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BOR - Papers in Press, published online ahead of print March 5, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013318
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BIOLOGY OF REPRODUCTION 69, 202–210 (2003)
DOI: 10.1095/biolreprod.102.013318
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Ischemia-Reperfusion of the Murine Testis Stimulates the Expression of Proinflammatory Cytokines and Activation of c-jun N-Terminal Kinase in a Pathway to E-Selectin Expression1

Jeffrey J. Lysiak2,3, Quoc An T. Nguyen3, Jennifer L. Kirby4, and Terry T. Turner3,4

Departments of Urology3 Cell Biology,4 University of Virginia Health Science System, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ischemia-reperfusion (IR) of the testis results in germ cell-specific apoptosis and can lead to aspermatogenesis. Germ cell-specific apoptosis after IR of the testis has been shown to be correlated with and dependent on neutrophil recruitment to the testis after IR. Studies that used E-selectin-deficient mice have demonstrated that E-selectin expression is critical for neutrophil recruitment to subtunical venules in the testis after IR and for the resultant germ cell-specific apoptosis. The present study investigates the in vivo signaling pathway that exists after IR that leads to neutrophil recruitment in the murine testis. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. Results demonstrate that the proinflammatory cytokines, tumor necrosis factor {alpha} (TNF{alpha}) and interleukin 1ß (IL-1ß), are stimulated after IR as is the phosphorylation of c-jun N-terminal kinase (JNK). The downstream transcription factors of JNK, ATF-2 and c-jun are also phosphorylated at specific times after IR of the testis. Activation of the JNK stress-related kinase pathway is correlated with an increase in E-selectin expression and neutrophil recruitment to the testis after IR. Intratesticular injection of IL-1ß also caused JNK phosphorylation and neutrophil recruitment to the testis. These results suggest that testicular IR injury stimulates IL-1ß expression, which leads to activation of the JNK signaling pathway and ultimately E-selectin expression and neutrophil recruitment to the testis. This provides the first evidence of a cytokine/stress-related kinase signaling pathway to E-selectin expression in vivo.

cytokines, kinases, signal transduction, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue ischemia-reperfusion (IR) can result in a complex pathologic insult to the affected organ. IR injury has been described in numerous organs, such as the brain [1], kidney [2], heart [3], intestine [4], liver [5], and testis [6]. Common to many types of IR injury and perhaps mediators of the pathology are recruitment and activation of neutrophils [7], proinflammatory cytokine generation [8], mitochondrial dysfunction [9], generation of reactive oxygen species (ROS) [10], and alterations in intracellular calcium homeostasis [11]. These events often culminate in apoptosis of cells in the affected organ, and this can ultimately lead to organ dysfunction [6, 12–14]. The generation of proinflammatory cytokines after IR injury and the intracellular signaling pathways that lead to the recruitment of neutrophils to the affected organ have not been fully delineated.

Testicular torsion or more specifically torsion of the spermatic cord results in an obstruction of blood flow to the testis that renders the tissue ischemic. In humans, testicular torsion is a medical emergency that usually requires surgical intervention to reperfuse the affected testis; however, even after reperfusion, testicular atrophy is a common outcome [15]. In a rat model of IR of the testis, permanent loss of spermatogenesis is observed despite the return of blood flow [16, 17] and maintenance of functional Leydig [18] and Sertoli [19] cells. This loss of spermatogenesis has been shown to be due to germ cell-specific apoptosis [6, 20]. Recent studies have confirmed this observation in a mouse model as well [21].

Coinciding with IR-induced germ cell-specific apoptosis is an increase in neutrophils [21] and ROS in the testis [22]. Recent studies have shown that the intratesticular neutrophils are essential for the observed germ cell-specific apoptosis after IR of the testis [21]. Further current data suggest that ROS produced by the recruited neutrophils perturb Bcl-2 family members in the germ cells and thus initiate apoptosis via the mitochondria pathway [20].

