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? METHOD OF STUDY: Blood samples were taken during the estrus cycle in rats (n = 11). Thereafter, rats were rendered pregnant (n = 6) or pseudopregnant (n = 5) and blood samples were taken at days 4, 8, 11, 15, and 20 of pregnancy and pseudopregnancy. White blood cell (WBC) count was measured and whole blood was stimulated with phorbol 12-myristate 13-acetate and calcium ionophore; interferon-gamma (IFNgamma) and interleukin-4 (IL-4) production as well as (sub)populations of lymphocytes were measured using flow cytometry. RESULTS: We observed an increase of WBC in the second week of pregnancy and a slowly decreasing percentage of lymphocytes during the course of pregnancy. The percentage IFNgamma producing T-lymphocytes after in vitro stimulation was increased during pregnancy (for Th-lymphocytes only in the second week of pregnancy, for Tc-lymphocytes at all days). This increased IFNgamma production in pregnant T-lymphocytes was accompanied by an increase during pseudopregnancy, and therefore may result from increased sex hormone concentrations. The percentage IFNgamma producing natural killer (NK) cells after in vitro stimulation was decreased on day 20 of pregnancy. No effect of pregnancy or pseudopregnancy was seen on percentage IL-4 producing lymphocytes after in vitro stimulation. CONCLUSION: In the rat the IFNgamma production after in vitro stimulation varies during pregnancy and is increased, rather than decreased, during pregnancy.

Monocyte activation, but not granulocyte activation, is inhibited in the presence of developing ovarian follicles.

This study was set up to evaluate the influence of ovarian factors on the acute phase of the endotoxin-induced glomerular inflammatory reaction. Six groups of rats with permanent jugular vein cannulas were used. This included three groups with increased progesterone and/or 17beta-oestradiol concentrations (day 14 pregnant rats, pseudopregnant rats and lactating rats), one group with the presence of developing ovarian follicles (cyclic rats), and two groups with both increased sex hormone concentrations and the presence of developing ovarian follicles (day 14 pregnant rats treated with FSH and day 21 pregnant rats). Rats were infused for 1h with either saline or endotoxin (1 microg/kg body weight) and sacrificed 4h after the infusion. Kidney sections were snap-frozen and prepared for immunohistochemistry. Endotoxin-induced glomerular granulocyte infiltration was increased only in the groups of rats with increased progesterone and/or 17beta-oestradiol concentrations. This could be due to endotoxin-induced ICAM-1 and/or VCAM-1 expression, which was observed in all endotoxin-treated groups and in all endotoxin-treated groups with increased sex hormone concentrations, respectively. It could also be due to an effect on granulocytes per se, since the number of endotoxin-induced CD11b-positive cells in the glomeruli was increased only in the groups with increased sex hormone concentrations. Endotoxin-induced glomerular monocyte infiltration, however, was seen only in those groups in which developing ovarian follicles were lacking (i.e. day 14 pregnant, pseudopregnant and lactating rats), suggesting that developing ovarian follicles produce anti-inflammatory factors. These factors did not have an effect on endothelial or leukocyte adhesion molecule expression. We hypothesize that the presence of elevated progesterone concentrations increased the endotoxin-induced glomerular granulocyte infiltration, while endotoxin-induced glomerular monocyte infiltration was inhibited in the presence of developing ovarian follicles.

Total white blood cell counts and LPS-induced TNF alpha production by monocytes of pregnant, pseudopregnant and cyclic rats.

Pregnancy in the rat may be associated with an activated innate immune system. Therefore, we investigated monocyte function as well as total white blood cell (WBC) counts during the follicular phase of the ovarian cycle, pregnancy and pseudopregnancy in the rat. Rats were equipped with a permanent jugular vein cannula, and 0.43 ml blood samples were taken from this cannula during the 4 days of the regular oestrus cycle of the rat (n=12). Thereafter, six rats were rendered pregnant, and the other six rats were rendered pseudopregnant according to standard methods. Blood samples were withdrawn from the cannula on days 4, 7 and 11 of pseudopregnancy and on days 4, 7, 11 and 20 of pregnancy. From each blood sample, 0.4 ml was stimulated with lipopolysaccharide (LPS) and monocyte intracellular cytokine production measured using flow cytometry. 30 microl of the blood was used to measure WBC counts and differential WBC counts. The results showed that the number of WBC was significantly increased only on day 11 of pregnancy compared with the follicular phase, and that this was due to the increased numbers of polymorphonuclear (PMN) cells. The percentage of TNF alpha-producing monocytes was increased on all days of pseudopregnancy and on day 11 of pregnancy. The fact that the percentage of monocytes producing TNF alpha upon an LPS stimulus was increased during the post-implantation phase of pregnancy and during pseudopregnancy as compared to the follicular phase may indicate that these conditions are proinflammatory conditions. For the post-implantation phase of pregnancy, this is once more stressed by the increased numbers of WBC and PMN.

Glomerular immunoglobulin deposits induce glomerular inflammation in pregnant but not in non-pregnant rats.

PROBLEM: Does an inflammatory stimulus evoke a more intense inflammatory response in pregnant rats as compared with non-pregnant rats? METHOD OF STUDY: Non-pregnant rats were injected with antibodies against the glomerular basement membrane (GBM), 14 days before pregnancy, to induce a subclinical glomerulonephritis. Part of the rats were rendered pregnant, the others remained non-pregnant throughout the experiment. Two experiments were performed: in experiment 1, pregnant and non-pregnant rats were killed at various intervals after the injection with antibody and parameters characteristic of a glomerular inflammation were evaluated using immunohistology on cryostat kidney sections and liver sections. In experiment 2, 24-hr urinary protein excretion was measured at various days after the injection in pregnant and non-pregnant rats. RESULTS: Experiment 1 revealed that a significant glomerular inflammation, as characterized by increased numbers of monocytes and LFA-1 positive cells per glomerulus, was only observed in pregnant rats with glomerulonephritis. Experiment 2 revealed that only pregnant rats with glomerulonephritis showed increased urinary protein excretion. CONCLUSION: The fact that glomerular inflammation coincides with proteinuria only in pregnant rats with glomerulonephritis, may suggest that these phenomena are causally related and promoted by the pregnant condition.