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. 2015 Mar;93(3):311-20.
doi: 10.1038/icb.2014.97. Epub 2014 Nov 18.

Mice lacking Axl and Mer tyrosine kinase receptors are susceptible to experimental autoimmune orchitis induction

Nan Li  1 , Zhenghui Liu  2 , Yue Zhang  2 , Qiaoyuan Chen  2 , Peng Liu  2 , C Yan Cheng  3 , Will M Lee  4 , Yongmei Chen  5 , Daishu Han  2
Affiliations

Mice lacking Axl and Mer tyrosine kinase receptors are susceptible to experimental autoimmune orchitis induction

Nan Li et al. Immunol Cell Biol. 2015 Mar.

Abstract

The mammalian testis is an immunoprivileged organ where male germ cell autoantigens are immunologically ignored. Both systemic immune tolerance to autoantigens and local immunosuppressive milieu contribute to the testicular immune privilege. Testicular immunosuppression has been intensively studied, but information on systemic immune tolerance to autoantigens is lacking. In the present study, we aimed to determine the role of Axl and Mer receptor tyrosine kinases in maintaining the systemic tolerance to male germ cell antigens using the experimental autoimmune orchitis (EAO) model. Axl and Mer double-knockout (Axl(-/-)Mer(-/-)) mice developed evident EAO after a single immunization with germ cell homogenates emulsified with complete Freund's adjuvant. EAO was characterized by the accumulation of macrophages and T lymphocytes in the testis. Damage to the seminiferous epithelium was also observed. EAO induction was associated with pro-inflammatory cytokine upregulation in the testes, impaired permeability of the blood-testis barrier and generation of autoantibodies against germ cell antigens in Axl(-/-)Mer(-/-) mice. Immunization also induced mild EAO in Axl or Mer single-gene-knockout mice. By contrast, a single immunization failed to induce EAO in wild-type mice. The results indicate that Axl and Mer receptors cooperatively regulate the systemic immune tolerance to male germ cell antigens.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
EAO score. (a) Histological EAO score. Paraffin sections of the testes were stained with hematoxylin–eosin. Images represent six EAO stages based on testicular inflammation as described in the Results. Arrows, focal inflammation; BV, blood vessel; TA, tunica albuginea; RT, rete testis; ST, seminiferous tubule; TR, tubuli recti. Scale bar =20 μm. (b) EAO stages in different mice. Mice with indicated genotypes were immunized with male germ cell homogenates emulsified in CFA. EAO stage of individual mice after 50 days was determined based on histological analysis. (c) Testis size. Representative testicular images of wild-type (WT) and Axl−/−Mer−/− (AM−/−) mice at 50 days after immunization (Imm.). Mice injected with CFA alone served as the control (Ctrl). Testis weight was measured in the indicated mice (right panel). Data represent mean values ±s.e.m. (n =12). **P<0.01. A full color version of this figure is available at the Immunology and Cell Biology journal online.
Figure 2
Figure 2
Macrophage infiltration. (a) Immunohistochemistry for macrophages. Testicular cryosections of WT and AM−/− mice at 50 days after immunization were immunostained with anti-F4/80 antibody (right panels). Testes of mice injected with CFA alone served as the control (left panels). (b) CD68± and CD163± macrophages. Immunohistochemical analyses of testicular sections of immunized AM−/− mice were performed for discriminating CD68± and CD163 ± macrophages using ED1 and ED2 antibodies. Insets in the upper right corner are the negative controls, in which pre-immune animal sera was used as primary antibodies. Scale bar =20 μm. Images represent at least three mice. (c and d) Quantitative analyses of macrophages in the testes of WT (c) and AM−/− (d) mice. Testicular interstitial cells were labeled with PE-conjugated anti-F4/80 antibody (anti-F4/80-PE) and analyzed by flow cytometry. Left panels represent flow cytometry density plots, whereas right panels represent absolute macrophage numbers per testis based on flow cytometry data. Data are mean values ±s.e.m. of five mice. **P<0.01. A full color version of this figure is available at the Immunology and Cell Biology journal online.
Figure 3
Figure 3
