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Increased PGE2 from human monocytes isolated in the luteal phase of the menstrual cycle. Implications for immunity?
Increased PGE2 from human monocytes isolated in the luteal phase of the menstrual cycle. Implications for immunity? Crystal A. Leslie*,# and Devendra P. Dubey** From the #ENRM Veterans Administration Hospital, Bedford, MA 01730, #Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, * *Division of Immunogenetics, Dana-Farber Cancer Institute and * *Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
The reproductive hormones are implicated in the well documented sexual dimorphism in cellular and i m m u n e responses. Prostaglandins (PGs) are mediators of the immune response with their concentration and relative amounts being pivotal to their impact. In resident peritoneal macrophages isolated from mice we had previously noted that the cells from females synthesized significantly more PG than males. In these experiments we investigated whether PG metabolism in the human monocyte was influenced by gender and by stage of the menstrual cycle. Monocytes isolated from the female and activated in vitro with LPS produced on average significantly more PG into the medium than the males. Among females, significantly more PG was found in the medium from cells isolated during the luteal phase of the cycle than during the early follicular phase. It was also in this luteal phase in which the female differed substantially from males. We suggest that the in vivo hormonal changes associated with the menstrual cycle modulate monocyte synthesis of PG and other i m m u n e modulators such as IL-1. This could be a key to understanding differences in vulnerability between males and females as well as within phases of the cycle, to i m m u n e and inflammatory insult.
Introduction H u m a n females compared to males have been reported to have enhanced cellular and humoral i m m u n e responses ~as well as an increased frequency
© 1994 Butterworth-Heinemann
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Prostaglandin fluctuations in monocytes: Leslie and Dubey of auto immune disease such as lupus and arthritis.283 In the NZB/NZW Fl mouse and in patients with lupus, androgens depress and estrogens augment4s5 the disease process and severity changes with age,2 the menstrual cycle,6 pregnancy’ and contraceptive use. R Although the gonadal steroids have been clearly implicated in this sexual dimorphism’ the cellular and molecular mechanism underlying these differences have not been defined. PGs are intercellular mediators operating at several levels of the immune response with their concentration and relative amounts being key to their impact.9 In resident peritoneal macrophages from mice we had previously noted that the cells from the females synthesized significantly more PG than males. Since the individual female mice showed a greater range of values some of which encompassed the male values, we postulated that PG synthesis was being modulated by hormone(s) released during the estrus cycle.‘oIn humans the monocyte is the major immune cell responsible for PGE production. I1In the following experiments we determined whether in the monocyte there are fluctuations in PC metabolism during the menstrual cycle. We report here that the hormonal status of the human female is associated with changes in PG metabolism, in that PGE, and PGI, are significantly increased in the medium from in vitro activated cultured monocytes isolated during the luteal phase of the cycle.
Materials and Methods Human Subjects Four healthy male and 4 healthy female subjects volunteered for this study. All were either students or technicians (age 26 to 31 years) who had signed a consent form. Approval for this study had been obtained from Bedford VA Medical Center Subcommittee on Human Studies. All women reported a history of normal menstrual cycles. Neither male or female subjects were on any medications on a regular basis. All were asked to notify the investigators about intake of any medications during the course of the study. During the experiment, 1 woman did report on one occasion taking 2 aspirin like drugs. However blood was withdrawn several days later and would not have interfered with PG synthesis in monocytes. Each female subject was paired with an age matched male control ( L 3yr) and each pair [A, B, C, and D) had blood withdrawn at 3 intervals (designated Pl, P2 and P3) during a month. Blood was withdrawn before 1 lam and a small sample used to prepare serum which was stored at -70°C for subsequent estradiol and progesterone determinations. The remaining 35ccs was used to prepare monocytes. Monocyte
Isolation
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by Ficoll-Paque (Pharmacia, Piscatway, NJ) as previously
42
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Prostaglandin fluctuations in monocytes: Leslie and Dubey described.12 The buffy coat was collected in 5Occs RPM1 1640 and centrifuged (200 g for 15 mins). The pellet was re suspended in 12 ccs of supplemented media containing 10% heat inactivated fetal calf serum, 2mM L-glutamine and antibiotics and centrifuged (18Og for 10mins). This pellet was re suspended in 3ccs supplemented culture media and cells counted by haemocytometery. Cells were washed twice and 10 million cells plated in wells precoated with FCS. After 2h incubation at 37°C in 5% CO,/95% air atmosphere, the supernatant containing non adherent cells was removed. Of total cells plated 75% were removed at this step. Four further washings removed another 12 to 15%. Before adherence, 13 to 14% of the cells stained with the mouse monoclonal (MO-2) antibody (detected using fluorescence isothiocyanate conjugated goat anti-mouse IgG) for the human monocyte’” and 84% stained with an antibody to T cells (OKT-3).14 In contrast, 91% to 93% of the washed adhered cells were positive for MO-2 while 90% of the non-adherent cells stained with the OKT-3 antibody. About 4 million monocytes were obtained at each blood withdrawal. Duplicate sets of monocytes were incubated in supplemented culture media for a further 20h in the absence or presence of LPS (5kg/ml). Media samples were frozen for subsequent PG and cytokine analysis. At the end of the incubation protein was determined on the washed adhered cells using a standard kit from Bio Rad. Timing
of the Cycle Stage
Our hypothesis was that the hormonal status of the female would affect PG production from the monocyte. Hence the 3 windows of time (Pl, P2 and P3) in the menstrual cycle during which the 4 females (and their paired male controls) had blood withdrawn were chosen to be within 3 endocrinologically defined periods of the menstrual cycle (i) a period (PI) in early menses with low serum estradiol and progesterone (ii) a period (P2) near the time of ovulation and (iii) a period P3 in the luteal phase of the cycle when both serum progesterone and estradiol levels are increased.15 To do this the 4 women had the length of at least one menstrual cycle recorded (Table 1). Female subjects A and D reported and were found to have cycles of fairly consistent length. Subject C reported and was found to have a somewhat shorter cycle and subject B was found to have an occasional longer cycle of 45 days. Female subjects verbally reported their first day of menses which was counted as day 1. Blood (35 to 4Occs) was withdrawn from each pair three times (Table 1); between days 2 to 4 (Pl); between days 10 to 15 (P2) and between days 21 to 26 (P3) Because of the relatively shorter cycles of female subjects C and D, slightly shorter intervals from menses were chosen for P2 and P3 .
