The effect of lactation in the post-partum arthritis of MRL-lpr/fasmice

L. G. Ratkay2,, J. Weinberg1 and J. D. Waterfield

Department of Oral Biological and Medical Science,
1 Department of Anatomy, University of British Columbia, Vancouver and
2 QLT Phototherapeutics, Vancouver, BC, Canada


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Objective. To investigate the effects of lactation on the post-partum arthritic flare in MRL-lpr/fas mice.

Methods. Three groups of mice were investigated. Group 1: females whose litters were weaned at termination of the experiment; group 2: females whose litters were weaned at parturition; group 3: females who were not bred. Clinical evaluation was carried out at 5-day intervals following parturition. Blood samples were also collected during the course of the experiment and assayed for corticosterone and prolactin. Histological evaluation of the joints was assessed at day 30.

Results. The incidence of swelling and erythema, the bimalleolar ankle width and the histopathology were significantly reduced by removal of the litters at parturition. This correlated well with a decrease seen in prolactin levels in these females. Corticosterone, an immunomodulatory glucocorticoid, did not play a significant role in the arthritic flare.

Conclusion. Our findings suggest that prolactin levels contribute to the inflammation seen in MRL-lpr/fas mice following parturition.

KEY WORDS: MRL-lpr/fas, Rheumatoid arthritis, Pregnancy, Post-partum arthritic flare, Lactation.


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The relationship of rheumatoid arthritis (RA) to pregnancy is well established. More than 75% of patients with RA enter remission during the course of gestation. However, most of these individuals will have relapsed by 3 months post-delivery [1]. The mechanism(s) responsible for the flare has yet to be determined. However, a number of predictors of response are under investigation, including the immunological challenge to fetal HLA antigens, changes in the glycosylation patterns of serum IgG and the hormonal changes involved in the pre- and post-parturition state [24].

Several experimental animal models have been developed to study both the remission of the disease during gestation and the subsequent exacerbation post-partum. In a model of collagen-induced arthritis using DBA/1 mice, treatment of the mice after parturition with oestradiol has been shown to protect them from a post-partum flare [5, 6]. It was also found that treatment with bromocriptine, a drug inhibiting the endogenous release of prolactin, had a similar effect [6]. Conversely, however, treatment of the animals with exogenous prolactin enhanced the course of the disease only during the immunization period, suggesting that prolactin does not play a significant role in the collagen-induced arthritis model [7]. In another experimental system, a proteoglycan-induced progressive polyarthritis in BALB/c mice, pregnancy had a beneficial effect on the clinical symptoms [8].

We have previously documented another model using female MRL-lpr/fas mice to study the post-partum flare seen in patients with RA [9]. In that study we found that within 30 days of parturition approximately 70% of these mice developed a post-partum exacerbation of their mild spontaneous arthritis, the arthritis becoming evident between 5 and 15 days after delivery. The arthritic condition was characterized histologically by a significant increase in subsynovial inflammation and synovial hyperplasia. Immunohistological evaluation demonstrated major histocompatibility complex (MHC) class II-bearing cells as well as the presence of CD3-, CD4-, and CD43-staining cells in the subsynovium [9]. Furthermore, we found that administration of pharmacological amounts of oestradiol on lactation days 2, 3, 9, 15, and 20 reduced the post-partum flare from 70 to 23% of the animals [9].

As the post-partum drop in oestrogen is accompanied by a rise in prolactin, we hypothesized that the hyperprolactinaemia of the breast feeding period may also play a role in the onset of arthritis in our mouse model. The present study was undertaken to investigate the effects of lactation on the onset of disease activity following parturition.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
MRL-lpr/fas mice (n = 24) were obtained from a breeding colony maintained in the Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada. The mice were maintained on a standard diet with water ad libitum. Pregnant mice were housed one per cage. The colony was routinely screened for Mycoplasma pulmonis, and Myc. arthritides, rodent coronaviruses (including hepatitis), and Sendai virus using the Murine ImmunoComb test (Charles River Laboratories, Wilmington, MA, USA). No antibodies were detected.

Breeding protocol
Ten-week-old virgin females were mated. In all experiments the females were separated from the males after mating. On day 1 of gestation, the females were divided into three groups: group 1: females whose litters were weaned at termination of the experiment (day 30 following parturition; n = 14 at day 1 of gestation of which n = 10 at termination); group 2: females whose litters were weaned at parturition (n = 17 at day 1 of gestation of which n = 10 at termination); group 3: females who were not bred (n = 10 at day 1 of gestation of which n = 4 at termination). Surviving females in all groups were terminated at 30 days after parturition, or the equivalent.

