45Ca2+ uptake. In this study, selection of a pHrange for testing had to be in the range compatible withcell viability. The increase of 45Ca2+ uptake in endo-toxin-challenged cells was proportional to the increase

Calcium Influx: Effect of Endotoxin

50Endotoxin

100Concentration

200(ug/ml)

Figure 2. 45Ca2+ uptake by endotoxin-treated cells (E coli 055: B5, TCA extract, Sigma). Cells were pre-incubated in normal growth mediumwith different concentrations ofendotoxin followed by a 2.5-minute incubation in *5 Ca2*-containing medium al a pH of 7.2 to 7.4. The cell densitywas 3 to 4 106 cells/flask. Each point represents the average ± standard deviation of three separate experiments.

Volume 55Number 11

IO

c

Possible Role ofCalcium 645

Calcium Influx: Effect of various Endotoxins

1. Control100

3 80 +

te

<ft

co

·*->ft3

60 +

40 +

30 +

1 2. E. coli 0127:B8butanol extract

3. E. coli 055:B5 C A extract

4. E. coli 055:B5

5. S. minisota C A extract

1 * 3 4 5Figure 3. Cells were pretreated with various endotoxins (200 ßg/ml) for 4 hours in normal growth medium followed by a 2.5-minute incubationin i5Ca2*-containing medium at a pH of 7.2 to 7.5. The cell density was 3 to 4 106 cells/flask. Each bar represents the mean ± standarddeviation.

Calcium Influx: Endotoxln-Challenged CellsEffect of Cell Density

_200 +ou

-(->E

O 150o

*«.

100 «

Pl=3

50 -

.75 3.5 7.0 14.0Cell Density (Millions)

Figure 4. Cells were pre-incubated with 200 ßg/ml endotoxin (E coli0127:B8, butanol extract, Sigma) in HEPES medium described inthe Material and Methods section, followed by a 2.5-minute incuba-tion in fresh HEPES medium at pH 7.4 containing^Ca2*. Each pointrepresents per cent ofcontrol ± standard deviation.

in medium pH, whereas the control cells reflected a

quite different behavior. The increase was greatest inendotoxin-challenged cells and least in control cells atpH 7.8; the nature of this pH effect is unknown but itsuggests that the potential for calcium influx-associated-cellular-damage is greatest in endotoxin-treated cells asthe pH is increased (Fig. 6).

DISCUSSIONIf one accepts the role of calcium in cell injury,

several observations from these data are of potentialimportance in inflammatory periodontal disease. Thefirst is that the increase in calcium influx in endotoxin-challenged cells was directly proportional to the con-centration of endotoxin, which suggests a possible dose-response direct injury to the plasma membrane or someinvolvement of endotoxin with calicum transport itself.Polymyxin Bs reversal of the endotoxin effect supportsthe role of the antibiotic in neutralizing the endotoxicityof lipopolysaccharides. Whether polymyxin acts bybinding directly to endotoxin alone or by interferingwith the interaction ofendotoxin with the plasma mem-brane could not be determined. It was also observedthat the effect of endotoxin varied with the bacterialsource and method of extraction; this variation was

only in degree and not in overall effect. These obser-vations are not new but merely confirm the wide rangeof results when comparing a series of experiments andthe data reported in the literature.

Another observation was that the calcium influx inendotoxin-challenged cells decreased as cell density in-creased. In a tissue culture system, this may only reflecta level of cellular metabolism in which the endotoxineffect is minimal or in which, as cell-to-cell contactincreases at high cell densities, there is a limitation ofcellular plasma membrane surface area available forendotoxin interaction. Lastly, the effect of pH appearedto be the key factor in cellular calcium influx. Inconsidering tooth-tissue-plaque, the direct toxic effects

646 Aleo, Padh, Subramoniam J. Periodontol.November, 1984

Calcium Influx : Endotoxi -Chailenged CellsEffect of Polymyxin Sulphate

a so

-t->ot*ft 60to

Í40H©

ß

§20-|«

«0°* 0

3.

rfii

i

1. Control2. Endotoxin treated

200 ug/mlPolymyxin 25 ug/mlPolymyxin 50 ug/ml(2+4)(2+3)

4.