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TABLE I. Menstrual cycle lengths for female subjects and days of blood withdrawal of menses for each pair
from start
Days Blood Withdrawal Subject
Cycle Length (days)
A B C D
30, 45, 23, 29,
33 31, 29, 31 28, 23 26, 32, 26
Pl
P2
P3
2 2 3 4
14 15 10 12
26 26 21 24
The 4 female subjects had the length of at least one cycle recorded prior to the start of blood sampling. Pl, P2 and P3, were the number of days from the start of menses on which blood was withdrawn and monocytes isolated from each pair. Pl, P2 and P3 were chosen to be within endocrinologically defined segments of the menstrual cycle (Table 2).
Prostaglandin and Cytokine Analysis PGs were assayed by RIA according to our published procedure.‘” The 6-keto-PGF,, (a measure off PG12) antibody (Seragen Inc., Boston, MA) crossreacted (co.01 %) with TXB1, PGD2, PGA, PGB and metabolites of PGE, and PGF2_. The cross reaction with PGF2, was 2.2%, 0.4% with PGE, and 0.06% with PGE,. The PGE, antibody prepared in rabbits in this laboratory crossreacted ~0.5 % with PGAz, PGBz, PGFZa, with metabolites of PGE2, PGFz, and 6-keto-PGF,,. The crossreaction with PGD, was 2%. Cytokines (IL- 1B and IL-6) were assayed using ELISA kits (Genzyme Corp., Boston, MA for IL-6 and Cistron Biotechnology, Pine Brook, NJ for IL-l p). Medium controls incubated under the same conditions contained negligible quantities of PG or cytokines. The data was expressed as the amount of PG or IL found in the medium after 20h incubation at 37°C. Progesterone and Estradiol Determinations Serum levels of progesterone house RIA.
and estradiol
were assayed by a standard in
Statistical Analysis Repeated measures analysis of variance (ANOVA) was used to test for period and gender effects as well as the interaction of period and gender.” For analysis restricted to females, single factor repeated measures were used to test for period effects. Individual comparisons were made using a t test. Level of significance was p 5 .05.
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Results Reproductive Hormones and Cycle Timing In the males serum estradiol and progesterone levels did not markedly change at each period. All 4 females showed low levels of estradiol and progesterone 2 to 4 days from start of menses (Pl) and increased amounts 21 to 26 days from start of menses (P3). Ten to 15 days from the start of menses (P2) all 4 female subjects had unchanged progesterone levels relative to PI while ~3estradiol levels had increased 3 to 4 fold (Table 2). The increase in progesterone from P2 to P3 for each of the 4 females ranged from 10 to 60 fold. By these endocrinological criteria, ls all 4 blood samples taken in P3 were from the luteal phase of the cycle, the samples taken in P2 were taken near ovulation and the samples taken in Pl were from the early follicular phase of the menstrual cycle. Thus all 4 female subjects were accurately reporting the start of their menses. Neither hormone was detectable in the medium (~2 x10-lo M and ~2 x lo-“M for progesterone and estradiol respectively). Prostaglandins The medium from the cultured monocytes after 20h incubation in the absence of LPS, contained only small amounts of PGE, (Figure 1) and PGI, (data not shown). There was no difference between males and females or within phases of the cycle. In the presence of LPS, both PGE, (Figure 1) and PGI, (data not shown] levels increased about 7 fold. The activated monocyte from the female produced a greater range of PG amounts than the male (3.2 to 18.6 ng PGE, for females versus 2.4 to 8.4 ng PGE, for males; 0.3 to 2.5 ng PGI, for females versus 0.3 to 1.3 ng for males). The mean PGE, and mean PGI, amounts (averaged over P 1, P2 and P3) found in the medium for the female of each pair was consistently and significantly greater than the male average (p< .05 for PGI, and p< .03 for PGE,). As TABLE2. Serum estradiol and progesterone
Cycle Period Pl P2 P3
levels in male and femal subjects at the period of blood withdrawal
Progesterone ng/ml + SE
Estradiol pg/ml + SE
Progesterone ng/ml +- SE
Estradiol pg/ml 5 SE
.35 & .13 .19 2 .Ol .42 + .09
45 2 1.5 39 i 2.9 43 c 3.8
.33 * .05 .26 ? .06 6.10 & 2.0
36 2 9 124 i 30 113 + 18
Blood was withdrawn within 24 days (Pl), 10-15 days (P2) and 21-26 days (P3) from the start of menses of each female and serum progesterone and estradiol assayed. The hormone levels in Pl , P2 and P3 were within the expected ranges for samples taken during menses, near ovulation and during the luteal phase of the cycle respectively.
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Prostaglandin fluctuations in monocytes: Leslie and Dubey 16 PGE2
I I I I I I I I I
12 LPS absent
8
m
Male
q
Female
LPS present
I
I I I
4
I I I
I 1
2
3
Period
1
FIGURE 1. Blood was withdrawn within 2-4 days (Pl), 10-15 days (P2) and 21-26 days (P3) from the start of menses of each female. Monocytes were isolated, incubated for 20h with LPS (5ug/ml) and the medium assayed for PGE, by RIA.
46
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Prostaglandin fluctuations in monocytes: Leslie and Dubey shown in Figure 1 for PGE2, this difference between males and females was due to an increase in PG in the m e d i u m from monocytes isolated later (P3) rather than earlier (P1) in the cycle. By ANOVA the increased amount of PGE~ in the m e d i u m from monocytes isolated from w o m e n compared to monocytes from m e n was significant (p< .038) and the effect of time of blood withdrawal (period effect) also differed for the 2 sexes (p< .025). Since this difference was significant only when comparing P1 with P3 (p< .039) this means that monocytes isolated from females during P3 (the luteal phase of the cycle) produced more PG than monocytes isolated from males at the same time. Monocytes isolated from females during other phases of the cycle (P1 and P2) did not produce significantly more PG than monocytes from males. An analysis restricted to females only also showed (Figure 1) a significant increase (p< .028) in PG levels in the luteal phase of the cycle relative to the two other phases (P1 and P2) or to P1 alone (p = .05) A similar significant pattern of PG production in P 1, P2 and P3 was found for PGI2 (Figure 2). Thus for total cylooxygenase products assayed (PGE~ plus PGI2) the females had higher levels than males (p< .035) with the significant increase relative to males (p<. 02) and among females (p< .045) occurring in P3 relative to P1. There was no variation from period to period in the ~g cell protein obtained per 10 million cells plated.
