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Secretion of tumor necrosis factor alpha by testicular macrophages
Journal of Reproductive Immunology, 23 (1993) 63-72 Elsevier Scientific Publishers Ireland Ltd.
63
JRI 00799
Secretion of tumor necrosis factor alpha by testicular macrophages J a m e s C. H u t s o n Department of Cell Biology and Anatomy, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 (USA) (Accepted for publication 14 August 1992)
Summary While it has been shown that culture medium from testicular macrophages can influence testosterone production when added to Leydig cells, the identity of the factor(s) responsible for this activity remains unknown. Since tumor necrosis factor ct (TNFo0 has been shown to be capable of influencing testosterone production by Leydig cells, a series of studies was conducted to determine if testicular macrophages produce TNFo~. It was found that testicular macrophages from adult rats produce a factor that is capable of lysing L929 cells, which are used as a traditional bioassay for TNFot. The TNFo~ activity in the macrophage-conditioned medium could be neutralized by the addition of anti-murine TNFa but not with the addition of preimmune IgG. While lipopolysaccharide (LPS) slightly increased the release of TNFct, neither follicle-stimulating hormone (FSH) nor testosterone had a similar effect. It was not determined if the isolation procedure had artificially 'activated' the macrophages. Medium from cultured Sertoli cells, Leydig cells and peritubular cells did not contain TNFot activity. These studies are consistent with the hypothesis that the paracrine interaction between testicular macrophages and Leydig cells is mediated in part by TNFet. Key words:
TNF alpha; rnacrophages; testis; Leydig cells; cytokines
Introduction Macrophages are relatively numerous in the interstitium of the testis Correspondence to: James C. Hutson, Department of Cell Biology and Anatomy, Texas Tech University Health Sciences Center, Lubbock, TX, USA. 0165-0378/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
64
(Christensen et al., 1969; Fawcett et al., 1973; Connell and Christensen, 1975; Clark, 1976; Sinha et al., 1977; Wing and Lin, 1977). The cells are thought to be authentic macrophages in that they: exhibit typical macrophage morphology, have Fc receptors, secrete superoxide anion and lysozyme, can phagocytose and kill pathogenic bacteria, rapidly attach to culture substrates, and possess specific macrophage antigens (Miller et al., 1983, 1984; Hutson, 1988, 1990; Wei et al., 1988). These cells, however, appear to be involved in functions beyond traditional host defence mechanisms characteristic of most macrophages. For example, macrophages appear in the testis in a temporal manner during the period of rapid and dramatic testicular development and maturation (Hutson, 1990). Interestingly, the increase in size of the macrophage population that occurs during this time can be induced to occur precociously by exogenous hCG (Raburn et al., 1991). Once the concentration of macrophages in the interstitium reaches equilibrium, Leydig cells then begin to form membrane digitations with macrophages just prior to the major steroidogenic activity associated with puberty (Hutson, 1992). These digitations are composed of cytoplasmic extensions of the Leydig cells inserted into coated channels of the macrophage (Christensen et al., 1969; Hutson, 1992). It appears that these two cell types are also functionally coupled since testicular macrophages release a factor(s) in culture that alters the secretion of testosterone by Leydig cells (Yee and Hutson, 1985a). While the factors responsible for this effect on Leydig cells have not been identified, it has been reported that TNFa, a known macrophage product, can alter the release of testosterone from Leydig cells (Calkins et al., 1990; Warren et al., 1990; Mauduit et al., 1991). However, the physiological significance of these results remains in question since: (1) testicular macrophages are known to secrete many proteins electrophoretically different than those of macrophages from other anatomical sites (Hutson and Stocco, 1989), (2) macrophages generally do not produce TNFc~ unless stimulated by endotoxin or other activating factors (Carswell et al., 1975) and (3) peritoneal macrophages, which are known to produce TNFc~, do not influence basal testosterone secretion by Leydig cells (Francis et al., 1991). Thus, the purpose of the present study was to determine the feasibility of the hypothesis that TNFa from testicular macrophages is involved in the interaction with Leydig cells by directly testing if they produce TNFo~. Materials and Methods Animals
Wistar Kyoto (WKY) inbred rats originally obtained from Charles River Breeding Laboratories (Wilmington, MA) were maintained as a local
65
breeding colony in a controlled environment within our institutional animal care facility under the direction of a licensed veterinarian. The animals were housed in plastic cages with wood shavings as bedding and provided rat chow and water continuously. The experimental protocol was reviewed and approved by an institutional animal care and use committee.
