Effects of Sex Hormones of Human Peripheral



Polymorphonuclear Leukocytes and Monocytes* Masaharu


The effects

Hitoshi Aoyama,

Masayuki Morishita,


Yoshifumi Iwamoto

the in vitro Chemotaxis of polymorphonuclear leuinvestigated using fMLP as the chemoattractant. PMNs, monocytes, and plasma were obtained from heparinized peripheral blood of healthy adults. Chemotaxis of PMNs or monocytes treated with sex hormones were tested using 48-well Chemotaxis microchambers. The correlation between sex hormone levels in plasma and the chemotactic ability of PMNs from the same donor was also investigated. The Chemotaxis of PMNs was enhanced by progesterone, while it was reduced by estradiol. Random migration of PMNs was also enhanced by progesterone and reduced by estradiol. The effect of estradiol on PMN Chemotaxis was inhibited by addition of antiestrogens or progesterone. Testosterone did not have a measurable effect on PMN Chemotaxis. A significant positive correlation was found between the concentration of progesterone in plasma of females and PMN chemotactic ability in vitro. For males, there was no significant relationship between plasma levels of sex hormones and PMN chemotactic ability. Estradiol and testosterone levels in plasma did not correlate with PMN chemotactic ability. Sex hormones had no effect on the Chemotaxis of monocytes. These results suggest that the altered PMN Chemotaxis associated with gingival inflammation may be due to the effects of sex hormones. J Periodontol 1992; 63:28-32. of sex hormones on

kocytes (PMNs)




Key Words: Leukocytes, polymorphonuclear; progesterone; neutrophils; estradiol; gin-


It is well-known that gingival inflammation is exaggerated during puberty and pregnancy.1 Altered levels of circulating sex hormones at puberty are considered to aggravate gingivitis induced by bacterial plaque. Morishita et al.2 examined the relationship between salivary concentrations of sex

hormones, which reflect the levels of sex hormones in

gingival condition of junior high school They reported that: 1) male students with greater tendency toward bleeding on probing had high salivary levels of estradiol; 2) students with lower pocket depth had high salivary levels of progesterone and; 3) students with low subgingival bacterial counts had high salivary progesterone levels. They suggested that unbalanced secretion of plasma,

and the



hormones; i.e.,


increase of estradiol and



might be one of the factors promoting gingivitis at puberty. However, the mechanisms of the effects of these hormones on gingival inflammation are not of progesterone,

yet known.

of Preventive

'Department Dentistry, Dentistry, Hiroshima, Japan.



School of

generally accepted that bacterial plaque induces gininflammation gival through interactions with host defense mechanisms. In such defense mechanisms, phagocytic cells such as polymorphonuclear leukocytes (PMN) and macrophages are suggested to play an important role in periodontal disease. Numerous studies have revealed that damage to periodontal tissue may be aggravated by depressed function of PMN. It has been demonstrated that defects in the function of phagocytes are closely associated with localized juvenile Periodontitis and other types of Periodontitis.3"5 In conditions with dysfunctional PMNs, such as Chediak-Higashi syndrome and diabetes mellitus, severe periodontal breakdown was reported.6'7 Furthermore, it has been reported that PMN Chemotaxis could be inhibited by several oral microorganisms involved in periodontal lesions and that the inhibitory effect on PMN Chemotaxis may contribute to the virulence of these periodontopathic microIt is


Therefore, we hypothesized that sex hormones may affect inflammation through their actions on the function of PMNs or monocytes. The purpose of this study was to

Volume 63 Number 1


the effects of sex hormones on the function of PMNs and monocytes, especially their Chemotaxis in vitro.


MATERIALS AND METHODS Isolation of Cells and Plasma Seventeen ml of venous blood was drawn from young healthy volunteers. PMN were separated from heparinized blood by Ficoll-Hypaque* sedimentation at 350 g for 30 minutes, and then washed in phosphate-buffered saline (PBS), pH 7.2. PMNs were suspended in Gey's balanced salt solution* containing 2% bovine serum albumin8 and 20mM HEPES (Gey's-BSA), pH 7.2 at a concentration of 2.5 106 cells/ ml. Morphological analysis showed that 96% of the cells were PMNs and 99% of the cells were viable by trypan blue exclusion. A small portion of heparinized blood was used to obtain plasma by centrifugation at 350 xg for 15 minutes and stored at 20°C until used for determination of sex hormone levels. Human peripheral monocytes were isolated by the method reported by Kumagai et al.9 Briefly, 20 ml of venous blood was drawn from each of 8 healthy adults, 4 males and 4 females, with a mean age of 21.5 years. Mononuclear leukocytes were separated from heparinized blood by FicollHypaque sedimentation, and suspended in Gey's-BSA, pH 7.0. Each cell suspension was introduced into a plastic dish coated with fetal bovine serum and incubated for 2 hours at 37°C. The dish was then rinsed twice with cold Gey'sBSA to remove the nonadherent cells. Adherent monocytes were recovered by further incubation for 15 minutes at 4°C with Gey's-BSA containing 0.2% EDTA, washed in PBS, and suspended in Gey's-BSA at a concentration of 5 106 cells/ml. Ninety-five percent of the cells were monocytes by Esterase activity and 99% of the cells were viable by trypan blue exclusion. -

