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The Journal of Immunology, 1999, 162: 2648-2654.
Copyright © 1999 by The American Association of Immunologists

Pregnancy Ameliorates Induction and Expression of Experimental Autoimmune Uveitis

Rajeev K. Agarwal*, Chi-Chao Chan*, Barbara Wiggert{dagger} and Rachel R. Caspi1,*

* Laboratory of Immunology and {dagger} Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Female patients suffering from autoimmune uveitis are reported to experience a temporary remission during pregnancy. Experimental autoimmune uveitis (EAU) is a model for human uveitis. Here we examine the effect of pregnancy on the development of EAU and its associated immunological responses. Susceptible C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP). EAU scores and Ag-specific responses were evaluated 21 days later. Mice immunized during pregnancy developed significantly less EAU than nonpregnant controls. Their lymph node cells and splenocytes produced a distinct pattern of cytokines in response to IRBP: reduced IFN-{gamma} and IL-12 p40, but unchanged levels of TNF-{alpha}, IL-4, IL-5, and IL-10. Anti-IRBP Ab isotypes revealed an up-regulation of IgG1, indicating a possible Th2 bias at the humoral level. Ag-specific proliferation and delayed hypersensitivity, as well as mitogen-induced IFN-{gamma} production, remained undiminished, arguing against an overall immune deficit. Interestingly, pregnant mice that received an infusion of IRBP-primed lymphoid cells from nonpregnant donors also developed reduced EAU, suggesting that pregnancy suppresses not only the generation, but also the function of mature uveitogenic effector T cells. Pregnant mice at the time of immunization exhibited elevated levels of TGF-ß, but not of IL-10, in the serum. We suggest that protection from EAU during pregnancy is due primarily to a selective reduction of Ag-specific Th1 responses with only marginal enhancement of Th2 function, and that these effects may in part be secondary to elevated systemic levels of TGF-ß.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune uveitis (EAU)2 is a prototypic organ-specific autoimmune disease in which the target tissue is the neural retina. EAU can be induced in susceptible animals by immunization with evolutionarily conserved retinal proteins such as interphotoreceptor retinoid binding protein (IRBP) or their peptides, or by adoptive transfer of CD4+, MHC class II-restricted T cells specific to those Ags 1, 2, 3, 4 .

CD4+ lymphocytes can exist as polarized subsets known as Th1 and Th2 that differ in the cytokines they produce and the effector functions they mediate 5, 6, 7 . Th1 type cells produce IFN-{gamma}, IL-2, and lymphotoxin, are involved in cell-mediated immunity, and promote Ab switching to IgG2a, whereas Th2 type cells produce IL-4, IL-5, IL-6, IL-10, and IL-13, mediate allergic responses, and promote Ab switching to IgG1. These two kinds of subsets cross-regulate each other, such that IFN-{gamma} inhibits the generation and proliferation of Th2 cells, whereas IL-4 and IL-10 inhibit the generation and function of Th1 cells 6, 8, 9 . EAU in genetically unmanipulated animals is dependent on a Th1 response; uveitogenic T cells show a Th1-like cytokine profile, and susceptible rodent strains typically mount a Th1-dominant response to the uveitogenic Ag 10, 11, 12 .

Pregnancy has been believed to be associated with immunosuppression, and it has been hypothesized that this is necessary to prevent immunological rejection of the allogeneic fetus 13, 14, 15 . More recently, it has been proposed that rather than blanket immunosuppression, pregnancy induces immune deviation promoting Th2 at the expense of Th1 type responses 14 . In general, administration of anti-inflammatory and Th2 cytokines (IL-4, IL-10, and TGF-ß) appears to promote fetal survival, whereas treatment with proinflammatory and Th1 type cytokines (TNF-{alpha}, IFN-{gamma}, and IL-2) can terminate pregnancy 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 . Thus, the immunological balance imposed by pregnancy would seem an antithesis to the immunological balance that promotes cell-mediated autoimmunity.