E-selectin is an endothelial cell adhesion molecule responsible for the tethering and slow rolling of neutrophils to endothelial cells [23, 24]. Previous studies have shown E-selectin to be expressed on endothelial cells following IR [25–27] or after treatment with tumor necrosis factor {alpha} (TNF{alpha}) [28] or interleukin 1ß (IL-1ß) [29]. Importantly, mice deficient in E-selectin exhibit a decrease in neutrophil recruitment to testicular subtunical venules after IR of the testis and a corresponding decrease in germ cell-specific apoptosis [21]. These results indicate that E-selectin is the key cell adhesion molecule for neutrophil recruitment to the endothelium of subtunical venules in the murine testis after IR and that neutrophils are important mediators of the germ cell-specific apoptosis observed [21].

Proinflammatory cytokines, in particular TNF{alpha} and IL-1ß, are increased after IR in some model systems [8, 30], and stress-related kinases c-jun N-terminal kinase (JNK) [31, 32] and p38 [14, 33] are activated. These events can lead to an increase in neutrophils [26, 34] or an increase in apoptosis [14, 31] in the affected organ, respectively. Further, TNF{alpha} stimulates E-selectin expression in human umbilical vein endothelial cells, and the intracellular signaling pathway responsible for the increase in E-selectin expression is via the JNK/p38 stress-related kinase pathway [35]. Alternatively, the NF{kappa}B pathway may be activated by proinflammatory cytokines and lead to the same result [26, 35].

To further understand the pathway(s) that leads to neutrophil recruitment in the testis after IR, we examined the role of proinflammatory cytokines and the stress-related kinase pathway after IR of the murine testis. The results demonstrate that neutrophil recruitment after testicular IR is preceded by a cytokine-dependent stimulation of E-selectin expression through the JNK stress-related kinase pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies

The protease inhibitors, leupeptin, PMSF, E-64, aprotinin, the phosphatase inhibitor sodium orthovanadate, and protein G sepharose were all obtained from Sigma (St. Louis, MO). Recombinant murine IL-1ß and recombinant murine TNF{alpha} were both purchased from R&D Systems (Minneapolis, MN). Antibodies (Abs) recognizing total JNK, total ATF-2, total c-jun, phospho-ATF-2, phospho-c-jun, and I{kappa}B-{alpha} were obtained from Cell Signaling Technology (Beverly, MA). The Ab recognizing phospho-JNK was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). This Ab is a monoclonal Ab raised against a peptide corresponding to a short amino acid sequence containing the phosphorylated Thr-183 and Tyr-185 residues of JNK1 of human origin also corresponding identically to JNK2 sequence. The Ab against E-selectin was also purchased from Santa Cruz Biotechnology Inc.

Experimental Testicular Torsion

This work was conducted in accordance with the Guiding Principals of the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. Adult male C57BL/6 mice were anesthetized with an intraperitoneal injection of 0.01 mg/g of sodium pentobarbital, and the testis was rotated as described by Lysiak et al. [21]. Briefly, the testis was exteriorized through a low midline laparotomy, the gubernaculum was divided, and the testis was freed from the epididymo-testicular membrane. The testis was rotated 720° for 2 h, during which time it remained in the abdomen with a closed incision. After 2 h the incision was reopened, the testis was counterrotated to the natural position, the gubernaculum was rejoined, and the testis was reinserted into the scrotum via the inguinal canal. Testes were examined at the time of repair for the apparent degree of ischemia and reperfusion. Sham-operated animals were treated identically except that on completion of the torsion maneuver the testis was immediately counterrotated.