Cytokines IL-6 and IL-lf~ were assayed only in the female subjects. Incubation of monocytes with LPS increased both cytokines. About 10 fold more IL-6 than IL-1 ~ was found in the medium. There was no association between the amount of IL-6 released and the particular phase of the cycle either in the absence of LPS (24.1 + 6.5, 26.7 + 5.3 and 20.4 + 5.0 in ng/20h + SE) or in the presence of LPS ( 56.2 _+ 7.0, 54.3 + 2.6 and 55.5 + 7.1 in ng/20h _+ SE) for P1, P2 and P3 respectively. For all w o m e n the amount of IL-I~ found in the m e d i u m peaked in P2 relative to the other periods (Table 3). This period effect was significant for monocytes incubated both in the absence (p< .041) or in the presence (p< .049) of LPS.
Discussion In a previous report we had noted in female mice that the amount of PG synthesized from the macrophage was greater than from male mice and was apparently influenced by the stage of the estrus cycle. 1° This new data demonstrates that PG accumulation in the m e d i u m from the activated h u m a n monocyte was also greater in females than in males and may be influenced by the hormone(s) released during the menstrual cycle. Thus, in monocytes isolated from the females and exposed in vitro to an inflammatory stimulus, more PG was found in the m e d i u m from cells
Prostaglandins 1994:47, J a n u a r y
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Prostaglandin fluctuations in monocytes: Leslie and Dubey 16 PGE2
1
I 12
LPS present
LPS absent
8
H
Male
0
Female
I I I I I I I I
I
1
2
3
Period
1
2
3
FIGURE 2. Blood was withdrawn within 2-4 days (Pl), 10-15 (P2) and 21-26 days (P3) from the start of menses of each female. Monocytes were isolated, incubated for 20h with LPS (5ug/ ml) and the medium assayed for 6-keto-PGF,,, (a measure of PGI,) by RIA.
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Prostaglandin fluctuations in monocytes: Leslie and Dubey TABLE 3. Release of interleukin-1 from monocytes isolated at endocrinologically of the menstrual cycle
defined phases
Cycle Period Pl P2 P3
LPS Absent
LPS Present
1.11 ? .48 ‘2.60 ? .84 .69 2 .29
6.27 ? 1.25 11.35 ” 2.22 6.46 ? 1.62
Blood was withdrawn from the 4 women during menses (Pi), near ovulation (P2) and during the luteal phase (P3) of the menstrual cycle. Monocytes were isolated, incubated for 20h with and without LPS (5ug/ml) and the medium assayed for IL-1 B by ELISA. For all 4 females both the spontaneous release of IL-1B and the release after LPS activation peaked near ovulation (P2). This increase was significant in both the absence (~~041) and present (pe.049) of LPS.
isolated in the luteal (P3) than in the early follicular phase (Pl) of the menstrual cycle. It was also in this luteal phase in which the female differed substantially from the males. Since in these human experiments monocytes from each female were isolated only once during the luteal phase of the cycle, further experiments would need to be done to determine the duration of this increase and when the peak amount of PG accumulates. In the absence of an in vitro stimulus very little PG was detected. Both arachidonate products assayed were increased 7 fold when cells were stimulated with LPS. However PGE, was present in much greater quantities than PGI,. It has previously been documented that in humans the monocyte is the major cell in the immune system responsible for PGE production.” Besides our report of differences in PG synthesis in macrophages from the male and female mouselo there is one other report in the rat.18 These authors were the first to report that LPS activated macrophages from females produced more PG than the males. In human neutrophils, both gender and menstrual cycle related variations in PG production have been documented.lY In the human monocyte from females the PG increase occurred in the luteal phase of the menstrual cycle in which granulosa cells, form a corpus luteum and produce progesterone and 17 hydroxyprogesterone. Since both PGE, and PGI, increased during this luteal phase, this implies that the reproductive hormone(s) involved, may exert their effect to up regulate a common precursor. There is little information on the influence of the ovarian steroids on PC metabolism in the monocyte. However in the sheep endometrium, progesterone induces expression of mRNA encoding for cyclooxygenase, the rate limiting enzyme in PG production.20 In this context it has been documented that certain cells, including macrophages and monocytes,z1,22 express not only a constitutive cyclooxygenase but a second regulated cylooxygenase (COX2) which is
Prostaglandins 1994147, January
Prostaglandin fluctuations in monocytes: Leslie and Dubey LPS inducible.23 We have noted that LPS activated macrophages from ovarectomized mice produces less mRNA for COX2 than macrophages from control mice (Leslie C.A. unpublished observation). In another experiment we found a marked reduction in PGE, ( + SE) synthesis from the macrophages of ovarectomized mice (6.7 + 3.4 ng/24h) relative to sham operated mice (19.4 + 4.2 ng/24h) and control female mice (14.6 + 2.9 rig/244). We therefore suggest that exposure of monocytes to an ovarian steroid such as progesterone, during the luteal phase of the cycle increases the amount or stability of COX2 mRNA when induced by an inflammatory stimulus. If monocytes are isolated in the luteal phase of the cycle and activated in vitro by LPS (or activated in vivo) more immunosuppresive PGE, will be produced than from monocytes isolated at other phases. This may be a mechanism which in vivo will favor implantation and fetal survival. Thus if fertilization occurs, the continued high levels of progesterone, may through a local increase of immunosuppresive PGE, by infiltrating macrophages24 protect the developing blastocyst from the maternal immune response. Interestingly it has been documented that in the human endometrium more PG is released during the luteal phase of the cycle.2s Gonadal steroids are known to modulate IL-1 activity in both human pelvic macrophages26 and human monocytes.27,28 In the 4 females quantitated here, IL-6 release from the monocyte was independent of the stage of the cycle. However monocytes isolated close to ovulation (P2), prior to any increase in serum progesterone but during the first estradiol surge, produced significantly more IL-ll3 than at other periods (Table 3). The reproductive hormones may therefore differentially regulate IL-l and IL-6 production in activated monocytes. 