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The Journal of Immunology, 2004, 173: 1051-1058.
Copyright © 2004 by The American Association of Immunologists

Requirements of NK Cells and Proinflammatory Cytokines in T Cell-Dependent Neonatal Autoimmune Ovarian Disease Triggered by Immune Complex1

Yulius Y. Setiady2,*, Patcharin Pramoonjago* and Kenneth S. K. Tung*,{dagger}

* Departments of Pathology and {dagger} Microbiology, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A model of neonatal autoimmune disease has been described recently in which an epitope-specific autoantibody to murine zona pellucida 3 induces severe ovarian disease in neonatal, but not adult, mice (neonatal AOD). The autoantibody forms immune complex with endogenous ovarian zona pellucida 3, and a pathogenic CD4+ T cell response is triggered. The basis for the predominant neonatal susceptibility has not been clarified. In this study innate immunity, including neonatal NK cells, in neonatal AOD was investigated. Neonatal spleen contained readily detectable NK1.1+TCRV{beta}–, but not NK1.1+TCRV{beta}+, cells. Ab depletion of NK1.1+TCRV{beta}– cells inhibited neonatal AOD development. Moreover, in adoptive transfer of neonatal AOD, recipient disease was ameliorated when either donor or recipient NK cells were depleted. Thus, NK cells operate in both induction and effector phases of the disease. IFN-{gamma} was produced by neonatal NK cells in vivo, and it may be important in neonatal AOD. Indeed, ovaries with neonatal AOD expressed high levels of IFN-{gamma} and TNF-{alpha} which correlated with disease severity, and the disease was inhibited by IFN-{gamma} or TNF-{alpha} Ab. Importantly, disease was enhanced by recombinant IFN-{gamma}, and treatment of T cell donors with IFN-{gamma} Ab also significantly reduced adoptive transfer of neonatal AOD. Finally, neonatal AOD was ameliorated in mice deficient in Fc{gamma}RIII and was enhanced in Fc{gamma}RIIB-deficient mice. We conclude that neonatal NK cells promote pathogenic T cell response at multiple stages during neonatal autoimmune disease pathogenesis. Also operative in neonatal AOD are other mediators of the innate system, including proinflammatory cytokines and Fc{gamma}RIII signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Autoantibody is an integral part of T cell-mediated autoimmune diseases, but is generally regarded as a secondary effect of an autoreactive T cell response. However, in vitro studies have shown that Ab, by forming immune complex with the Ag, can also significantly affect the T cell response. Engagement of immune complex with FcRs on APCs results in a >1000-fold increase in Ag uptake (1), induces maturation and cytokine production by dendritic cells (DC)3 (2, 3), and promotes the T cell response. Ab also affects Ag processing by APC and selection of the cognate T cell peptides for presentation (4). It has been demonstrated that autoantibodies from human with autoimmune diseases can modify autoreactive T cell response in vitro (5, 6, 7). In a recent model of T cell-mediated neonatal autoimmune ovarian disease (neonatal AOD) that is triggered by an autoantibody, the in vivo relevance of these in vitro findings has been verified (8).

The neonatal AOD model was discovered in the course of a study of adult AOD induced by immunization with an oocyte-specific zona pellucida 3 (ZP3) peptide (ZP3330–342) in CFA (9). Adult AOD is dependent on pathogenic autoreactive T cells. To study the role of autoantibodies in AOD, a chimeric peptide, CP2, that contained a foreign T cell epitope and the ZP3 B cell autoepitope (aa 335–342) was produced (10). Immunization of CP2 in CFA elicited a strong ZP3 Ab response without a concomitant T cell response to ZP3, and the mice were free of AOD (10). Thus, autoantibodies and autoreactive B cell per se are insufficient to induce AOD in adults.

Although adult female mice injected with CP2 did not develop AOD, severe AOD was found in their female progeny 2 wk after birth (8). Neonatal AOD, triggered by maternal ZP3 autoantibodies transferred in milk, was dependent on the B cell epitope specificity of the autoantibodies, the MHC haplotype, and functional Fc{gamma}R. Strikingly, neonatal AOD occurrence also requires the presence of T cells in neonates. Therefore, ZP3 immune complex can trigger activation of autoreactive T cells, probably via Fc{gamma}R-positive APC. In parallel to the neonatal AOD study, Greeley et al. (11) reported that T cell-dependent diabetes in NOD mice is significantly ameliorated by prevention of transfer of maternal autoantibodies to both female and male progeny. These findings have prompted the hypothesis that neonatal autoimmunity induced by maternal Ab, which results in progressive and destructive disease, such as diabetes in NOD mice and the congenital heart block in the setting of lupus Abs to Ro and La, may include de novo autoreactive T cell activation.