Relative-Quantitative Reverse Transcription-Polymerase Chain Reaction

To examine the mRNA expression of the proinflammatory cytokines IL-1ß and TNF{alpha} and the endothelial cell adhesion molecule E-selectin, reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA isolated from the testis at either 0.5 or 4 h after IR of the testis. Briefly, 4 µg of RNA was reverse transcribed using Superscript Preamplification System (Life Technologies, Rockville, MD) according to the manufacturer's instructions. The RT product was diluted 1:10, and multiplex PCR was performed with primers specific for IL-1ß and 18S rRNA, TNF{alpha} and 18S rRNA, and E-selectin and 18S rRNA. The primers for the 18S rRNA were purchased from Ambion (Austin, TX). Initial pilot experiments were performed to determine the ratio of competing primers to primers for the 18S rRNA so the reaction product was in the same linear range as the target products. The primers for IL-1ß, TNF{alpha}, and E-selectin were designed with the aid of Gene Runner (Hastings Software, Inc., Hastings, NY) and purchased from GIBCO BRL Custom Primers (Life Technologies). The IL-1ß forward primer was 5'-TTCCAGGATGAGGACATGAGCACC-3', and the IL-1ß reverse primer was 5'-CAGGAAGACAGGCTTGTGCTCTGC-3'. The TNF{alpha} forward primer was 5'-AACCTCCTCTCTGCCGTCAAGAGC-3', and the TNF{alpha} reverse primer was 5'-CTGTGAGGAAGGCTGTGCATTGC-3'. The E-selectin forward primer was 5'-GAAGCCAGTGCATACTGTCAGCG-3', and the E-selectin reverse primer was 5'-TCCACTCTCCAGAGGACGTACACC-3'. PCR was performed with a denaturation temperature of 94°C, annealing temperatures of 58°C for the IL-1ß primers, 59°C for the TNF{alpha} primers, and 58°C for the E-selectin primers, and an elongation temperature of 68°C for 30 cycles. The RT-PCR reactions were performed in a Biometra thermal cycler (Whatman Biometra, Gottingen, Germany). The RT-PCR products were cloned into pCR-Blunt-II Topo (Invitrogen), and sequence identity was confirmed by sequence analysis at the Protein and Nucleic Acid Research Core at the University of Virginia. Subsequent agarose gels were imaged using a ChemiImager 4400 (Alpha Innotech Corporation, San Leandro, CA), reaction products were analyzed using ImageQuant software (Molecular Dynamics Inc., Sunnyvale, CA), and the band densities of the reaction products for the specific target mRNAs, IL-1ß, TNF{alpha}, and E-selectin were normalized to the band density of the internal 18S RNA.

Western Blot Analysis

Western blot analyses of the stress-related kinase JNK, the transcription factors ATF-2 and c-jun, and their phosphorylated forms were performed by isolating testicular proteins at 0.0, 0.5, 2.0, and 4.0 h after reperfusion of the testis or sham operations. Briefly, the testes were removed, snap frozen in liquid nitrogen, and stored at -80°C until use. Whole testes were ground with a mortar and pestle chilled in liquid nitrogen, and the resultant powder resuspended in RIPA buffer (0.1% SDS, 1 mM EDTA, 100 mM Tris, 0.15 NaCl, 1.0% deoxycholate, 1.0% Triton X-100, pH 7.4) with the addition of protease inhibitors (100 µM leupeptin, 1 mM PMSF, 10 µM E-64, 20 µg aprotinin) and phosphatase inhibitors (1 mM sodium orthovanadate). The protein suspension was vortexed, incubated on ice for 15 min, and centrifuged at 14 000 x g for 15 min at 4°C and the supernatant was collected. The protein concentration in the supernatant was determined using the bicinchoninic assay kit (Pierce, Rockford, IL), and 50 µg of protein per lane was subjected to SDS-PAGE. The gel contents were electrotransferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), blocked with 5% nonfat dried milk and 0.1% Tween 20 in PBS, incubated with an Ab that recognized the phospho-specific form of the protein or an Ab that recognized the total protein, washed, and incubated for 1 h at room temperature with the appropriate peroxidase-conjugated secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA). Immunocomplexes were detected with enhanced chemiluminescence (SuperSignal West Pico; Pierce). Densitometry and ImageQuant analysis were subsequently performed.

Immunoprecipitation and Immunoblotting

Immunoprecipitation and immunoblotting for E-selectin were performed on testicular proteins isolated 24 h after IR of the testis. Briefly, testicular proteins were isolated as described herein and the protein concentration determined. One hundred micrograms of protein was incubated with 1 µg of polyclonal E-selectin Ab in RIPA buffer overnight at 4°C with constant agitation. The next day, 25 µl of protein G-sepharose was added and the tubes incubated for 1 h at room temperature with constant agitation. The immune complexes were collected by centrifugation, washed, resuspended in gel loading buffer, boiled, and subjected to SDS-PAGE. The gel contents were electrotransferred to nitrocellulose membranes (Bio-Rad) blocked with 5% nonfat dried milk and 0.1% Tween 20 in PBS and incubated overnight with the polyclonal anti-E-selectin Ab at 4°C. The next day membranes were washed and incubated with peroxidase-conjugated goat anti-rabbit Ab (Jackson ImmunoResearch Laboratories), and immunocomplexes were detected with enhanced chemiluminescence (Pierce).

