Testicular Injury Induces Cell-Mediated Autoimmune Response to Testis

0022-5347/95/1534-1316$03.00/0 IkEJOURNAL OF UROLOGY Copyright 0 1995 by AMERICANURO~OCICAL AssOc~"toN,INC.

Vol. 153,1316-1320,April 1995 Printed in U.S.A.

TESTICULAR INJURY INDUCES CELL-MEDIATED AUTOIMMUNE RESPONSE TO TESTIS YASUKI SAKAMOTO,* TETSURO MATSUMOTO, YOSHIMITSU MIZUNOE, MASASHI HARAOKA, MISAO SAJSUMOTO AND JOICHI KUMAZAWA From the Department of Urology, Faculty of Medicine, Kyushu University

ABSTRACT

Studies on testis autoimmunity are needed for a better understanding of immunological male infertility. Evidence has accumulated that the delayed type hypersensitivity (DTH) response plays a key role in the induction andor maintenance of experimental autoimmune orchitis (EAO), an animal model for human immunological male infertility or aspermatogenesis. We report here that an antigen-specific DTH response to autologous testicular cells (TC) could be induced by bilateral testicular injury (trauma) in mice. Pretreatment of traumatized mice with a high dose of cyclophosphamide (CY) enhances the DTH response in a dose-dependent manner. The DTH response induced by testicular injury reaches its peak on the ninth day. We have shown that the local passive transfer of the footpad reaction to normal recipients by T cells further defines the DTH reaction. These characteristics resemble those of the previously reported DTH response to syngeneic TC induced by subcutaneous immunization with viable syngeneic crude TC. Our present injury model mimics clinical testicular trauma; therefore, this testicular injury model can be very useful in studying the immunological mechanism of EAO and of human immunological male infertility. KEY WORDS: testis, autoimmunity, infertility

Approximately 15% of the 40 million couples in the United

States are infertile, and one-third of this infertility is believed to be due primarily to the male partner.' There are quite a few idiopathic male infertility patients, but it is believed that there are some immunologically mediated cases of infertility. Autoimmune responses to spermatozoa have been detected in men with infertility from unknown causes and in vasectomized men. On the other hand, inflammatory testis damage of a n autoimmune nature does not seem to occur very frequently in humans, even though a n autoimmune pathogenesis may be postulated for both granulomatous orchitis and the atrophy that follows mumps orchitis in some individuals. However, the recent finding of immune deposits in the testes of infertile men suggests that many cases of male infertility may be the consequence of earlier episodes of testis autoimmunity.' Therefore, animal models of testis autoimmunity are useful in the study of male infertility. Delayed type hypersensitivity (DTH), one of the cell-mediated immunities, has been detected in animals showing experimental autoimmune orchitis (EX01 and is believed to Delayed play a key role in the development of the footpad reaction (DFR) against syngeneic testicular cells (TC) was elicited in C3WHe mice immunized subcutaneously with viable syngeneic TC without adjuvant^.^ Delayed footpad reaction was augmented in a dose-dependent manner by pretreatment of immunized mice with a high dose of cyclophosphamide (CY).6*7We have reported in detail that EAO could be induced in these immunized mice, especially in those pretreated with 100 mg./kg. of CY, with the presence of DTH against syngeneic TC and of anti-TC antibodies5 In the present study, we have chosen testicular injury as a triggering mechanism of autoimmunity against testicular

antigen. Because of the testicular injury, destruction of the testis-blood barrier occurs; the normally sequestered TC leak into and around the testis and might then work as endogenous antigens. We were able to induce the DTH response to autologous TC when the testes were injured bilaterally. Active immunization by injection of TC was not carried out in this study. MATERIALS A N D METHODS