2y In P3 after the isolated monocytes had been exposed in vivo to luteal phase levels of progesterone, IL1 release decreased. Others have shown that 10m7to 10m8M progesterone, the concentration reached in vivo during the luteal phase is sufficient to markedly inhibit IL-l mRNA30 as well as IL-1 activity.28 Our experiments do not enable us to determine for how long before or after P2, IL-ll3 is up regulated. However using an activity assay for IL-I, Cannon and Dinarello”’ reported in humans increased plasma levels after ovulation. Monocytes and macrophages process and present antigens to B and T cells32 and amplify the immune response by producing IL-l, a fundamental event in the initiation of the immune/inflammatory response.“” They are unique among cell types in containing high levels of arachidonate16,34 and are the principal source of eicosanoids affecting other immune cells.“” Of great interest is the ability of PGE, to down regulate monocyte peptide signals such as IL- 1 which are necessary to promote a full blown immune response.36 High concentrations of PGE, in vitro inhibit mitogen responsiveness, clonal proliferation, antigenic stimulation, lymphokine production, the proliferative response of IL-2 dependent T cells, monocyte
50
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Prostaglandin fluctuations in monocytes: Leslie and Dubey cytotoxic activity, natural killer cell activity and B cell proliferation.37)38 Chronic overproduction of PGE can cause immunosuppresion and inhibiting PC synthase is known to stimulate cellular immune responses in animals and humans.“‘+0 The documented increase in PGE, synthesis from both spleens of old mice41+2 and PBMC from elderly people4” relative to young controls is associated with a decline in immune function with increasing age.42,44 Thus, a possible consequence of cyclical changes in amount of PGE, and IL-l produced from an activated monocyte or macrophage in vivo would be to make premenopausal females more vulnerable to immune stress at certain times of their cycle and less vulnerable at other times. This is certainly true in mice where quantitative differences in immune function with the cycle phase have been reported.4s In women there is a dearth of such information although in one unique study it has been documented that Candidal vaginitis occurs most frequently during the luteal phase, the same time of maximal reduction of cellular responsiveness to Candidia.4” Further work by this group4’ established that in the presence of luteal phase levels of progesterone there was a 50% decrease in C. albicans induced lymphocyte proliferation. Removal of the monocytes from the PBMC obviated the ability of progesterone to inhibit this response strongly suggesting that the inhibition of lymphocyte proliferation was a monocyte mediated mechanism.“’ This is very consistent with our observation that human monocytes isolated during the luteal phase (after exposure to luteal phase levels of progesterone) produce increased amounts of immunosuppresive PGE,. In view of the significance of PGs and cytokines from the monocyte to modulate the immune response, understanding how normal physiologic changes associated with the menstrual cycle alters this balance could be a clue to understanding differences in vulnerability, within phases of the cycle, and between males and females, to immune and inflammatory insult. We are suggesting that activation and suppression of monocyte PG and cytokine synthesis by the precisely timed high amplitude fluxes of the ovarian hormones released during the menstrual cycle may provide the key to the mechanism underlying the biological puzzle of sexual dimorphism in immunity.
Acknowledgments Reprint requests to Crystal A. Leslie, Research (1.51), ENRM VA Hospital, 200 Springs Rd., Bedford, MA 01730. This work was supported by the US Department of Veterans Affairs and National Institute of Health Grant AI 268 17. We thank Dr. Deborah Andersen, Fearing Research Laboratory, Harvard Medical School for hormone determinations, Dr. Jennifer Andersen of l
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Prostaglandin fluctuations in monocytes: Leslie and Dubey Boston University School of Medicine N. Mirza for technical assistance.
for the statistical
analysis and Dr.
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Kunkel S.L., SW. Chensue, and S.M. Phan. Prostaglandins as endogenous mediators of interleukin-I production. J. Immunol. 236: 186. 1986. Kinsella, J.E., and B. Lokesh. Dietary lipids, eicosanoids and the immune system. Critical Care Med. 18: S94. 1990. Goodwin, J.S., A.D. Bankhurst, and RI? Messner. Suppression of human T cell mitogenesis by prostaglandins. J. Exp. Med. 146: 1719. 1977. Rodgers, T.G. In: Prostaglandins and Immunity. (J.S. Goodwin ed) Martinus Nijhoff, Boston, 1985, p.79. Wasserman, J., H. Blomgren, S. Rotstein , B. Petrini, and S. Hammarstrom. Immunosuppresion in irradiated breast cancer patients: in vitro effect of cyclooxygenase inhibitors. Bull N.Y. Acad. Med. 65: 36. 1989. Meydani, S.N., M. Meydani, C.P. Verdon, J.B. Blumberg, and K.C. Hayes. Vitamin E supplementation suppresses prostaglandin E, synthesis and enhances the immune response of aged mice. Mech. Aging Devel. 34: 191. 1986. Bartocci, A., F.M. Maggi, R.D. Welker, and F. Veronese. In: Prostaglandins and Cancer (T.J. Powles, Backman R.S., Honn X.V., Ramwell P. eds) Alan Riss, N.Y. ,1982, p.725. Meydani, S.N., P.M. Barklund, S. Liu, et al. Effect of vitamin E supplementation on immune responsiveness of healthy elderly subjects. Am J. Clin. Nutr. 52: 557. 1990. Rosenstein, M.M., and H.R. Stauser. Macrophage induced T cell mitogen suppression with age. J. Reticuloendoth. SC. 27: 159. 1980. Krzych, U., H.R. Strausser, J.P. Bressler, and A.L. Goldstein. Quantitative differences in immune responses during the various stages of the estrous cycle in female BALB/c mice. J. Immunol. 221:1603. 1978. Kalo-Klein, A., and S.S. Witkin. Candidia albicans: cellular immune system interactions during different stages of the menstrual cycle. Am. J. Qbstet. Gynecol. 161: 1132. 1989. Kalo-Klein, A., and S.S. Witkin. Regulation of the immune response to Candida albicans by monocytes and progesterone. Am. J Qbstet. Gynaecol. 164:1351. 1991.