Clinical evaluation
Clinical evaluation was carried out as previously described [10]. Briefly, mice were examined ‘blind’ 7 days prior to parturition and every 5 days thereafter for 30 days for the visual appearance of arthritis and scored as positive if erythema and swelling of a fore or hind paw(s) was observed. Bimalleolar ankle widths were also determined using a micrometer.

Histological evaluation
After the animals were terminated the tarsometatarsal joints were dissected and fixed in 10% buffered formalin. The joints were then decalcified for 48 h in 10% formic acid. The tissues were subsequently processed for paraffin embedding. Serial 5 µm thick sections were stained with haematoxylin and eosin (H&E). Histopathological alterations of the joints were evaluated ‘blind’ for the presence of the following features: subsynovial inflammation; synovial hyperplasia; cartilage erosion and pannus formation; and bone destruction. Within each parameter scores were assessed from 0 to 2 (0, no; 1, mild; and 2, severe involvement) with a total score of 8 being possible.

Blood sampling
The mouse colony facility was closed for 12 h prior to testing to prevent artificial elevations in hormone levels due to human disturbance. The samples were taken between 1700 and 1800 h to control for variations in circadian rhythm. The samples were collected 5 days prior to parturition (-5 days), within 24 h following parturition (0 days) and 10 and 30 days post-parturition. The animals were anaesthetized briefly with halothane and blood samples collected from the retro-orbital plexus. The tubes were left to coagulate for 1 h at room temperature and then centrifuged at 8000g for 5 min at 4°C. The serum was harvested and stored at -20°C.

Measurement of corticosterone and prolactin
Corticosterone was measured by the method of Weinberg and Bezio [11]. The radioimmunoassay (RIA) was based on competition of exogenous murine corticosterone with purified tritiated corticosterone. Briefly, 30 µl of serum was extracted twice with 270 µl of absolute ethanol from which the total corticosterone (bound plus free) was measured. The antisera for use in the RIA was obtained from Immunocorp (Montreal, PQ, Canada). [1,2,6,7-3H]-corticosterone was obtained from Dupont (New England Nuclear, Mississauga, Ontario, Canada), while unlabelled corticosterone standards were obtained from Sigma (St Louis, MO, USA). Dextran-coated charcoal was used to absorb and precipitate the free steroids after incubation. Samples were counted in Formula 989 (Dupont) using a liquid scintillation counter.

Prolactin was measured by enzyme-linked immunosorbent assay (ELISA). Ninety-six-well ELISA plates (Falcon Plastics, Becton-Dickinson, Mississauga, Ontario, Canada) were coated overnight at 4°C with 50 µl of a 1/10 dilution, in phosphate-buffered saline (PBS), of mouse serum. After washing with PBS, the non-saturated binding sites of the plates were blocked with 100 µl of 2% bovine serum albumin (BSA)/PBS for 2 h at room temperature followed by three sequential washes with PBS. Fifty microlitres of rabbit anti-mouse prolactin (Cedarlane Laboratories, Hornby, Ontario, Canada) was resuspended in 2 ml of antibody buffer (1% BSA, 0.05% Tween 20 in PBS). A 1/10 dilution was then prepared and 75 µl added to each well. The plates were then incubated for 1 h at room temperature. After washing twice with PBS/Tween 20 the plates were incubated for a further 1 h at room temperature with 50 µl of a 1/5000 dilution (in antibody buffer) of alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Calbiochem, La Jolla, California, USA). The plates were then washed again with PBS/Tween 20 and developed by adding 50 µl/well (0.5 mg/ml) of p-nitrophenyl phosphate (Sigma) in diethanolamine buffer. The optical densities were measured after a 45 min/37°C incubation at 405 nm by a Titertek ELISA recorder (Flow Laboratories, Mississauga, Ontario, Canada).

Statistical analysis
Data are expressed as mean ± standard error of the mean (S.E.M.). Statistical differences among groups were determined using appropriate analyses of variance (ANOVA) and either the Kolmogorov–Smirnov multiple comparison test or Tukey's multiple comparison test.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effect of lactation on post-partum flare
The effect of lactation was studied by comparing lactating females (litters not removed at parturition) with non-lactating females (litters removed at parturition) and virgin (not bred) females.