Lymphocyte infiltration. (a) Flow cytometry analysis. Testicular interstitial cells were isolated from control and immunized Axl−/−Mer−/− mice and gated for lymphocyte analyses. SSC, side scattering; FSC, forward scattering. (b) CD4+ cells. Testicular interstitial cells were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 antibody (anti-CD4-FITC) and analyzed by flow cytometry. (c) CD8+ cells. Testicular interstitial cells were labeled with PE-Cy5-conjugated anti-CD8 antibody (anti-CD8-PE-Cy5) and analyzed using flow cytometry. (d) B cells. Testicular interstitial cells were labeled with PE-Cy5-conjugated anti-B220 antibody (anti-B220-PE-Cy5) and analyzed using flow cytometry. Absolute numbers of lymphocytes per testis were determined based on flow cytometry data (right panels). Flow cytometry density plots represent five mice. Data are mean values ±s.e.m. of five mice. **P<0.01. A full color version of this figure is available at the Immunology and Cell Biology journal online.
Figure 4
Figure 4
Lymphocytes in renal lymph nodes (RLNs). (a) RLN size. WT and Axl−/−Mer−/− (AM−/−) mice were immunized. After 50 days, RLNs were collected for analysis. Images represent RLNs from five mice. Scale bar =1 mm. (b) Lymphocytes in RLNs of AM−/− mice. RLN cells were isolated from the control and immunized AM−/− mice. Cells were labeled with anti-CD4-FITC, anti-CD8-PE-Cy5 and anti-B220-PE-Cy5. Lymphocyte populations were gated (upper left panel). Ratios of CD4+ cells (upper right panel) and CD8+ and B cells (lower panels) were determined using flow cytometry. (c) Lymphocytes in RLNs of WT mice. RLN cells of control and immunized WT mice were analyzed as in b. Data are mean values ±s.e.m. of five mice. *P<0.05. FITC, fluorescein isothiocyanate; FSC, forward scattering; SSC, side scattering. A full color version of this figure is available at the Immunology and Cell Biology journal online.
Figure 5
Figure 5
Expression of pro-inflammatory cytokines in the testis. (a) Cytokine expression at messenger RNA (mRNA) levels. Total RNAs were extracted from testes of individual control and immunized WT and Axl−/−Mer−/− (AM−/−) mice. Relative mRNA levels of major pro-inflammatory cytokines, including TNF-α, IL-6 and MCP-1, were determined using real-time qRT-PCR. (b) Protein levels of cytokines in the testis. The testes of control and immunized AM−/− mice were lysed by grinding in PBS. Cytokine levels in the lysates were measured using ELISA. (c) Distribution of cytokines in the testis. Immunohistochemistry of testis cryosections from control (upper panels) and immunized (lower panels) AM−/− mice were performed using specific antibodies against TNF-α, IL-6 and MCP-1. Arrows and arrowheads point to Sertoli cells and interstitial cells, respectively. Images are the representatives of at least three independent experiments in three mice. Scale bar =20 μm. Data represent mean values ±s.e.m. of three experiments. **P<0.01. A full color version of this figure is available at the Immunology and Cell Biology journal online.
Figure 6
Figure 6
Autoantibody production and BTB permeability. (a) Autoantibodies in sera. Sera of control and immunized Axl−/−Mer−/− (AM−/−) mice were collected by tail bleeding. Male germ cells were isolated from 10-week-old WT mice. Western blots on germ cell lysates were performed using sera (1:500 dilution) of AM−/− mice as primary antibodies. (b) Distribution of autoantibodies. Testicular cryosections of 10-week-old WT mice were immunostained with sera (1:100 dilution) of control (left panel) and immunized (right panel) AM−/− mice. Insets in the upper right corners are negative controls. Negative controls were stained using sera of WT mice (without immunization) as primary antibodies. (c) Autoantibody deposition. Testicular sections of control (left panel) and immunized (middle panel) AM−/− mice were directly stained with anti-mouse IgG antibodies. Signals in germ cells (arrow) of immunized AM−/− mice indicate deposition of autoantibodies. Arrows indicate elongating spermatids. Testis of WT mice without immunization served as the negative control (upper right corners). Heavy (H) and light (L) chains of IgG in testes of immunized AM−/− mice were confirmed by Western blot using anti-mouse IgG antibodies (right panel). β-Actin was used as the loading control for western blot. (d) BTB permeability. Biotin was injected underneath the testicular capsules of control (lower panels) and immunized (upper panels) AM−/− mice. After 30 min, paraffin sections were stained with fluorescein isothiocyanate-conjugated streptavidin (left panels), after which the sections were counterstained with 4′, 6-diamidino-2-phenylindol (DAPI) (middle panels). Right panels are merged biotin and DAPI images. Arrowheads, arrows and asterisks indicate interstitial spaces, basal and adluminal compartments of the seminiferous tubules, respectively. Images represent at least three experiments of three mice. Scale bar =20 μm. A full color version of this figure is available at the Immunology and Cell Biology journal online.
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