Henk Moes

The immune response during the luteal phase of the ovarian cycle: increasing sensitivity of human monocytes to endotoxin

OBJECTIVE To test the hypothesis that during the luteal phase of the human ovarian cycle, as compared with the follicular phase, the percentage of cytokines producing peripheral monocytes after in vitro stimulation with endotoxin is increased. DESIGN Prospective study. SETTING Academic research institution. PATIENT(S) Women with regular menstrual cycles. INTERVENTION(S) Blood samples were collected between days 6 and 9 of the menstrual cycle (follicular phase) and between days 6 and 9 of the menstrual cycle following the LH surge (luteal phase). MAIN OUTCOME MEASURE(S) Percentages of tumor necrosis factor (TNF)-alpha-, interleukin (IL)-1 beta-, and IL-12-producing monocytes as well as total white blood cell (WBC) count, differential WBC counts, and plasma 17 beta-estradiol and progesterone concentrations. RESULT(S) Mean plasma 17 beta-estradiol and progesterone concentrations, percentage of TNF-alpha- and IL-1 beta-producing monocytes, WBC counts, and granulocyte cell count were significantly increased in the luteal phase as compared with the follicular phase of the ovarian cycle. The percentage of IL-12-producing monocytes, monocyte count and lymphocyte count did not vary between the 2 phases of the ovarian cycle. CONCLUSION(S) Together with an increase in progesterone and 17 beta-estradiol during the luteal phase, there is an increase in percentage TNF-alpha- and IL-1 beta-producing peripheral monocytes after in vitro stimulation with endotoxin as compared with the follicular phase of the ovarian cycle. Whether this increased sensitivity of monocytes for proinflammatory stimuli during the luteal phase is due to increased plasma levels of progesterone or 17 beta-estradiol needs further investigation.

Association between macrophage activation and function of micro-encapsulated rat islets

Aims/hypothesisSurvival of microencapsulated islet grafts is limited, even when inflammatory reactions against the capsules are restricted to a small portion of less than 10%.MethodsThis study investigates both in vivo in rat recipients and in vitro whether cellular overgrowth on this minority of the capsules contributes to limitations in the functional survival of the 90% of the encapsulated islets which remain free of any cellular overgrowth.ResultsIn successful rat recipients of an allogenic microencapsulated islet graft we found that the vast majority of cells in the capsular overgrowth were activated ED-1 and ED-2 positive macrophages which were found in numbers of approximately 1500 per capsule. Co-culture of encapsulated islets with 1500 (nr8383) rat-macrophages per capsule showed that the activation of macrophages was caused by islet-derived bioactive factors since TNF-α and IL-1β secretion by macrophages was induced by islet-containing capsules and not by empty capsules. This activation of macrophages was associated with a decrease in function of the encapsulated islets as evidenced by a quantitatively reduced (35%) insulin response in static incubation and a slower response in perifusion.Conclusion/interpretationPresent research aims to design strategies for the temporary inhibition of macrophage activation since macrophages are predominantly present in the first two months after implantation. These strategies will serve as a pertinent basis for future clinical application of microencapsulated islets.

The Effect of Testosterone on Cytokine Production in the Specific and Non-specific Immune Response

Problem:  Cytokine production of monocytes and lymphocytes differs between males and females. This difference is characterized by a decreased percentage of interleukin (IL)‐2‐producing lymphocytes and an increased percentage of IL‐12, IL‐1β and tumour necrosis factor (TNF)‐α‐producing monocytes in males compared with females. In the present study, we investigated whether testosterone may explain these differences.

Species differences in the effect of pregnancy on lymphocyte cytokine production between human and rat

In the present study, we evaluated whether lymphocyte cytokine production during human and rat pregnancy shifts toward T helper cell type 2 (Th2) cytokine production. Therefore, blood samples were taken during the follicular and luteal phase and during pregnancy in rats and humans. Whole blood was ex vivo‐stimulated with phorbol 12‐myristate 13‐acetate and calcium ionophore and intracellular interferon‐γ (IFN‐γ) and interleukin (IL)‐4 production, and the percentage of cells in the various lymphocyte populations was measured using flow cytometry. Rats and humans adapted their immune responses to pregnancy but have different strategies: During human pregnancy, the percentage of lymphocytes producing IFN‐γ was decreased, and the percentage IL‐4‐producing lymphocytes was not affected. The rat adapts its immune response to pregnancy by decreasing the total number of the various lymphocyte populations, and the percentage of IFN‐γ‐ or IL‐4‐producing lymphocytes was not affected or increased (% IFN‐γ‐producing cytotoxic lymphocytes). It is speculated that during rat pregnancy, there is no need to decrease the number of IFN‐γ‐producing lymphocytes, as in nonpregnant rats, the total number of IFN‐γ‐producing lymphocytes after stimulation is relatively low, and there is no necessity for a further decrease. In nonpregnant humans, the percentage IFN‐γ‐producing lymphocytes is much higher and probably dangerous for pregnancy, and therefore, this percentage needs to decrease during pregnancy. In conclusion, although the data from humans concur with the Th1/Th2 paradigm, the data from rats do not concur with this paradigm. The present studies therefore challenge the classical Th1/Th2 paradigm during pregnancy.