Mice. Inbred male C3WHeN mice (10 to 14 weeks of age at the time of study initiation) were supplied from the Institute for Animal Experiment, Faculty of Medicine, Kyushu University. Testicular injury. The mice were anesthetized with an intraperitoneal injection of 50 mg./kg. of pentobarbital. The testes were injured bilaterally in the following way: 10 to 20 needle punctures were made to the testes to pierce through the tunica albuginea, and the testes were crushed fully by a needle-holder. Treatment with CY. Cyclophosphamide (Endoxan, Shionogi and Co., Osaka, Japan) was dissolved in phosphate buffered saline (PBS) at a concentration of 20 mg./ml. and injected intraperitoneally in a dose of 100 or 200 mg./kg. 2 days before testicular injury. Preparation of TC or liver cells (eliciting antigens). The viable TC of syngeneic mice were used as the eliciting antigens; liver cells were used as a control. The testes and liver were removed from mice after complete bleeding and squeezed with a plunger in cold Hanks' balanced salt solution (HBSS). Cell suspensions were passed through a Cell Strainer (Becton Dickinson and Company, Franklin Lakes, New Jersey) to remove residual large fragments. After washing, the cells were resuspended in HBSS and adjusted to Accepted for publication October 21, 1994. designated concentrations after viability counting by the * Reprints: Department of Urology, Faculty of Medicine, Kyushu trypan blue exclusion method. University, 3-1-1Maidashi. Higashi-ku, Fukuoka 812,Japan. Donor preparation for local passive transfer. The testes of This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, donors were crushed 12 days previously. In some experiJapan. ments, donors were treated with CY alone or were not 1316

TESTICULAR INJURY INDUCES AUTOIMMUNE RESPONSE TO TESTIS

treated a t all. Peritoneal exudate cells (PEC) were harvested 5 days after an intraperitoneal injection of 0.5 ml. incomplete Freunds adjuvant (IFA; GIBCO, Grand Island, New York) as previously reported.' Treatment of T cells. Monoclonal anti-Thy-1.2 (Olac 1976 Ltd., London, United Kingdom) was mixed in a 1:3200 dilution in RPMI 1640 (GIBCO). Peritoneal exudate cells were incubated at 1 X 10'/ml. diluted antiserum for 45 minutes at 37C. The cells were then centrifuged and resuspended in a 1:3 dilution of rabbit complement (C) in RPMI 1640 prepared just before use. After 30 minutes at 37C, cells were washed and transferred with 5 X lo6 TC. Measurement of DTH. Delayed type hypersensitivity was assessed by measuring delayed footpad reaction (DFR) after testicular injury as described previously.' In brief, at various times after treatment, mice received an injection of 1 X lo6 autologous TC in 0.05 ml. of HBSS into the right hind footpad. The left hind footpad received HBSS only. In the standard protocol for local transfer, 1 X lo7 PEC with 5 X lo6 of viable TC or liver cells in a volume of 0.05 ml. were injected into the right hind footpad. Footpad thickness was measured with a dial gauge caliper 24 hours after challenge. Reactions were expressed as the difference in thickness between the left and right footpads. The results were expressed as the mean f SD of 5 to 10 animals. Statistics. The statistical significance of the data was determined according to Student's t test using the values for footpad swelling. A p value less than 0.05 was taken as significant. RESULTS

Znduction of antigen-specific DFR to autologous TC by bilateral testicular injury. The testes of mice were crushed as described in Materials and Methods. Footpad elicitation was carried out with 1 X lo6 of viable autologous TC in 0.05 ml.