Immunohistochemical Analysis of Tissue Sections

To obtain testes to be sectioned for immunohistochemical analysis for phospho-JNK and I{kappa}B-{alpha}, mice were killed at 2 h after reperfusion. Animals were subjected to perfusion fixation via intracardiac infusion of 4% paraformaldehyde for 20 min at which time the testes were removed, immersed in 4% paraformaldehyde for 6 h, paraffin embedded, and sectioned.

Tissue sections were deparaffinized, rehydrated, and washed in PBS. Slides for phospho-JNK immunolocalization were placed in unmasking solution (Vector Laboratories Inc., Burlingame, CA), microwaved for 20 min, and allowed to cool for 1 h at room temperature. Subsequently, endogenous peroxidase activity was inhibited in all sections by incubating the sections in 3% H2O2 methanol solution for 15 min. Slides were then washed, blocked, and incubated with the phospho-JNK or I{kappa}B-{alpha} Ab overnight at 4°C. The next day slides were washed and incubated with the appropriate biotinylated secondary Ab (Vector Laboratories Inc.) for 1 h at room temperature. Immunocomplexes were visualized with avidin-biotin-peroxidase complex (Vector Laboratories Inc.) with diaminobenzidene (Sigma) as the chromogen. All slides were lightly counterstained with hematoxylin.

Immunostaining for E-selectin was performed on cryosections of testes. Briefly, animals were killed 24 h after reperfusion of the testes, and the testes were immediately frozen in liquid nitrogen. Cryosections were fixed for 20 min in acetone and washed, and endogenous peroxidase activity was inhibited. The sections were then blocked and incubated with the polyclonal E-selectin Ab overnight at 4°C. The next day slides were washed and incubated with the biotinylated secondary Ab, and complexes were visualized with the avidin-biotin-peroxidase complex with diaminobenzidene as the chromogen.

Histological Sections

To examine for the recruitment of neutrophils to subtunical venules of the testis, animals were killed 24 h after reperfusion of the testis or after intratesticular injections of IL-1ß and TNF{alpha}; the testis was then removed and fixed in Bouin fixative. Histological sections of the testis were stained with hematoxylin-eosin and examined. Images of subtunical venules were captured with a Nikon Coolpix camera attached to a Nikon microscope (Image Systems, Columbia, MD).

Intratesticular Injections of IL-1ß and TNF{alpha}

To determine if the increase in the proinflammatory cytokines detected by RT-PCR after reperfusion of the testis was directly responsible for the phosphorylation of JNK and the recruitment of neutrophils to the testis, subtunical intratesticular injections of the cytokines was performed. Briefly, animals were anesthetized with an intraperitoneal injection of 0.01 mg/g of sodium pentobarbital and a midline incision performed. The testis was delivered into the abdomen and injected with 100 ng of IL-1ß, 400 ng of TNF{alpha}, or a combination of both in a 20-µl volume via a glass micropipette sharpened to a 50-µm tip. Control injections were made with vehicle (saline) alone. The testis was then delivered into the scrotum, the incision sutured, the animal killed 0.5 h later, and the capsule of the testis collected for Western blot analysis for phospho-JNK and total JNK. Other animals were killed at 24 h for histological assessment of neutrophil recruitment.

Statistical Analysis

All statistical evaluations were performed either by ANOVA followed by the Tukey range test or the Student t-test (P < 0.05) after evaluation of each data set by the Chauvenet criterion for homogeneity. In one case (TNF{alpha} plus IL-1ß injections relative to sham controls), the data were subjected to log transformation because the percentage of increase in JNK phosphorylation was over such a broad range.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Evaluation of IL-1ß and TNF{alpha} Expression after IR of the Testis

Results from relative-quantitative RT-PCR on total RNA isolated from the mouse testis 0.5 h after IR or a sham operation of the testis revealed a significant increase in the mRNA for TNF{alpha} (Fig. 1A). There was also a trend toward an increased expression of IL-1ß mRNA (Fig. 1B).