One of the most intriguing observations about neonatal AOD is that the disease occurred only when ZP3 Ab-positive milk feeding was initiated during the first 5 days of life. This neonatal time window did not appear to depend on neonatal deficiency of CD4+CD25+ regulatory T cells because the infusion of adult CD4+CD25+ T cells failed to protect neonates from neonatal AOD. We considered it more likely that the neonatal innate system may favor the promotion of a T cell response, but may resist CD4+CD25+ T cell regulation. However, our current understanding of the neonatal innate system, including NK cell function, is limited.

In this study we examined the role of neonatal NK cells in the pathogenesis of neonatal AOD. Neonatal NK cells with the capacity to produce IFN-{gamma} were found to constitute a significant fraction of neonatal spleen cells. In the presence of immune complex and Fc{gamma}RIII, neonatal NK cells were found to promote the induction and effector function of pathogenic T cells, possibly through production of proinflammatory cytokines. This study has therefore demonstrated for the first time a pathogenic role of neonatal NK cells in autoimmune disease pathogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and reagents

C57BL/6 (B6), A/J, and (C57BL/6 x A/J)F1 (B6AF1) mice were obtained from the National Cancer Institute (Frederick, MD). FcR{gamma} subunit and Fc{gamma}RIIB-deficient B6 mice were purchased from Taconic Farms (Germantown, NY), and Fc{gamma}RIII-deficient B6 mice were obtained from The Jackson laboratory (Bar Harbor, ME). The mice were housed in a specific pathogen-free facility under the animal care and use committee guidelines of University of Virginia.

To generate autoantibodies to native antigenic epitopes of mouse ZP3, we used a chimeric peptide named CP2 (NCAYKTTQANKQAQIHGPR) that contained a bovine RNase T cell epitope 94–104 (underlined) linked to the modified native self B cell epitopes of ZP3335–342 (QFQIHGPR), in which Phe336, a critical residue of the T cell epitope, was replaced by an alanine. The peptide was synthesized by Multiple Peptide Systems (San Diego, CA) with >90% purity as determined by HPLC.

IFN-{gamma}-neutralizing mAb (clone XMG1.2) and TNF-{alpha}-neutralizing mAb (clone MP6-XT3) and Abs used for IFN-{gamma} detection in the ELISA were obtained from BD Pharmingen (San Diego, CA). Recombinant mouse IFN-{gamma} and IL-12 were obtained from BD Pharmingen and R&D Systems (Minneapolis, MN), respectively. For NK cell depletion, PK136 mAb (BD Pharmingen) against NK1.1 Ag and rabbit polyclonal Abs against asialo-GM1 (AGM1) (Cedarlane Laboratories, Hornby, Canada) were used.

Immunization and neonatal AOD induction by milk feeding or serum transfer

Immunization was performed as described previously (8). In short, 6- to 8-wk-old B6 or B6AF1 mice were injected with 50 nmol of CP2 in CFA in one footpad and at the base of the tail. One month after immunization, the ZP3 Ab titer was determined by indirect immunofluorescence staining of a frozen ovarian section.

For neonatal AOD induction by milk feeding, CP2-immunized B6 female mice were impregnated by normal A/J males. The neonatal AOD was constantly observed when the fluorescence serum ZP3 autoantibodies titers of milk donors exceeded 1/1000. In some cases, naive B6AF1 pups were fostered to the ZP3 autoantibody-positive postpartal dams starting from day 3 of age. For neonatal AOD induction by serum transfer, sera with a fluorescence ZP3 autoantibody titer at or exceeding 1/3000 were pooled from CP2-immunized male or female mice, and 100 µl of serum was injected i.p. into naive neonatal B6AF1 mice on days 3 and 5. All ZP3 autoantibodies recipients were euthanized at 14 days of age.