Isolation of macrophages Five rats/experiment were killed by cervical dislocation and the testes removed and perfused with phosphate-buffered saline (without calcium and magnesium, PBS) to remove contaminating blood cells. The testes were gently decapsulated without cutting seminiferous tubules and then digested in a solution of collagenase (Sigma Type I, 100 units/ml in 50 ml PBS). The digestion took place at 34°C in a shaking water bath at 120 cycles/min for 20 min in a plastic Erlenmeyer 250 ml flask (Coming Glass Works, Coming, NY). At the end of the digestion, the flask was inverted several times until the tubules loosened but remained attached at the rete testis. The flask was then placed on ice for 2 min to allow the tubules to settle. The supernatant, containing the crude interstitial cells, was then centrifuged at 400 x g for 10 min at 4 ° C. The cells were suspended in PBS acclimated to 34°C and plated into 60-mm Coming Petri dishes (Coming Glass Works, Corning, NY). The dishes were placed on a glass plate (commonly used for cooling an electrophoresis gel) through which water was circulated to maintain the surface temperature at 34°C. After 4 min, the unattached cells were removed by vigorous washings with PBS. Three milliliters of complete medium was then added (Dulbecco's modified Eagle's medium, containing 2.2 g/1 sodium bicarbonate, 10 mM HEPES, 10% horse serum (donor herd), 100 units/ml penicillin and 100/~g/ml streptomycin, (all from Sigma Chemical Co., St. Louis, MO). The pH of this medium was strictly controlled between 7.0 and 7.4 at all times. Preliminary studies indicated that testicular macrophages are seriously influenced by extremes of pH. Macrophages prepared by this technique are greater than 95°/~ positive for Fc receptor, secrete superoxide anion and lysozyme and phagocytose and kill pathogenic bacteria (Hutson, 1988; Wei et al., 1988). The cells were then treated for 24 h with 100 ng/ml oFSH-17 (N1DDK), 100 ng/ml testosterone (Sigma Chemical Co., St. Louis, MO) or 1 /~g/ml LPS (Sigma Chemical Co., St. Louis, MO) for 24 h at 34°C in a humidified atmosphere containing 95% air and 5% CO2. The medium was then filtered (0.2 #m) and either stored frozen at -16°C or tested immediately in the TNFot bioassay as described below.
Isolation of Sertoli cells and peritubular cells Sertoli cells and peritubular cells were isolated from testes of six 20-23day-old Kyoto pups per experiment as previously described (Tung et al.,
66
1984). Briefly, the testes were decapsulated and sequentially digested in 0.25% trypsin, 0.1% collagenase and 0.1% hyaluronidase (Sigma Chemical Co., St. Louis, MO). The Sertoli cell preparation was taken from the final hyaluronidase digestion while the peritubular cells were obtained in the collagenase supernatant. The Sertoli cells were plated into 60-mm Petri dishes (Corning Glass Works, Corning, NY) and cultured in 3 ml complete medium without serum but with and without 100 ng/ml oFSH-23. The peritubular cells were plated similarly except that 10% defined bovine calf serum (Hyclone, Logan, UT) was added. Peritubular cells were treated with or without 100 ng/ml testosterone (Sigma Chemical Co., St. Louis, MO). Sertoli cells and peritubular cells were maintained in culture as described above for testicular macrophages.
