| | IL-13 induces the expression of the alternative activation marker Ym1 in a subset of testicular macrophagesReceived 5 April 2007; received in revised form 20 October 2007; accepted 15 January 2008. published online 10 March 2008. Abstract Macrophages are thought to play an important role in the maintenance of immune privilege in the testis, which functions to prevent immune responses to developing sperm. Two populations of macrophages are known to exist in the testis, one of which exhibits immunosuppressive activity. Macrophages that are alternatively activated with either IL-4 or IL-13 have been shown to be anti-inflammatory and promote wound healing. Expression of the Ym1 protein is an established marker of alternatively activated macrophages. Testicular macrophages were examined for expression of Ym1 protein, and it was found to be highly expressed in a subpopulation of CD11b+ cells. Furthermore, we have shown that Ym1 protein expression in the testis is dependent upon IL-13R signaling, and that IL-13 is produced in the testis. These data suggest that IL-13 plays a role in testicular immune privilege by the maintenance of an alternatively activated macrophage population. 1. Introduction  Immune privilege in the testis is essential to maintain immunological tolerance to male germ cells during their development into spermatozoa (Naz, 2004). Immunity to sperm through the production of antisperm antibodies is thought to contribute to infertility, with 9–36% of infertility in couples being attributed to an immunological mechanism (Naz, 2004). Immune privilege in the testis is maintained by the blood–testis barrier, constitutive expression of anti-inflammatory cytokines and by testicular macrophages (Naz, 2004). In addition, testicular macrophages present in the interstitial tissue are thought to be essential for male reproductive function by regulating testosterone production by Leydig cells (Nes et al., 2000). Using 2D electrophoresis, testicular macrophages were shown to be distinct from peritoneal macrophages in the number and type of secreted proteins (Hutson and Stocco, 1989). In both rats and mice, two distinct populations of testicular macrophages have been described. In rats, the major population expresses CD163, while a subpopulation comprising 15–20% of total macrophages are CD163− but express CD63 (Fijak and Meinhardt, 2006). In mice, using density gradient separation, a subpopulation of macrophages was shown to produce TGF-β and exhibit immunosuppressive activities, while the majority population were immunogenic (Bryniarski et al., 2004). The factors in the testis that maintain their unique phenotypes are not known. Classic activation of macrophages by IFN-γ is known to be important in cellular immunity to intracellular pathogens and are proinflammatory, playing roles in extracellular matrix destruction and apoptosis. These features of classic activation tend to drive chronic inflammation and tissue injury (Gordon, 2003, Mosser, 2003). Alternative activation of macrophages by IL-4 was first described as the upregulation of both the mannose receptor and MHC class II expression, and a reduction in proinflammatory cytokine secretion (Stein et al., 1992). IL-13, a cytokine that shares the IL-4Rα subunit in its receptor (Hilton et al., 1996), was shown also to alternatively activate macrophages (Doherty et al., 1993). Although IL-4 and IL-13 are both produced by Th2 cells and have biological redundancy, they exhibit also unique functions (Gordon, 2003, McKenzie et al., 1999). Alternatively activated macrophages are involved in the resolution of inflammation and wound healing by promoting extracellular matrix construction, cell proliferation and angiogenesis (Gordon, 2003, Mosser, 2003). Recently, additional markers of alternatively activated macrophages have been described, including the lectin-binding protein Ym1 (Chang et al., 2001, Raes et al., 2002). Ym1 is a member of a family of genes that shares sequence homology to chitinases (Jin et al., 1998). In the mouse, four Ym1-like genes have been identified, with Ym1–Ym3 sharing ∼95% sequence identity (Jin et al., 1998). Due to the sequence similarity, Ym1 and Ym2 are difficult to differentiate on the RNA and protein level. However, there are differences in tissue localization, with Ym1 highly expressed in the lung and spleen. Ym2 is highly expressed in the stomach, but minimally expressed in the lung and spleen (Jin et al., 1998, Nio et al., 2004). Ym1 protein was first identified in motheaten