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Cell-Mediated Immunity and Postpartum Thyroid Dysfunction: A Possibility for the Prediction of Disease?¹

Municipal Health Service Southeast Brabant (J.L.K.), Valkenswaard; Department of Immunology (M.H.-M., H.A.D.) Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands; Clinical Laboratories (H.L.V.), St. Joseph Hospital Veldhoven, 5500 MB Veldhoven; Department of Social and Behavioral Sciences (V.J.P.), University of Tilburg, 5000 LE Tilburg; and Department of Endocrinology (W.M.W.), Academic Medical Center, University of Amsterdam, 1100 DD Amsterdam, The Netherlands

Address correspondence and requests for reprints to: J. L. Kuijpens, M.D., Department of Public Health, Municipal Health Service Southeast Brabant, P.O. Box 135, 5550 AC Valkenswaard, The Netherlands.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Postpartum (pp) thyroid dysfunction (PPTD) is thought to be caused by an autoimmune (AI) destruction of thyroid follicles during the pp period. The chronic thyroid AI process [already present in pregnancy, as shown by the positivity for thyroid peroxidase antibodies (TPO-Ab)] becomes overt disease in the pp period, and one assumes that this exacerbation represents a rebound phenomenon after a general immunosuppression during pregnancy. The presence of TPO-Ab in pregnancy has been suggested as a predictor for later PPTD development. Apart from B cells, e.g. production of autoantibodies, various functions of the cell-mediated immune (CMI) system, including those of peripheral T cells, monocytes, and dendritic cells (DC), are also disturbed in AI states.

The objectives of the present study were: determining alterations in various CMI parameters in pregnancies followed by PPTD vs. those not followed by PPTD; and determining the usefulness of these parameters in the prediction of PPTD.

In a prospective study (region: Kempenland, southeast Netherlands), a random sample of 291 women were tested at 12 and 32 weeks gestation and 4 weeks pp for TPO-Ab. Women were followed until 9 months pp, for developing PPTD. PPTD was defined as both: an abnormal TSH, and fT₄ pp women developing PPTD and/or being positive for TPO-Ab (n = 26); and thyroidological uneventful control women of the same cohort, matched for age and parity (n = 21), were tested for thyroid-stimulating antibodies, percentages of peripheral blood lymphocyte subsets using fluorescence-activated cell sorter analysis (CD3, CD4, CD8, CD16, CD56, major histocompatibility complex-class II), for monocyte polarization, and for cluster capability of monocyte-derived DC.

Results were: 1) 31 women (10.7%) were positive for TPO-Ab (TPO-Ab⁺) in gestation (12 and/or 32 weeks); 2) 15 women (5.2%) developed PPTD, of whom 10 were TPO-Ab⁺ in gestation; 3) pregnancy-related CMI alterations consisted of low percentages of CD16⁺CD56⁺ natural killer (NK) cells and a low DC cluster capability at 12 weeks gestation (these functions were normalized at 32 weeks gestation); 4) the TPO-Ab⁺ PPTD⁺ women (4 hyper, 5 hypo, and 1 hyper/hypo) were characterized by a persistently low percentage of NK cells, a lowered monocyte polarization, and a raised percentage of major histocompatibility complex-class II⁺CD3⁺ T cells; 5) the TPO-Ab^- PPTD⁺ women (all 5 hyper) had neither thyroid-stimulating antibodies nor CMI alterations, apart from those normally seen in pregnancy; 6) 21 women were positive for TPO-Ab in pregnancy but did not develop PPTD (they had the same lowered NK cell percentages and monocyte polarization as the TPO-Ab⁺ PPTD⁺ cases, but they had normal percentages of activated peripheral T cells and a lower titer of TPO-Ab); 7) determination of the number of NK cells and monocyte polarization hardly contributed to the prediction of PPTD (as compared with TPO-Ab status), because of strong interindividual variation and close association with the presence of TPO-Ab; and 8) combining TPO-Ab assays with testing for activated T cells was the most optimal parameter for the prediction of TPO-Ab⁺ cases of PPTD in our small test set.