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  		FIG. 1. Relative-quantitative RT-PCR displaying the increase in TNF{alpha} mRNA (A) and IL-1ß mRNA (B) at 0.5 h after IR of the testis. Bars represent mean ± SEM; n = 6 testes for each group; * denotes statistically significant (P < 0.05)

Activation of JNK after IR of the Testis

Since it is known that proinflammatory cytokines can activate stress-related kinase, we examined for the phosphorylation (activation) of JNK at specific time points after IR of the testis. Mice were killed at 0.0, 0.5, 2.0, and 4.0 h after reperfusion or sham operation of the testis and examined for the phosphorylation of JNK. Phosphorylation of both the 54-kDa (JNK54) and 46-kDa (JNK46) JNK isoforms increased significantly by 2.0 h after reperfusion of the testis (Fig. 2A and B), whereas total immunoreactive JNK remained relatively constant (Fig. 2A). The largest increase seen was with the JNK54 isoform, which increased 71 times at 2 h relative to its sham control (Fig. 2C), whereas the combination of JNK54 and JNK46 increased only 4.5 times the sham control at the same time (Fig. 2B). A significant increase in the phosphorylation of JNK54 occurred at 0.5, 2.0, and 4.0 h after reperfusion of the testis, but it was declining by 4 h (Fig. 2C). These data indicate that after IR of the testis there is a phosphorylation of both JNK54 and JNK46, but the increase in phosphorylation of the 54-kDa isoform is more marked. For this reason, all further analyses of JNK phosphorylation focused on JNK54.



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  		FIG. 2. A) Representative Western blots of phospho-JNK (p-JNK) and total JNK at various times after IR of the testis (S = sham and T = torsion). B) Relative increase in phosphorylation of JNK isoforms after IR. C) Relative increase in phosphorylation of the 54-kDa isoform after IR. Histogram bars represent mean ± SEM; n = 5 testes for each group; bars sharing the same letter are not statistically different from one another (P < 0.05)

Immunohistochemical analysis was performed on testis sections after IR to determine in which cell type JNK was phosphorylated. Immunoreactive phospho-JNK was localized to the endothelial cells of venules and arterioles in the testis (Fig. 3A and B). Other cells of the testis, including all germ cells, Sertoli cells, and Leydig cells, were negative for phospho- JNK immunoreactivity. The NF{kappa}B complex was localized by immunohistochemical detection of the NF{kappa}B inhibitory protein I{kappa}B-{alpha}. Immunoreactive I{kappa}B-{alpha} was localized to Sertoli cells and some spermatogonia but was absent from testicular endothelial cells (Fig. 3C and D).



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  		FIG. 3. Immunolocalization of phospho-JNK and I{kappa}B-{alpha} in the mouse testis 2 h after IR. A) Negative control for phospho-JNK, omission of primary Ab. B) Phospho-JNK immunoreactivity localized to the endothelium of venules (arrows) and arterioles (arrowheads). C) Negative control for I{kappa}B-{alpha} omission of primary Ab. D) I{kappa}B-{alpha} is localized to Sertoli cells (arrows) and potentially some spermatogonia (arrowheads). Endothelial cells of the testis are negative for I{kappa}B-{alpha}. Original magnification x219

Activation of ATF-2 and c-jun after IR of the Testis

Western blot analysis for the transcription factors ATF-2 and c-jun was performed to determine if transcription factors downstream of JNK are phosphorylated. A significant increase in the phosphorylation of ATF-2 was noted at 0.5 and 2.0 h after reperfusion of the testis compared with sham controls (Fig. 4A). Total ATF-2 protein remained unchanged. An increase in the phosphorylation of c-jun was also observed after IR of the testis (Fig. 4B). The increase in c-jun phosphorylation was seen at 2.0 and 4.0 h after IR of the testis compared with sham controls. An increase in total c-jun protein was also observed at 2.0 and 4.0 h after IR.