Prostaglandins 1994:47, January
The reproductive hormones are implicated in the well documented sexual dimorphism in cellular and i m m u n e responses. Prostaglandins (PGs) are mediators of the immune response with their concentration and relative amounts being pivotal to their impact. In resident peritoneal macrophages isolated from mice we had previously noted that the cells from females synthesized significantly more PG than males. In these experiments we investigated whether PG metabolism in the human monocyte was influenced by gender and by stage of the menstrual cycle. Monocytes isolated from the female and activated in vitro with LPS produced on average significantly more PG into the medium than the males. Among females, significantly more PG was found in the medium from cells isolated during the luteal phase of the cycle than during the early follicular phase. It was also in this luteal phase in which the female differed substantially from males. We suggest that the in vivo hormonal changes associated with the menstrual cycle modulate monocyte synthesis of PG and other i m m u n e modulators such as IL-1. This could be a key to understanding differences in vulnerability between males and females as well as within phases of the cycle, to i m m u n e and inflammatory insult.
Introduction H u m a n females compared to males have been reported to have enhanced cellular and humoral i m m u n e responses ~as well as an increased frequency
© 1994 Butterworth-Heinemann
Prostaglandins 1994:47, J a n u a r y
41
Prostaglandin fluctuations in monocytes: Leslie and Dubey of auto immune disease such as lupus and arthritis.283 In the NZB/NZW Fl mouse and in patients with lupus, androgens depress and estrogens augment4s5 the disease process and severity changes with age,2 the menstrual cycle,6 pregnancy’ and contraceptive use. R Although the gonadal steroids have been clearly implicated in this sexual dimorphism’ the cellular and molecular mechanism underlying these differences have not been defined. PGs are intercellular mediators operating at several levels of the immune response with their concentration and relative amounts being key to their impact.9 In resident peritoneal macrophages from mice we had previously noted that the cells from the females synthesized significantly more PG than males. Since the individual female mice showed a greater range of values some of which encompassed the male values, we postulated that PG synthesis was being modulated by hormone(s) released during the estrus cycle.‘oIn humans the monocyte is the major immune cell responsible for PGE production. I1In the following experiments we determined whether in the monocyte there are fluctuations in PC metabolism during the menstrual cycle. We report here that the hormonal status of the human female is associated with changes in PG metabolism, in that PGE, and PGI, are significantly increased in the medium from in vitro activated cultured monocytes isolated during the luteal phase of the cycle.
Materials and Methods Human Subjects Four healthy male and 4 healthy female subjects volunteered for this study. All were either students or technicians (age 26 to 31 years) who had signed a consent form. Approval for this study had been obtained from Bedford VA Medical Center Subcommittee on Human Studies. All women reported a history of normal menstrual cycles. Neither male or female subjects were on any medications on a regular basis. All were asked to notify the investigators about intake of any medications during the course of the study. During the experiment, 1 woman did report on one occasion taking 2 aspirin like drugs. However blood was withdrawn several days later and would not have interfered with PG synthesis in monocytes. Each female subject was paired with an age matched male control ( L 3yr) and each pair [A, B, C, and D) had blood withdrawn at 3 intervals (designated Pl, P2 and P3) during a month. Blood was withdrawn before 1 lam and a small sample used to prepare serum which was stored at -70°C for subsequent estradiol and progesterone determinations. The remaining 35ccs was used to prepare monocytes. Monocyte
Isolation
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by Ficoll-Paque (Pharmacia, Piscatway, NJ) as previously
42
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Prostaglandin fluctuations in monocytes: Leslie and Dubey described.12 The buffy coat was collected in 5Occs RPM1 1640 and centrifuged (200 g for 15 mins). The pellet was re suspended in 12 ccs of supplemented media containing 10% heat inactivated fetal calf serum, 2mM L-glutamine and antibiotics and centrifuged (18Og for 10mins). This pellet was re suspended in 3ccs supplemented culture media and cells counted by haemocytometery. Cells were washed twice and 10 million cells plated in wells precoated with FCS. After 2h incubation at 37°C in 5% CO,/95% air atmosphere, the supernatant containing non adherent cells was removed. Of total cells plated 75% were removed at this step. Four further washings removed another 12 to 15%. Before adherence, 13 to 14% of the cells stained with the mouse monoclonal (MO-2) antibody (detected using fluorescence isothiocyanate conjugated goat anti-mouse IgG) for the human monocyte’” and 84% stained with an antibody to T cells (OKT-3).14 In contrast, 91% to 93% of the washed adhered cells were positive for MO-2 while 90% of the non-adherent cells stained with the OKT-3 antibody. About 4 million monocytes were obtained at each blood withdrawal. Duplicate sets of monocytes were incubated in supplemented culture media for a further 20h in the absence or presence of LPS (5kg/ml). Media samples were frozen for subsequent PG and cytokine analysis. At the end of the incubation protein was determined on the washed adhered cells using a standard kit from Bio Rad. Timing
of the Cycle Stage
Our hypothesis was that the hormonal status of the female would affect PG production from the monocyte. Hence the 3 windows of time (Pl, P2 and P3) in the menstrual cycle during which the 4 females (and their paired male controls) had blood withdrawn were chosen to be within 3 endocrinologically defined periods of the menstrual cycle (i) a period (PI) in early menses with low serum estradiol and progesterone (ii) a period (P2) near the time of ovulation and (iii) a period P3 in the luteal phase of the cycle when both serum progesterone and estradiol levels are increased.15 To do this the 4 women had the length of at least one menstrual cycle recorded (Table 1). Female subjects A and D reported and were found to have cycles of fairly consistent length. Subject C reported and was found to have a somewhat shorter cycle and subject B was found to have an occasional longer cycle of 45 days. Female subjects verbally reported their first day of menses which was counted as day 1. Blood (35 to 4Occs) was withdrawn from each pair three times (Table 1); between days 2 to 4 (Pl); between days 10 to 15 (P2) and between days 21 to 26 (P3) Because of the relatively shorter cycles of female subjects C and D, slightly shorter intervals from menses were chosen for P2 and P3 .
Prostaglandins 1994:47,
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43
Prostaglandin
fluctuations
in monocytes:
Leslie and Dubey
TABLE I. Menstrual cycle lengths for female subjects and days of blood withdrawal of menses for each pair
from start
Days Blood Withdrawal Subject
Cycle Length (days)
A B C D
30, 45, 23, 29,
33 31, 29, 31 28, 23 26, 32, 26
Pl
P2
P3
2 2 3 4
14 15 10 12
26 26 21 24
The 4 female subjects had the length of at least one cycle recorded prior to the start of blood sampling. Pl, P2 and P3, were the number of days from the start of menses on which blood was withdrawn and monocytes isolated from each pair. Pl, P2 and P3 were chosen to be within endocrinologically defined segments of the menstrual cycle (Table 2).