We conclude that TPO-Ab⁺ pregnant women who develop PPTD show several CMI abnormalities other than those seen in normal pregnant women, such as persistently lower percentage of NK cells, a lowered monocyte polarization, and a raised percentage of activated T cells. The latter seems rather specific for the actual PPTD development and is not found in TPO-Ab⁺ (but PPTD) uncomplicated pregnancies. TPO-Ab^- (but PPTD⁺) women had no signs of CMI abnormalities (apart from those specific for the pregnancy state). Although studied cases are low in number, our data are suggestive for the existence of two forms of PPTD: a TPO-Ab⁺ (AI) form (two-thirds of patients, classical PPTD pattern); and a TPO-Ab^- (non-AI) form (one-third of patients, only hyper). Such assumption implies that, at best, two-thirds of PPTD cases can be predicted using either humoral and/or cellular immune tests.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
THE SYNDROME of postpartum (pp) thyroid dysfunction (PPTD) is characterized by periods of hyperthyroidism and/or hypothyroidism in the first pp year; the hyperthyroidism is usually very mild; the hypothyroidism, however, is attended with symptoms such as lack of energy and depression (1, 2, 3). The incidence of PPTD ranges from 4–8% (4, 5, 6).

PPTD is considered to be part of the spectrum of autoimmune (AI) thyroid diseases and caused by an AI destructive lymphocytic thyroiditis (3, 6, 7). Basic to autoreactive processes are immunodysregulations such as defective tolerance mechanisms and an enhanced autoagression towards target antigens. As signs of such immunodysregulations, various humoral and cell-mediated immune (CMI) abnormalities have been reported in thyroid and other endocrine AI patients: autoantibodies [in the case of lymphocytic thyroiditis, thyroid peroxidase antibodies (TPO-Ab)] are present in serum, and there is often a rise in the number of activated [major histocompatibility complex (MHC)-class II⁺) peripheral T cells, a lowering of the number of peripheral natural killer (NK) cells, a lowered monocyte polarization, and an impaired T cell cluster capability of monocyte-derived dendritic cells (DC) (8, 9).

The presence of TPO-Ab has been reported as the most prominent risk factor for developing PPTD: about 70% of women developing PPTD are positive for TPO-Ab (TPO-Ab⁺) during pregnancy or pp, as compared with approximately 10% in the normal female population (4, 5, 6). The positive predictive value of TPO-Ab positivity, however, is about 40–60%: only one of two TPO-Ab⁺ women develops PPTD (4, 5). Therefore, TPO-Ab testing alone is not a sufficiently reliable method for identifying women at risk for PPTD.

The objectives of the study reported here are to determine possible alterations in the number of subsets of T cells, activated T cells and NK cells in the peripheral blood, and the capability of monocytes to polarize and of monocyte-derived DC to form cellular clusters in pregnancies followed by PPTD vs. those pregnancies not followed by PPTD. In addition, the possible usefulness of these alterations for the prediction of PPTD was studied. Therefore, we performed a prospective study of a sample of women who were followed during pregnancy and in the first pp year. Serial testing of thyroid function, serum TPO-Abs, and the mentioned CMI functions was carried out.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Permission for the study was obtained from the Medical Ethics Committee of the Academic Medical Centre, University of Amsterdam.

Between January 1994 and April 1996, a prospective study on thyroid dysfunction (TD) in gestation and in the pp was performed. Four hundred forty-eight consecutive women, at their first visit to the local midwife or the obstetrical department of the St. Joseph Hospital, Veldhoven, were invited to participate. Informed consent was obtained from 310 women (69%; none on thyroid drugs at entry); 291 women completed the study and 19 (2 TPO-Ab⁺ (10.5%)) were withdrawn from analysis. All women with TPO-Ab in gestation (TPO-Ab⁺; irrespective of developing PPTD), all women with PPTD (PPTD⁺) and those negative for TPO-Ab (TPO-Ab^-) were introduced in the study, together with age- and parity-matched controls (TPO-Ab^-, PPTD^-).