Histopathologic grading of neonatal AOD

Mouse ovaries were fixed in Bouin’s fixative and embedded in paraffin, and 5-µm-thick sections were stained with H&E. The severity index of neonatal AOD was determined without knowledge of the identity of the mice under study. A severity grade of one to four was assigned to each of the following histopathologic changes: 1) oophoritis or ovarian inflammation, 2) depletion of growing oocytes, and 3) depletion of primordial oocytes. An oophoritis grade of 1 denotes one or two foci of leukocyte infiltration, grades 2–3 represents an incremental numbers of multifocal inflammatory foci; and grade 4 is diffuse inflammation. Loss of each type of oocytes was graded 1 when occasional oocytes were lost from the follicle and were graded 4 when all oocytes disappeared. Grades 2 and 3 represent partial and incremental oocyte loss between grades 1 and 4. Because the loss of oocytes results in a reduction of ovarian function, their grades were weighed as follows: the grade for growing oocyte loss was multiplied by 1.5, and those of primordial oocyte loss were multiplied by 2. Accordingly, the total disease severity index was calculated as: oophoritis grade + (growing oocyte loss grade x 1.5) + (primordial oocyte loss grade x 2.0). and the total neonatal AOD scores ranged from 1 to 18.

T cell enrichment and transfer, in vivo cytokine neutralization, and NK cell depletion

For adoptive transfer, T cells were enriched from the spleens of 2-wk-old ZP3 Ab recipients using T cell enrichment column (R&D Systems, Minneapolis, MN). T cells with purity of >90% were injected i.p. into 3-day-old normal B6AF1 pups at 2 x 106 cells/mouse. The cell recipients were studied 11 days after cell transfer.

To neutralize cytokine and deplete NK cell in vivo, the appropriate mAb was injected i.p. at 20 µg/injection every 3 days starting on day 3. A 50- to 100-µl volume of Ab was injected through a 30-gauge needle inserted s.c. from the chest to the level of the abdomen and then into the peritoneal cavity. After injection, the needle was withdrawn very slowly to avoid leakage of Ab. All mice were studied at 14 days of age.

To verify depletion of NK cell function, NK cell-depleting Ab was injected i.p. at 20 µg/pup into 5-day-old pups. Two days later, the pups were injected i.p. with LPS at 20 µg/pup, and sera were collected 6 h later. Serum IFN-{gamma} levels were measured by ELISA.

Flow cytometric analysis

Single-cell suspensions were prepared from the spleen or lymph nodes. Contaminating RBC were lysed using ACK lysing buffer (BioSource International, Camarillo, CA). After two washes, Fc{gamma}RIIB/III were blocked by incubation with 2.4G2 Ab at 4°C for 20 min. The cells were incubated for 20 min on ice with the desired fluorochrome-conjugated mAbs or isotype control Ig at 0.5 µg/106 cells. The cells were washed twice with PBS/1% BSA, fixed in 2% paraformaldehyde, and analyzed by FACScan (BD Biosciences, San Jose, CA) using the CellQuest program (BD Biosciences).

RNA extraction and real-time PCR

Quantitative real-time RT-PCR was performed using a sequence detector (model 7700; Applied Biosystems, Foster City, CA). Total RNA from one ovary of each mouse receiving ZP3 autoantibodies or control Ab was extracted using the RNeasy minikit (Qiagen, Valencia, CA). The cDNA was then generated with 1 µg of total RNA using the TaqMan RT kit (Applied Biosystems). Briefly, total RNA was added to 50 µl of RT reaction mixture (1x RT buffer, 5.5 mM MgCl2, 500 µM dNTP, 2.5 µM random hexamers, 0.4 U/µl RNasin (RNase inhibitor), and 1.25 U/µl Multiscribe reverse transcriptase) at 25°C for 10 min, 48°C for 30 min, and finally 98°C for 5 min to inactivate reverse transcriptase. Real-time PCR were performed using TaqMan Pre-Developed Assay reagents for cytokine gene expression kits (Applied Biosystems) according to the manufacturer’s instructions. The results were analyzed using SDS software (Applied Biosystems). The final data were normalized to the expression of GADPH, a housekeeping gene.