Prostaglandin and Cytokine Analysis PGs were assayed by RIA according to our published procedure.‘” The 6-keto-PGF,, (a measure off PG12) antibody (Seragen Inc., Boston, MA) crossreacted (co.01 %) with TXB1, PGD2, PGA, PGB and metabolites of PGE, and PGF2_. The cross reaction with PGF2, was 2.2%, 0.4% with PGE, and 0.06% with PGE,. The PGE, antibody prepared in rabbits in this laboratory crossreacted ~0.5 % with PGAz, PGBz, PGFZa, with metabolites of PGE2, PGFz, and 6-keto-PGF,,. The crossreaction with PGD, was 2%. Cytokines (IL- 1B and IL-6) were assayed using ELISA kits (Genzyme Corp., Boston, MA for IL-6 and Cistron Biotechnology, Pine Brook, NJ for IL-l p). Medium controls incubated under the same conditions contained negligible quantities of PG or cytokines. The data was expressed as the amount of PG or IL found in the medium after 20h incubation at 37°C. Progesterone and Estradiol Determinations Serum levels of progesterone house RIA.
and estradiol
were assayed by a standard in
Statistical Analysis Repeated measures analysis of variance (ANOVA) was used to test for period and gender effects as well as the interaction of period and gender.” For analysis restricted to females, single factor repeated measures were used to test for period effects. Individual comparisons were made using a t test. Level of significance was p 5 .05.
44
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Prostaglandin
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Leslie and Dubey
Results Reproductive Hormones and Cycle Timing In the males serum estradiol and progesterone levels did not markedly change at each period. All 4 females showed low levels of estradiol and progesterone 2 to 4 days from start of menses (Pl) and increased amounts 21 to 26 days from start of menses (P3). Ten to 15 days from the start of menses (P2) all 4 female subjects had unchanged progesterone levels relative to PI while ~3estradiol levels had increased 3 to 4 fold (Table 2). The increase in progesterone from P2 to P3 for each of the 4 females ranged from 10 to 60 fold. By these endocrinological criteria, ls all 4 blood samples taken in P3 were from the luteal phase of the cycle, the samples taken in P2 were taken near ovulation and the samples taken in Pl were from the early follicular phase of the menstrual cycle. Thus all 4 female subjects were accurately reporting the start of their menses. Neither hormone was detectable in the medium (~2 x10-lo M and ~2 x lo-“M for progesterone and estradiol respectively). Prostaglandins The medium from the cultured monocytes after 20h incubation in the absence of LPS, contained only small amounts of PGE, (Figure 1) and PGI, (data not shown). There was no difference between males and females or within phases of the cycle. In the presence of LPS, both PGE, (Figure 1) and PGI, (data not shown] levels increased about 7 fold. The activated monocyte from the female produced a greater range of PG amounts than the male (3.2 to 18.6 ng PGE, for females versus 2.4 to 8.4 ng PGE, for males; 0.3 to 2.5 ng PGI, for females versus 0.3 to 1.3 ng for males). The mean PGE, and mean PGI, amounts (averaged over P 1, P2 and P3) found in the medium for the female of each pair was consistently and significantly greater than the male average (p< .05 for PGI, and p< .03 for PGE,). As TABLE2. Serum estradiol and progesterone
Cycle Period Pl P2 P3
levels in male and femal subjects at the period of blood withdrawal
Progesterone ng/ml + SE
Estradiol pg/ml + SE
Progesterone ng/ml +- SE
Estradiol pg/ml 5 SE
.35 & .13 .19 2 .Ol .42 + .09
45 2 1.5 39 i 2.9 43 c 3.8
.33 * .05 .26 ? .06 6.10 & 2.0
36 2 9 124 i 30 113 + 18
Blood was withdrawn within 24 days (Pl), 10-15 days (P2) and 21-26 days (P3) from the start of menses of each female and serum progesterone and estradiol assayed. The hormone levels in Pl , P2 and P3 were within the expected ranges for samples taken during menses, near ovulation and during the luteal phase of the cycle respectively.
Prostaglandins
1994:47,
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45
Prostaglandin fluctuations in monocytes: Leslie and Dubey 16 PGE2
I I I I I I I I I
12 LPS absent
8
m
Male
q
Female
LPS present
I
I I I
4
I I I
I 1
2
3
Period
1
FIGURE 1. Blood was withdrawn within 2-4 days (Pl), 10-15 days (P2) and 21-26 days (P3) from the start of menses of each female. Monocytes were isolated, incubated for 20h with LPS (5ug/ml) and the medium assayed for PGE, by RIA.
46
Prostaglandins 1994:47, January
Prostaglandin fluctuations in monocytes: Leslie and Dubey shown in Figure 1 for PGE2, this difference between males and females was due to an increase in PG in the m e d i u m from monocytes isolated later (P3) rather than earlier (P1) in the cycle. By ANOVA the increased amount of PGE~ in the m e d i u m from monocytes isolated from w o m e n compared to monocytes from m e n was significant (p< .038) and the effect of time of blood withdrawal (period effect) also differed for the 2 sexes (p< .025). Since this difference was significant only when comparing P1 with P3 (p< .039) this means that monocytes isolated from females during P3 (the luteal phase of the cycle) produced more PG than monocytes isolated from males at the same time. Monocytes isolated from females during other phases of the cycle (P1 and P2) did not produce significantly more PG than monocytes from males. An analysis restricted to females only also showed (Figure 1) a significant increase (p< .028) in PG levels in the luteal phase of the cycle relative to the two other phases (P1 and P2) or to P1 alone (p = .05) A similar significant pattern of PG production in P 1, P2 and P3 was found for PGI2 (Figure 2). Thus for total cylooxygenase products assayed (PGE~ plus PGI2) the females had higher levels than males (p< .035) with the significant increase relative to males (p<. 02) and among females (p< .045) occurring in P3 relative to P1. There was no variation from period to period in the ~g cell protein obtained per 10 million cells plated.