All women were visited at home at 12 and 32 weeks gestation, and in the pp period at regular intervals (starting 4 weeks pp, every 8 weeks, until 36 weeks). Signs and symptoms of hyper- or hypothyroidism (3) were recorded at every visit; depression was assessed at every visit using the Research Diagnostic Criteria (10).

Venous blood samples were collected into Vacutainer tubes: every visit, 8 mL for thyroid function and TPO-Ab testing, and at 12 and 32 weeks gestation and 4 weeks pp, 40–50 mL heparinized blood for peripheral blood mononuclear cell separation.

Methods

Thyroid function tests. The thyroid function was assessed by measuring the concentration of TSH (reference interval: 0.15–2.0 mU/L, defined for nonpregnant women, 20–40 yr old, originating from the same region; Kodak Amerlite TSH-30, Kodak Clinical Diagnostics Ltd., Amersham, UK) and by measuring the free T₄ concentration (fT₄; reference interval: 8.7–19.6 pmol/L; Kodak Amerlite MAB FT₄ Assay). PPTD was defined as an abnormal TSH in combination with an abnormal fT₄ in the pp period.

TPO-Ab were measured using the Immunometric Enzyme Combikit (Orgentec GMBH, Mainz, Germany); a concentration more than 50 U/mL was defined as positive (TPO-Ab⁺).

TSAb were measured using the TRAK-Assay (Brahms Diagnostica GMBH, Berlin, Germany); reference values are: <9 U/L antibody, negative; 9–14 U/L, borderline; 15 U/L, positive.

Peripheral mononuclear cell separation. Peripheral blood mononuclear cell separation was performed at the Clinical Laboratories of the St. Joseph Hospital, Veldhoven, using Ficoll Paque density gradient centrifugation (density, 1.077 g/mL; Pharmacia, Uppsala, Sweden). The cells were washed twice in PBS. Aliquots of 40–80 x 10⁶ cells were stored in a solution of 20% dimethylsulphoxide in RPMI 1640 supplemented with 10% FCS (Gibco, Breda, The Netherlands). The actual immunological tests were performed at the Department of Immunology, Erasmus University, Rotterdam.

Monocyte polarization assay for peripheral blood monocytes. This assay has proven to reflect the responsiveness of blood monocytes to chemoattractants and is a measure of preactivated monocytes in the peripheral blood; it is disturbed in endocrine AI diseases (11, 12). Monocytes were isolated from separated and deep-frozen lymphoid cells. An enrichment for the monocytes in the Ficoll-Isopaque isolated fraction was obtained by Percoll gradient centrifugation (13): after washing, the Ficoll-isolated pellet containing both monocytes and lymphocytes was resuspended in RPMI 1640 10% FCS and carefully layered on top of an equal volume of Percoll 1.063 (Pharmacia, Diagnostics AC). After centrifugation (40 min, 450 g) the cells were collected from the interface, washed twice in medium (10 min, 500 g), and counted: the suspension now contained 70–95% monocyte-specific esterase-positive cells. This suspension was directly used for the monocyte polarization assay or for the maturation of monocytes to obtain DC.

For monocyte polarization, aliquots (0.2 mL) of the Percoll-purified cell suspension, containing 200,000 monocytes, were added to 12 by 75 mm polypropylene tubes (Falcon Labware Division of Becton Dickinson, Oxford, CA) containing 0.05 mL of either medium or N-formylmethionyl-leucyl-phenylalanine (fMLP) in medium, to reach a final concentration of 10 nm. The tubes were incubated at 37 C in a water bath for 15 min. The incubation was stopped by addition of 0.25 mL ice-cold 10% Formaldehyde in 0.05% PBS (pH 7.2). The cell suspensions were kept at 4 C, until counting, in a hemocytometer using an ordinary light microscope (magnification 250X). The test was read blindly by two persons; 200 cells were counted, manually, from each tube. A cell was termed polarized if any of the following occurred: elongated or triangular shape, broadened lamellopodia, and/or membrane ruffling. The responsiveness of a monocyte population was expressed as the percentage of polarized monocytes in the presence of fMLP, minus the percentage of polarized monocytes in the absence of fMLP. The percentage of polarized monocytes was calculated as follows: (% cells polarized/% monocyte-specific esterase positive cells) x 100%.