Statistical analyses

Statistical differences were determined by nonparametric Wilcoxon test or nonparametric Mann-Whitney test. Significance was considered at p ≤ 0.05 in all experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neonatal NK cells are critical for neonatal AOD

The spleens of 3- or 9-day-old mice contained a significant number of NK1.1+ cells that did not express TCRV{beta} (Fig. 1A). In contrast, very few, if any, NKT cells (NK1.1+TCRV{beta}+ cells) were detectable in the neonatal spleen. The average ratio of NK cell to {alpha}{beta}TCR+ T cells in the 3-day-old mice was 0.6, and it declined to 0.2 by day 9 as T cell numbers increased.



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  		FIGURE 1. IFN-{gamma}-producing NK cells are detectable in neonatal mice. A, Cytofluorometric analysis of spleen cells from 3- and 9-day-old mice. B, To examine neonatal NK cell function, NK cells were depleted from 5-day-old pups with anti-NK1.1 mAb (PK136) or anti-AGM1 polyclonal Ab, followed by LPS (20 µg/pup) injection on day 7. Sera were collected 6 h later, and the serum level of IFN-{gamma} was determined by ELISA. The control group contains data from mice treated with rabbit Ig or mouse IgG. IFN-{gamma} levels in the two treatments with control Ab were comparable. Each group contained 13–17 mice. C, NK cell depletion in vivo with anti-AGM1 Ab and anti-NK1.1 Ab. Ten-day-old mice were injected with the corresponding Ab, and 2 days later, lymph node cells were stained with anti-CD3 and anti-DX5 Abs.

 
The functional capacity of neonatal NK cells was investigated in 7-day-old mice given LPS. Six hours after injection of LPS (plus control IgG), the serum IFN-{gamma} level reached 30.5 ± 6.3 ng/ml. To demonstrate that NK cells were a source of the serum IFN-{gamma}, the mice were treated with anti-NK1.1 Ab or anti-AGM1 Ab before LPS injection, and as shown in Fig. 1B, serum IFN-{gamma} levels were reduced to 9.6 ± 2.4 and 1.6 ± 0.4 ng/ml, respectively. The AGM1 Ab was found to be more efficient in depleting neonatal NK cells (CD3–DX5+ cells) than the NK1.1 Ab (60% reduction (n = 8) vs 49% (n = 9); Fig. 1C); this may account for the difference in serum IFN-{gamma} reduction of the two treatment groups (Fig. 1B). Altogether, neonatal NK cells are well represented in neonatal mice, and they produce IFN-{gamma} in response to LPS in vivo.

We next determined the effect of NK cell depletion on the development of neonatal AOD induced by ZP3 autoantibodies. The incidence of neonatal AOD was 27% (three of 11 mice) in the recipients of ZP3 autoantibody-positive milk after NK cell depletion by NK1.1 Ab; this was significantly less than the disease incidence of 80% in control mice (Fig. 2A). The effect of rabbit Ab to AGM1 was even more dramatic. None of the mice with NK cell depletion developed neonatal AOD, whereas 100% of the control mice developed severe neonatal AOD (Fig. 2B). The possibility that the effect was due to NKT cell depletion is unlikely because NKT cells are not depleted by AGM1 Ab treatment (Fig. 1C) (12, 13, 14).



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  		FIGURE 2. NK cells have a pivotal role in neonatal AOD. A and B, Neonatal AOD severity in ZP3 Ab recipients treated with anti-NK1.1 mAb (PK136), anti-AGM1 Ab, or control Abs every 3 days from day 3 and studied on day 14. The disease was induced by ZP3 Ab-positive serum transfer in B and by ZP3 Ab-positive milk feeding in A. C, Normal ovarian histology of ZP3 Ab recipients with NK cell depletion. Note growing follicles (long arrow) and primordial follicles (arrowhead) in the ovary free of inflammation (magnification, x200). D, Ovary from ZP3 Ab recipient treated with rabbit polyclonal Ab shows loss of oocytes and heavy mononuclear infiltration (arrow; magnification, x200). Inset, Ovarian ZP immune complexes found in ZP3 Ab recipients.

 
NK cells operate in two stages of neonatal AOD induction

Neonatal NK cells may participate in the induction phase of neonatal AOD by responding to ZP3 Ag-Ab complexes and, through cytokines, activate DC and promote a T cell response. Alternatively, they may cooperate with effector T cells downstream by facilitating tissue inflammation and/or cytolysis. To dissect these possibilities, we studied the effect of neonatal NK cell depletion on the adoptive transfer of neonatal AOD. As shown in Fig. 3, the spleen T cells from mice with neonatal AOD adoptively transferred severe ovarian inflammation to normal syngeneic pups. To investigate neonatal NK cell requirement in the effector phase of neonatal AOD, NK cells were depleted from the T cell recipients by AGM1 Ab. As shown in Fig. 3A, this treatment reduced the disease incidence in T cell recipients from 62% (eight of 13) to 29% (four of 14; p = 0.016). Thus, the neonatal NK cells participate in promoting inflammation triggered by autoreactive T cells manifested in the cell recipients that are devoid of immune complexes.