Treatment

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of HBSS 9 days after testicular injury. As shown in fig. 1, a positive DFR was detected when elicited with syngeneic TC in testis-injured mice compared with the control mice (p <0.001). No significant DFR was detected when the testisinjured mice were challenged with the third party antigen. Dose-dependentaugmenting effectof CYpretreatment on DFR to autologous TC. To rule out the possibility that a direct effect of CY on spermatogenesis might cause an enhanced release of antigen and enhancement of the overall immune response to the testis, including DTH, normal mice were treated with 100 or 200 mg&. of CY. No augmentation of DFR was observed (fig. 2). The testes of mice were crushed with 100 or 200 mg&. of CY 2 days before testicular injury or without CY pretreatment. Elicitation of DFR was carried out with 1 x lo6 of viable autologous TC in 0.05 ml. of HBSS 9 days after testicular injury. When the injured mice were pretreated with 100 mg.Acg. of CY, DFR tended to be enhanced compared with the injured mice without CY-pretreatment. However, the difference was not statistically sigdicant (p <0.065). When the mice were pretreated with 200 mg&. of CY, DFR was augmented sigmficantly compared with mice pretreated with 100 mg.kg. of CY (p = 0.001). Time course of DFR to autologous TC. We compared the degrees of DFR in relation to the days after testicular injury. The testes of mice were crushed, and DFR was elicited with 1 X lo6 of autologous TC on 3 , 6 , 9 , 1 4 , 2 8 and 40 days after injury. Separate groups were used for each experimental day. A positive DFR was detected as early as day 3 (p C0.005). Delayed footpad reaction reached a peak on day 9 and remained high as long as day 40 compared with the negative control (p <0.001) (fig. 3). Local passive transfer of DFR by PEC and its antigen specificity. Peritoneal exudate cells were harvested from donors whose testes were crushed 12 days previously. When PEC were transferred to the footpads of normal recipients

Eliciting antigens

24-hr footpad swelling (xO.lmm) FIG. 1. Induction of antigen-specific DFR to autologous TC by bilateral testicular injury. Testes of C3WHeN mice were crushed. Footpad elicitation was camed out with 1 x 106 autologous TC or liver cells 9 days after testicular injury.

TESTICULAR INJURY INDUCES AUTOIMMUNE RESPONSE TO TESTIS

Treatment

24-hr footpad swelling (xO.1mm) FIG.2. Delayed footpad reaction to autologous TC with or without CY pretreatment. Testes of mice were crushed by retreatment with 0, 100,or 200 mg.kg. of CY 2 days before testicular injury. As a control, normal mice were treated with 100 or 200 mg./Rg. of CY. Footpad elicitation was carried out with 1 X lo6 autologous TC 9 days after testicular injury. ients. Figure 5 shows that DFR was reduced to the negative control level by treatment of the transferred cells with a n t i 4 serum plus C compared with the untreated cells (p <0.001). Treatment with C alone had no effect on the transfer. DISCUSSION

04

1

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9

14

26

40

Days after testicular injury

FIG.3. Kinetics of DFR induced by bilateral testicular injury. Testes of mice were crushed. Footpad elicitation was camed out with 1 x lo6 autologous TC at various times after testicular inj;ury Footpad elicitation of normal mice carried out with 1 X 10 autologous TC (W). Separate groups were used for each day.

together with syngeneic TC, significant DFR was detected (p <0.001)(fig. 4). However, the PEC elicited no significant DFR when transferred together with syngeneic liver cells. Peritoneal exudate cells from normal or CY-treated donors could not elicit DFR in recipients, even when transferred with syngeneic TC. T cell dependency of the activity to transfer DFR. Whole PEC harvested from donors whose testes were crushed 12 days previously were treated with monoclonal a n t i 4 serum and complement (C) or C alone as described in Materials and Methods and transferred with syngeneic TC to normal recip-