Of the 77 healthy control individuals tested during the last 2 yr, a mean of 30% polarized monocytes was found (SD, 8%; range, 18–70%). There were no differences between females and males: females, a mean of 33% (SD, 9%; n = 36); males, a mean of 32% (SD, 13%; n = 41). Nor were differences found between individuals less than 50 yr and more than 50 yr of age: respectively, a mean of 34% (SD, 11; n = 66) and a mean of 31% (SD, 5%; n = 11). The interassay variation never exceeded 17% (n = 13), the intraassay variation never exceeded 15% (n = 77). On the basis of these outcomes, fMLP-induced polarization values of less than 20% polarized monocytes are considered to be abnormal.

The maturation of DC from blood monocytes, clustering of DC. DC were prepared from peripheral blood monocytes, according to the method described by Mooy et al. (14). Cells from the Percoll-isolated monocytic fractions were exposed to T₃ in suspension culture for 30 min. Thereafter, the cells were washed (culture fluid was added slowly to prevent osmotic lysis of the cells) and further cultured under nonadhering conditions for 16 h in polypropylene tubes (5% CO₂ and 37 C, 100% humidity). This procedure yields 40–60% cells with a dendritic morphology, showing class-II MHC positivity, a decreased expression of the monocytic CD14 determinant, a decreased phagocytic capability, but an enhanced stimulator capability in the mixed lymphocyte reaction. The full technical details of this method are given in Mooy et al. (14). Fifty thousand DC prepared from peripheral blood monocytes exposed to T₃ were allowed to cluster with 5,000 allogenic lymphocytes isolated from healthy controls (4 h, 37 C, 5% CO₂) in 250-µL flat-bottomed wells. The lymphocytic isolation was performed, according to standard procedures, with Ficoll-Isopaque and Nylon wool adherence (Leuko-Pak, Fenwall Laboratories, IL). Formed cellular clusters were counted using an inverted microscope, and values were expressed as the number of clusters per 6 microscopic fields (X250). A cluster was defined as an accumulation of 4–25 cells. Of the 25 healthy control individuals tested during the last 2 yr, a mean of 187 clusters was found (SD, 54; range, 150–230). On the basis of these outcomes, values of less than 150 clusters are considered as abnormal.

Fluorescence-activated cell sorter analysis. Peripheral blood mononuclear cell subsets were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The technique has been described in detail elsewhere (15). Mononuclear cells were incubated with monoclonal antibodies for detection of surface markers. The monoclonal antibodies were conjugated with fluorescein isothiocynate (FITC) or phycoerythrin (PE) for double-marker analysis. The following markers were used: the B cell markers CD19 (Leu-12 PE, Becton Dickinson, San Jose, CA) and CD20 (B1 FITC, Coulter Clone, Hialeah, FL), the T cell markers CD3 (Leu-4, FITC, Becton Dickinson), CD4 (Leu-3 PE, Becton Dickinson) and CD8 (Leu-2 PE, Becton Dickinson), the T and B cell marker CD5 (Leu-1 FITC, Becton Dickinson), the human leucocyte antigen (HLA)-DR marker (L243 PE, Becton Dickinson), the NK cell markers CD56 (Leu-19 PE, Becton Dickinson) and CD16 (Leu-11c PE, Becton Dickinson), and the monocyte marker CD14 (MY4 PE, Coulter Clone). Percentages of the total mononuclear cells were calculated. Reference values were obtained from 14 concomitantly tested nonpregnant TPO-Ab^- women, 20–40 yr old: CD19⁺ CD20⁺ B cells 10.9% (SD = 5.6), CD4⁺ CD3⁺ T helper (TH) cells 40.4% (SD = 5.8), CD8⁺ CD3⁺ T suppressor/cytotoxic cells 23.4% (SD = 6.9), activated (HLA-DR⁺/CD3⁺) T cells 6.4% (SD = 3.1), and CD16⁺ CD56⁺ NK cells 7.9% (SD = 1.8).

