Cytokine production by peripheral blood monocytes during the normal human ovulatory menstrual cycle

C. Willis1,2,3, J.M. Morris3,4, V. Danis3 and E.D.M. Gallery2,3,5

1 Department of Biomedical Sciences at Cumberland Campus, Sydney University, NSW, 2141 and Departments of 2 Renal Medicine and 4 Obstetrics and Gynaecology, and 3 Kolling Institute of Medical Research, Sydney University at Royal North Shore Hospital, St Leonards, NSW 2065, Australia

5 To whom correspondence should be addressed at: Department of Renal Medicine, Sydney University at Royal North Shore Hospital, St Leonards, NSW 2065, Australia. e-mail: eileeng{at}med.usyd.edu.au


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: There is considerable evidence that hormone-driven changes resembling an inflammatory response occur in the vascular compartment during the menstrual cycle, and peripheral blood monocytes may be central in the process. We investigated whether there is a cyclical change in intrinsic production of pro-inflammatory cytokines by monocytes in the ovulatory menstrual cycle, and whether there is a circulating factor that influences the pattern of cytokine production in a cyclical manner. METHODS: Monocytes were purified by density-gradient centrifugation followed by countercurrent centrifugal elutriation, from the blood of normal women (n = 10) pre- and post-ovulation. Monocytes were cultured under basal conditions with bacterial lipopolysaccharide (LPS), and to determine the effects of circulating factors, incubations were also conducted in the presence of autologous serum. Concentrations of interleukin (IL)-1{alpha}, IL-1{beta}, IL-6, tumour necrosis factor (TNF)-{alpha} and IL-1 receptor antagonist (IL-1Ra) were measured by sandwich ELISA. RESULTS: The majority of IL-1{alpha} and IL-1{beta} was cell associated, while the other cytokines were almost entirely secreted. Basal levels of IL-1{alpha}, IL-1{beta}, and TNF-{alpha} were significantly increased following ovulation, while there was no significant change in levels of secretion of IL-6 or IL-1Ra. These effects were present in unstimulated cells, suggesting prior activation in vivo. Cytokine production was increased in response to LPS; however, there was no consistent effect of autologous serum. CONCLUSIONS: Intrinsic production of pro-inflammatory cytokines by monocytes is increased following ovulation.

Key words: cytokine/human/monocyte/ovulation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many changes suggesting inflammation are seen in the vascular compartment during the normal human menstrual cycle, in parallel with alteration in female sex hormone levels. Systemic changes that have been described following ovulation include: (i) an increase in circulating levels of granulocytes, monocytes and lymphocytes (Mathur et al., 1979Go; Faas et al., 2000Go); (ii) increased serum concentration of the pro-inflammatory cytokines interleukin (IL)-1 (Cannon and Dinarello, 1985Go) and tumour necrosis factor (TNF)-{alpha} (Brannstrom et al., 1999Go), but not IL-6 (Angstwurm et al., 1997Go), IL-10 (Maskill et al., 1997Go) or IL-2 (Brannstrom et al., 1999Go); (iii) changes in phenotype and secretory activity of some leukocytes to a more pro-inflammatory, pro-migratory profile (Leslie and Dubey, 1994Go; Polan et al., 1994Go); and (iv) changes in the phenotype of endothelium to facilitate leukocyte adhesion and migration (Tabibzadeh et al., 1994Go; Freitas et al., 1999Go). Physical changes that are seen commonly in the luteal phase of the ovulatory menstrual cycle also suggest an inflammatory response (Finn, 1986Go). These changes include oedema, a rise in body temperature and leukocyte migration from the vascular compartment into the endometrium (Starkey et al., 1991Go). The peripheral blood monocyte is a likely candidate for a central role in this sequence of events. Monocytes secrete pro-inflammatory cytokines including ILs and TNF, and these cytokines can activate other leukocytes and endothelial cells to a pro-adhesion, pro-migratory phenotype, and stimulate them to secrete vasoactive substances. Monocytes are known to be activated by estrogen and progesterone (Li et al., 1993Go), and demonstration of specific receptors for estrogen (Ben-Hur et al., 1995Go; Stephano et al., 1999Go) on monocytes suggests that these immune cells are potentially susceptible to cyclical changes in circulating hormone concentrations. If monocytes are under female sex hormone control, a degree of activation would be expected following ovulation, when both estrogen and progesterone levels rise. This study was undertaken to examine the effects of ovulation on production of pro-inflammatory cytokines by peripheral blood monocytes, and to determine whether there was an effect due to circulating factors. We hypothesized that monocytes are responsible for the inflammatory changes of the luteal phase of the menstrual cycle and that soluble factors in the circulation stimulate monocytes to produce inflammatory cytokines. The aims of the study were: (i) to determine the production of the inflammatory cytokines IL-1{alpha}, IL-1{beta}, TNF-{alpha} and IL-6 from monocytes isolated from blood in both follicular and luteal phases of the mentrual cycle from normal ovulating women; (ii) to determine the effect of stimulation by lipopolysaccharide (LPS) and autologous serum on monocyte production of these cytokines; and (iii) to determine whether there are compensating anti-inflammatory changes that occur following ovulation by measuring IL-1 receptor antagonist (IL-1Ra).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
The investigative protocol for this study was approved by the Human Research Ethics Committee of the Royal North Shore Hospital. All subjects who participated gave informed consent. Subjects (n = 10) were normotensive female volunteers, aged 24–44 years, with regular 28-day menstrual cycles, in good general health and not taking hormonal contraception or any other medication. Dates for blood collection were determined from the first day of menses: pre-ovulation samples were collected between days 8 and 12; post-ovulation samples collected between days 18 and 22.

