European Journal of Clinical Investigation ( 1 992) 22, 659-66 1

In vitro immune modulation of polymorphonuclear leukocyte adhesiveness by sodium fluoride J. L. GOMEZ-UBRIC, J. LIEBANA, J. GUTIERREZ & A. CASTILLO Department of Microbiology, School of Odontology, University of Granada, E- 1807I Granada, Spain Received 6 December 1991 and in revised form 8 May 1992; accepted 21 May 1992

Abstract. We investigated the influence of sodium fluoride on polymorphonuclear leukocyte (PMN) adhesiveness in a healthy subject with low serum levels of fluoride. The PMN were separated from venous blood, and the percentages of adhered and unadhered cells were determined in ziitro in plastic culture plates. The cells were cultured with five different fluoride concentrations ranging from 6.25 to-? p~ to 4.0 p~ in the presence and absence of autologous serum. PMN adhesiveness in both the presence and in the absence of autologous serum was 98.5%; the addition of fluoride had no effect on the results in the absence of serum. However, in the presence of autologous serum, PMN adhesiveness decreased significantly with the addition of fluoride ( P < 0 . 0 5 ) from 0.5 p ~ The . decrease was smaller ( 1 . 1 I%) at a concentration of 0.125 PM, and larger at 12 times this concentration of fluoride (52.7%). We conclude that sodium fluoride reduces PMN adhesiveness in a dose-dependent manner. The effect is not direct, but should be modulated by a seric factor. Keywords. Adhesiveness, fluoride, polymorphonuclear leukocyte. Introduction Fluoride is widely used in odontostomatology to prevent dental caries. Several different mechanisms have been proposed to explain its anticariogenic action [ 1-41. However, little is known of its effect on cells of the immune system, or its influence in another major group of odontostomatological processes-periodontitis. These processes can be classified basically into diseases accompanied by host immune overresponse or underresponse, and transitional forms [5]. The involvement of polymorphonuclear leukocytes (PMN) in the genesis of periodontitis is well documented [6101. This report describes the effect of fluoride in vitro on the first sign of PMN activity during the inflammatory process: adhesion to the vascular endothelium [Ill. Correspondence: Dr Jose Liebana Ureiia. Departmento de Microbiologia, Facultad de Medicina. Avda. de Madrid I I , E-I8071 Granada. Spain.

Material and methods All analyses described below were performed in triplicate on three occasions separated by intervals of 1 month. In each experience we studied PMN adhesiveness in the presence and absence of autologous serum, and in the absence and presence of fluoride at different concentrations. Subject All assays were performed with venous blood from a healthy 22-year-old male with no immunological or periodontal alterations. The subject had taken no medication during the 60 days preceding the study, and at no time throughout the study showed clinical signs of infection. Any immunological, clinical of hematological alteration detected during the experience led us to discard the results from previous samples, and start a new cycle of blood sampling until three consecutive normal samples were obtained. Sariipling procedure Blood (40 ml) was drawn from a forearm vein. An aliquot of 20 ml was processed immediately for assay, and the rest of the sample was used for control assays. Control assays Serum concentration of fluoride was determined with the Villa method [12]. We also determined serum concentrations of C3, C4, and CH50, leukocyte count and differential leukocyte formula. Separation of’ P M N Polymorphonuclear leukocytes were separated by dextran sedimentation (Pharmacia Fine Chemicals, Uppsala, Sweden, 17-0320-02) in 6% phosphate buffered saline (PBS) (Oxoid Ltd, UK, BR-I4a), followed by density gradient centrifugation with Ficoll sodium diatrizoate (Pharmacia LK B Biotechnology, Uppsala, Sweden, 17-0840-03). Red blood cells remaining in the cell sediment were removed by hypotonic lysis [ 131.