Even though only antisperm antibodies (humoral immunity) have been taken into account by many urologists or andrologists in assessing immunological male infertility, many reports imply that a DTH response, that is cell-mediated immunity, plays an important role in the onset and/or maintenance of EA0.3-5 A DTH response to syngeneic TC could be induced in mice with EAO induced by immunization. We were able to induce an antigen-specific DTH response to autologous TC (T cell-mediated testis autoimmunity) by mechanical injury to the testis. There are three reasons why we have chosen this testicular injury as a trigger to induce a DTH response. (1)Even though this is not a natural trauma to the testis, this kind of mechanical injury induced by needle puncture and crushing is similar to clinical piercing of the testis. (2) Based on our previous studies on the DTH response to syngeneic TC induced by immunization, subcutaneous, but not intravenous immunization, induces a DTH response.' Suppressor T cells (T,) for DTH are induced by intravenous i m m ~ n i z a t i o nIt. ~seems that quite a few of the traumatized TC spread out of the blood-testis barrier in and around the testis and only a few enter the blood circulation. (3) Unlike other studies on immunization with artificial adjuvants, such as complete Freunds adjuvant (CFA),'?'' our previous immunization model was studied by using freshly prepared syngeneic crude viable TC without any adjuvant^.^ The histopathology of the injured testis shows the complete destruction of seminiferous tubules and the blood-testis barrier. Therefore, traumatized TC spreading in and around the testis might work as endogenous antigens, and access of testis

1319

TESTICULAR INJURY INDUCES AUTOIMMUNE RESPONSE TO TESTIS

24-hr footpad swelling (xO.1mm) FIG. 4. Antigen-specific local transfer of DFR by PEC. Peritoneal exudate cells were harvested from donors whose testes were crushed 12 days before, from donors who were treated with CY only, or from normal donors. Ten million IFA-induced PEC were transferred to normal syngeneic recipients together with 5 x lo6 syngeneic viable TC or 5 X 10' syngeneic liver cells. Harvest and transfers were performed concurrently for all groups. Antigen-specific footpad swelling was determined 24 hours after transfer.

,

Do nor treatment (Testicular injury)

Treatment of PEC 1

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Untreated

+ +

Anti-fl -t C i

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6

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24-hr footpad swellinq (x0.1mm) FIG. 5. Abolition of DFR by treatment with a n t i 4 serum plus C. Ten million PEC harvested from normal donors or from donors whose testes were crushed 12 days before were treated with C alone, with a n t i 4 plus C, or were not treated at all. After each treatment, those cells were transferred together with 5 x lo6 syngeneic TC per mouse. Footpad swelling was determined 24 hours &r transfer.

antigens to the macrophages or dendritic cells in and around the testis should be sufficient to elicit the response. This model might be very similar to subcutaneous rather than mtravenous immunization with viable syngeneic crude TC. Cyclophosphamide pretreatment of the testis-injured mice enhanced the DTH response to autologous TC in a dosedependent manner. We have already proved histopathologi-

cally that CY treatment alone does not directly damage spermatogene~is;~ therefore, no DTH response to TC was induced or enhanced by CY treatment alone. This augmenting phenomenon is very similar to our previous immunization model.' This is probably due to specific elimination of CY-sensitive T, for DTH, as we have previously shown.7 In OUT immunization model, we have demonstrated the presence of

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TESTICULAR INJURY INDUCES AUTOIMMUNE RESPONSE TO TESTIS

T, for DTH at the induction stage and have also shown that CY pretreatment abolished the induction of such T,. The time course of the appearance of the DTH response to autologous TC was consistent with that of the classical DTH response. The response reached a peak on the ninth day and remained high for at least 40 days of observation. For further evidence that this response is a DTH, we have conducted the local passive transfer of PEC from testis-injured mice together with syngeneic TC and showed the generation of effector T cells for DTH in a way similar to our previous report.' In a preliminary experiment, interstitial inflammation and inhibition of maturation of sperm in the seminiferous tubules were detected in the contralateral testis 28 days after unilateral testicular injury. Details of such observations will be reported in the next paper of this series. In this paper, we did not investigate antibody. Many investigators have reported antibody production against TC and perm.'^-'^ Even though there are more reports of passive EL40 by T cell^,'^.'^ there are some reports of EAO in guinea pigs induced by passive transfer of serum." Therefore, EL40 probably can be induced by antibody as well as sensitized T cells. We intend to examine antibody production against TC in the next series. This model, in which recognition of autologous TC was induced by a more natural trigger (almost a clinical testicular trauma), is believed to be a significant cause of autoimmune orchitis and can be used as a clinical testicular trauma model to determine whether the injured (traumatized) testis should be removed for the maintenance of fertility. REFERENCES