Isolation of Leydig cells Leydig cells were isolated from testes of 4 adult rats/experiment as previously described (Hutson, 1988). Briefly, the testes were digested in 50 ml of collagenase (100 units/ml) in PBS and the cells in the supernatant fractionated on Percoll density gradients. Leydig cells found in the most dense band (those located just above band of red cells at a density of 1.09-1.12) were used in these studies. Preparations obtained by this technique are approximately 80-85% positive for 3/3-steroid dehydrogenase activity. The cells were plated into 35-mm Petri dishes (Primaria, Falcon Plastics, Oxnard, CA) in 2 ml complete medium with or without 100 ng/ml o-LH. Cells were maintained in culture as described above. TNFa assay This assay was performed as previously described based on the known sensitivity of L929 cells to TNFo~ (Baarsch et al., 1991). L929 cells (American Type Culture Collection, Rockville, MD) were plated in 96-well plates (Corning Glass Works, Corning, NY) on the morning of the assay at a concentration of 10 000 cells/well in 200/A complete medium. At the beginning of the assay, the medium was replaced with control medium (Dulbecco's modified Eagle's medium plus 1% horse serum (Sigma Chemical Co., St. Louis, MO, donor herd), 2.2 g/1 sodium bicarbonate, 10 mM HEPES, 100 units/ml penicillin and 100 t~g/ml streptomycin and 3 /~g/ml actinomycin D (Sigma Chemical Co., St. Louis, MO) plus various dilutions of medium previously exposed to testicular macrophages, Sertoli cells, peritubular cells or Leydig cells. Test substances (FSH, LH, testosterone and LPS) and control medium that had not been exposed to cells were also tested individually. Medium from macrophages was also incubated with rabbit polyclonal anti-murine recombinant TNFot (10 #g/ml) or preimmune IgG (10 #g/ml) (Endogen, Boston, MA) for 48 h at 4°C prior to testing in the assay.
67
The L929 cells were then incubated in the presence of dilutions of the conditioned media or test substances at 37°C for 20-24 h. MTT (3-(4,5dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) (Sigma Chemical Co., St. Louis, MO) was then added at a concentration of 2.5 mg/ml PBS and the cells incubated an additional 1 h at 37°C in a humidified atmosphere of 100% air. MTT is a water-soluble yellow tetrazolium salt that is converted by mitochondrial dehydrogenases only by living cells to a water-insoluble blue precipitate. The MTT was then removed and the blue precipitate solubilized with ethanol. Cell viability was then assessed spectrophotometrically at -4550. The percentage viable cells was calculated by comparison with cells treated only with control medium. Cell viability was also assessed microscopically by determining the percentage of cells containing blue reaction product. Statistical methods The means of the various treatment groups were compared for significant differences (95% confidence level) using a computer program for analysis of variance and Scheffe's F-test (StatView SE+, Abacus Concepts, Inc. Berkeley, CA). Results
It was found that conditioned medium from testicular macrophages had TNFot activity and induced optimal lysis of L292 cells at 12-24 h depending upon the density of the macrophage cultures (Fig. 1). The L929 cells exposed 100
80 o~
60
~ ~
40
~'~
20
~g
0
,
HOURS Fig. 1. Testicular macrophage conditioned medium was diluted 1:]0 and added to L929 cells for various times. The ceils were then j~ubat~d with M l " r and the percentage of viable (blue) cells determined
microscopically.