P M N adhesiveness stirdi~s The vascular endothelium was simulated in tlitro with individual culture plates (Cluster6, Costar, Cambridge 659

660

J. L. GOMEZ-UBRIC et al. Table 1. Percentage reduction in polymorphonuclear leukocyte adhesiveness in the presence of sodium fluoride and autologous serum. Control value 98.5%

Percentage reduction adhesiveness Fluoride (PM)concentration

1st Determination

2nd Determination

6.25 0.125 0.25 0.5

0 1.1 3.4 15.2 24.2 37.7 36 39.8

0 1.2 3. I 13.7 44.7 48.5 65.1 65. I

I 1.5 L

4

MA, USA). One million PMN in 1 ml Hank balanced salt solution (HBS) (Gibco, Paisley, UK, 04104175M) with and without 25% autologous serum were placed in each Cluster Costar culture plate and incubated for 45 min at 36 f 1°C in an atmosphere of 5 % COz. Unadhered PMN were freed by several vigorous washings with Hank solution, and the results were expressed as the percentage of inoculated cells that were adhered to the bottom of the well, according to the formula (Yo PMN adhesiveness = 100 - number of unadhered PMN/total number of PMN added to each well x 100 [14]. EfSect offluoride on P M N adhesiveness The effect of fluoride on PMN adhesiveness was assessed by comparing the percentages of adhered and unadhered leukocytes in the presence and absence of autologous serum. Sodium fluoride (Sigma, St Louis, MO, USA, S-1504) was dissolved in isotonic saline solution. The concentrations of fluoride studied were all within the physiological range. We tested 1.0 p~ (0.019 ppm) (optimal plasma concentration obtained with optimal dietary intake of fluoride), and 6.25 lo-', 0.125, 0.25, 0.5, 1.5, 2.0, and 4.0 p~ concentrations. Statistical treatment The results from experiments with and without fluoride were analyzed statistically with Wilcoxon's test for nonpaired data.

Results All results are given as the mean of three determinations. Control assays Serum fluoride concentrations were 0.32 p ~ 0.27 , pM, and 0.30 p~ in the first, second and third analyses respectively. The results of all other analyses were within normal limits for the subject's age and sex. Adhesiveness in the absence of autologous serum In the absence of autologous serum and fluoride, PMN adhesiveness was 98.5%. The addition of different

3rd Determination 0

I 3.2 16.7 35.5 51 50.8 52.7

Mean 0 1.1 3.2 15.2 34.8 44.4 50.6 52.7

concentrations of fluoride to the culture medium had no significant effect on adhesiveness. Adhesiueness in the presence of autologous serum

Adhesiveness in the presence of autologous serum but without fluoride was also 98.5%. This figure was significantly decreased (P< 0.05) in comparison with controls in all cultures to which fluoride was added ; lowest and highest from concentrations 0.5 p ~ the concentrations assayed led to reductions of 1 . I "Ar and 52.7% respectively (Table I). Larger decreases in PMN adhesiveness were associated with higher concentrations of fluoride from 0.5 pM.

Discussion Because of the pathogenic significance of PMN in the genesis of periodontitis, stimulation of neutrophil function in diseases characterized by immune underresponse, and delaying PMN migration to the subgingival sulcus in forms with overresponse, would be of considerable preventive and therapeutic advantage. Fluoride, a widely used compound in odontostomatology, could be used as an immune modulator of PMN response. Gabler and Leong [ 151 have shown that fluoride suppresses nonoxidative metabolism in PMN, a finding that probably accounts for the inhibition of phagocytosis and antimicrobial activity of PMN exposed to fluoride compounds. However, these authors used much higher concentrations of fluoride than we did, and concluded that there was no evidence that plasma levels of fluoride in persons living in areas with fluoridated water were related with the inhibition of PMN functions. They further surmised that serum concentrations 1000 times higher than those found in their subjects would be necessary to bring about a 50% reduction in PMN functions. Wilkinson [16], who studied the influence of sodium fluoride on leukocyte locomotion and chemotaxis, concluded that a serum concentration 100 times higher than that normally found in subjects inhabiting optimally fluoridated areas would be necessary to interfere with these processes. Our findings, in contrast, strongly suggest that physiological concentrations of fluoride can sig-