Cytokines IL-6 and IL-lf~ were assayed only in the female subjects. Incubation of monocytes with LPS increased both cytokines. About 10 fold more IL-6 than IL-1 ~ was found in the medium. There was no association between the amount of IL-6 released and the particular phase of the cycle either in the absence of LPS (24.1 + 6.5, 26.7 + 5.3 and 20.4 + 5.0 in ng/20h + SE) or in the presence of LPS ( 56.2 _+ 7.0, 54.3 + 2.6 and 55.5 + 7.1 in ng/20h _+ SE) for P1, P2 and P3 respectively. For all w o m e n the amount of IL-I~ found in the m e d i u m peaked in P2 relative to the other periods (Table 3). This period effect was significant for monocytes incubated both in the absence (p< .041) or in the presence (p< .049) of LPS.
Discussion In a previous report we had noted in female mice that the amount of PG synthesized from the macrophage was greater than from male mice and was apparently influenced by the stage of the estrus cycle. 1° This new data demonstrates that PG accumulation in the m e d i u m from the activated h u m a n monocyte was also greater in females than in males and may be influenced by the hormone(s) released during the menstrual cycle. Thus, in monocytes isolated from the females and exposed in vitro to an inflammatory stimulus, more PG was found in the m e d i u m from cells
Prostaglandins 1994:47, J a n u a r y
47
Prostaglandin fluctuations in monocytes: Leslie and Dubey 16 PGE2
1
I 12
LPS present
LPS absent
8
H
Male
0
Female
I I I I I I I I
I
1
2
3
Period
1
2
3
FIGURE 2. Blood was withdrawn within 2-4 days (Pl), 10-15 (P2) and 21-26 days (P3) from the start of menses of each female. Monocytes were isolated, incubated for 20h with LPS (5ug/ ml) and the medium assayed for 6-keto-PGF,,, (a measure of PGI,) by RIA.
48
Prostaglandins 1994:47, January
Prostaglandin fluctuations in monocytes: Leslie and Dubey TABLE 3. Release of interleukin-1 from monocytes isolated at endocrinologically of the menstrual cycle
defined phases
Cycle Period Pl P2 P3
LPS Absent
LPS Present
1.11 ? .48 ‘2.60 ? .84 .69 2 .29
6.27 ? 1.25 11.35 ” 2.22 6.46 ? 1.62
Blood was withdrawn from the 4 women during menses (Pi), near ovulation (P2) and during the luteal phase (P3) of the menstrual cycle. Monocytes were isolated, incubated for 20h with and without LPS (5ug/ml) and the medium assayed for IL-1 B by ELISA. For all 4 females both the spontaneous release of IL-1B and the release after LPS activation peaked near ovulation (P2). This increase was significant in both the absence (~~041) and present (pe.049) of LPS.
isolated in the luteal (P3) than in the early follicular phase (Pl) of the menstrual cycle. It was also in this luteal phase in which the female differed substantially from the males. Since in these human experiments monocytes from each female were isolated only once during the luteal phase of the cycle, further experiments would need to be done to determine the duration of this increase and when the peak amount of PG accumulates. In the absence of an in vitro stimulus very little PG was detected. Both arachidonate products assayed were increased 7 fold when cells were stimulated with LPS. However PGE, was present in much greater quantities than PGI,. It has previously been documented that in humans the monocyte is the major cell in the immune system responsible for PGE production.” Besides our report of differences in PG synthesis in macrophages from the male and female mouselo there is one other report in the rat.18 These authors were the first to report that LPS activated macrophages from females produced more PG than the males. In human neutrophils, both gender and menstrual cycle related variations in PG production have been documented.lY In the human monocyte from females the PG increase occurred in the luteal phase of the menstrual cycle in which granulosa cells, form a corpus luteum and produce progesterone and 17 hydroxyprogesterone. Since both PGE, and PGI, increased during this luteal phase, this implies that the reproductive hormone(s) involved, may exert their effect to up regulate a common precursor. There is little information on the influence of the ovarian steroids on PC metabolism in the monocyte. However in the sheep endometrium, progesterone induces expression of mRNA encoding for cyclooxygenase, the rate limiting enzyme in PG production.20 In this context it has been documented that certain cells, including macrophages and monocytes,z1,22 express not only a constitutive cyclooxygenase but a second regulated cylooxygenase (COX2) which is
Prostaglandins 1994147, January
Prostaglandin fluctuations in monocytes: Leslie and Dubey LPS inducible.23 We have noted that LPS activated macrophages from ovarectomized mice produces less mRNA for COX2 than macrophages from control mice (Leslie C.A. unpublished observation). In another experiment we found a marked reduction in PGE, ( + SE) synthesis from the macrophages of ovarectomized mice (6.7 + 3.4 ng/24h) relative to sham operated mice (19.4 + 4.2 ng/24h) and control female mice (14.6 + 2.9 rig/244). We therefore suggest that exposure of monocytes to an ovarian steroid such as progesterone, during the luteal phase of the cycle increases the amount or stability of COX2 mRNA when induced by an inflammatory stimulus. If monocytes are isolated in the luteal phase of the cycle and activated in vitro by LPS (or activated in vivo) more immunosuppresive PGE, will be produced than from monocytes isolated at other phases. This may be a mechanism which in vivo will favor implantation and fetal survival. Thus if fertilization occurs, the continued high levels of progesterone, may through a local increase of immunosuppresive PGE, by infiltrating macrophages24 protect the developing blastocyst from the maternal immune response. Interestingly it has been documented that in the human endometrium more PG is released during the luteal phase of the cycle.2s Gonadal steroids are known to modulate IL-1 activity in both human pelvic macrophages26 and human monocytes.27,28 In the 4 females quantitated here, IL-6 release from the monocyte was independent of the stage of the cycle. However monocytes isolated close to ovulation (P2), prior to any increase in serum progesterone but during the first estradiol surge, produced significantly more IL-ll3 than at other periods (Table 3). The reproductive hormones may therefore differentially regulate IL-l and IL-6 production in activated monocytes. 