Uveitis patients, as well as patients suffering from other cell-mediated autoimmune conditions, have been observed to go into remission during pregnancy 24, 25, 26, 27, 28, 29, 30 . However, this observation is supported mostly by anecdotal evidence rather than by systematic studies that would implicate the pregnant state directly in the suppression of disease parameters. Therefore, we undertook to examine the effect of pregnancy on the induction of ocular autoimmunity in the mouse EAU model. Our results indicate that mice challenged for EAU induction during pregnancy, whether by active immunization or by adoptive transfer of primed effector cells, develop significantly reduced disease scores. The data support the interpretation that protection from disease stems from a selective inhibition of type 1 responses without a major deviation toward type 2 response and without induction of an overall immune deficit. These effects may, at least in part, be secondary to systemic elevation of circulating TGF-ß.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Timed pregnant and nonpregnant C57BL/6 mice were obtained from Harlan (National Cancer Institute, Frederick, MD). Pregnant mice were received as 12-day pregnant, were rested overnight, and were entered into the experiments as 13-day pregnant. Pups were euthanized within 24 h of birth (~21 days). All the mice were housed under specific germ-free conditions and were used following the guidelines of the National Institutes of Health and the Association for Research in Vision and Ophthalmology.

Reagents

IRBP was purified from bovine retinas as described earlier 31, 32 . The preparations were aliquoted and were stored at -70°C until use. Pertussis toxin (PTX), {alpha}-methyl-D-mannopyranoside and BSA were purchased from Sigma (St. Louis, MO). PHA was purchased from Murex Biotech Limited (Dartford, U.K.). Horseradish peroxidase-streptavidin was from Southern Biotechnologies Associates (Birmingham, AL). Mycobacterium tuberculosis strain H37RA and CFA was from Difco (Detroit, MI).

Immunization

Mice were immunized s.c. with 100–200 µg of IRBP in 200 µl of emulsion with CFA (1:1, v/v) that had been supplemented with M. tuberculosis to a final concentration of 2.5 mg/ml. PTX was given i.p. as an additional adjuvant at the same time (1.0 µg in 100 µl). Pregnant mice were immunized toward the end of the second trimester (day 13).

Histopathology and EAU grading

Eyes were enucleated 21 days after immunization, fixed in 4% phosphate-buffered glutaraldehyde for 1 h, and stored in 10% phosphate-buffered formaldehyde until processing. Tissues were embedded in methacrylate. Sections (4–6 µm) were cut through the pupillary optic nerve plane and were stained with hematoxylin and eosin. Eight to 10 sections, cut at different planes, were examined for each eye in a masked fashion by one of us (C.-C.C.). The incidence and severity of EAU were scored on an arbitrary scale of 0 to 4 in half-point increments, according to a semiquantitative system described earlier 33 . Briefly, the minimal criterion to score an eye as positive by histopathology was inflammatory cell infiltration of the ciliary body, choroid, or retina (EAU grade 0.5). Progressively higher grades were assigned for the presence of discrete lesions in the tissue, such as vasculitis, granuloma formation, retinal folding and/or detachment, photoreceptor damage, and retinal atrophy. The grading system takes into account lesion type, size, and number.

Delayed-type hypersensitivity (DTH)

DTH was performed by ear assay. Two days before the termination of the experiment, the pinna of one ear was injected with 10 µg of IRBP in 10 µl with the aid of a 30-gauge needle. The pinna of the other ear was similarly injected with vehicle alone (PBS). Ear thickness was measured 48 h later with a spring-loaded micrometer. The response is expressed as Ag-specific swelling in micrometers, calculated as the difference between the thickness of the IRBP-injected ear and the PBS-injected ear.