68
to macrophage-conditioned medium changed from firmly-attached slightly flattened fibroblastic cells to poorly-attached rounded cells over this time (Fig. 2). Therefore, the assay was subsequently terminated between 18 and 20 h. It was found in these pilot studies that viable cell number correlated well with absorbance at A550 as previously reported (Baarsch et al., 1991). Therefore, we used results from the spectrophotometric assay to illustrate subsequent studies. The lytic activity of testicular macrophage-conditioned medium was not significantly stimulated by addition of FSH or testosterone, while LPS had a very slight but statistically significant influence (Fig. 2). These factors had no influence on viability of L929 cells when tested alone as vehicle controls. Lytic activity of medium from macrophages was significantly reduced by addition of rabbit anti-mouse recombinant TNFot but not by the addition of preimmune IgG (Fig. 3). Preliminary studies also indicated that a monoclonal antibody to recombinant human TNFot was unable to neutralize the lyric activity of the rat TNFot activity from testicular macrophages in accordance with known species specificity (data not illustrated). Conditioned medium from Sertoli cells, Leydig cells and peritubular cells did not contain T N F a activity (Fig. 4). Conversely, conditioned medium from both peritubular cells and Leydig cells stimulated growth of L929 cells in the presence of actinomycin D (Fig. 4). The addition of testosterone or LH to peritubular cells or Leydig cells, respectively, had no influence on proliferation of L929 cells.
100 o
g
75-
o 50-
25
0 o
P, M a c r o p h a g e Conditioned M e d i u m
Fig. 2. This graph illustrates the effects of various test substances on the production of TNFc~ by testicular macrophages treated with 1 #g/ml LPS, 100 ng/ml testosterone (T), or 100 ng/ml follicle-stimulating hormone (FSH). LPS, FSH and T, in the absence of macrophage conditioned medium, were also tested (3 bars to the far right o f the graph). Values represent the mean ± S.E.M. of four culture dishes• Asterisks indicate a statistical significant difference from the control at P >_ 0.05.
69 125" 100 ¢j
1 1111 1111
75
50
25 0
~
~
.~..c2.
~: 4: 4-: T . . ~.
Preimmune IgG
Anti-TNF alpha
i
o
i
I
Macrophage Conditioned Medium Fig. 3. The neutralizing influence of 10 t~g/ml a n t i - T N F or preimmune I g G on TNFc~ activity. Values are the mean ± S.E.M. (n = 4). Asterisks indicate a statistical significant difference from the control at P _> 0.05.
Discussion Three important parameters are necessary to establish that a single factor such as TNFo~ is an important factor in a complex mixture of factors such as conditioned medium from macrophages. First, addition of the factor by
175
-
150 m
o
125
~o o~
0 ,:.: ,:.: .~.
o
~H I
i
Sertoli cells
"~ ".: .. I
I
--
•
I
.~. J
LH I
Peritubular cells
,:.: ~ I
T I
...~.
I
I
Leydig cells
Fig. 4. The cellular specificity of TNFc~ secretion by various testicular cell types. Sertoli cells, peritubular cells and Leydig cells were cultured for 24 h and the medium assayed for T N F a activity. Values are the mean ± S.E.M. (n = 4). Asterisks indicate a statistical significant difference from the control at P _ 0.05.
70
itself should produce a response similar to the response observed in the conditioned medium. Secondly, it should be demonstrated that the putative factor is present within the conditioned medium. And finally, removal or neutralization of the factor from the conditioned medium should reduce the activity of the medium. The first criterion, that purified TNFc~ has an influence on Leydig cells, has been established previously (Calkins et al., 1990; Warren et al., 1990; Mauduit et al., 1991). The results of the present study satisfy the second criterion, that the conditioned medium in fact contains TNFo~. The final criterion, to demonstrate a change in the activity of conditioned medium by removal of TNFo~ remains to be completed. It was surprising to find that LPS had little influence on the secretion of TNFot since endotoxin has been shown to have a stimulatory action on macrophages from other tissues (Carswell et al., 1975; Francis et al., 1991). It is possible that the testicular macrophage is artificially activated by the isolation procedure even though care was taken to be sure the solutions used in isolating and culturing the macrophages were sterile and low in endotoxin. However, collagenase or other unknown factors could have 'activated' these cells. It was also interesting to learn that