It can be seen from Fig. 1aGo that the incidence of swelling and erythema seen at the end of the post-partum period in animals with litters was 100%. Lactating females exhibited a significantly increased incidence of swelling and erythema at 15 days post-partum (P < 0.005) compared with that seen at day 0; both measures remaining significantly elevated until termination at day 30 post-partum. The clinical onset of the post-partum flare was successfully prevented (32%) by the removal of litters at the time of parturition (P < 0.01). This incidence was not different from the 25% incidence seen in the non-mated female control group. Bimalleolar ankle measurements showed similar patterns (Fig. 1bGo). In addition, subsynovial inflammation, synovial hyperplasia and the overall histological scores increased significantly post-partum in animals with litters compared with unbred animals (Fig. 2Go, P < 0.01). However, there were no significant increases in cartilage erosion, pannus formation, or bone destruction. Removal of the litters significantly lowered subsynovial inflammation as well as the overall histological scores (P < 0.01). There was also some decrease in the level of synovial hyperplasia following the removal of litters, but this did not reach statistical significance.



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FIG. 1. The effect of lactation on the post-partum flare of arthritis in MRL-lpr/fas mice. (a) Incidence of swelling and erythema. (b) Changes in bimalleolar ankle width during the 30-day post-partum period. Elevated clinical parameters of arthritis occurred in lactating animals ({square}) from 15 to 30 days post-partum. This elevation was prevented in non-lactating animals ({blacklozenge}). Unmated, age-matched females served as controls ({circ}). The values corresponding to * are significantly different from the lactating group's value: (a) P < 0.01, Kolmogorov–Smirnov's multiple comparison test; (b) P < 0.01, Tukey's multiple comparison test. Changes in ankle width are relative to day 0 (parturition) measurements.

 


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FIG. 2. The effect of lactation on histopathological conditions of the tarsometatarsal joints at 30 days following parturition. The scoring system is described in the Methods section. (A) subsynovial inflammation; (B) synovial hyperplasia; (C) pannus formation and cartilage destruction; (D) bone destruction; (E) overall score. The values corresponding to * are significantly different from the lactating group's value (P < 0.01, Tukey's multiple comparison test).

 

Correlation of prolactin and corticosterone levels with post-partum flare
In order to test whether prolactin levels decreased in the females whose litters were removed at the time of parturition, the mice were bled at selected time intervals. The levels of serum prolactin were measured in those mice from which enough sera was successfully obtained. Table 1Go shows that the day 10 post-partum prolactin levels were significantly higher in those animals retaining their litters when compared with either the control group (P < 0.001) or the group of females whose litters had been removed (P < 0.02), while on day 30, the post-partum prolactin levels were significantly higher in those animals retaining their litters when compared with the levels of females whose litters had been removed (P < 0.03). It was interesting to note that prolactin levels increased in the pregnant animals just prior to parturition (day -5).


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TABLE 1. Effect of litter removal on prolactin levels in MRL-lpr/fas mice

 
We also tested the possibility that a change in the levels of hormones of the hypothalamic–pituitary–adrenal (HPA) axis may be involved in the pregnancy-related disease activity as immune functions are affected by changes in glucocorticoid levels. We investigated this possibility by measuring the plasma levels of corticosterone during the course of the experiment in those mice from which enough sera was successfully obtained. Table 2Go shows that at days 10 and 30 following parturition there was no significant difference in corticosterone levels in those animals retaining their litters when compared with the group of females whose litters had been removed (P > 0.05). As was noted with prolactin, corticosterone levels increased in all pregnant animals prior to parturition (day -5).


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TABLE 2. Effect of litter removal on corticosterone levels in MRL-lpr/fas mice

 


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During gestation RA generally remits, while following delivery a relapse is often seen. To date studies attempting to resolve this phenomenon have failed to provide a satisfactory explanation. Studies involving both pregnant females with RA as well as studies using a number of animal model systems have provided evidence for several possible mechanisms; these include the immunomodulating properties of sex steroids [12], immunosuppression mediated by the pregnancy-associated proteins alpha 2-glycoprotein [13] and {alpha}-fetoprotein [1], fetal–maternal disparity in class II HLA antigen [2] and/or production of transforming growth factor-beta (TGF-ß) [14]. Moreover, changes in glycosylation patterns of immunoglobulin molecules have been reported during pregnancy [3]. A number of investigators have also suggested that a defect in the HPA axis may be involved in the pregnancy-related changes in disease activity [6, 1517].