5.6.

f

1 2 3 4 5 6Figure 5. Polymyxin sulfate (PBS) was dissolved in saline (1.25 or 6.25 mg/ml). To study the effect ofPBS binding to endotoxin, PBS wasdissolved in endotoxin containing saline (5 mg/ml) to give a ratio of 1:4 or 1:8 PBS.endotoxin and incubated for 15 minutes at 37°C. Then 0.2ml of this solution was added to each culture (4.8 ml) which gave a final concentration of200 ßg/ml endotoxin and 25 or 50 ßg/ml PBS. The celldensity was 3.5 x 106/flask, pH 7.8. Data are normalized to per cent ofcontrol and are mean ± standard deviation ofduplicates or triplicates.

Calcium Influx: Endotoxi -Chailenged CellsInfluence of pH

2 60

50o

ß.

»40

30+

S 20+•fr*

ê

]]ControlliliExperimental

6.6 7.0 7.4 7.8pH

Figure 6. Cells were pre-incubaled with 200 ßg/ml endotoxin (E coli 0127: B8, butanol extract, Sigma) in HEPES medium at different pHs,followed by a 2.5-minute incubation in fresh HEPES medium containing 4>CaCl2. The cell density was approximately 7 x 106 cells/flask. Eachbar represents the mean ± standard deviation.of endotoxin may depend upon the ultimate effects onplaque pH of the many variables found in the plaqueenvironment.

Several excellent reviews cover plaque, plaque for-

mation, metabolism and the influence of plaque ondental caries and periodontal disease. Suffice it to saythat the dissolution and destruction of tooth structureby acids result from the metabolism ofcarbohydrate by

Volume 55Number 11

plaque microflora. In other situations, the formation ofbase and a high pH in plaque results from the metab-olism of nitrogenous substances, most notably urea.28,29Kleinberg and Hall30 reported that base formation inplaque is associated with calculus formation and softtissue destruction resulting in inflammatory periodon-tal disease. They also correlated the depth of gingivalcrevices with supragingival plaque pH and found thatthe deeper crevices were in those areas of the dentitionwhere plaques had a higher pH. They concluded thatthe factors influencing higher pH are probably respon-sible for deeper crevices.

Although the regulation of acid-base metabolism ofdento-gingival plaque is admittedly complex and in-volves many factors, the fact remains that a higher pHenvironment is associated with a deeper gingival crev-

ice; the deeper crevice denoting tissue destruction. ThepH of supragingival calculus associated with thesedeeper crevices was reported to range somewhere be-tween pH 7-8.26 This pH range correlates well with thecalcium influx findings of this study. The cellular influxof calcium was greatest in endotoxin-challenged cells inthis pH range, reaching a maximum at pH 7.8. If,indeed, an increased calcium influx is involved in cellinjury ultimately leading to cell death, the conditionsdescribed in plaque associated with inflammatory peri-odontal disease appear to be ideal for the increasedinflux of calcium in endotoxin-challenged tissues andsuggest a possible mechanism for the direct toxic actionof endotoxin.

ACKNOWLEDGMENTSThe authors wish to acknowledge the valuable technical assistance

of Mr. Alex Mucha and Miss Susan Howell.

REFERENCES1. Gewürz, H.: Alternate pathways to activation of the comple-

ment system. D. G. Ingram (ed), Biological Activities ofComplement,pp 56-88. New York, S. Karger, 1972.

2. Snyderman, R.: Humoral and bacterial mediators of inflam-mation. S. E. Mergenhagen and H. W. Sherp (eds). ComparativeImmunology of the Oral Cavity, pp 183-191, United States Depart-ment of Health, Education and Welfare, 1973.

3. Hausmann, E.: Potential pathways for bone résorption in hu-man periodontal disease. J Periodontol 45: 338, 1974.

4. Taichman, N. S., and Courant, P.: The production of hemor-rhagic necrosis in the hamster cheek pouch by bacterial endotoxinand catecholamines. Arch Oral Biol 10: 541, 1965.

5. Wall, L. M., Wall, S. M., Mergenhagen, S. E., and Martin, GR.: Collagenase production by endotoxin-activated macrophages.Proc Nat Acad Sci USA 71, 3598, 1974.

6. Rosenstreich, D. L., Glode, L. M., and Mergenhagen, S. E.:Action ofendotoxin on lymphoid cells. J Infect Dis (suppl.) 136: 239,1977.

7. Aleo, J. J., DeRenzis, F. ., Farber, P. ., and Varboncoeur,A. P.: The presence and biologic activity of cementum-bound endo-toxin. J. Periodontol 45: 671, 1974.

8. Aleo, J. J., DeRenzis, F. ., and Färber, P. .: In vitro attach-ment of human fibroblasts to root surfaces. J Periodontol 46: 639,1975.