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  		FIGURE 3. NK cell requirement in neonatal AOD induction. A, To investigate NK cells in the disease effector phase, splenic T cells from mice with neonatal AOD were transferred into naive neonatal recipients treated with anti-AGM1 Ab or control Ig. B, To examine the disease induction phase, pups were treated with ZP3 Ab as well as anti-AGM1 Ab (or rabbit Ig). Their splenic T cells were then transferred into naive NK cell-sufficient pups, which were studied 11 days later.

 
To determine whether neonatal NK cells also influence pathogenic T cell induction, we depleted NK cells from the T cell donors (Fig. 3B). As shown in Fig. 2A, this treatment effectively reduced the severity and incidence of neonatal AOD in the T cell donors. More importantly, neonatal NK cell depletion of the T cell donors led to significant reduction of ovarian disease in the T cell recipients. Only four of 15 (27%) recipients of T cells from NK cell-depleted mice developed neonatal AOD, whereas 60% (nine of 15) of the recipient mice in the control group developed neonatal AOD (p = 0.019).

Therefore, neonatal NK cells participate in multiple stages of neonatal AOD pathogenesis. They assist in the induction of the effector T cell response as well as in the functional expression of activated effector T cells.

Severity of neonatal AOD correlates with ovarian expression of IFN-{gamma} and TNF-{alpha}

To investigate other mechanisms of innate immunity that may also require neonatal NK cell action, we studied the influence of proinflammatory cytokines, including IFN-{gamma} and TNF-{alpha}, on neonatal AOD development. mRNA isolated from the ovaries of mice that received ZP3 autoantibody-positive milk and the control mice were studied for the expression of these cytokines by real-time RT-PCR. Significantly higher levels of IFN-{gamma} and TNF-{alpha} mRNA were detected in the ovaries of ZP3 autoantibody recipients than in control ovaries (Fig. 4A). The experimental to control ratio was 11.0 for IFN-{gamma} and 4.4 for TNF-{alpha}. When the cytokine mRNA levels of individual mice were analyzed against ovarian disease severity, a highly significant correlation was found for both cytokines (Fig. 4, B and C).



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  		FIGURE 4. Expressions of IFN-{gamma} and TNF-{alpha} are elevated in ovaries with neonatal AOD, with correlation to disease severity. Total RNA, extracted from ovaries of 14-day-old mice that received ZP3 Ab-positive milk (n = 26) or from mice fed milk from dams immunized with CFA (n = 10), were reverse transcribed using random primer and subjected to real-time PCR. A, Absolute cytokine mRNA levels in the ovaries of experimental ({blacksquare}) and control ({square}) mice. B and C, Correlation between ovarian disease severity and IFN-{gamma} (B) and TNF-{alpha} (C) expression levels in individual mice with neonatal AOD.

 
Neonatal AOD was inhibited by neutralizing Abs to IFN-{gamma} or TNF-{alpha} and was promoted by rIFN-{gamma} or IL-12

We next documented the pathogenic role of IFN-{gamma} and TNF-{alpha} in neonatal AOD by treatment with neutralizing Ab to the cytokines. As shown in Fig. 5A, mild neonatal AOD developed in two of nine (22%) ZP3 Ab recipients treated with IFN-{gamma} Ab and in three of 11 (27%) mice treated with TNF-{alpha} Ab. In contrast, severe neonatal AOD developed in 17 of 18 (94%) recipients of ZP3 Ab-positive milk and control rat IgG.