Statistical analysis. For statistical testing, the -square test and Student’s t test were performed. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
From the 291 women completing the prospective study, 31 (10.7%) were TPO-Ab⁺ at 12 and/or 32 weeks gestation (Table 1). In total, 15 women (5.2%) developed PPTD; of these, 10 were TPO-Ab⁺ and 5 were TPO-Ab^-. From the remaining 276 women, 21 were TPO-Ab⁺ and 255 were TPO-Ab^-; the latter were considered as thyroidological uneventful pregnancies. CMI testing was carried out for 47 selected women: 21 (blindly selected) TPO-Ab^- and PPTD^- women (thyroidological uneventful pregnancies = controls), the 5 TPO-Ab^- (but PPTD⁺) women, the 21 TPO-Ab⁺ (but PPTD^-) women, and the 10 TPO-Ab⁺PPTD⁺ women. None of these 47 women was positive for TSAb. No differences between the groups were found concerning educational level, family history for thyroid disease, alcohol use during pregnancy, pregnancy complications, and gender and birth weight of the child.

In all groups (irrespective of thyroid status), alterations were found, during pregnancy, in the percentage of peripheral CD16⁺ CD56⁺ NK cells and in the DC cluster capability (Fig. 1). The DC cluster capability was decreased at 12 weeks gestation and returned to normal in all groups within 32 weeks (Fig. 1). Percentages of CD16⁺ CD56⁺ NK cells were significantly decreased at 12 weeks gestation. In TPO-Ab^- women, a gradual return to normal levels occurred (Fig. 1); in TPO-Ab⁺ women (irrespective of PPTD development), however, percentages of NK cells remained low (Fig. 1). The percentages of CD19⁺ CD20⁺ B cells and the number of CD3⁺, CD4⁺, and/or CD8⁺ T cells were within the reference limits for all tested groups at all three time points.

View larger version (47K): [in this window] [in a new window]   Figure 1. The percentages of CD16+ CD56+ NK cells (norm: 7.9 ± 1.8%), activated (MHC-class II+) CD3+ T cells (norm: 6.4 ± 3.1%) (Fig. 1a), the percentage of monocytes capable to polarize under the influence of the chemoattractant fMLP (norm: 30 ± 8%), and the capability of monocyte-derived DC to form cellular clusters (norm: 187 ± 54 clusters/6 microscopic fields) (Fig. 1b) in normal (thyroidological uneventful) pregnancies (I), in PPTD cases without TPO-Abs in gestation (II), and in pregnancies positive for TPO-Abs (III), at 12 and 32 weeks of gestation (G12 and G32). The latter group was subdivided: those women developing PPTD (IIIb) and those not developing PPTD (IIIa). Shaded areas represent values found in healthy, nonpregnant controls. Mean () ± SD values are given. Accentuated bars represent values significantly different from those of healthy, nonpregnant controls (P < 0.05, Student’s t test, INSTAT programme). P4, 4th week pp; a, see Fig. 2.

In the women who later developed PPTD, two different patterns of CMI alterations were found that correlated with the presence or absence of TPO-Ab: 1) in TPO-Ab^- PPTD⁺ women, no CMI alterations were found, except for those related with the pregnancy state (see Fig. 1); and 2) in all TPO-Ab⁺ women, persistent decreases were found in NK cell numbers and the monocyte polarization assay, regardless of the development of PPTD (Fig. 1). The percentages NK cells remained significantly decreased in TPO-Ab⁺ women, as compared with nonpregnant healthy women [but not in comparison with the healthy pregnant (TPO-Ab^- PPTD^-) women]. The monocyte polarization also remained lower in TPO-Ab⁺ women and significant, in comparison with both healthy pregnant and nonpregnant women. The percentage of activated (MHC-class II⁺ CD3⁺) T cells was, in general, not raised in TPO-Ab⁺ women. However, in TPO-Ab⁺PPTD⁺ women, percentages were significantly higher at all time points studied, as compared with PPTD^- TPO-Ab⁺ women (Figs. 1 and 2).