9. Lucas, R. M., Chen, S. Y., and Aleo, J. J.: Histochemical study

Possible Role ofCalcium 647

of strain L fibroblasts exposed to endotoxin. J Periodontol 50: 20,1979.

10. Bradley, S. G.: Cellular and molecular mechanisms of actionof bacterial endotoxins. Annu Rev Microbiol 33: 67, 1979.

11. Bona, C, Chedid, L., and Lancensans, .: In vitro attachmentof radioactive endotoxins to lysosomes. Infect Immun 4: 532, 1971.

12. Springer, G. F., Adye, J. C, Bezkorovainy, ., and Murthy,J. R.: Functional aspects and nature of the lipopolysaccharide-recep-tor of human erythrocytes. J Infect Dis 128: 202, 1973.

13. Springer, G. F., Adye, J. C, Mergenhagen, S. E., and Rosen-streich, D. L., D. Schlessinger (ed). Microbiology, 326. Washington,DC American Society for Microbiology. 1977.

14. Kubasova, T., Somosy, Z., Bertok. L., and Köteles, G. J.:Effect of endotoxin and radio-detoxified endotoxin on cell mem-

branes in vitro. Acta Microbiol Acad Sci Hung 29, 235, 1982.15. Färber, J. L.: The role of calcium in cell death. Life Sci 29,

1289, 1981.16. Farber, J. L., El-Mofty, S. K., Schanne, F. A. X., et al.:

Intracellular calcium homeostasis in galactosamine-intoxicated ratliver cells. Arch Biochem Biophys 178: 617, 1977.

17. Schanne, F. A. X., Kane, A. B., Young, E. E., and Farber, J.L.: Calcium dependence of toxic cell death: a final common pathway.Science 206: 700, 1979.

18. Casini, A. F., and Färber, J. L.: Dependence of the carbontetrachloride-induced death of cultured hepatocytes on the extracel-lular calcium concentration. Ann J Pathol 105: 138, 1981.

19. Chenery, R., George, M., and Krishna, G.: The effect ofionophore A23187 and calcium on carbon tetrachloride-inducedtoxicity in cultured rat hepatocytes. Toxicol Appi Pharmacol 60: 241,1981.

20. Bygrave, F. L.: Mitochondrial calcium transport. Curr TopBioenergó: 259, 1977.

21. Marcum, J. M., Dedma, B., Brinkley, R., and Mean. A. R.:Control of microtubule assembly-disassembly by calcium-dependentregulator protein. Proc Nat Acad Sci USA 75: 3771, 1978.

22. Wong, P. Y. K., and Cheung, W. Y.: Calmodulin stimulateshuman platelet phospholipase A2. Biochem Biophys Res Commun90:473, 1979.

23. Bradford, M. M.: A rapid and sensitive method for the quan-titation of microgram quantities of protein utilizing the principle ofprotein-dye binding. Anal Biochem 72: 248, 1976.

24. Morrison, D. C, and Jacobs, D. M.: Bending of polymyxin to the lipid A portion of lipopolysaccharides. Immunochemistry 13:813, 1976.

25. Cooperstock, M. S.: Inactivation of endotoxin by polymyxinB. Antimicrob Agents Chemother 6: 422, 1974.

26. Kleinberg. I.: Regulation of the acid-base metabolism of thedento-gingival plaque and its relation to dental caries and periodontaldisease. Int Dent J 20: 451, 1970.

27. Stephan, R. M.: The effect of urea in counteracting the influ-ence of carbohydrates on the pH of dental plaques. J Dent Res 22:63, 1943.

28. Kleinberg. I., Kanapka, J. ., Chatterjee, R., et al.: Metabolismof nitrogen by the oral mixed bacteria. I. Kleinberg, S. A. Ellison andI. D. Mandel (eds). Saliva and Dental Caries, pp 357-377, New York,Information Retrieval, Inc, 1978.

29. Kleinberg, I., and Hall, G.: pH and depth of gingival crevicesin different areas of the mouths of fasting humans J Periodont Res 4:109, 1969.

30. Kleinberg, I., Kanapka, J. ., and Craw, D.: The effect ofsaliva and salivary factors on the metabolism of the mixed oral flora. . M. Stiles, W. J. Loesche, and T. C. O'Brien (eds), MicrobialAspects ofDental Caries, pp 433-464, Washington, DC, InformationRetrieval, Inc, 1976.

Send reprint requests to: Dr. Joseph J. Aleo, Department ofPathology, Temple University School of Dentistry, 3223 North BroadSt, Philadelphia, PA 19140.