Chemotaxis Assay Chemotaxis of PMNs or monocytes was assessed using 48well Chemotaxis microchamber11 with N-formyl-methionylleucyl-phenylalanine (fMLP) as the chemoattractant at concentrations of 10-6M for PMNs or 10~8M for monocytes.10-11 Random migration of PMNs was assessed by the absence of fMLP in the lower wells. Progesterone, 17-beta-estradiol (estradiol) and testosterone were used as representative sex hormones, and clomiphene citrate (clomiphene) and nafoxidine§ were used as antiestrogens. All were initially dissolved in ethanol followed by serial dilutions made in Gey's-BSA so that the final ethanol concentration in incubation mixtures was 0.1% for PMNs or 0.05% for monocytes. PMN and monocyte samples were preincubated at 37°C fFlow Laboratories, McLean, VA. *Gibco Laboratories, Gaithersburg, MD. 5Sigma Chemical Co., St. Louis, MO. Neuro-Probe, Bethesda, MD.


for 15 minutes in Gey's-BSA with sex hormones at the designated concentrations or with ethanol alone as the control. Then, 50 µ of the cell suspension was placed in each of the upper wells of the Chemotaxis microchamber and incubated at 37°C with 5% C02 in air. Six wells were used for each condition. Incubation times were 60 minutes for PMN and 2 hours for monocytes. The pore sizes of the membrane filters were 3µ for PMN and 5µ for monocytes.* After incubation, PMN or monocytes on the membrane filters were fixed and stained. The number of cells migrated through the membrane filter were counted under a microscope with a magnification of 400 x. Ten highpower fields were counted for each well. Sex Hormone Levels The concentrations of estradiol, progesterone, and testosterone in plasma were determined in triplicate for each sample using commercially available direct radioimmunoassay kits.1



between the numbers of migrated PMNs or done using Wilcoxon's signed rank test and the significance of the difference between males and females was determined using Mann-Whitney's U-test. Spearman's rank correlation coefficient was calculated to determine if a correlation existed between the number of migrated PMNs and the concentrations of sex hormones in plasma. As shown in Tables 1, 2, and 3 and in Figures 1 and 2, the chemotactic ability of PMNs or monocytes treated with sex hormones was expressed as a percentage of each concurrently tested control.

Comparisons monocytes


RESULTS The numbers of control PMNs counted for the Chemotaxis assay and the random migration assay were 142.0 ±32.2 cells/0.0861 mm2 (mean ± s.d., 32) and 12.9 ±6.0 cells/0.0861 mm2 (mean ± s.d., 16) respectively. The chemotactic ability of PMNs was significantly reduced in the presence of estradiol at either 0.4 ng/ml (by 27%; < 0.01) or at 2.0 ng/ml (by 22%; < 0.01) (Table 1, Fig. 1). Random migration was also reduced by 0.4 ng/ml of estradiol (Table 1). Progesterone significantly enhanced PMN chemotactic ability and random migration at a concentration of 200 ng/ml (Table 2, Fig. 1). Testosterone did not have any significant effect on either Chemotaxis or random migration of PMNs at all concentrations tested (Fig. 1). The chemotactic abilities of PMNs treated with 0.4ng/ml of estradiol and/or clomiphene, nafoxidine, progesterone are shown in Table 3. Two antiestrogens, clomiphene and nafoxidine, by themselves did not significantly influence PMN Chemotaxis. The reduced chemotactic ability of PMNs treated with 0.4 ng/ml of estradiol was restored to the con=


#Nuclepore, Pleasanton, 1Sorin


Biomedica, Vercelli, Italy

Table 1. Effects of Estradiol


PMN Chemotaxis



(ng/ml) 0 (control)



100.0 95.1 73.2 78.2 94.2

0.04 0.4 2.0 20.0

8, % of control;


Effects of sex hormones on chemotaxis of human peripheral polymorphonuclear leukocytes and monocytes.

The effects of sex hormones on the in vitro chemotaxis of polymorphonuclear leukocytes (PMNs) and monocytes were investigated using fMLP as the chemoa...
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