2y In P3 after the isolated monocytes had been exposed in vivo to luteal phase levels of progesterone, IL1 release decreased. Others have shown that 10m7to 10m8M progesterone, the concentration reached in vivo during the luteal phase is sufficient to markedly inhibit IL-l mRNA30 as well as IL-1 activity.28 Our experiments do not enable us to determine for how long before or after P2, IL-ll3 is up regulated. However using an activity assay for IL-I, Cannon and Dinarello”’ reported in humans increased plasma levels after ovulation. Monocytes and macrophages process and present antigens to B and T cells32 and amplify the immune response by producing IL-l, a fundamental event in the initiation of the immune/inflammatory response.“” They are unique among cell types in containing high levels of arachidonate16,34 and are the principal source of eicosanoids affecting other immune cells.“” Of great interest is the ability of PGE, to down regulate monocyte peptide signals such as IL- 1 which are necessary to promote a full blown immune response.36 High concentrations of PGE, in vitro inhibit mitogen responsiveness, clonal proliferation, antigenic stimulation, lymphokine production, the proliferative response of IL-2 dependent T cells, monocyte
50
Prostaglandins 1994:47, January
Prostaglandin fluctuations in monocytes: Leslie and Dubey cytotoxic activity, natural killer cell activity and B cell proliferation.37)38 Chronic overproduction of PGE can cause immunosuppresion and inhibiting PC synthase is known to stimulate cellular immune responses in animals and humans.“‘+0 The documented increase in PGE, synthesis from both spleens of old mice41+2 and PBMC from elderly people4” relative to young controls is associated with a decline in immune function with increasing age.42,44 Thus, a possible consequence of cyclical changes in amount of PGE, and IL-l produced from an activated monocyte or macrophage in vivo would be to make premenopausal females more vulnerable to immune stress at certain times of their cycle and less vulnerable at other times. This is certainly true in mice where quantitative differences in immune function with the cycle phase have been reported.4s In women there is a dearth of such information although in one unique study it has been documented that Candidal vaginitis occurs most frequently during the luteal phase, the same time of maximal reduction of cellular responsiveness to Candidia.4” Further work by this group4’ established that in the presence of luteal phase levels of progesterone there was a 50% decrease in C. albicans induced lymphocyte proliferation. Removal of the monocytes from the PBMC obviated the ability of progesterone to inhibit this response strongly suggesting that the inhibition of lymphocyte proliferation was a monocyte mediated mechanism.“’ This is very consistent with our observation that human monocytes isolated during the luteal phase (after exposure to luteal phase levels of progesterone) produce increased amounts of immunosuppresive PGE,. In view of the significance of PGs and cytokines from the monocyte to modulate the immune response, understanding how normal physiologic changes associated with the menstrual cycle alters this balance could be a clue to understanding differences in vulnerability, within phases of the cycle, and between males and females, to immune and inflammatory insult. We are suggesting that activation and suppression of monocyte PG and cytokine synthesis by the precisely timed high amplitude fluxes of the ovarian hormones released during the menstrual cycle may provide the key to the mechanism underlying the biological puzzle of sexual dimorphism in immunity.
Acknowledgments Reprint requests to Crystal A. Leslie, Research (1.51), ENRM VA Hospital, 200 Springs Rd., Bedford, MA 01730. This work was supported by the US Department of Veterans Affairs and National Institute of Health Grant AI 268 17. We thank Dr. Deborah Andersen, Fearing Research Laboratory, Harvard Medical School for hormone determinations, Dr. Jennifer Andersen of l
Prostaglandins 1994:47, January
51
Prostaglandin fluctuations in monocytes: Leslie and Dubey Boston University School of Medicine N. Mirza for technical assistance.
for the statistical
analysis and Dr.
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Grossman, C. Interactions between the gonadal steroids and the immune system. Science 227: 257. 1985. Masi, A.T., and T.A. Medsge. In: Arthritis and Allied Conditions 1D.J. MC Carty ed) Lea and Febiger, Philadelphia, 1979, p.11. Roubinian, J.R., N. Talal, J.S. Greenspan, JR. Goodman, and P.K. Siiteri. Delayed androgen treatment prolongs survival in murine lupus. J. Clin. Inv. 63: 902. 1979. Sakane, T., A.D. Steinberg, and I. Green. Studies of immune function of patients with systemic lupus erythematosus. Arthritis Rheum. 22: 657.1978. Rose, E., and D.M. Pillsbury. Lupus erythmatosus and ovarian function, observations on possible relationship with report of 6 cases. Ann. Intern. Med. 22: 1022. 1944. Mund, A., J. Swison, and N. Rothfield. Effect of pregnancy on the course of systemic lupus erythmatosus. JAMA. 183: 917. 1963. Jungers, P., M. Dougsdos, C. Pelissier, et al. Influence of oral contraceptive therapy on the activity of systemic lupus erythematosus. Arthritis Rheum. 25: 618. 1982. Kunkel, S. The importance of arachidonate metabolism by immune and nonimmune cells. Lab. Invest. 58: 119. 1988. Leslie, C.A., W.A. Gonnerman, and E.S. Cathcart. Gender differences in eicosanoid production from macrophages of arthritis-susceptible mice. J. Immunol. 138: 413. 1987. Goodwin, J., and J.L. Ceuppens. Regulation of the immune response by prostaglandins. J. Clin. Immunol. 3: 295. 1983. Dubey, D.P., LYunis, C.A. Leslie, C. Mehta, and E.J. Yunis. Homozygosity in the major histocompatibility complex region influences natural killer cell activity in man. Eur. J. Immunol. 17: 61. 1987. Todd, R.F., L.M. Nadler, and S.F. Schlossman. Antigens on human monocytes identified by monoclonal antibodies. J. Immunol. 226: 1435. 1981. Kung, P., G. Goldstein, E.L. Reinherz, and SF. Schlossman. Monoclonal antibodies defining distinctive human T cell surface antigens. Science 206: 347. 1979. Schechter, D., G.A. Bachman, J. Vaitukaitis, D. Philips, and D. Saperstein. Perimenstrual symptoms: Time course of symptom intensity in relation to endocrinologically defined segments of the menstrual cycle. Psychosomatic Med. 51: 173. 1989. Leslie, C.A., W.A. Gonnerman, D. Ullman, K.C. Hayes, C. Franzblau, and E. S. Cathcart. Dietary fish oil modulates macrophage fatty acids and decreases arthritis susceptibility in mice. J. Exp. Med. 162: 1336. 1985. Snedecor G.W., and W.G. Cochran. In: Statistical Methods. 6th edition. Iowa University Press, Ames, Iowa 1967, p. 1.
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