Confirmation of ovulation
Ovulation was confirmed by a rise in serum progesterone levels. Concurrent estradiol (E2) levels were measured to ensure that subjects were not within a day of ovulation. These were measured by standard, commercially available radioimmunoassay in the Reproductive Medicine section of Pacific Laboratory Medical Service (PaLMS), Northern Sydney Area Health Service. Values are expressed as pmol/l. Ovulation was deemed to have occurred if serum progesterone levels were >25 pmol/l, and there was no concurrent E2 surge.

Purification of peripheral blood monocytes and monocyte culture experiments
Blood (100 ml) was collected using 0.38% sodium citrate as anticoagulant. Monocyte isolation and purification was achieved by density gradient centrifugation and monocyte enrichment by counter current elutriation centrifugation, as described previously (Hawkins et al., 1993Go). Recovery of monocytes was >85% and monocyte enrichment after countercurrent elutriation was >90% as determined by immunostaining with CD68. Cell viability was in excess of 95% as determined by Trypan Blue exclusion.

Monocytes (1 x 106 per tube in a total volume of 0.4 ml) from all subjects (N = 10) were cultured for 20 h, in X-Vivo 15 (Bio Wittaker, Walkersville, MD, USA), with and without bacterial LPS (1, 10, 100 ng/ml). In six of the subjects, the effect of autologous serum (5% and 10% v/v) on cytokine production was assessed. In each case, serum used for these incubations was collected at the same time as the cells. After collection of supernatants for estimation of secreted cytokines, the cells were disrupted by sonication and used to determine cell-associated levels. Samples were stored at –20°C for up to 8 weeks prior to assay.

Cytokine ELISA
Samples were batched so that pre- and post-ovulatory samples were measured on the same plate to reduce potential effects of inter assay variability. IL-1{alpha}, IL-1{beta}, IL-1Ra, IL-6 and TNF-{alpha} were measured by specific sandwich enzyme-linked immunosorbent assay (ELISA) as described previously (Danis et al., 1991Go). The cytokines measured have been shown to be stable in storage at –20°C for up to 6 months (Danis et al., 1995Go). Serum was measured at the concentrations used in the experiments (5 and 10%) to determine whether there were detectable levels of the cytokines in the experimental medium.

Statistical analysis
Results are shown as mean and SEM. Within-group comparisons were done by Wilcoxon signed rank test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Confirmation of ovulation
Progesterone levels in samples collected between days 8 and 12 of the menstrual cycle were 2.2 ± 0.8 pmol/l, and those between days 18 and 22 were 39.1 ± 3.8 pmol/l. All luteal-phase samples were >25 pmol/l. There was no difference in serum E2 levels between pre- and post-ovulatory samples (382 ± 112 versus 399 ± 44 pmol/l).