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  		FIG. 4. Representative Western blots of phospho-ATF-2 (p-ATF-2) and total ATF-2 (A) and phospho-c-jun (p-c-jun) and total c-jun (B) at various times after IR of the testis (S = sham; T = torsion). Histograms bars represent mean ± SEM (n = 5 testes for each group) relative increases in phosphorylation. Bars sharing the same letter are not significantly different (P < 0.05)

E-selectin Expression after IR of the Testis

Previous studies have shown that E-selectin is essential for neutrophil recruitment to subtunical venules of the testis after IR [21]. Relative-quantitative RT-PCR demonstrated a significant increase in E-selectin mRNA expression 4 h after IR of the murine testis (Fig. 5). Immunohistochemical staining of murine testes for E-selectin revealed immunoreactivity in endothelial cells from a sham-operated mouse (Fig. 6B), suggesting that there is a low level of E-selectin normally occurring in the murine testis. To confirm this immunoprecipitation, immunoblotting experiments for E-selectin from sham-operated animals and from animals 24 h after IR were performed. Results revealed a weak specific band for E-selectin from sham-operated animals, which was increased approximately 4-fold in four of six testes after IR but failed to respond in two of the six (Fig. 6C). This led to a difference between the two groups that approached statistical significance (P = 0.07).



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  		FIG. 5. Relative quantitative RT-PCR displaying an increase in E-selectin mRNA 4.0 h after IR of the testis. Bars represent mean ± SEM; n = 8 testes for each group; * denotes statistically significant (P < 0.05)



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  		FIG. 6. Immunohistochemical analysis for E-selectin in frozen sections revealed no specific immunoreactivity in the no primary negative control (A) and specific immunostaining of endothelial cells in a sham-operated mouse (B). Seminiferous tubule (ST). Immunoprecipitation/immunoblot for E-selectin 24 h after IR of the testis (C). An E-selectin-specific band is seen at approximately 100 kDa and is increased after torsion (T) compared with sham (S). Histogram displays the results of E-selectin immunoreactivity. Original magnification x150

Recruitment of Neutrophils to the Testis after IR

One of the hallmarks of IR injury is the recruitment of neutrophils to the affected organ [7, 25–27]. Twenty-four hours after repair of torsion, neutrophil accumulation in subtunical venules had increased (Fig. 7). This is consistent with previous findings [6, 21] but was done to provide contemporaneous determination that the stimulation of E-selectin expression was leading to neutrophil recruitment and to provide a proper comparison for the cytokine injection studies.



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  		FIG. 7. Representative sections of the testis 24 h after either a sham operation (A) or IR (B). Note the accumulation of neutrophils in the subtunical venule after IR (arrows). Original magnification x219

Intratesticular Injections of IL-1ß and TNF{alpha}

After IR of the testis, there was an increase in the expression of TNF{alpha} mRNA and a trend toward increased expression of IL-1ß mRNA (Fig. 1). To determine if either TNF{alpha} or IL-1ß is responsible for the phosphorylation of JNK and the subsequent recruitment of neutrophils to the testis, murine testes were injected with TNF{alpha}, IL-1ß, or a combination of both. Injection of TNF{alpha} alone had no significant effect on the phosphorylation of JNK54, whereas injection of IL-1ß alone did significantly increase the phosphorylation of JNK54 (Fig. 8). Injection of both TNF{alpha} and IL-1ß also consistently caused greater JNK54 phosphorylation than did sham injections, although the degree of the increase relative to the sham value was highly variable (Fig. 8).



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  		FIG. 8. Representative Western blots of phospho-JNK (p-JNK) and total JNK after either sham injections or injections with TNF{alpha}, IL-1ß, or both TNF{alpha} and IL-1ß. Histogram displays the relative increase in phosphorylation of the 54-kDa JNK isoform (p-JNK54) over total JNK54. Bars represent mean ± SEM; n = 5 testes for each group; * denotes statistically significant (P < 0.05)

Sham injection of the testis caused little stimulation of neutrophil recruitment to the testis (Fig. 9A), and TNF{alpha} injections gave only slightly more (Fig. 9B). Injection with IL-1ß alone caused a large infiltration of neutrophils into the testis (Fig. 9C). Injection of TNF{alpha} and IL-1ß also caused a large infiltration of neutrophils, but in these testes there was accompanying disruption of the seminiferous epithelium (Fig. 9D).