We have recently reported a new model of post-partum flare of RA in MRL-lpr/fas mice. Within 30 days of parturition, approximately 70% of mice develop a post-partum exacerbation of their mild spontaneous arthritis [9]. Administration of physiological levels of oestradiol on days 2, 3, 9, 15, and 20 post-partum delayed and reduced the flare in these animals. These findings are in agreement with those of Mattsson et al. [6] and Jansson et al. [18] who found that treatment of collagen-induced arthritis in DBA/1 and B10.RIII mice with oestradiol after parturition protected the mice from the post-partum flare. In a subsequent study on the MRL-lpr/fas mice, we reported a change in N-acetylglucosamine expression in IgG following parturition which correlated significantly with the timing of the post-partum flare [19]. Increases in IgG glycoforms terminating in N-acetylglucosamine have been shown to correlate with disease severity, decreasing during pregnancy and rising post-partum [3, 20]. It has been postulated that agalactosylated IgG may be responsible for inducing rheumatoid factor production and immune complex formation [21].

Pregnancy is accompanied by a number of hormonal changes, including a rise in progesterone and oestrogen. As mentioned above, we and others have noted that the post-partum drop in oestrogen might be important in mediating the flare of disease activity seen in both experimental arthritis and RA. During pregnancy, oestrogen production stimulates the growth and replication of the pituitary lactotrophs thereby causing increased prolactin secretion. High levels of oestrogen inhibit the action of prolactin at the breast. Lactation does not commence until oestrogen levels decline after parturition. Evidence suggesting a role for prolactin in the post-partum flare comes from utilizing the drug bromocriptine, an inhibitor of prolactin. Administration of this drug in the collagen-induced arthritis model utilizing DBA/1 mice demonstrated suppression of the post-partum exacerbation of the disease activity [22]. However, it is unclear why the administration of oestrogen, a sex hormone which increases circulating prolactin levels, provides protection from the post-partum flare in DBA/1 mice [6] unless prolactin exhibits differential effects during different periods of arthritis development [7].

In the present study we determined the effect of the removal of the litters of MRL-lpr/fas mice on the development of the post-partum flare. We found that both the incidence of swelling and erythema as well as the bimalleolar ankle width were significantly reduced by removal of the litters at the time of parturition. This correlated well with the determination of subsynovial inflammation, synovial hyperplasia and the overall histological scores in this group of animals. Prolactin levels as measured by ELISA decreased in the females whose litters were removed at the time of parturition, suggesting that prolactin in the MRL-lpr/fas mice contributes to the pro-inflammatory effects. It was interesting to note that although removal of the litters had an effect on the arthritic flare it did not have an effect on the incidence of glomerulonephritis as measured by increased protein in the urine (control: 1 g/l, lactating females: 0.91 g/l, and non-lactating females: 0.99 g/l). In humans the relationship of post-partum flare to lactation is not clear [23].

We also investigated whether corticosterone, a glucocorticoid, may have an effect on the post-partum flare noted in our model. The rationale for this is that glucocorticoids, hormones which primarily have an immunosuppressive effect, increase during pregnancy and are suppressed during nursing, allowing immune function to recover promptly [24, 25]. We investigated the possible role of glucocorticoids by measuring the plasma levels of corticosterone during the course of the experiment. There was no significant difference in the levels of corticosterone at 10 or 30 days following parturition in those animals retaining their litters when compared with the group of females whose litters had been removed. Thus, corticosterone does not appear to play a significant role in the arthritic flare seen in the post-partum period.

Although we have demonstrated a role for prolactin in the post-partum flare, other factors such as sex hormone changes and glycosylation patterns also appear to be involved, suggesting that the enhanced clinical symptoms seen in the MRL-lpr/fas mouse model may prove to be multifactorial in nature [9, 19].


    Notes
 
Correspondence to: L. G. Ratkay, QLT Phototherapeutics, 520 W6th Avenue, Vancouver, BC, Canada V5Z 4H5. Back


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Submitted 27 July 1999; revised version accepted 20 December 1999.



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