Cytokine production by peripheral blood monocytes
Group data are given for all cytokines examined in Table I. Major findings for the pro-inflammatory IL-1{alpha} and IL-1{beta} and their naturally occurring antagonist IL-1Ra, then the ubiquitous IL-6, followed by TNF-{alpha}, are summarized and discussed individually below. The majority of the IL-1{alpha} and IL-1{beta} was cell-associated and very little was secreted. By contrast, there was very little cell-associated IL-Ra, IL-6 or TNF-{alpha} (data for TNF-{alpha} not shown). For all cytokines examined, levels of medium containing 5 and 10% serum were the same as basal media.


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Table I. Response of monocytes to stimulation
 
Individual values for unstimulated production, pre- and post-ovulation, of the five cytokines examined are shown in Figure 1A–C. LPS was used as a known stimulus of cytokine production by monocytes, and a typical set of dose–response curves (for IL-1{alpha}) is shown for LPS in Figure 2. Basal production of cell associated IL-1{alpha} was significantly higher, and more variable, in samples collected post-ovulation (P < .01). LPS at all doses used (1, 10, 100 ng/ml), stimulated IL-1{alpha} production and secretion. This effect was greatest for cell-associated IL-1{alpha} with a 3–5-fold increase (P < 0.05) both pre- and post-ovulation in response to an LPS concentration of 10 ng/ml (Table I). There was no significant change in the levels of IL-1{alpha} in the presence of autologous serum.



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Figure 1. The effect of ovulation on cytokine production by peripheral blood monocytes: (A) cell-associated IL-1{alpha} and IL-1{beta}; (B) cell-associated and secreted IL-1Ra; (C) secreted IL-6 and TNF-{alpha}. Individual values are shown with significance between pre- and post-ovulation values. Data are from 10 healthy individuals. Horizontal bar = mean.

 


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Figure 2. The effect of various doses of LPS on peripheral blood monocytes IL-1{alpha} production. Values are shown as mean (SEM). Data from 10 healthy individuals studied pre- and post-ovulation.

 
As for IL-1{alpha}, basal production of IL-1{beta} was higher and more variable in cells collected following ovulation, and this was reflected in both cell-associated (P < 0.01) and secreted (P < 0.05) levels. LPS stimulated IL-1{beta} production and secretion by monocytes both pre-ovulation (P < 0.05) and post-ovulation (P < 0.01). Again, there was no significant effect of autologous serum.

In contrast to IL-1{alpha} and IL-1{beta}, antagonist IL-1Ra was predominantly secreted rather than cell associated. However, while basal levels of cell-associated IL-1Ra were significantly increased post-ovulation (P < 0.05) compared with pre-ovulatory levels, secreted levels were not significantly different. The stimulatory effect of LPS was reflected in secreted levels only. The effect of autologous serum was very variable. Although there was no significant effect on IL-1Ra levels, there was a trend towards increased production, a pattern different to that of IL-1{alpha} (Figure 3A and B).



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Figure 3. (A) Monocyte IL-1{alpha} production and (B) IL-1Ra secretion in response to autologous serum. Values [mean (SEM)] for six healthy individuals studied pre- and post-ovulation are shown for medium-only control, 5 and 10% (v/v) serum.

 
For IL-6, cell-associated levels were significantly increased post-ovulation. However, the trend towards increased basal secreted levels did not reach statistical significance. LPS stimulation of IL-6 production was very variable and was reflected in significant increase compared with basal levels in both secreted (P < 0.01) and cell-associated (P < 0.05) levels in pre-ovulation. Variation in response was again a feature of the effect of autologous serum, and group data failed to achieve statistical significance.

Basal secretion of TNF-{alpha} was significantly increased post-ovulation compared with pre-ovulation (P < 0.01). While there was an increase overall in the presence of LPS, the change failed to reach significance compared with basal levels both pre- and post-ovulation. There was little discernible effect of autologous serum on secretion of TNF-{alpha}.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytokines are soluble proteins produced and secreted as part of the immune response to a variety of tissue insults including inflammation, malignancy and autoimmune disorders. Extremely high systemic levels of inflammatory cytokines seen in disorders such as rheumatoid arthritis or septic shock demonstrate clearly how detrimental inflammatory mediators can be if not properly regulated. The pattern of cytokine production demonstrated in this work is part of the normal physiological changes associated with the ovulatory menstrual cycle. This involvement of the immune system indicates a role for the peripheral blood monocyte, precursor to the tissue macrophage, in a non-specific immune response to ovulation.