View larger version (33K): [in this window] [in a new window]   Figure 2. Of each TPO-Ab+ pregnant woman at G12, the mean of the percentages of MHC-class II+ CD3+ T cells at G12, G32, and P4 was calculated and used as the value (percentage) of MHC-class II+ CD3+ T cells representative for that pregnant woman (G12+G32+P4/3). The figure represents the means (± SD) of these latter values in TPO-Ab+ women not developing PPTD (n = 16) and in TPO-Ab+ women who did develop PPTD (n = 10). The difference is statistically significant (Student’s t test, P < 0.05, INSTAT programme). The hatched area represents values found in nonpregnant, healthy controls.

The five PPTD⁺ women without humoral and cellular immune alterations all had a period with hyperthyroidism at different pp time points but no signs of hypothyroidism while symptoms were relatively mild. None of these women had indications for an ingestion of excess iodine or T₄; one woman had a (probably viral) infection preceding the hyperthyroidism.

From the 10 TPO-Ab⁺ CMI abnormality⁺ PPTD⁺ women, 4 had only hyperthyroidism, 5 had only hypothyroidism, and 1 had hyperthyroidism followed by hypothyroidism. Women in this group had a significantly higher frequency of TSH more than 2.0 mU/L at 12 weeks gestation, as compared with the TPO-Ab^- PPTD cases (30% vs. 0%). They also had more signs and symptoms. Although cases are low in number, data are nevertheless given as an illustration. The number of signs and symptoms of hyperthyroidism reported during the consecutive pp home visits was comparable within the TPO-Ab⁺ and the TPO-Ab^- PPTD group: weight loss in 13% vs. 16%, increased sweating in 18% vs. 24%, heat intolerance in 3% vs. 20%, nervousness in 5% vs. 4%, palpitations in 5% vs. 0%, and increased appetite in 3% vs. 4%, respectively. The number of signs and symptoms characteristic of hypothyroidism, however, was higher in the TPO-Ab⁺ PPTD cases vs. the TPO-Ab^- PPTD cases: lassitude/fatigue in 26% vs. 8%, cold intolerance in 16% vs. 4%, hoarseness in 13% vs. 0%, dry/fragile hair in 13% vs. 0%, paresthesia in 11% vs. 0%, and lethargy in 11% vs. 0%. Depression was diagnosed in 17% of home visits in TPO-Ab⁺ and in 12% of TPO-Ab^- PPTD cases, respectively. Two of the TPO-Ab⁺ PPTD cases were treated for several months with l-T₄ because of the indicated symptoms of hypothyroidism.

In our study group of 291 women, 12 women (6 TPO-Ab⁺) had one or more pp periods with elevated TSH only in the absence of fT₄ abnormalities [i.e. not cases of PPTD in our definition (see Materials and Methods)]. None of these women experienced more complaints of either hyper- or hypothyroidism, as compared with the healthy control pp women.

Prediction

With regard to our second objective, we determined the usefulness of the various CMI parameters in predicting PPTD, in general, i.e. both the TPO-Ab⁺ plus the TPO-Ab^- cases. Table 2 shows that the predictive values of monocyte polarization, activated T cells, or NK cells are low at 12 weeks gestation, and actually not much higher than those of TPO-Ab status. CMI values at 32 weeks gestation or 4 weeks pp also were not better in predicting PPTD. The combination of TPO-Ab status and abnormalities in the monocyte polarization assay, the number of activated T cells or NK cells at 12 weeks gestation, slightly increased the positive predictive value; but sensitivity decreased, in comparison with TPO-Ab status alone (Table 2).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