Lymphocyte proliferation assay

Spleens and draining lymph nodes (inguinal and iliac) were collected on day 21 after immunization and were pooled within groups. Single cell suspensions were prepared in DMEM (HyClone, Logan, UT) medium supplemented with 2 mM L-glutamine, 5 x 10-5 M 2-ME, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 50 µg/ml gentamicin sulfate, 20 mg/ml {alpha}-methyl-D-mannopyranoside, and 1.5% fresh frozen syngeneic mouse serum. Triplicate cultures of 5 x 105 cells/200 µl/well were incubated in 96-well round-bottom tissue culture plates with IRBP (50 µg/ml) or PHA (1 µg/ml). The cells were cultured for 48 h at 37°C with 10% CO2 and were pulsed with 1 µCi [3H]thymidine/well for an additional 18 h. The cultures were harvested using a PHD Cell Harvester (Cambridge Technology, Watertown, MA), and radioisotope incorporation was determined using standard liquid scintillation.

Determination of cytokine production

Processing and culturing of draining lymph nodes and spleens were as described previously for the proliferation assay, except for the following changes. The cell suspensions were cultured in 96-well flat-bottom plates with 1 x 106 cells/200 µl/well. Supernatants cultures were collected at 48 h, and cytokines (IFN-{gamma}, IL-4, IL-5, IL-10, IL-12 p40, and TNF-{alpha}) were determined by ELISA. IFN-{gamma} and IL-4 were measured using mAb pairs from PharMingen (San Diego, CA) as described previously 34 . IL-5, IL-10, and TNF-{alpha} were measured using a kit from Endogen (Woburn, MA). IL-12 p40 and TGF-ß1 (total) were estimated using an ELISA kit from Genzyme (Cambridge, MA). TGF-ß2 (total) was determined with the aid of an ELISA kit from R&D Systems (Minneapolis, MN).

Primary cell culture for adoptive transfer

Donor nonpregnant mice were immunized with 125 µg of IRBP in CFA along with 1.5 µg of PTX. After 15 days, draining lymph nodes and spleens were harvested and pooled, and single cell suspensions were prepared as described above. Bulk cultures containing 10 x 106 cells/ml were stimulated for 72 h with 30 µg/ml of IRBP with or without IL-12 (50 ng/ml) in DMEM supplemented as above, except that syngeneic serum was reduced to 1%. The cultures were transferred to a new flask after 24 and 48 h to remove excess macrophages. After 3 days of culture, dead cells were removed by passing over Lympholyte M (Accurate Chemical, Westbury, NY). Cells were washed twice, counted, and 40 x 106 cells were injected i.p. into each recipient mouse. After 10 days, eyes were harvested for evaluation of EAU by histopathology as described above.

Assay for IRBP-specific Ab IgG isotypes

Anti-IRBP IgG2a and IgG1 subclasses were determined by isotype-specific ELISA in sera of individual mice, as described for another Ag 35 . Briefly, 96-well flat-bottom microtiter plates (Costar, Cambridge, MA) were coated with IRBP (1 µg/ml). Nonspecific binding was prevented by incubating the plates with PBS with 5% BSA and 1% neonate bovine agamma serum (Biocell, Rancho Dominguez, CA). After overnight incubation with serial dilutions of serum samples, the plates were incubated with horseradish peroxidase-conjugated goat anti-IgG subclass-specific Abs (Southern Biotechnology Associates). The plates were developed with orthophenylene diamine dihydrochloride substrate (Sigma), and the absorbence was read at 490 nm. The concentrations of anti-IRBP Ab were determined using standard curves generated by coating the wells with anti-isotype Abs and adding polyclonal Ig standards of the pertinent isotype.

Statistical analysis, reproducibility, and data presentation

Experiments were repeated at least twice and in most cases three or more times. The response patterns were highly reproducible. Statistical analysis of EAU scores was performed by frequency analysis, using Snedecor and Cochran’s test for linear trend in proportions 36 . Disease severity for each animal was calculated as the average of both eyes. Analysis of DTH and lymphocyte proliferation was by independent Student’s t test. Probability values <=0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pregnant mice are less susceptible than controls to induction of EAU by active immunization

Pregnant mice were immunized with a uveitogenic regimen of IRBP as described in Materials and Methods. Histopathologic examination of eyes harvested on day 21 after immunization in five separate experiments revealed that pregnant mice developed significantly lower EAU scores than nonpregnant controls (p = 0.036) (Fig. 1Go). The disease scores developed by nonpregnant mice are typical of the C57BL strain, which is moderately susceptible to EAU. Histology of eyes from unimmunized mice (or from mice immunized with CFA and PTX alone) invariably showed disease scores of zero. Mortality among pregnant mice tended to be higher than among controls (up to 50%), which we attribute to the immediate shock of CFA and pertussis administration, combined with the physiological stress of pregnancy, because any deaths tended to occur within the first few days after immunization. The surviving mice subsequently appeared healthy and gave birth normally at term.