FSH had no activity on TNFo~ secretion since FSH has been shown to bind to specific receptors on testicular macrophages and elicit changes in amino acid incorporation into acid precipitable material (Yee and Hutson, 1985b,c). The rationale for testing the effects of testosterone on the secretion of TNFot was based on the possibility that a feedback loop may exist between macrophages and Leydig cells. That is, the secretion of TNFc~ by macrophages might have altered testosterone production by Leydig cells which would in return modify macrophage activity. While such a loop was not found for TNFot and testosterone, other factors may be involved. TNFot exists in two forms; one is associated with the cytoplasmic membrane and the other is soluble (Kriegler et al., 1988). The present findings tested only the TNFc~ that was released into the culture medium. It is tempting to speculate that the bound form is also involved in influencing Leydig cells in vivo since the testicular macrophage and Leydig cell are so closely associated in situ. The presence of the membrane digitations between these two cells (Christensen et al., 1969; Hutson, 1992) would be a strategic site for such a mechanism to exist. The secretion of TNFot appeared to be specific to macrophages in that Sertoli cells, peritubular cells and Leydig cell did not secrete TNFot. However, it should be pointed out that the Sertoli cells and peritubular cells were isolated from immature animals while the macrophages were isolated from adult animals. It is yet to be determined if the secretion of TNFot is agedependent in any of the cell types tested. The unexpected observation that peritubular cells and Leydig cells produce a factor or factors that increase the
71
number of L929 cells is intriguing. It may be possible that this activity is due to the production of one or several growth factors by these cells. It is interesting that Sertoli cells lacked similar activity since they have been shown to produce a factor capable of stimulating the proliferation of other cell lines (Feig et al., 1980). The physiological significance of TNFot secretion by testicular macrophages remains to be established. While LH is generally thought to be the primary hormone involved in regulating testosterone production by Leydig cells, it should be noted that LH activity can be drastically reduced in culture in the presence of TNFo~ (Mauduit et al., 1991). Thus only when the entire spectrum of autocrine, paracrine and endocrine regulatory mechanisms is understood, can conclusions concerning relative importance of any one signal be appreciated. However, the present studies together with previous findings indicating that Leydig cells respond to TNFc~ (Calkins et al., 1990; Mauduit et al., 1991) provide additional information supporting the hypothesis that TNFot from testicular macrophages plays a role in Leydig cell regulation.
Acknowledgements The pituitary hormones were generous gifts from the NIDDK National Pituitary Program. This work was supported by a grant from the NICHD (HD26733).
References Baarsch, M.J., Wannemuehler, M.J., Molitor, T.W. and Murtaugh, M.P. (1991) Detection of tumor necrosis factor a from porcine alveolar macrophages using an L929 fibroblast bioassay. J. Immunol. Methods 140, 15-22. Calkins, J.H., Guo, H., Sigel, M.M. and Lin, T. (1990) Tumor necrosis factor-or enhances inhibitory effects of interleukin-I/3 on Leydig cell steroidogenesis. Biochem. Biophys. Res. Commun. 166, 1313-1318. Carswell, E.A., Old, L.J., Kassel, R.L., Green, S., Fiore, N. and Williamson, B. (1975) An endotoxininduced serum factor that causes necrosis of tumors. Proc. Natl. Acad. Sci. USA 72, 3666-3670. Christensen, A.K. and Gillim, S.W. (1969) The correlation of fine structure and function in steriodsecreting cells, with emphasis on those of the gonads. In: The Gonads (McKerns, K.W. ed.), pp. 415-488. Appleton-Century-Crofts, New York. Clark, R.V. (1976) Three-dimensional organization of the testicular interstitial tissue and lymphatic space in the rat. Anat. Rec. 184, 203-225. Connell, C.J. and Christensen, A.K. (1975) The ultrastructure of the canine testicular interstitial tissue. Biol. Reprod. 12, 368-382. Fawcett, D.W., Neaves, W.B. and Flores, M.N. (1973) Comparative observations on intertubular lymphatics and the organization of the interstitial tissue of the mammalian testis. Biol. Reprod. 9, 500-532. Feig, L., Bellve, A.R., Erickson, N.H. and Klagsbrun, M. (1980) Sertoli cells contain a mitogenic polypeptide. Proc. Natl. Acad. Sci, USA 77, 4774-4778.
