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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cytokine/human/monocyte/ovulation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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., 1993). 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, IL-1
, IL-1Ra, IL-6 and TNF-
were measured by specific sandwich enzyme-linked immunosorbent assay (ELISA) as described previously (Danis et al., 1991
). The cytokines measured have been shown to be stable in storage at 20°C for up to 6 months (Danis et al., 1995
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 and IL-1
and their naturally occurring antagonist IL-1Ra, then the ubiquitous IL-6, followed by TNF-
, are summarized and discussed individually below. The majority of the IL-1
and IL-1
was cell-associated and very little was secreted. By contrast, there was very little cell-associated IL-Ra, IL-6 or TNF-
(data for TNF-
not shown). For all cytokines examined, levels of medium containing 5 and 10% serum were the same as basal media.
|
|
|
In contrast to IL-1 and IL-1
, 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
(Figure 3A and B).
|
Basal secretion of TNF- 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-
.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated that monocyte production and/or secretion of the inflammatory cytokines IL-1, IL-1
, IL-6 and TNF-
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
or IL-1
mRNA by northern blotting (Danis and Millington, 1994
). 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
and IL-1
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 and IL-1
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., 2001
). Although there was also an increase in basal production of IL-6 and TNF-
in luteal-phase monocytes, the heightened LPS sensitivity was restricted to production of IL-1
and IL-1
. 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., 1995) and on the surface (Stephano et al., 1999
) 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., 1972
) and monocytes are known to have glucocorticoid receptors (Aittomaki et al., 2000
). Previous work has shown that estrogen and progesterone at physiological levels modulate cytokine secretion by mixed PBMCs (Li et al., 1993
) and adherent monocytes (Polan et al., 1988
) 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
, IL-1
and TNF-
can up-regulate their own production (Danis et al., 1995
); 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., 1990). 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 and IL-1
(both of which were increased post-ovulation) as well as granulocyte macrophage colony-stimulating factor (GM-CSF), IL-10 and TGF-
have all been found to stimulate IL-1Ra production (Granowitz et al., 1992
; Danis et al., 1995
). 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
(Danis et al., 1995
).
The only known physiological effect of IL-1Ra is to modulate the production of IL-1 and IL-1
, 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
and IL-1
appear to up-regulate their own production in a positive feedback loop, and IL-1Ra can block this feedback loop (Conti et al., 1992
). The addition of IL-1Ra to LPS-stimulated monocytes has been shown to reduce production of both IL-1
and IL-1
(Granowitz et al., 1992
), while in GM-CSF-stimulated monocytes IL-1Ra down-regulated IL-1
production without affecting IL-1
production (Danis et al., 1995
). 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., 1997). 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-, IL-1
and IL-1
), 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 cellpericyte contacts, also thought to be cytokine mediated (Bulletti et al., 1998
).
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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Angstwurm, M., Gartner, R. and Zeigler-Heitbrock, H. (1997) Cyclic plasma IL-6 levels during normal menstrual cycle. Cytokine, 9, 370374.[CrossRef][ISI][Medline]
Ben-Hur, H., Mor, G., Insler, V., Blickstein, I., Amir-Zaltsman, Y., Sharp, A., Globerson, A. and Kohen, F. (1995) Menopause is associated with a significant increase in blood monocyte number and a relative decrease in the expression of estrogen receptors in human peripheral monocytes. Am. J. Reprod. Immunol., 34, 363369.[ISI][Medline]
Bouman, A., Moes, H., Jan Heineman, M., De Leij, L. and Faas, M.M. (2001) The immune response during the luteal phase of the ovarian cycle: increased sensitivity of human monocytes to endotoxin. Fertil. Steril., 76, 555559.[CrossRef][ISI][Medline]
Brannstrom, M., Friden, B., Jasper, M. and Norman, R. (1999) Variations in peripheral blood levels of immunoreactive tumor necrosis factor (TNF
) throughout the menstrual cycle and secretion of TNF
from the human corpus luteum. Eur. J. Obstet. Gynecol. Reprod. Biol., 83, 213217.[CrossRef][ISI][Medline]
Bulletti, C., Zeigler, D.D., Albonetti, A. and Flamigni, C. (1998) Paracrine regulation of menstruation. J. Reprod. Immunol., 39, 89104.[CrossRef][ISI][Medline]
Cannon, J.G. and Dinarello, C.A. (1985) Increased plasma interleukin-1 activity in women after ovulation. Science, 227, 12471249.[ISI][Medline]
Conti, P., Feliciani, C., Barbacane, R., Panara, M., Reale, M., Placido, C., Sauder, D., Dempsey, R. and Amerio, P. (1992) Inhibition of interleukin-1 mRNA expression and interleukin-1
and
secretion by a specific human recombinant interleukin-1 receptor antagonist in human peripheral blood mononuclear cells. Immunology, 77, 245250.[ISI][Medline]
Danis, V. and Millington, M. (1994) Gentle separations suit the cell. Todays Life Sci., 6, 4044.