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  		FIGURE 5. Proinflammatory cytokines are critical in neonatal AOD induction. A, IFN-{gamma}- or TNF-{alpha}-neutralizing Ab significantly inhibits neonatal AOD. Abs were injected every 3 days into recipients of ZP3 Ab-positive milk starting from day 3, and mice were studied on day 14. B, Injection of recombinant mouse IFN-{gamma} or recombinant mouse IL-12 greatly increased the severity of neonatal AOD. Recombinant cytokines (100 ng/pup) were injected with ZP3 antiserum of low Ab titer on days 3 and 5, and mice were studied on day 14. C, Adoptive transfer of T cells from ZP3 Ab-treated pups that were also given anti-IFN-{gamma} Ab (or rat IgG). Disease severity in T cell recipients was assessed 11 days after adoptive transfer.

 
To confirm the results based on Ab treatment, we next examined the effect of recombinant proinflammatory cytokine injection on neonatal AOD. In this experiment mild neonatal AOD was induced by a pool of antiserum with low ZP3 autoantibody titer (Fig. 5B). When the ZP3 Ab recipients were coinjected with recombinant mouse IFN-{gamma}, all 12 neonatal mice developed very severe neonatal AOD with complete ovarian atrophy. Similar results were obtained when the mice were coinjected with recombinant murine IL-12, a cytokine known to activate NK cells (Fig. 5B).

Therefore, the proinflammatory cytokines, including IFN-{gamma}, TNF-{alpha}, and IL-12, are clearly operative in the pathogenesis of neonatal AOD. However, this study did not clarify whether NK cells were the source of the pathogenic cytokines.

Role of IFN-{gamma} in the induction phase of neonatal AOD

Activated NK cells and Th1 cells are principal producers of IFN-{gamma} in an immune response. To address the role of IFN-{gamma} production by NK cells in neonatal AOD, we studied the requirement for IFN-{gamma} during the induction phase of pathogenic T cells. IFN-{gamma} was neutralized in T cell donors before disease transfer. As shown in Fig. 5C, only one of nine mice that received T cells from IFN-{gamma} Ab-treated cell donors developed AOD, whereas five of seven recipients of T cells from mice treated with control Ab developed AOD (p = 0.011). Therefore, neonatal AOD was inhibited when IFN-{gamma} was neutralized only during the inductive phase of the disease.

Neonatal AOD induction is dependent on Fc{gamma}RIII

IFN-{gamma} has been reported to promote stimulatory Fc{gamma}R functions by decreasing the expression of inhibitory Fc{gamma}RIIB and increasing the expression of stimulatory Fc{gamma}RIIA on human monocytes (15). In the recent study we evaluate the roles of stimulatory and inhibitory Fc{gamma}R in neonatal AOD. Our previous study has indicated that Fc{gamma}R-positive cells are critical for neonatal AOD induction (8), and the inhibiting effect of 2.4G2 mAb was confirmed in the present study (Fig. 6A). Although this mAb can block the inhibitory Fc{gamma}RIIB and the stimulatory Fc{gamma}RIII, NK cells are known to express predominantly Fc{gamma}RIII, not Fc{gamma}RIIB (16). To examine the role of Fc{gamma}RIII further, we compared the contributions of Fc{gamma}RIIB vs Fc{gamma}RIII by studying neonatal AOD in wild-type C57BL/6 (B6) mice and B6 mice deficient in the FcR common {gamma}-chain, only Fc{gamma}RIII, or only Fc{gamma}RIIB.



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  		FIGURE 6. Neonatal AOD induction is dependent on Fc{gamma}RIII, but not Fc{gamma}RIIB, function. A, Injection of mAb against Fc{gamma}RIII and Fc{gamma}RIIB (2.4G2) completely inhibited neonatal AOD. B, Incidence and disease severity of neonatal AOD in B6AF1 and B6 wild-type mice are compared with the disease in B6 pups deficient in FcR{gamma}-chain, Fc{gamma}RIII, or Fc{gamma}RIIB. The disease was induced by ZP Ab-positive milk starting on day 3, and mice were studied on day 14.