mice due to its crystallization in the lung induced by macrophage dysregulation, which resulted in a fatal lung injury (Guo et al., 2000). Although the exact function of Ym1 is not known, it is speculated that when produced at sites of inflammation it binds N-acetylglucosamines acting as a selectin (Chang et al., 2001); however, more recent evidence by this same group refutes this finding (Tsai et al., 2004). Thus, the role of Ym1 in Th2-type immunity is not clear. In this study, we have investigated whether testicular macrophages exhibit the characteristics of alternatively activated macrophages by examining Ym1 expression. Using real-time RT-PCR and a novel intracellular staining technique, we found that a subpopulation of testicular macrophages, comprising 12–20% of all CD11b+ cells, express high levels of Ym1 protein in an IL-13-dependent manner. These data suggest that IL-13 is a critical factor in the maintenance of immune privilege in the testis by regulating testicular macrophage function. 2. Materials and methods 2.1. Mice B10.PL, C57BL/6J, BALB/cJ, C57BL/6-Il4tm1Nnt/J (IL-4−/−) and BALB/c-Il4ratm1Sz/J (IL-4αR−/−) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were housed at the Biomedical Research Center of the Medical College of Wisconsin. All animal protocols were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. 2.2. Macrophage isolation Thioglycolate-induced peritoneal macrophages were obtained as described (Raes et al., 2002) and incubated in DMEM media (Cellgro™, Mediatech, Hernon, VA) with 10% FBS supplemented with IL-4 (200 U/ml; Genzyme, Cambridge, MA) or IFN-γ (150U/ml; eBioscience) combined with LPS (100ng/ml; Sigma–Aldrich, St. Louis, MO) for 24h before staining for Ym1. Tissue-resident mononuclear cells were isolated from lungs, liver and testis as previously described for the brain and spinal cord (Ponomarev et al., 2005). Prior to testicular homogenization, the capsules were removed as described (Bryniarski et al., 2005). Briefly, deeply anesthetized mice were perfused with 25ml of ice-cold PBS and lungs and testis were harvested, homogenized and strained through a 75μm nylon filter. Mononuclear cells were purified using 40%/70% discontinous Percoll gradient (Sigma–Aldrich, St. Louis, MO). 2.3. Flow cytometry Ex vivo isolated mononuclear cells (1×106) were washed in 1ml staining buffer (PBS containing 2% FBS and 0.01% NaN3), Fc receptors were blocked with anti-mouse FcR (2.4G2) for 15min on ice, and the cells were stained with anti-CD11b-PE-Cy5 (eBioscience, San Diego, CA) for 20min on ice and then washed with staining buffer. For the detection of cytoplasmic Ym1, we utilized the Cytofix/Cytoperm™ kit for intracellular cytokine staining from BD Bioscience (San Diego, CA). For permeablization, cells were centrifuged and gently resuspended in 100μl of Cytofix/Cytoperm solution and incubated for 20min on ice. The cells were then washed in 1ml of Perm/Wash solution and blocked with 10% goat serum (Zymed, San Francisco, CA) and 20% FBS in Perm/Wash solution for 30min at room temperature. After blocking, the cells were washed again with 1ml Perm/Wash solution and resuspended in 100μl Perm/Wash solution containing rabbit polyclonal anti-Ym1 Ab, kindly provided by Dr. Shioko Kimura (National Cancer Institute, Bethesda, MD), which recognizes both Ym1 and Ym2 proteins (Ward et al., 2001) or rabbit serum as a control (Sigma–Aldrich, St. Louis, MO). After 1.5h incubation at room temperature, cells were washed with 1ml Perm/Wash solution and then incubated in 100μl Perm/Wash solution containing biotinylated anti-rabbit IgG (Jackson Immuno Research, West Grove, PA) for 20min at room temperature. Cells were washed with 1ml Perm/Wash solution and then incubated in 100μl Perm/Wash solution containing streptavidin–PE (Caltag, Burlingame, CA) for 15min at room temperature. Finally, the cells were washed twice with 1ml Perm/Wash solution, fixed in 1% paraformaldehyde and analyzed on LSR II Flow Cytometer (BD Bioscience) or image files were automatically acquired using the Imagestream 100 imaging cytometer (Amnis Inc., Seattle, WA). Single color controls were used to calculate a spectral crosswalk matrix to compensate images. The resulting compensated images were analyzed using IDEAS statistical analysis software (Amnis Inc.). Brightfield area versus brightfield aspect ratio features were used to eliminate debris and clumps from the analysis. 