We consider it unlikely that the TPO-Ab^- PPTD cases suffered from Graves’ disease; arguments are that all were TSAb negative and that Graves’ disease patients do show CMI abnormalities (11). In fact, the etiology of our TPO-Ab^- PPTD cases is obscure; it is possible that one case was the result of a subacute thyroiditis after a viral infection (16). In previous studies, comparable proportions of TPO-Ab^- cases of PPTD were found. Pop et al. (5) described, in an earlier study from our region, that 28% of PPTD cases were negative for MsAb in the third trimester of pregnancy and pp (all hyper). Gerstein (4) found, in his review of studies on the incidence of PPTD, also a group of cases with hyperthyroidism negative for MsAb, and he also came to the conclusion that these cases could not be considered as Graves’ disease cases.

Adaptation of the immune system of the mother is required for acceptation of the fetus and maintaining pregnancy, because the fetus expresses, next to maternal, also paternal HLA-molecules. Despite extensive research, the precise mechanisms of the immune tolerance for the fetus remain unclear (17). Both local placental and systemic alterations in the immune system are responsible (17). In very early pregnancy, an accumulation of NK cells (with distinctive phenotype: CD56⁺CD16^- CD3^-) and of macrophages/DC occurs in the decidua; these cells are supposed to play a role in the acceptation of the early pregnancy and in the regulation of the local immune responses (17, 18, 19, 20, 21). It is well established that these local immune adaptations are associated with various alterations in the number and activity of peripheral leucocytes, amongst which are a decreased number and activity of NK cells (17, 22). In our study, we confirmed that the percentages of blood NK cells were lowered during early pregnancy. Our study is special in that it is the first study describing a diminished function of the monocyte-derived DC in the first trimester of human pregnancy. Morphological changes, indicating alterations in the function of skin-localized DC (Langerhans cells) of pregnant guinea pigs, have been described before (23).

Recent research suggests that a normal pregnancy is accompanied by a shift from predominant TH-1- to a predominant TH-2-driven immune response characterized by a decreased production of type-1 cytokines (e.g. interleukin-2 and -IFN) and an increased production of type-2 cytokines (22, 24). Because interleukin-2 is a stimulator of NK cells and -IFN induces DC activation and MHC-class II expression, this TH-1 to TH-2 shift might explain both the lowered numbers and activity of peripheral NK cells and the diminished peripheral DC clustering.

We found percentages of activated T cells and the CD4/CD8 ratio in normal pregnancies within reference limits. Results from other studies are conflicting, in this respect: both lowered and increased numbers of activated T cells and altered CD4/CD8 ratios have been found (22, 25). These differences in outcomes are probably the result of a difference in methods of identifying the lymphocyte subsets [we used the very sensitive double labelling technique (see Materials and Methods) (15)].

Although the mechanisms of the adaptive immune alterations in pregnancy, thus, are far from clear, it is the generally held view that the pregnancy state represents a state of relative immunosuppression and that, in the pp period, the function of the immune system is restored with a rebound phenomenon. Indeed the fall in the titer of TPO-Abs in pregnant women with a pp rise is suggestive of such an assumption, and TPO-Abs likely contribute to the development of PPTD (26, 27, 28). However, the prime pathogenetic role of TPO-Abs in thyrocyte destruction is questionable, and TH-1 cell mediated processes are now considered to be the most important mechanisms of action in thyroid and other destructive endocrine AI diseases. The data reported here show that TPO-Ab⁺ women already have alterations in various CMI functions, from the first trimester of pregnancy onwards, about 1 yr before a possible onset of clinical symptoms. These alterations included a lowered fMLP-induced monocyte polarization and a persistently lower number of NK cells. We assume that the lowered monocyte polarization and the lowered NK cell numbers reflect a state of subclinical chronic AI thyroiditis. Identicle CMI abnormalities have been found in other clinically overt AI endocrine diseases (8, 11, 12, 29). The persistently lowered number of NK cells might be a representation of an AI-related immunodysregulation, because NK cells are not only involved in target cell lysis but also in the regulation of the immune response (30). The lowered polarization of blood monocytes towards fMLP might also represent such immunodysregulation. The polarizing capability of monocytes not only reflects the chemotactic ability, it also reflects the activation state of the peripheral monocytes and their recruitment capacity into peripheral tissues (31), both important functions in immunoregulation.