We have demonstrated that monocyte production and/or secretion of the inflammatory cytokines IL-1{alpha}, IL-1{beta}, IL-6 and TNF-{alpha} is significantly greater in the luteal phase compared with the follicular phase of the menstrual cycle. Since these effects were present in unstimulated cells, this suggests prior activation in vivo. It is possible but unlikely that luteal-phase monocytes are selectively activated in response to the isolation procedure of countercurrent centrifugal elutriation. Previous studies from our unit have demonstrated that monocytes freshly isolated by countercurrent centrifugal elutriation have no detectable IL-1{alpha} or IL-1{beta} mRNA by northern blotting (Danis and Millington, 1994Go). This is the first demonstration of simultaneous increases within the menstrual cycle in the levels of these four pro-inflammatory cytokines in a purified, non-adherent population of monocytes. The increase in IL-1{alpha} and IL-1{beta} was not accompanied by an increased production of their natural antagonist IL-1Ra. The results support our hypothesis that the inflammatory events which follow ovulation may be mediated by monocyte cytokine production.

In addition to basal levels of production of IL-1{alpha} and IL-1{beta} being higher in luteal-phase monocytes, they were also more powerfully stimulated by a given dose of LPS than proliferative phase monocytes. This is similar to findings described recently (Bouman et al., 2001Go). Although there was also an increase in basal production of IL-6 and TNF-{alpha} in luteal-phase monocytes, the heightened LPS sensitivity was restricted to production of IL-1{alpha} and IL-1{beta}. The cyclical nature of cytokine production, the subtle nature of systemic evidence of their increased production and the specificity of responses of individual cytokines to stimulation all emphasize the tight control of the regulatory mechanisms involved.

The precise mechanism(s) underlying the interaction between monocyte activation and cycling female sex hormones has not been delineated. It is not known whether the increases in cytokine production observed in this study are due to direct activation of immune cells by hormones or represent an indirect or secondary effect. Demonstration of the presence of estrogen receptors in the nuclei (Ben-Hur et al., 1995Go) and on the surface (Stephano et al., 1999Go) of monocytes suggests that monocytes may respond directly to this sex hormone. Progesterone receptors have not been demonstrated on monocytes, but this does not exclude the possibility of a direct effect, as progesterone binds to glucocorticoid receptors (Rosenau et al., 1972Go) and monocytes are known to have glucocorticoid receptors (Aittomaki et al., 2000Go). Previous work has shown that estrogen and progesterone at physiological levels modulate cytokine secretion by mixed PBMCs (Li et al., 1993Go) and adherent monocytes (Polan et al., 1988Go) in vitro. The response demonstrated in this work with a non-adherent, purified population of monocytes may indicate a direct interaction between female sex hormones and the immune cells, and such interaction may result in initiation of the production of one or all of the pro-inflammatory cytokines measured. It is known that cytokines IL-1{alpha}, IL-1{beta} and TNF-{alpha} can up-regulate their own production (Danis et al., 1995Go); therefore, an autocrine positive feedback loop mechanism may magnify and prolong an initial response.

During the normal menstrual cycle in vivo there are many factors that could modulate the basal activity of monocytes. These include soluble circulating factors and/or direct cellular interactions amongst monocytes, other leukocytes and endothelial cells. To investigate soluble factors in blood as a possible source of activators of monocytes seen post-ovulation, in vitro incubation experiments were performed with autologous serum. At the concentrations tested (5 and 10%) there was no significant effect of autologous serum on pro-inflammatory cytokine production by monocytes either pre- or post-ovulation. This lack of response of monocytes to autologous serum is in contrast to the effect of pooled normal or pathological (rheumatoid factor positive) human serum, which stimulated monocyte IL-1 production (Danis et al., 1990Go). Pooled serum is likely to contain different concentrations of known (growth factors, immune complexes, hormones and cytokines) and unknown stimulants to those of autologous serum, and many of these have been shown to alter cytokine production and secretion by monocytes. The lack of response to autologous serum by monocytes in production of inflammatory mediators demonstrated in this study suggests that in healthy subjects their own serum provides a neutral or benign environment which does not disturb the homeostasis of monocytes, either in the quiescent state pre-ovulation or the more activated state evident post-ovulation. The different patterns of secretion of the anti-inflammatory cytokine IL-1Ra and the pro-inflammatory cytokines following stimulation with autologous serum may represent another aspect of these homeostatic mechanisms. Interactions between monocytes and other cells in the vascular compartments were not investigated in the experiments reported here, as we focused on intrinsic monocyte function.