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  		FIGURE 1. EAU scores in mice immunized during pregnancy. Mice were immunized with 100–200 µg of IRBP in CFA and 1.0 µg of PTX. Eyes were harvested on day 21 for histopathology. Scores were assigned on a scale of 0 to 4, according to the extent of inflammation and tissue damage. The data are a compilation of four different experiments. The statistical analysis was by linear trend in proportions.

 
Pregnancy protects from EAU induced by adoptive transfer of uveitogenic effector cells

IRBP-primed cells were generated from nonpregnant donors immunized with a uveitogenic regimen of IRBP and competent effector cells were generated by in vitro stimulation with IRBP for 72 h in bulk cultures. Some cultures were incubated in the presence of IL-12, which in previous studies has been shown to generate fully polarized Th1 effector cells 12, 37 . Naive 13-day pregnant and naive nonpregnant syngeneic recipients were infused with 40 million cells, and after 10 days their eyes were evaluated for EAU by histopathology. Irrespective of the presence or absence of IL-12 during in vitro culture, the pregnant recipients developed considerably less EAU than the nonpregnant ones (p = 0.014) (Fig. 2Go). Unlike with active immunization, EAU challenge by adoptive transfer did not result in any mortality in the pregnant group.



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  		FIGURE 2. Pregnant mice are less susceptible to adoptively transferred EAU. Lymph node cells and splenocytes obtained from IRBP-primed nonpregnant mice were cultured with IRBP and 40 x 106 cells were injected i.p. into pregnant and nonpregnant recipients. The graph represents a compilation of two experiments. In one experiment, the cells were cultured in the presence of Ag and IL-12 and in the second with Ag only. The scores were read in eyes collected 10 days after cell transfer. Because the presence or absence of IL-12 in culture did not seem affect the pathology scores of the recipients, the two experiments were combined for the purpose of analysis. Scores of the two recipient groups were significantly different (linear trend in proportions).

 
Mice immunized during pregnancy exhibit cellular responses of normal magnitude

DTH responses were measured in pregnant and nonpregnant mice by ear challenge. Both pregnant and nonpregnant mice had DTH responses to IRBP of similar magnitude (Fig. 3Go). Furthermore, both pregnant and nonpregnant mice also exhibited similar proliferative responses in spleens (Fig. 4Go) as well as in draining lymph nodes (not shown).



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  		FIGURE 3. DTH response to IRBP in mice immunized during pregnancy. Nineteen days after immunization mice were challenged with 10 µg of IRBP in the right ear and 10 µl of PBS in the left ear. After 48 h, specific DTH was measured as the difference between right and left ears. There was no statistical difference between the groups (Student’s t test).

 


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  		FIGURE 4. Lymphocyte proliferation of splenocytes to IRBP and PHA in mice immunized during pregnancy. Spleens were harvested 21 days after immunization, were pooled within each group, and were stimulated with IRBP or with PHA. Shown are net cpm after subtraction of background. There was no statistical difference between the two groups (Student’s t test).