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  		FIG. 9. Representative sections of testes 24 h after sham (A), TNF{alpha} (B), IL-1ß (C), or TNF{alpha} plus IL-1ß (D) injection. Arrows indicate a large infiltration of neutrophils in the testis after IL-1ß injection and after the injection of IL-1ß and TNF{alpha} (seminiferous tubule: ST). Original magnification x150


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
IR of either the mouse [21] or rat [6] testis results in germ cell-specific apoptosis contemporaneous with an increase in neutrophil margination and diapedesis. The recruitment of neutrophils to the testis after IR has been shown to be essential for the observed pathology, and E-selectin has been reported to play a critical role in the recruitment of neutrophils to the testis after IR [21]. The present study was designed to investigate the possible involvement of proinflammatory cytokines and stress-related signaling pathways that may lead to expression of E-selectin following IR of the murine testis. Results demonstrate that the proinflammatory cytokine, IL-1ß, is involved in the recruitment of neutrophils to the testis after IR, and it is correlated with an activation of the stress-related kinase JNK and the transcription factors downstream of JNK, ATF-2 and c-jun. To our knowledge, this is the first fully in vivo description of a proinflammatory stress-related kinase signaling pathway that leads to E-selectin expression and neutrophil recruitment.

Up-regulation of the proinflammatory cytokines IL-1ß and TNF{alpha} has been associated with IR in numerous model systems [8, 30]. These proinflammatory cytokines are also known to recruit neutrophils at sites of inflammation by stimulating the expression of endothelial cell adhesion molecules [28, 29]. Karmann et al. [36] reported that TNF{alpha} and IL-1ß are capable of stimulating E-selectin expression in human umbilical vein endothelials through stimulation of a JNK-ATF-2/c-jun pathway. After IR of the murine testis, an increase in expression of TNF{alpha} mRNA and a trend upward for IL-1ß mRNA are observed (Fig. 1). This is mRNA expression not protein; however, as predicted from these results, injection of IL-1ß caused a stimulation of JNK phosphorylation (Fig. 8) and neutrophil recruitment (Fig. 9). This supports our contention that the cytokine is an upstream regulator of the stress-related kinase pathway. The exact cell types(s) that express either TNF{alpha} or IL-1ß or both after IR of the testis are currently not known. Cell populations in the testis previously shown to secrete IL-1ß or TNF{alpha} under different in vitro and in vivo conditions include resident macrophages [37, 38] and germ cells [39].

Intracellular signaling pathways that may mediate stress from various stimuli have yet to be studied in detail in the testis. The stress-related kinase, JNK, plays a critical role in the cellular response to many types of cellular stress, including UV irradiation [40], inflammatory cytokines [41], and IR [42]. In the present study phosphorylated JNK was detected with a monoclonal Ab that specifically reacts with the 46- and 54-kDa isoforms of phosphorylated JNK1, 2, and 3 of the mouse; thus, the specific JNK gene(s) expressed in the testis after IR was not identified.

Western blot analysis of testicular proteins for JNK revealed that both JNK54 and JNK46 are present in the murine testis; however, JNK54 predominated (Fig. 2). JNK54 phosphorylation was not evident in sham control testes but peaked at 2 h after IR and decreased by 4 h (Fig. 2). A low level of phosphorylation of JNK46 was detected in the sham controls, suggesting that JNK46 may be constitutively phosphorylated. An increase in JNK46 phosphorylation compared with sham controls was observed after testicular IR, but because of constitutive phosphorylation the relative increase detected after IR was not as great as with JNK54. Phosphorylation of JNK54 was only seen after IR, and its expression was significantly elevated within 0.5 h after IR (Fig. 2C). The reason the two isoforms differ in their respective phosphorylation states is unclear, but it is thought that different intracellular signaling pathways may lead to a preferential phosphorylation of one isoform over the other. Hreniuk et al. [31] reported a similar phosphorylation pattern for JNK following IR of cardiac myocytes in vitro. Myocytes exposed to 4 h of ischemia followed by reoxygenation showed an increase in JNK phosphorylation 15 min after reoxygenation that peaked at 2 h and declined by 4 h.