72 Francis, G., Newman, R., Betz, J. and Poth, M. (1991) Macrophage conditioned media but not tumor necrosis factor inhibits in vitro testosterone production by rat Leydig cell enriched cultures. Abstract presented at the 73 annual meeting of the Endocrine Society, June 19-22, p. 52. Hutson, J.C. (1988) Leydig cells do not have Fc receptors. J. Androl. 10, 159-165. Hutson, J.C. (! 990) Changes in the concentration and size of testicular macrophages during development. Biol. Reprod. 43, 885-890. Hutson, J.C. (1992) Development of interdigitations between Leydig cells and macrophages. Cell Tissue Res. 267, 385-389. Hutson, J.C. and Stocco, D.M. (1989) Comparison of cellular and secreted proteins of macrophages from the testis and peritoneum on two-dimensional polyacrylamide gels. Regional Immunol. 2, 249-253. Kriegler, M., Perez, C., DeFay, K., Albert, I. and Lu, S.D. (1988) A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: Ramifications for the complex physiology of TNF. Cell 53, 45-53. Miller, S.C., Bowman, B.M. and Rowland, H.G. (1983) Structure, cytochemistry, endocytic activity and immunoglobulin (Fc) receptors of rat testicular interstitial-tissue macrophages. Am. J. Anat. 168, 1-13. Miller, S.C., Bowman, B.M. and Roberts, L.K. (1984) Identification and characterization of mononuclear phagocytes isolated from rat testicular interstitial tissues. J. Leuk. Biol. 36, 679-687. Mauduit, C., Hartman, D.J., Chauvin, M.A., Revol, A., Morera, A.M. and Benahmed, M. (1991) Tumor necrosis factor t~ inhibits gonadotropin action in cultured porcine Leydig cells: Site(s) of action. Endocrinology 129, 2933-2940. Raburn, D.J., Coquelin, A. and Hutson, J.C. (1991) Human chorionic gonadotropin increases the concentration of macrophages in neonatal rat testis. Biol. Reprod. 45, 172-177. Sinha, A.A., Erickson, A.W. and Seal, U.S. (1977) Fine structure of Leydig cells in crabeater, leopard and ross seals. J. Reprod. Fertil. 49, 51-54. Tung, P.S., Skinner, M.K. and Fritz, I.B. (1984) Fibronectin synthesis is a marker for peritubular cell contaminants in Sertoli cell-enriched cultures. Biol. Reprod. 30, 199-211. Warren, D.W., Pasupuleti, V., Lu, Y., Platler, B.W. and Horton, R. (1990) Tumor necrosis factor and interleukin-I stimulate testosterone secretion in adult male rat Leydig cells in vitro. J. Androl. 11, 3530-360. Wei, R.Q., Yee, J.B., Straus, D.C. and Hutson, J.C. (1988) Bactericidal activity of testicular macrophages. Biol. Reprod. 38, 830-835. Wing, T.Y. and Lin, H.S. (1977) The fine structure of testicular interstitial cells in the adult golden hamster with special reference to seasonal changes. Cell Tissue Res. 183, 385-393. Yee, J.B. and Hutson, J.C. (1985a) Effects of testicular macrophage-conditioned medium on Leydig cells in culture. Endocrinology 116, 2682-2684. Yee, J.B. and Hutson, J.C. (1985b) Biochemical consequences of FSH binding to testicular macrophages. Biol. Reprod. 32, 872-879. Yee, J.B. and Hutson, J.C. (1985c) In vivo effects of FSH on testicular macrophages. Biol. Reprod. 32, 880-883.