 
Mice immunized during pregnancy have reduced Ag-specific type 1 lymphokine response

Lymph node cells and splenocytes of mice immunized for EAU induction on day 13 of pregnancy were collected 21 days after immunization and were stimulated in vitro with IRBP or PHA. Cytokines were measured by ELISA in supernatants collected 48 h after stimulation. Type 1 cytokines were assessed by measurement of IFN-{gamma}, IL-12, and TNF-{alpha}, and type 2 cytokines were represented by IL-4, IL-5, and IL-10. The results showed that IRBP-elicited IFN-{gamma} production was depressed in mice immunized during pregnancy compared with nonpregnant controls (p < 0.0001); however, PHA-induced IFN-{gamma} production was unaltered (Fig. 5Go, a and b). Similarly to IFN-{gamma}, the level of IRBP-induced IL-12 p40 was depressed in pregnant mice (p < 0.002) (Fig. 5Goc). IRBP-induced TNF-{alpha} production in the same supernatants was equivalent in pregnant and control mice (Fig. 5God), as was PHA-induced TNF-{alpha} (data not shown). Ag-specific production of all three type 2 cytokines tested was comparable in mice immunized during pregnancy and in controls (Fig. 6Go). Thus, in pregnant mice, depressed levels of type 1 cytokines were not accompanied by an obvious immune deviation at the cytokine level as when tested 21 days after immunization. Both splenocytes (Figs. 5Go and 6Go) and lymph node cells (data not shown) presented identical cytokine response patterns.



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  		FIGURE 5. Type 1 and proinflammatory cytokine production in mice immunized during pregnancy. Lymphoid cells collected 21 days after immunization were pooled within groups and were stimulated with IRBP or with PHA. Supernatants collected 48 h after stimulation were assayed by ELISA. The data for each cytokine are a composite of three to four separate experiments, with each point representing one experiment. To compensate for interexperiment variability, the values in each experiment were normalized to the nonpregnant group. Each point represents one experiment.

 


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  		FIGURE 6. Type 2 cytokine production in mice immunized during pregnancy. The experimental conditions and the analysis were as in Fig. 1Go.

 
Serum anti-IRBP Ab isotypes

Because Ab isotype switching to IgG2a and IgG1 is promoted by IFN-{gamma} and IL-4, respectively, the titers of these Ab isotypes can serve as a measure of the type of response that develops to the Ag in vivo. We, therefore, assayed the anti-IRBP Abs of each isotype in sera of individual pregnant and nonpregnant mice (Fig. 7Go). IRBP-specific IgG1 Abs in mice immunized during pregnancy were significantly elevated (p = 0.012). This suggested that during the evolution of the Ab response there must have been increased production of IL-4 in vivo. IgG2a titers remained unaffected. The ratio of Ag-specific IgG1/IgG2a for nonpregnant mice was ~3, whereas the ratio for pregnant mice was elevated to 11.



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  		FIGURE 7. IgG1 and IgG2a isotypes analyses of the anti-IRBP Ab response. Ig isotypes bound to IRBP-coated plates were measured by isotype-specific ELISA. Each symbol in the scatter graph corresponds to an individual mouse. The horizontal line indicates the group average. IgG1 ratio in pregnant vs nonpregnant mice was 11 (p = 0.012), whereas IgG2a ratio was 3.5 (p > 0.05).

 
Pregnant mice have elevated circulating TGF-ß levels in the serum

Sera were collected from pregnant mice on day 13 of pregnancy and were assayed for IL-10 and for TGF-ß content by ELISA in comparison to nonpregnant age-matched controls. The results showed that, with the exception of one pregnant animal, both groups had similar levels of IL-10, which in most cases hovered around the limit of detectability (Fig. 8Goa). Interestingly, the pregnant females had consistently elevated serum TGF-ß1 that was in some individuals up to twice that found in controls (Fig. 8Gob). This difference was highly statistically significant. TGF-ß2 levels in the same sera were overall much lower than those of TGF-ß1 (Fig. 8Goc). Pregnant mice had a 50% increase in serum TGF-ß2 levels; however, the difference did not attain statistical significance. Because platelets are a significant source of TGF-ß, platelet counts were performed in 18 nonpregnant and 18 pregnant mice on day 13 of pregnancy. The pregnant mice were found to have a 28% increase in their platelet counts (average of 868 x 103 vs 677 x 103 platelets/µl, significant at p < 0.01) which could have accounted for at least some of the elevation in their serum TGF-ß.