 
The severity and incidence of neonatal AOD are influenced by the genetic background of ZP3 autoantibody recipient mice (Y. Y. Setiady, E. Samy, and K. Tung, unpublished observations). Although 85% (11 of 13) B6AF1 mice that received ZP3 Ab-positive milk developed neonatal AOD, only 35% (six of 17) B6 mice developed the disease (Fig. 6B). Despite the low incidence of neonatal AOD in wild-type B6, a significant reduction of neonatal AOD was found in B6 deficient in FcR{gamma} and Fc{gamma}RIII. Thus, two of 26 (8%) B6 mice deficient in FcR{gamma} and none of the 14 B6 mice deficient in Fc{gamma}RIII developed neonatal AOD. In contrast, the disease incidence and severity in B6 mice deficient in Fc{gamma}RIIB were higher than those in wild-type B6 pups (p = 0.041). We conclude that Fc{gamma}RIII is required to promote neonatal AOD, whereas engagement of Fc{gamma}RIIB has the opposite effect.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently a novel model of autoimmune ovarian disease was described that affects neonatal mice, but spares the adult mice (8). Neonatal AOD was induced by B cell epitope-specific maternal or serum autoantibodies to the ovarian Ag ZP3. Immune complexes were deposited in ovarian ZP, and the disease process was dependent on the presence of Fc{gamma}R. However, the occurrence of neonatal AOD also required de novo activation of pathogenic CD4+ T cells, presumably by the tissue immune complexes. Thus, neonatal mice rendered T cell deficient were resistant to disease, whereas the CD4+ T cells from mice with neonatal AOD were able to adoptively transfer severe disease to naive neonatal recipients. We have since found that neonatal AOD development depends on T cell costimulation and requires Th1 cytokines (E. T. Samy, Y. Setiady, and K. Tung, unpublished observations).

An intriguing finding in neonatal AOD was the requirement for ZP3 Ab to reach the neonate within the first 5 days of life. To investigate this neonatal time window, we found that depletion of CD4+CD25+ T cells would allow the pups that received the ZP3 Ab beyond the neonatal window (after 9 days of age) to develop disease; however, the disease was not preventable by infusion of adult CD4+CD25+ T cells. It was postulated that the neonatal environment may be resistant to the CD4+CD25+ T cell action. For example, innate immune cells, including NK cells, macrophages, and DC, are known to influence the adaptive immune response as well as to inhibit the regulatory function of CD4+CD25+ T cells (17). Therefore, in this study we investigated the neonatal innate system, specifically the role of NK cells in neonatal AOD.

NK cells were readily detected in 3-day-old spleens using NK1.1 Ab in numbers that surpass those of NKT cells but approximate those of T cells. The finding is consistent with the report by Ortaldo et al. (18). After day 3, the relative number of NK cells in the spleen declined as T cell numbers increased. Neonatal NK cells produce similar amounts of IFN-{gamma} as adult NK cells in response to LPS stimulation (19). Most importantly, neonatal NK cells were documented to be required for the pathogenesis of neonatal AOD. When they were depleted from the neonatal mice by NK1.1 or AGM1 Ab, disease development was completely inhibited. This provides the first demonstration that murine neonatal NK cells are functional and that they support the development of T cell-mediated autoimmune disease.

Because neonatal AOD has complex mechanisms, involving initially immune complex and subsequently the CD4+ T cell response, the activated NK cells may participate in different stages of disease when the immune complex may or may not be present. To differentiate between these alternatives, we studied NK cells in the adoptive transfer of neonatal AOD, when the immune complexes would be present in the cell donors, but not in the T cell recipients. Our results clearly showed that the recipient disease was ameliorated regardless of whether the NK cells were removed from the T cell donors or the T cell recipients. Therefore, NK cells are involved in both the inductive phase and the effector phase of neonatal AOD.

Previous studies of the role of NK cells in adult autoimmune disease have reported somewhat controversial results (reviewed in Ref. 20). Reports of disease amelioration upon NK cell depletion (21, 22, 23, 24, 25, 26) were not confirmed in other studies (27, 28, 29, 30, 31, 32). However, the research was based on several disease models that required different methods of induction, and they may have different pathogenetic mechanisms. An exception is EAE in C57BL/6 mice immunized with the myelin oligodendrocyte gp35–55 peptide in adjuvant. Zhang et al. (27) immunized the mice shortly after NK cell depletion with Ab and found disease enhancement, whereas Shi et al. (21) depleted NK cells with Ab 2 days before immunization and reported disease reduction. Besides the timing of NK cell depletion, the studies were conducted in different laboratories and may have been influenced by different environmental factors that can impact autoimmune disease development (33).