Immunolocalization of phospho-JNK revealed immunoreactivity in the endothelial cells of the testis after testicular IR (Fig. 3A and B); I{kappa}B-{alpha}, detected as a marker for the NF{kappa}B complex, was absent from endothelial cells (Fig. 3C and D). This is consistent with the hypothesis that JNK activation in testicular endothelial cells is the primary pathway that leads to stimulation of E-selectin expression.

Activation of JNK in other tissues has been shown to stimulate E-selectin expression through activation of the transcription factors ATF-2 and c-jun [35, 36]. Activation of JNK and p38 by TNF{alpha} lead to an activation of the transcription factors ATF-2 and c-jun, which subsequently form a heterodimer and associate with the positive regulatory domain II site of the E-selectin promoter [35]. Also, Reimold et al. [43] used mice producing mutant ATF-2 protein to demonstrate a decreased induction of a number of adhesion molecules, including E-selectin, in those animals. In the present study, phosphorylation of both ATF-2 and c-jun was observed after IR of the testis at time points that correspond to the phosphorylation of JNK (Fig. 4). Correlated with this was an increase in E-selectin mRNA expression (Fig. 5) and protein presence in testicular endothelial cells (Fig. 6). Subsequently, neutrophils were recruited to subtunical venules in the testis (Fig. 7). Total c-jun protein was also increased at 2.0 and 4.0 h after reperfusion of the testis (Fig. 4). It has been previously demonstrated that phosphorylation of c-jun increases the stability of the protein [44]; thus, the observed increase in total c-jun protein is mostly likely due to phosphorylation.

To determine if the increases in TNF{alpha} and IL-1ß were responsible for the stimulation of the JNK pathway and, ultimately, neutrophil recruitment to the testis, intratesticular injections of TNF{alpha} and/or IL-1ß were performed. Surprisingly, injection of TNF{alpha} alone did not cause an increase in the phosphorylation of JNK54. Injection of IL-1ß, on the other hand, caused a significant increase in the phosphorylation of JNK54. The injection of TNF{alpha} and IL-1ß gave a response that was highly variable but consistently stimulatory to JNK54 phosphorylation relative to sham-injected controls (Fig. 8). Injection with IL-1ß or TNF{alpha} plus IL-1ß also recruited large numbers of neutrophils to the testis (Fig. 9). Thus, it appears that even though TNF{alpha} is up-regulated to a greater extent than IL-1ß after IR of the testis, IL-1ß may be the more biologically active in this setting.

The involvement of proinflammatory cytokines and stress-related kinase pathways in IR injury are receiving much attention. Results of the present study demonstrate for the first time that after IR of the murine testis TNF{alpha} and IL-1ß and the activation of the JNK pathway are correlated with the expression of E-selectin and neutrophil recruitment to the testis. Future studies in our laboratory will develop procedures for in vivo infusion of specific JNK inhibitors into the testis to specifically block JNK activation after IR of the testis. Testicular torsion of the murine testis provides an excellent in vivo model system to investigate the many pathological effects, such as apoptosis, proinflammatory cytokine expression, activation of intracellular stress-related signaling pathways, cell adhesion molecule expression, and neutrophil recruitment, brought about after IR. Understanding the intracellular signaling pathways activated after IR of the testis may lead to the design of specific therapies that will aid in rescuing testicular function and spermatogenesis after IR.


   ACKNOWLEDGMENTS
 
The authors acknowledge the assistance of the Cell Science Core of the Center for Research in Reproduction (NICHD Specialized Cooperative Centers Program in Reproductive Research; U54 HD28934) at the University of Virginia.


   FOOTNOTES
 
1 Supported by the National Institutes of Health grant RO1-DK-53072 (to T.T.T.), the Medical Scientist Training Program National Institutes of Health grant 2T32 GM07267 (to J.L.K.), and a grant from the AFUD/AUA Research Scholar Program (to J.J.L.). Back

2 Correspondence: Jeffrey J. Lysiak, Department of Urology, Box 800422, University of Virginia Health Science System, Charlottesville, VA 22908. FAX: 434 924 8311; jl6n{at}virginia.edu Back

Received: 12 November 2002.

First decision: 5 December 2002.

Accepted: 5 February 2003.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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