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  		FIGURE 8. Serum titers of IL-10 (a), TGF-ß1 (b), and TGF-ß2 (c) in 13-day pregnant mice. Sera of individual mice were assayed by ELISA. Each point represents one mouse. The horizontal line shows the calculated average of the group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present investigation, we show that mice immunized for induction of EAU during pregnancy are less susceptible to the induction and development of disease. Despite exhibiting an apparently unreduced immune capacity (DTH and lymphocyte proliferation), they are able to control not only the generation, but also the function of uveitogenic effector T cells. This is indicated by reduced disease scores both in a situation in which the uveitogenic effector must be generated during pregnancy but exerts its function after parturition (active immunization) and in a situation in which the effector has been generated under normal physiologic conditions, but exerts its function during pregnancy (adoptive transfer). The protection is accompanied by a selective inhibition of Ag-specific Th1 response, as indicated by reduced IL-12p40 and reduced IFN-{gamma} production to IRBP, but without evidence for an overall switch to a Th2-type response. Although the relative increase in the IgG1 Ab isotype may indicate a temporary up-regulation of IL-4, promoting a switch to the IgG1 isotype in the absence of an increase in Ag-specific IL-4, IL-5, or IL-10 production, this does not constitute compelling evidence for a lasting Th2 shift. Because uveitogenic effector T cells are of Th1 type phenotype 38, 39 , a selective suppression of the Th1 arm of the immune response can explain the observed protection from EAU.

This raises a question regarding the mechanism by which Th1 function is down-regulated in the absence of a Th2 shift. The present results are reminiscent of our previous work on genetic susceptibility and resistance to EAU, in which we showed that resistant strains of rats and mice are invariably low Th1 responders to the uveitogen, but that they are not necessarily dominant Th2 responders. In those studies, it was not clear what the alternative pathway to resistance might be, because studies in other Th1-dependent models of autoimmunity, EAE, and experimental diabetes 40, 41 indicated that the pathway to resistance involves a shift toward Th2. The present data reveal that although the pregnant females did not show elevated levels of IL-10 in the serum, they did show significantly elevated levels of circulating TGF-ß1 and a trend toward increased levels of circulating TGF-ß2. This may implicate TGF-ß as a possible mechanism in a non-Th2 pathway to resistance.

The question that must be considered is whether the statistically significant difference in TGF-ß is also biologically significant. Although the increment in TGF-ß1 in pregnant mice was on average only 50% over control and did not exceed double the control levels, this may well reflect a "spillover" of much higher local levels in the tissues. TGF-ß1 has repeatedly been shown to have protective and regulatory effects in autoimmunity. Administration of exogenous TGF-ß1 protects from EAE in actively immunized as well as adoptively transferred hosts 42, 43, 44 . Furthermore, endogenous TGF-ß is involved in the protective effects of oral tolerance 45 , and TGF-ß1-null mice invariably die an early death from autoimmune and inflammatory phenomena, indicating an important and nonredundant role for this regulatory cytokine 46, 47 . On the other hand, TGF-ß2 has been specifically associated with immune privilege and immune deviation 48, 49 . Although the levels of circulating TGF-ß2 in our mice were much lower than those of TGF-ß1 (picograms vs nanograms), its effects could be cumulative or synergistic with those of TGF-ß1. The present data, which support Th1 inhibition rather than a Th2 shift as the protective mechanism, can be viewed as being in line with the relative values of these two forms of TGF-ß.

Although our data do not exclude the contribution of hormonal influences specific to pregnancy in the protective effect, treatment of rats with pregnancy levels of progesterone had little or no effect on the development of EAU 50 . On the other hand, estrogen exacerbated disease at least in part by affecting the Th1/Th2 ratio 50 . Others reported exacerbating effects of estrogen in murine lupus, attributable to its enhancement of prolactin secretion 51 .