Comparison of adult NK cells in adult autoimmunity with neonatal NK cells in neonatal autoimmunity would also be misleading because many differences exist between neonatal and adult NK cells. For example, the cytolytic activity of purified neonatal NK cells against the classical NK cell targets was barely detectable until the mice reached 2–3 wk of age (34, 35). Jamieson et al. (36) documented that progenitors of neonatal NK cells underwent more rapid division than adult cells. The expression of receptors for MHC class I or class I-like molecule of the Ly49 and CD94/NKG2 families on adult NK cells was also far more diverse than that detected on neonatal NK cells. In particular, neonatal NK cells were reported to express predominantly CD94/NKG2A (37, 38), and the Ly49 receptors were not detectable until after 1 wk (18). It has even been suggested that NK cells from adult vs neonates may differ in their capacity of self/nonself discrimination.

NK cells may affect neonatal AOD by modifying the APC function of DC. Indeed, NK cells have recently been shown in vitro to induce DC maturation and cytokine production, and these events can, in turn, enhance T cell activation (39, 40, 41, 42, 43). Interaction of NK cells and DC can be bidirectional; and activated DC have been shown to induce proliferation, activation, and cytokine production by NK cells (39, 40, 41, 44, 45). The two cells may communicate by cell contact or via proinflammatory cytokines such as IFN-{gamma} and TNF-{alpha} (39, 40, 41, 44). In this context, it is of interest that the same cytokines are found in the present study to strongly influence neonatal AOD pathogenesis. When IFN-{gamma} was blocked, neonatal AOD development was abrogated, and the capacity to adoptively transfer disease by CD4+ T cells was inhibited. The latter finding, which indicated that IFN-{gamma} is involved during the process of T cell induction, provides strong support for the NK cell as the source of the proinflammatory cytokines.

Our study has shown that a balance between stimulatory and inhibitory Fc{gamma}R is critical in neonatal AOD; Fc{gamma}RIII engagement promotes neonatal AOD, whereas Fc{gamma}RIIB engagement has the opposite effect. These two Fc{gamma}Rs share similar specificities, but have opposite functions in promoting inflammation; Fc{gamma}RIII is proinflammatory, whereas Fc{gamma}RIIB opposes the stimulatory signals (46). Because the immune complex is responsible for triggering neonatal AOD, it may engage the Fc{gamma}R on NK cells, DC, or both. Because NK cells express predominantly Fc{gamma}RIII, this may explain the dominant effect of Fc{gamma}RIII deficiency on neonatal AOD development. In contrast, both Fc{gamma}RIIB and Fc{gamma}RIII are expressed on DC, and they could modulate the outcome of T cell activation in response to the DC that process the immune complexes. The finding that Fc{gamma}RIIB-deficient DC exhibit a lower threshold of activation by immune complex could explain the enhanced severity of neonatal AOD in Fc{gamma}RIIB-deficient mice (2). Finally, Fc{gamma}R is widely expressed in granulocytes, monocytes, and macrophages and with the attachment of cytophilic Ab to these cells, they may also contribute to neonatal AOD development.

Although our study investigated neonatal NK cells in an autoimmune response, the results could have broader implications and are applicable to in vivo investigation of the neonatal immune response in general. Many studies that compared neonatal and adult mice using in vitro approaches have described deficiency or immaturity in neonatal T cell and neonatal APC function (47, 48, 49, 50). In contrast, many in vivo studies of T and B cell responses to Ag with adjuvant or viral infections have reported comparable responses in neonatal and adult mice (51, 52, 53). Perhaps some of the discrepancies between the in vitro and in vivo findings would be reconciled if the participation of neonatal NK cells is included in the equation. As neonatal NK cells are now documented to have an important role in promoting neonatal autoimmunity, they are likely to also enhance neonatal APC function in other types of immune responses. Our study, therefore, emphasizes the importance of incorporating the interaction of innate and adaptive immunities in research on the ontogeny of immune response.


   Acknowledgments
 
We are grateful to Sharon Mangawang and Joyce Nash for expert technical assistance, and to Michael Brown for helpful suggestions. The histology was conducted at the Molecular Reproduction Research Center.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI51420, HD44415, and P50AR45222. Back

2 Address correspondence and reprint requests to Dr. Yulius Y. Setiady, Department of Pathology, P.O. Box 800214, University of Virginia, Charlottesville, VA 22908. E-mail address: js5bk{at}virginia.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; AGM1, asialo-GM1; AOD, autoimmune ovarian disease; CP2, chimeric peptide 2; ZP3, zona pellucida 3. Back

Received for publication February 26, 2004. Accepted for publication May 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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