The increase we have found in basal production of cell-associated IL-1Ra post-ovulation has not previously been reported. Levels of IL-1Ra increased following LPS stimulation, but it is not known whether this was a primary or secondary effect. LPS is a known stimulus for the production of pro-inflammatory cytokines, and interaction between monocytes and other cytokines is known to affect the production of IL-1Ra. Monocyte-derived IL-1{alpha} and IL-1{beta} (both of which were increased post-ovulation) as well as granulocyte– macrophage colony-stimulating factor (GM-CSF), IL-10 and TGF-{beta} have all been found to stimulate IL-1Ra production (Granowitz et al., 1992Go; Danis et al., 1995Go). Another possible source of variation in production of IL-1Ra is gene polymorphism, where a specific polymorphism is associated with high IL-1Ra production and a corresponding low production of IL-1{alpha} (Danis et al., 1995Go).

The only known physiological effect of IL-1Ra is to modulate the production of IL-1{alpha} and IL-1{beta}, and to inhibit their effects by competitive inhibition of binding between IL-1 and the specific IL-1 receptor. The effect varies according to the type of stimulus used. Both IL-1{alpha} and IL-1{beta} appear to up-regulate their own production in a positive feedback loop, and IL-1Ra can block this feedback loop (Conti et al., 1992Go). The addition of IL-1Ra to LPS-stimulated monocytes has been shown to reduce production of both IL-1{alpha} and IL-1{beta} (Granowitz et al., 1992Go), while in GM-CSF-stimulated monocytes IL-1Ra down-regulated IL-1{alpha} production without affecting IL-1{beta} production (Danis et al., 1995Go). Under physiological conditions, as demonstrated in the present study, IL-1Ra could contribute to regulation of immune responses by counteracting the biological activity of these ILs.

The increased levels of monocyte-derived pro-inflammatory cytokines that we have described in the vascular compartment following ovulation would be likely to facilitate change of the endothelium to a pro-adhesive, pro-migratory phenotype. As well as the up-regulation of expression of adhesion molecules, cytokines stimulate endothelial cell production of chemotactic factors that augment leukocyte adhesion and migration (Jones et al., 1997Go). Peripheral blood monocytes are a source of pro-inflammatory cytokines, and their increased production may be pivotal in the cascade of events that initially facilitates migration of cells from the vascular compartment into the endometrium during the luteal phase of the menstrual cycle and eventually may result in the tissue and vascular changes characteristic of menstruation.

The monocyte-derived cytokines that we have shown to be increased in the second half of the menstrual cycle (particularly TNF-{alpha}, IL-1{alpha} and IL-1{beta}), are known to induce endothelial cell retraction, resulting in increased microvascular permeability. IL-1 also induces matrix remodelling. These changes, tightly temporally regulated, are likely to be causally involved in the endometrial perivascular changes seen in the luteal phase of the menstrual cycle. The organ-specific nature of the vascular changes suggests an input from sex hormone receptors. The local vascular changes which then precede menstruation include fragmentation of the vascular basement membrane and reduction of endothelial cell–pericyte contacts, also thought to be cytokine mediated (Bulletti et al., 1998Go).

It is clear from our results that there is peripheral blood monocyte activation following ovulation. The monocyte-derived cytokines up-regulated in this hormonal milieu could induce many of the local endometrial changes that follow ovulation, and are therefore likely to be causally involved in these changes. This provides evidence for tightly regulated physiological interaction between immune and reproductive systems


    Acknowledgements
 
The authors are grateful to Bas Rijs for his excellent technical assistance. This study was supported by Sydney University Research Grant (URG, to C.W.) and Faculty of Heath Sciences Research Grant (CRG, to C.W.). Thanks are also due to PaLMS for measurement of S. progesterone levels.


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 Discussion
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Submitted on September 11, 2002; resubmitted on November 29, 2002; accepted on February 7, 2003.