It is noteworthy that in contrast to the suppression of disease, there was no suppression of DTH or proliferative responses in the pregnant mice, especially because others have reported a diminution of the DTH response 52 . Suppression of disease without apparent down-regulation of cellular immunity to IRBP has been observed by us previously 66 and is likely to stem from the fact that the immunogen used in our studies is bovine, rather than mouse, IRBP. Although disease expression is by definition a manifestation of the response to conserved self epitopes, DTH and lymphocyte proliferation are likely to reflect responses mainly to immunodominant xenogeneic epitopes. Thus, the immunosuppression induced by pregnancy appears to discriminate among Ags with different levels of "foreignness." Although the response to conserved self Ags with low avidity to receptors on immunocompetent cells (EAU) is measurably down-regulated, responses to nonconserved Ags having high avidity interactions (DTH and lymphocyte proliferation) enjoy undiminished expression. It is tempting to speculate that such an immune dichotomy could permit preferential suppression of responses to conserved tissue Ags (fetus) as opposed to nonconserved microbial Ags (pathogenic microorganisms).

Our data, indicating a selective suppression of Th1-specific cytokines with little or no evidence for a Th2 response shift, are at variance with the report of Krishnan et al. 53 , who showed up-regulation of Th2 type cytokines in spleen and lymph node cells of pregnant mice in response to Leishmania. This difference may stem in part from the immunogen and in part from the timing of immunization in relation to the assessment of immune functions. Whereas Krishnan et al. 53 initiated and completed their study while the mice were still pregnant, we challenged the mice during pregnancy, but assessed the immunological responses that were acquired during pregnancy after parturition. Although our data do not exclude an early, transient Th2 bias (and the elevation in IgG1 Abs may even support it), it clearly did not result in the acquisition of a stably Th2-dominated immunity to IRBP.

In our experiments, the Th1-low response pattern acquired during pregnancy and the reduced disease scores persisted even after parturition and the return of normal hormonal balance. It should be pointed out that, in contrast to the situation in mice, uveitis in patients is reported to relapse during the postpartum period 30, 54, 55 . This relapse might be explained by the release from suppression in patients of clones primed before pregnancy, as well as by the emergence of new clones that have been primed after parturition. There are two alternative explanations why such a relapse was not observed in the mice. The prosaic one is that we did not wait long enough to see it. The postpartum period in humans lasts for months, not days 56 . However, a more plausible possibility is that, due to the immediate removal of the pups from their mother after birth, the postpartum rise in prolactin, normally related to lactation and suckling, was prevented. Prolactin is immunostimulatory to lymphocytes and has been connected to the enhancement of autoimmunity in animals and in humans, supporting the notion that a relapse of autoimmunity in the postpartum period might be precipitated by rising levels of prolactin 51, 57, 58, 59, 60 . In keeping with this notion, breast feeding has been found to be a risk factor for exacerbation of rheumatoid arthritis 61 . Furthermore, bromocriptine (a prolactin inhibitor) ameliorates the development of autoimmune uveitis, diabetes, and encephalitis in female mice and rats 62, 63, 64 . Interestingly, it was recently reported that prolactin antagonizes the suppressive activity of TGF-ß on lymphocytes 65 . This could help to explain why, if TGF-ß is involved in remission and prolactin is involved in the relapse, the unopposed inhibitory effects of TGF-ß continued into the postpartum period in the present experiments.

In conclusion the present data provide an experimental basis for the clinical observations on remission during pregnancy of diseases such as arthritis, uveitis, and multiple sclerosis, and help explain the interrelationship between the pregnant state and cell-mediated autoimmune disease.


   Acknowledgments
 
We thank Shu-Hui Sun, Luiz Rizzo, and Phyllis Silver for their valuable contributions to different parts of the study.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Rachel R. Caspi, Laboratory of Immunology, National Eye Institute, National Institutes of Health, Building 10, Room 10N222, 10 Center Drive, MSC 1857, Bethesda, MD 20892-1857. E-mail address: Back

2 Abbreviations used in this paper: EAU, experimental autoimmune uveitis; DTH, delayed-type hypersensitivity; EAE, experimental allergic encephalomyelitis; IRBP, interphotoreceptor retinoid-binding protein; PTX, pertussis toxin. Back

Received for publication August 17, 1998. Accepted for publication November 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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