tal allergic orchitis induced by unilateral intratesticular bacterial infection in guinea-pigs. Immunology, 49 45, 1983. 4. Itoh, M., Hiramine, C. and Hojo, K.: A new murine model of autoimmune orchitis induced by immunization with viable syngeneic testicular germ cells alone. I. hmunological and histological studies. Clin. Exp. Immunol., 83. 137, 1991. 5. Sakamoto, Y., Himeno, K., Sanui, H., Yoshida, S. and Nomoto, K.: Experimental allergic orchitis in mice. I. A new model induced by immunization without adjuvants. Clin. Immunol. Immunopathol., 37: 360, 1985. 6. Yoshida, S.,Nomoto, K., Himeno, K. and Takeya, K.: Immune response to syngeneic or autologous testicular cells in mice. I. Augmented delayed footpad reaction in cyclophosphamidetreated mice. Clin. Exp. Immunol., 3 8 211, 1979. 7. Sakamoto, Y. and Nomoto, R: Suppression of delayed-type hypersensitivity to syngeneic testicular cells in mice: involvement of suppressor T cells which act at the induction stage. Cell. Immunol., 102 168,1986. 8. Sakamoto, Y., Sanui, H., Yoshida, S. and Nomoto, K.: Local passive transfer of delayed type hypersensitivity to syngeneic testicular cells in mice. Clin. Exp. Immunol., 6 6 353,1984. 9. Kohno, S.,Munoz, J. A., Williams, T. A., Teuscher, C., Bernard, C. C. A. and Tung, K. S. R: Immunopathology of murine experimental allergic orchitis. J. Immunol., 130 2675, 1983. 10. Freund, J.,Lipton, M. M. and Thompson, G. E.: Aspermatogenesis in the guinea pig induced by testicular tissue and adjuvants. J. Exp. Med., 91:711,1953. 11. Rapaport, F. T., Sampath, A., Kano, K., McCluskey, R. T. and Milgrom, F.: Immunological effects of thermal injury. I. Inhibition of spermatogenesis in guinea-pigs. J. Exp. Med., 130 1411, 1969. 12. Kosuda, L. L. and Bigazzi, P. E.: Autoantibodies to acrosomal antieen of snermatozoa in vasectomized mice. Invest. Urol.. 1 6 i30,i97k 13. Mahi-Brown. C. A.Yule. T. D. and Tune. K. S. K.: AdoDtive transfer of murine autoimmune orchitis to naive recipients with immune lymphocytes. Cell. Immunol., 106: 408,1987. 14. Itoh, M., Mukasa, A, Tokunaga, Y., Hiramine, C. and Hojo, K.: New experimental model for adoptive transfer of murine autoimmune orchitis. Andrologia, 2 3 415,1991. 15. Toullet, F. and Voisin, G. A.: Passive transfer of autoimmune aspermatogenic orchiepididymitis(AIAO) by antispermatozoa sera. Influence of the type of autoantigen and of the class of antibody. Clin. Exp. Immunol., 26 549,1976. I ,

1. Sigman, M. and Lipshultz, L. L.: Evaluation of the subfertile male. In: Infertility of the Male. Edited by L. L. Lipshultz and S. S. Howar&, 2nd ed. St. Louis: Mosby-Year Book, pp. 179210,1991. 2. Kosuda, L. L. and Bigazzi, P. E.: Animal models of testis autoimmunity. In: Immunology of the Male Reproductive System. Edited by P. E. Bigazzi. New York Marcel Dekker, pp. 253352,1987. 3. Sanui. H.. Yoshida. S.. Himeno. K. and Nornoto. ... K: Ewerimenr I

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