SODIUM FLUORIDE AND POLYMORPHONUCLEAR LEUKOCYTE nificantly reduce leukocyte adhesiveness in vitro from 0.5 PM. Logically, the effect seen might be expected to be much weaker in tivo, since the decrease in granulocyte adhesiveness would be apparent in any inflamed or infected tissue, and there has been no evidence of an increase in the incidence of infectious processes in areas with fluoridation. However, although the in oivo effect may be weaker, it is not likely to be so small as to annul the possible preventive and therapeutic action of physiological concentrations of fluoride in patients with periodontitis with immune over-response. We should nonetheless caution that in patients with immune under-response, fluoride may have the undesirable effect of further reducing PMN migration to the subgingival sulcus. Our findings have the following implications in normal subjects: Sodium fluoride ( I ) significantly inhibits leukocyte adhesiveness, ( 2 ) has no direct inhibitory action on PMN adhesiveness, but rather influences this characteristic indirectly through a seric factor, and (3) reduces adhesiveness in a dosedependent manner. Acknowledgments We thank Ms Karen Shashok for translating the original manuscript into English. This study was supported in part by the Education and Science Council of the Andalusian Regional Government through Project no. 082. References I Derand T. Lodding A, Pettersson LG. Effect of topical F solutions on caries-like lesions in root surfaces. Caries Res 1989;23:135-40. 2 Hamilton IR. Biochemical effects of fluoride on oral bacteria. J Dent Res 1990;69(Spec Iss):660- 7.

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3 Lagerlof F, Saxegaard E, Barkvoll P. Effects of inorganic orthophosphate and pyrophosphate on dissolution of calcium fluoride in water. J Dent Res 1988;67:447-9. 4 Saxegaard E, Rolla G . Kinetics of acquisition and loss ofcalcium fluoride by enamel in vivo. Caries Res 1989;23:406- 1 I . 5 Suzuki, J. Diagnosis and classification of the periodontal diseases. In: Brals M, ed. Dental Clinics of North America, vol 32. Philadelphia: W. 8. Saunders, 1988: 195 -216. 6 Guarnieri C, Zucchelli G, Bernardi F, Scheda M, Frezza R. I granulociti polimorfonucleati neutrofili nella difesa e nel danno dei tessuti parodontali. Minerva Stomatol 1989;38:783-94. 7 Kinane DF, Cullen CF, Johnston FA, Evans CN. Neutrophil chemotactic behaviour in patients with early-onset forms of periodontitis (I). Leading front analysis in Boyden chambers. J Clin Periodontol 1989;16:242-6. 8 Nisengard R, Newman MG, Sanz M. The host response: basic concepts. In: Carranza FA, ed. Clinical Periodontology. Philadelphia: W. B. Saunders, 1990:330-42. 9 Van Dyke TE, Offenbacher S, Kalmar J, Arnold R R . Neutrophil defects and host-parasite interaction in the pathogenesis of localized juvenile periodontitis. Adv Dent Res 1988;2:(Suppl2): 254-8. 10 Zafiropoulos G G , Flores de Jacoby L, Czerch V, Kolb G. Markitziu A, Maveman K. Neutrophil function in patients with localized juvenile periodontitis and with rapidly progressive periodontitis. J Biol Buccale 1988; 16:I5 I --6. I I Gutierrez-Fernandez J, Maroto MC, Martin A, Rodriguez L, Piedrola G , Zamora E. Alteraciones de 10s leucocitos polimorfonucleares en el curso de la sepsis. Medicina lntensiva 1988;12:408- I I . 12 Villa. A. E. Rapid method for determining very low fluoride concentrations using an ion-selective electrode. Analyst 1988;113:1299 303. 13 Ginsburg 1, Borinski R, Sadovnic M , Eilman Y , Rainsford K. Poly L-histidine: A potent stimulator of superoxide generation in human blood leukocytes. Inflammation 1987;11:253 -77. 14 Gutierrez J, Maroto MC, Pitdrola G . Granulocyte abnormalities in parenteral drug-addicts. The influence of HIV infection. Allergoll Immunopath 1989;17:25 1-5. 15 Gabler WL, Leong PA. Fluoride inhibition of polymorphonuclear leukocytes. J Dent Res 1979;58:1933-9. 16 Wilkinson PC. Effects of fluoride on locomotion of human blood leukocytes in vitro. Arch Oral Biol 1983;28:415-8.

In vitro immune modulation of polymorphonuclear leukocyte adhesiveness by sodium fluoride.

We investigated the influence of sodium fluoride on polymorphonuclear leukocyte (PMN) adhesiveness in a healthy subject with low serum levels of fluor...
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