It is remarkable that we found the percentages of activated MHC-class II⁺ T cells already increased in the first trimester of pregnancy in TPO-Ab⁺ women who later developed PPTD. This parameter of enhanced T cell activity might indicate that effector functions of T cells are out of balance in future PPTD patients, and our observations thus might be compatible with a view that TH-1 cells play an important pathogenic role in thyrocyte destruction. However, a pattern of suppression during pregnancy and a rebound phenomenon in the pp period was not evident in the number of MHC class II⁺ T cells in the PPTD patients (neither was that seen for NK cells and monocyte polarization disturbances). It is important to note, in this respect, that Stagnaro-Green et al. (32) found increased percentages of activated T cells (CD4⁺DR⁺) in TPO-Ab⁺ women (including those with later PPTD) in the second trimester of pregnancy, with a peak at 3 months pp. Clearly, future studies should more closely investigate numbers of activated T cells, in relation to PPTD complicated pregnancies.

If our assumption of two forms of PPTD holds true, then identifying women at risk for the TPO-Ab⁺ (AI) form of PPTD is more important than identifying those with the TPO-Ab negative form, because our data suggest that women with the TPO-Ab⁺ form are prone to develop more symptomatology of hypothyroidism (lassitude, hoarseness, fragile hair, paresthesia, and depression). Moreover, and more importantly, the literature indicates that they have a higher chance for permanent hypothyroidism (33, 34). Also, reinvestigation of the here reported TPO-Ab⁺ PPTD⁺ cases after 2–3 yr shows that 25–30% of the cases now have a permanent hypothyroidism (to be published). Also, when prophylactic treatment with T₄ is considered, to prevent symptoms during the hypothyroid period, a correct identification is essential to prevent under- and/or overtreatment. On the basis of the here reported results, the most feasible strategy to identify women at risk for the TPO-Ab⁺ form of PPTD might be a testing for TPO-Ab followed by measuring of percentages of activated T cells, in TPO-Ab⁺ women only. In fact, a practical approach for implementing a screening program would be the testing of all pregnant women, at 12 weeks gestation, for the identification of TPO-Ab⁺ women, because at 12 weeks, all women in our country are regularly tested for blood group/Rhesus, rubella-antibodies, Hepatitis B surface antigen, and syphilis. Moreover, at that time, the prevalence and titers of TPO-Abs are the highest. Thereafter, the TPO-Ab⁺ women could be tested and followed for percentages of activated T cells, which are probably high throughout pregnancy (see Figs. 1 and 2). The financial costs of this strategy are limited: about 10% of 20- to 40-yr-old women have TPO-Ab, and only these women need follow up with tests for activated T cells. In the Netherlands, this accounts for 18,000 women per year. Further study to evaluate the feasibility of such screening strategy and the effect of treatment on symptoms, however, is clearly needed.

In conclusion, the results of this study are an indication of the existence of two forms of PPTD: a TPO-Ab⁺ (AI) form (about two-thirds of cases) and a TPO-Ab^- (non-AI) form. The first form shows the more severe symptomatology, and a possible strategy for identifying women at risk for this form of PPTD would perhaps be a TPO-Ab test, in combination with a follow-up for activated T cells. However, further research on larger cohorts of pregnant women, to confirm our findings, is clearly needed.

View larger version (43K): [in this window] [in a new window]   Figure .

	Acknowledgments

	Footnotes

 
¹ This study was supported by grants from the Dutch Prevention Fund (002822380) and NWO-Medical Sciences (900–540-167).

Received November 12, 1997.

Revised February 9, 1998.

Accepted February 17, 1998.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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