Pancreatic beta-cell function and islet-cell proliferation: Effect of hyperinsulinaemia

Pancreatic beta-cell function was studied in adult female rats, in which endogenous insulin demand was fully met by SC infusion of human insulin (4.8 IU/24 h) for 6 days, resulting in hyperinsulinaemia and severe hypoglycaemia. The amount of pancreatic endocrine tissue declined by 40%, (pro)insulin mRNA, as determined by in situ hybridisation by 95%, and the amount of stored insulin by 90%. Islet-cell proliferation as determined by 24 h of BrdU infusion declined by 60%. Basal glucose levels normalized within 2 days after the insulin treatment was ended, whereas about 1 week was needed to restore the amount of pancreatic insulin, glucose-induced insulin release, and glucose tolerance to normal values. The amount of endocrine tissue recovered within 48 h and mRNA abundance within 96 h after discontinuation of the insulin infusion, whereas at that time islet-cell proliferation still showed a sixfold increase, before returning to control levels after 1 week. These results show that after a period of suppression of beta-cell function, recovery of insulin synthetic capacity does not immediately result in normalization of insulin stores and insulin release. Under these conditions, episodes of hyperglycaemia may occur, which may act as a stimulus for islet-cell proliferation.

Developing ovarian follicles inhibit the endotoxin-induced glomerular inflammatory reaction in pseudopregnant rats

Problem:  We tested the hypothesis that developing ovarian follicles produce factors inhibiting the endotoxin induced inflammatory response.