Danis, V.A., Kulesz, A.J., Nelson, D.S. and Brooks, P.M. (1990) Cytokine regulation of human monocyte interleukin-1 (IL-1) production in vitro. Enhancement of IL-1 production by interferon (IFN) gamma, tumour necrosis factor-alpha, IL-2 and IL-1, and inhibition by IFN-alpha. Clin. Exp. Immunol., 80, 435443.[ISI][Medline]
Danis, V.A., Franic, G.M., Rathjen, D.A. and Brooks, P.M. (1991) Effects of granulocytemacrophage colony-stimulating factor (GM-CSF), IL-2, interferon-gamma (IFN-), tumour necrosis factor-alpha (TNF-
) and IL-6 on the production of immunoreactive IL-1 and TNF-
by human monocytes. Clin. Exp. Immunol., 85, 143150.[ISI][Medline]
Danis, V.A., Millington, M., Hyland, V.J. and Grennan, D. (1995) Cytokine production by normal human monocytes: inter-subject variation and relationship to an IL-1 receptor antagonist (IL-1Ra) gene polymorphism. Clin. Exp. Immunol., 99, 303310.[ISI][Medline]
Faas, M.M., Bouman, A., Moes, H., Heineman, M.J., De Leij, L. and Schuiling, G. (2000) The immune response during the luteal phase of the ovarian cycle: a Th2-type response? Fertil. Steril., 74, 10081013.[CrossRef][ISI][Medline]
Finn, C. (1986) Implantation, menstruation and inflammation. Biol. Rev., 61, 313328.[ISI][Medline]
Freitas, S., Meduri, G., Nestour, E.L. and Bausero, P. (1999) Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium. Biol. Reprod., 61, 10701082.
Granowitz, E.V., Vannier, E., Poutsiaka, D.D. and Dinarello, C.A. (1992) Effect of interleukin-1 (IL-1) blockade on cytokine synthesis: II. IL-1 receptor antagonist inhibits lipopolysaccharide-induced cytokine synthesis by human monocytes. Blood, 79, 23642369.[Abstract]
Hawkins, T., Jones, M. and Gallery, E. (1993) Secretion of prostanoids by platelets and monocytes in normal and hypertensive pregnancies. Am. J. Obstet. Gynecol., 168, 661667.[ISI][Medline]
Jones, R., Kelly, R. and Critchley, H. (1997) Chemokine and cycloxygenase-2 expression in human endometrium coincides with leukocytes accumulation. Hum. Reprod., 12, 13001306.[ISI][Medline]
Leslie, C.A. and Dubey, D.P. (1994) Increased PGE2 from human monocytes isolated in the luteal phase of the menstrual cycle. Implications for immunity? Prostaglandins, 47, 4154.[CrossRef][Medline]
Li, Z.G., Danis, V.A. and Brooks, P.M. (1993) Effect of gonadal steroids on the production of IL-1 and IL-6 by blood mononuclear cells in vitro. Clin. Exp. Rheumatol., 11, 157162.[ISI][Medline]
Maskill, J., Laird, S., Okon, M., Li, T. and Blakemore, A. (1997) Stability of serum interleukin-10 levels during the menstrual cycle. Am. J. Reprod. Immunol., 38, 339342.[ISI][Medline]
Mathur, S., Mathur, R., Goust, J., Williamson, H. and Fudenberg, H. (1979) Cyclic variations in white cell subpopulations in the human menstrual cycle: correlations with progesterone and estradiol. Clin. Immunol. Immunopathol., 13, 246253.[ISI][Medline]
Polan, M.L., Daniele, A. and Kuo, A. (1988) Gonadal steroids modulate human monocyte interleukin-1 (IL-1) activity. Fertil. Steril., 49, 964968.[ISI][Medline]
Polan, M.L., Loukides, J.A. and Honig, J. (1994) Interleukin-1 in human ovarian cells and in peripheral blood monocytes increases during the luteal phase: evidence for a midcycle surge in the human. Am. J. Obstet. Gynecol., 170, 10001007.[ISI][Medline]
Rosenau, W., Baxter, J., Rousseau, G. and Tomkins, G. (1972) Mechanisms of resistance to steroids: glucocorticoid receptor defect in lymphoma cells. Nat. New Biol., 237, 2024.[ISI][Medline]
Starkey, P., Clover, L. and Rees, M. (1991) Variations during the menstrual cycle of immune cell populations in human endometrium. Eur. J. Obstet. Gynecol. Reprod. Biol., 39, 203207.[ISI][Medline]
Stephano, G., Prevot, V., Beauvillian, J., Fimiani, C., Welters, I., Cadet, P., Breton, C., Pestel, J., Salzet, M. and Bilfinger, T. (1999) Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor. J. Immunol., 163, 37583763.
Tabibzadeh, S., Kong, Q. and Babaknia, A. (1994) Expression of adhesion molecules in human endometrial vasculature throughout the menstrual cycle. J. Clin. Endocrin. Metab., 79, 10241032.[Abstract]
Submitted on September 11, 2002; resubmitted on November 29, 2002; accepted on February 7, 2003.