2.4. Cytokine quantitation by real-time RT-PCR Total RNA was extracted from lungs, liver and testis of PBS-perfused mice using TRIzol (Invitrogen, Carlsbad, CA) after mechanical homogenization. cDNA was synthesized as previously described using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) (Dittel et al., 1997). Ym1, FIZZ1, arginase-1, IL-4 and IL-13 mRNA was quantitated by real-time RT-PCR using SYBR-Green as the detection agent. Amplifications were performed with the iCycler iQ™ (Bio-Rad, Hercules, CA) in 25μl reaction volumes containing cDNA, primers and iQ Supermix (Bio-Rad) according to manufacturer instructions. Thermal cycling proceeded with one amplification cycle of denaturation at 94° C for 5min, followed by 45 cycles of 94°C for 1min, 55°C for 45s and 75°C for 1min. Threshold values of Ym1, IL-4 and IL-13 expression were normalized to GAPDH expression using standard curves generated for each sample by a series of four consecutive 10-fold dilutions (1 to 1×103) of the cDNA template. All reactions were performed in triplicate and the data analyzed using iQ Cycler analyzing software. The following sequence-specific primers were used in real-time PCR—GAPDH, forward: 5′-TTCACCACCATGGAGAAGGC-3′, reverse: 5′-GGCATGGACTGTGGTCATGA-3′; Ym1 (Raes et al., 2002), which amplifies both Ym1 and Ym2, forward: 5′-GGGCATACCTTTATCCTGAG-3′, reverse: 5′-CCACTGAAGTCATCCATGTC-3′; FIZZI (Raes et al., 2002) forward: 5′-TCCCAGTGAATACTGATGAGA-3′, reverse: 5′-CCACTCTGGATCTCCCAAGA-3′; arginase-1 (Raes et al., 2005) forward: 5′-ATGGAAGAGACCTTCAGCTAC-3′, reverse: 5′-GCTGTCTTCCCAAGAGTTGGG-3′; IL-4 (Overbergh et al., 1999) forward: 5′-ACAGGAGAAGGGACGCCAT-3′, reverse: 5′-GAAGCCCTA CAGACGAGCTCA-3′; and IL-13 (Overbergh et al., 1999) forward: 5′-AGACCAGACTCCCCTGTGCA-3′, reverse: 5′-TGGGTCCTGTAGATGGCATTG-3′. Specificity of the RT-PCR reaction was controlled by the generation of melting curves; PCR efficiencies were 100±15% and correlation coefficients were 0.97–0.99. 3. Results 3.1. Ym1 and other markers of alternatively activated macrophages are highly expressed in the testis Since it is known that several distinct populations of macrophages exist in the testis, we determined first whether a population of alternatively activated macrophages was present by examining expression of Ym1 by real-time RT-PCR. Because Ym1 is known to be highly expressed in the lung, we used this tissue as a positive control, and found that the message level for Ym1 in the lung equaled that of the GPDH housekeeping gene in B10.PL mice (Fig. 1A). Because Ym1 is induced by Th2 cytokines, we examined also its expression in the Th1-prone C57BL/6 mouse and the Th2-prone Balb/c mouse (A), and found expression to be identical to that in the B10.PL mouse. When we examined the testis, Ym1 was found to be highly expressed in the three strains of mice, indicating that the testicular microenvironment is supportive of an alternatively activated macrophage phenotype. To further confirm that alternatively activated macrophages are present in the testis, we used real-time RT-PCR to detect the expression of arginase-1 and FIZZ1, both considered markers of this macrophage subset (Edwards et al., 2006, Munder et al., 1999, Raes et al., 2002). Using B10.PL mice, we found that both proteins were highly expressed in the testis at a level similar to Ym1 (B). The primers used in this study amplify both Ym1 and Ym2 but, based on the reported expression of Ym2 essentially exclusively in the stomach, we are likely detecting only Ym1 (Jin et al., 1998, Nio et al., 2004). To confirm this, we examined also our samples using PCR primers specific for Ym1 or Ym2, as described (Welch et al., 2002), and only detected message for Ym1 (data not shown). 3.2. A subpopulation of testicular macrophages expresses Ym1 protein We developed next an intracellular flow cytometry staining technique to detect Ym1 protein in macrophages. To validate our staining technique, we used peritoneal macrophages as a negative control (Fig. 2A) and lung macrophages as a positive control (B). When CD11b+ cells were examined, we confirmed that thioglycolate-induced peritoneal macrophages were negative for Ym1 protein (A), while 57% of the lung macrophages were positive (B). We also examined macrophages from the liver to assess the relative expression level of tissue macrophages and found that they expressed Ym1 at a similar level as lung macrophages (C). In the testis, a subpopulation of macrophages, which constituted 20% of the CD11b+ cells, highly expressed Ym1 protein (C), with over a 30-fold increase in the mean fluorescence intensity compared to the lung