Monocyte cytokine production during pregnancy

With great interest, we have read the article, “Monocytes are progressively activated in the circulation of pregnant women” [1]. In their article, the authors showed that there is progressive, generalized, inflammatory cell activation, characterized by progressive up-regulation of CD11a, CD54, and CD64 during pregnancy. In addition, granulocyte numbers were increased, and lymphocyte numbers decreased during pregnancy. In this comprehensive paper, the authors not only showed phenotypical activation of the inflammatory cells but also functional activation of these cells, as they showed an increase in the percentage of monocytes spontaneously producing interleukin (IL)-12 in third-trimester pregnant women as compared with nonpregnant women and increased IL-1 production by stimulated second-trimester pregnant monocytes as compared with stimulated, nonpregnant monocytes. Moreover, stimulated third-trimester pregnant granulocytes produced more IL-8 than nonpregnant granulocytes. These data are in line with the hypothesis that pregnancy is a proinflammatory condition [2–4]. However, for adhesion molecule expression and intracellular cytokine production, not all data in this paper are consistent with data in other papers about the same subject [3–6], including a previous paper by Luppi et al. [7]. Indeed, studying their data in detail, Luppi et al. [7] showed some surprising results: First, a relatively high percentage of unstimulated monocytes produce IL-12 [mean percentage of IL-12-producing cells at the end of pregnancy, 33% (Fig. 5 in their paper)], and in an individual case, 71% of circulating monocytes are producing IL-12 (Fig. 4 of their paper). Similar results were found for tumor necrosis factor (TNF; 20% of circulating monocytes during pregnancy produce TNF, and in an individual case, up to 43% of circulating monocytes produce TNF). Only when male volunteers were injected with endotoxin did we observe cytokine production in unstimulated cells [8]; however, the percentage of cytokine-producing cells was never more than 10%, and even these low percentages of circulating, cytokine-producing cells were pyrogenic in these males. Spontaneous production of IL-12 and TNFduring normal pregnancy is a very dangerous situation, especially in light of the well-known, harmful effects of type 1 cytokines, such as IL-12 and TNF, for the fetus [9]. Therefore, from a biological point of view, it is not very likely that during normal pregnancy, 20–30% of the monocytes are spontaneously producing IL-12 and/or TNF. Indeed, we [4] and others [3, 5] have also looked at monocyte intracellular cytokine production during third-trimester pregnancy or immediately after pregnancy and did not find significant percentages of monocytes spontaneously producing TNFor IL-12 (i.e., percentages were below 5%). Additionally, we have measured intracellular monocyte cytokine production (TNF, IL-12, or IL-1 ) in various reproductive phases in humans and have never observed production of cytokines in unstimulated cells [8, 10]. This prompted us to question the methodology used by Luppi et al. [7]. The other surprising finding in the paper by Luppi et al. [7] was the fact that IL-12 production decreased after endotoxin or phorbol 12-myristate 13-acetate/ionomycin stimulation of monocytes of third-trimester women. This is in sharp contrast to all other papers studying endotoxin-induced IL-12 production, as it is common knowledge that endotoxin is a stimulator of IL-12 production [11]. Endotoxin not only stimulates monocyte IL-12 production in nonpregnant individuals or in males [8] but also during pregnancy [3–5]. A remarkable finding concerning the methodology of the present paper was the use of the isotype controls for analysis of intracellular cytokine production. Isotype controls are classically used to set the lower limits for staining positivity, such as done by Luppi et al. [7]. There has, however, been discussion as to the limitations of the use of isotype controls in flow cytometry [5, 12]. Issues to be considered are the fact that antibodies, including isotype controls, are currently produced by hybridomas. They will structurally differ from each other, even within the same immunoglobulin (Ig) isotype and may therefore have different binding properties [5, 12]. Second, different companies use different protocols to produce, purify, and chemically conjugate antibodies with fluorochromes. Therefore, the fluorochrome-to-antibody ratio may differ among different companies [5, 12]. This indicates that if we use isotype controls, the choice of the isotype control should be made on the basis of an identical Ig subclass from the same species, and it is also very important to purchase isotype controls from the same company as the antibody itself [5, 12]. Unfortunately, Luppi et al. [7] did not meet all above-mentioned criteria. This holds, for instance, for anti-IL-12 and its isotype control, which were purchased from different companies. We question, therefore, whether differences in results between the paper of Luppi et al. [7] and the other papers on unstimulated and stimulated IL-12 production by monocytes [3, 5] may be a result of use of an inappropriate isotype control by Luppi et al. [7]. Possibly, this may also hold

EFFECT OF INSULIN ON ENDOCRINE PANCREAS FUNCTION DURING LATE PREGNANCY IN THE RAT

To partly or completely satisfy the increasing demand for insulin, pregnant rats were infused SC with human insulin (2.4 or 4.8 IU/day) from day 14 to day 20 of gestation. Cyclic control rats underwent the same procedure of 6 days of insulin-treatment. During the treatment all groups of rats were hypoglycaemic, but foetal survival was not affected. The low dose treatment prevented the characteristic rise of the insulin response to a glucose challenge during pregnancy, both in vivo and in vitro, while the high dose treatment suppressed the insulin response, as well as the pancreatic insulin content. The insulin responses and insulin contents of pregnant rats were higher than those of the corresponding cyclic control rats. These results support the hypothesis that during gestation the increased insulin demand, due to the actions of placental hormones, is the cause of the increased insulin secretion. However, it cannot be excluded that direct effects of placental hormones on the islets of Langerhans are also involved.

Cytokine production of in vitro stimulated peripheral lymphocytes during the course of pregnancy and pseudopregnancy in the rat.

Problem  Does maternal lymphocyte cytokine production after in vitro stimulation vary with the stage of pregnancy in the rat?

Insulin Treatment Prevents Adaptation of the Endocrine Pancreas to Pregnancy

During pregnancy in the rat the insulin sensitivity of the maternal tissues decreases, thereby increasing the demand for insulin (Leturque et al. 1989). At the same time the insulin production and release increases (Koiter et al. 1989). These phenomena are associated with a transient increase in islet-cell proliferation (Dunger et al. 1989). It has been suggested that placental lactogens directly stimulate β-cell function and the proliferation of islet cells (Nielsen et al. 1986; Parsons et al. 1992). Alternatively, placental lactogens, by increasing the demand for insulin (Freinkel 1985), indirectly cause the increase in insulin production and cell-proliferation in the islets. This hypothesis of increased insulin demand was tested by investigating how insulin production and cell-proliferation change when exogenous insulin was infused in pregnant rats at such a rate that the demand for insulin was fully met.