and liver. We confirmed also that the testicular CD11b+ cells expressed the macrophage marker F4/80 (data not shown). To visualize the morphology of the alternative activated macrophage populations in the lung, liver and testis, we utilized an Imagestream 100 imaging cytometer (Amnis Inc., Seattle, WA) to obtain images of CD11b+ macrophages that were Ym1-positive and -negative (Fig. 3). In the lung and liver, the morphology and size of both macrophage populations was similar (Fig. 3). However, in the testis, the Ym1+ macrophage population appeared distinct from the Ym1− population, being both larger and exhibiting a ‘macrophage-like’ morphology. These findings support further the presence of an alternatively activated population of macrophages in the testis that is morphologically distinct from other CD11b+ macrophages present. 3.3. Ym1 expression in the testis requires expression of the IL-13R Since both IL-4 and IL-13 have been shown to induce expression of Ym1, we next examined the requirement for these cytokines in the lung and testis. We examined first whether IL-4 played a role in Ym1 expression using IL-4-deficient mice. We confirmed that Ym1 was expressed in the lung and testis of wild-type (WT) C57BL/6 mice (Fig. 4A). As with B10.PL mice (Fig. 2), 46% of lung and 12% of testicular CD11b-gated macrophages expressed Ym1 protein (Fig. 4A). In the IL-4-deficient mice, the percentage of lung macrophages decreased to 29%, while the testicular macrophages population was unaltered (Fig. 4A). The average of three experiments using IL-4−/− mice is shown in Fig. 4C, with no significant difference between the WT and IL-4−/− mice in either tissue. These data suggest that IL-4 does not regulate the expression of Ym1 in the testis. Thus, to confirm a role for IL-13, we conducted the identical experiment utilizing mice deficient in the IL-4αR subunit, common to both the IL-4 and IL-13 receptors. The WT Balb/c mice expressed similar levels of Ym1 protein in the lungs and testis as C57BL/6 mice (Fig. 4B). In the absence of both the IL-4 and IL-13 receptor, Ym1 protein expression was substantially reduced in the lungs, and completely absent in the testis. The average of three similar experiments is shown in Fig. 4D. The reduction in Ym1 protein expression in both the lungs and testis in the IL-4/IL-13R−/− mice was statistically significantly reduced (Fig. 4D). These data indicate that IL-13 is the predominant cytokine required for Ym1 expression in the lung and testis. We examined next whether IL-13 is produced in the testis using real-time RT-PCR. We detected similar levels of IL-13 message in both C57BL/6 and Balb/c mice (Fig. 5). We also detected IL-4 in the testis, but it was expressed near the sensitivity level for real-time RT-PCR and thus was not detectable in all mice examined (data not shown). These data indicate that the testis has the capacity to regulate and maintain a subpopulation of alternative activated macrophages via IL-13 and its receptor. 4. Discussion In this study, we have found that a subpopulation of testicular macrophages exhibited the phenotype of alternatively activated macrophages expressing high levels of Ym1, arginase-1 and FIZZ1 message, and Ym1 protein. Although both IL-4 and IL-13 message was detectable in the testis, Ym1 expression was dependent only upon IL-13R signaling. These data support a role for IL-13 in the maintenance of the testicular immunosuppressive environment. In the testis, it is known that at least two heterogeneous populations of macrophages exist. In rats, the two populations are distinguished by cell surface antigen expression and have been shown to produce different immune mediators (Fijak and Meinhardt, 2006). In mice, density gradients were used to identify an immunogenic population able to function as efficient antigen-presenting cells and a second less prevalent immunosuppressive population able to inhibit T cell proliferation in a TGF-β-dependent manner (Bryniarski et al., 2004). In this study, we have identified Ym1 as an additional marker to distinguish two mouse testicular macrophage populations. Ym1 expression indicates that the minor testicular macrophage population exhibits properties of alternatively activated macrophages. Our data showing that IL-13 is produced in the testis (Fig. 4), and that IL-13R signaling is required for Ym1 protein expression (Fig. 3), are consistent with this finding. The presence of IL-4αR, which is shared with the IL-13 receptor, has been previously reported in the testis (Debinski and Gibo, 2000). The cellular source of Th2 cytokines in the testis is unknown, and is not likely Th2 cells, since they are absent from the testis. The likely source is from mast cells present in the interstitial space and known to produce IL-13 (Fijak and Meinhardt, 2006, Galli et al., 2005). Interestingly, IL-13 has been shown to induce production of TGF-β in lung macrophages in a model of pulmonary fibrosis (Lee et al., 2001), and alternatively activated macrophages produce TGF-β (Kodelja et al., 1997, Song et al., 2000). Thus, IL-13 is likely an important cytokine in the maintenance of the testicular immunosuppressed microenvironment. The constitutive expression of Ym1 in both the lungs and testis suggests that it plays an important role in maintaining an immunosuppressive and anti-inflammatory environment in both organs. The lung is constantly exposed to potentially proinflammatory stimuli from the environment, a delicate balance that is easily perturbed in chronic diseases such as severe asthma (Moore and Peters, 2006). Many studies have shown independent roles for IL-4 and IL-13 in lung pathology (McKenzie et al., 1999, Mentink-Kane and Wynn, 2004). Of interest to our study is the finding that IL-13 is the cytokine that primarily contributes to Ym1 expression in the lungs (Fig. 3). In the testis, an immunosuppressive or anti-inflammatory environment is essential to maintain immune tolerance to developing sperm. The breaking of tolerance has the potential to allow antisperm antibody development in males leading to immunoinfertility. Antisperm antibodies are present in 8–21% of infertile males (Naz, 2004), making the mechanisms of immunoinfertility of scientific interest. Since IL-4αR−/− mice are fertile, macrophages exhibiting an alternatively activated phenotype are not essential for sperm development. Thus, we speculate that Ym1+ macrophages play an important role in maintaining the anti-inflammatory environment in the testis to prevent inflammation that would be detrimental to sperm development. This hypothesis is testable since IL-13R signaling is required for expression of Ym1 (Fig. 3). Although Ym1 has been shown to be a member of the chitinase family with lectin-binding properties, the exact function of the protein still remains enigmatic. A recent study using the cholesterol-lowering drug simvastatin showed that treated dendritic cells were able to promote the development of Th2-type responses by CD4 T cells in vitro (Arora et al., 2006). This included the induction of IL-4 and IL-13 production. Interestingly, addition of anti-Ym1 antibody to the cultures abolished the Th2-promoting effect of the simvastatin (Arora et al., 2006); it was concluded that Ym1 production by dendritic cells was required for the Th2 polarization. Whether this mechanisms exists in vivo remains to be shown, but the high level of Ym1 protein in the lung (Fig. 2) is consistent with that organ's Th2-type immune responses. In summary, we have shown that a subpopulation of testicular macrophages exhibits an alternatively activated phenotype as shown by their IL-13-dependent expression of Ym1 protein. We show also that IL-13 is produced within the testis. Thus, these studies suggest that IL-13 plays an important role in modulating the unique testicular macrophage phenotype that is important for the normal function of the testis, including testosterone production and spermatogenesis. Acknowledgments We thank Dr. Shioko Kimura for the rabbit polyclonal anti-Ym1/2 antibody, and Shelley Morris-Islo for assistance with the mice. References Arora et al., 2006. 1.Arora M, Chen L, Paglia M, Gallagher I, Allen JE, Vyas YM, et al. Simvastatin promotes Th2-type responses through the induction of the chitinase family member Ym1 in dendritic cells. Proc. Natl. Acad. Sci. U.S.A. 2006;103:7777–7782. MEDLINE |
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a BloodCenter of Wisconsin, Blood Research Institute, 8727 Watertown Plank Road, Milwaukee, WI 53226, USA b Flow and Imaging Cytometry Facility, Immune Disease Institute, Harvard Medical School, Boston, MA 02115, USA c Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, USA  Corresponding author at: BloodCenter of Wisconsin, Blood Research Institute, 8727 Watertown Plank Road, P.O. Box 2178, Milwaukee, WI 53226, USA. Tel.: +1 414 937 3865; fax: +1 414 937 6284.
PII: S0165-0378(08)00004-1 doi:10.1016/j.jri.2008.01.001 © 2008 Elsevier Ireland Ltd. All rights reserved. | |
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