63
JRI 00799
Secretion of tumor necrosis factor alpha by testicular macrophages J a m e s C. H u t s o n Department of Cell Biology and Anatomy, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 (USA) (Accepted for publication 14 August 1992)
Summary While it has been shown that culture medium from testicular macrophages can influence testosterone production when added to Leydig cells, the identity of the factor(s) responsible for this activity remains unknown. Since tumor necrosis factor ct (TNFo0 has been shown to be capable of influencing testosterone production by Leydig cells, a series of studies was conducted to determine if testicular macrophages produce TNFo~. It was found that testicular macrophages from adult rats produce a factor that is capable of lysing L929 cells, which are used as a traditional bioassay for TNFot. The TNFo~ activity in the macrophage-conditioned medium could be neutralized by the addition of anti-murine TNFa but not with the addition of preimmune IgG. While lipopolysaccharide (LPS) slightly increased the release of TNFct, neither follicle-stimulating hormone (FSH) nor testosterone had a similar effect. It was not determined if the isolation procedure had artificially 'activated' the macrophages. Medium from cultured Sertoli cells, Leydig cells and peritubular cells did not contain TNFot activity. These studies are consistent with the hypothesis that the paracrine interaction between testicular macrophages and Leydig cells is mediated in part by TNFet. Key words:
TNF alpha; rnacrophages; testis; Leydig cells; cytokines
Introduction Macrophages are relatively numerous in the interstitium of the testis Correspondence to: James C. Hutson, Department of Cell Biology and Anatomy, Texas Tech University Health Sciences Center, Lubbock, TX, USA. 0165-0378/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
64
(Christensen et al., 1969; Fawcett et al., 1973; Connell and Christensen, 1975; Clark, 1976; Sinha et al., 1977; Wing and Lin, 1977). The cells are thought to be authentic macrophages in that they: exhibit typical macrophage morphology, have Fc receptors, secrete superoxide anion and lysozyme, can phagocytose and kill pathogenic bacteria, rapidly attach to culture substrates, and possess specific macrophage antigens (Miller et al., 1983, 1984; Hutson, 1988, 1990; Wei et al., 1988). These cells, however, appear to be involved in functions beyond traditional host defence mechanisms characteristic of most macrophages. For example, macrophages appear in the testis in a temporal manner during the period of rapid and dramatic testicular development and maturation (Hutson, 1990). Interestingly, the increase in size of the macrophage population that occurs during this time can be induced to occur precociously by exogenous hCG (Raburn et al., 1991). Once the concentration of macrophages in the interstitium reaches equilibrium, Leydig cells then begin to form membrane digitations with macrophages just prior to the major steroidogenic activity associated with puberty (Hutson, 1992). These digitations are composed of cytoplasmic extensions of the Leydig cells inserted into coated channels of the macrophage (Christensen et al., 1969; Hutson, 1992). It appears that these two cell types are also functionally coupled since testicular macrophages release a factor(s) in culture that alters the secretion of testosterone by Leydig cells (Yee and Hutson, 1985a). While the factors responsible for this effect on Leydig cells have not been identified, it has been reported that TNFa, a known macrophage product, can alter the release of testosterone from Leydig cells (Calkins et al., 1990; Warren et al., 1990; Mauduit et al., 1991). However, the physiological significance of these results remains in question since: (1) testicular macrophages are known to secrete many proteins electrophoretically different than those of macrophages from other anatomical sites (Hutson and Stocco, 1989), (2) macrophages generally do not produce TNFc~ unless stimulated by endotoxin or other activating factors (Carswell et al., 1975) and (3) peritoneal macrophages, which are known to produce TNFc~, do not influence basal testosterone secretion by Leydig cells (Francis et al., 1991). Thus, the purpose of the present study was to determine the feasibility of the hypothesis that TNFa from testicular macrophages is involved in the interaction with Leydig cells by directly testing if they produce TNFo~. Materials and Methods Animals
Wistar Kyoto (WKY) inbred rats originally obtained from Charles River Breeding Laboratories (Wilmington, MA) were maintained as a local
65
breeding colony in a controlled environment within our institutional animal care facility under the direction of a licensed veterinarian. The animals were housed in plastic cages with wood shavings as bedding and provided rat chow and water continuously. The experimental protocol was reviewed and approved by an institutional animal care and use committee.
Isolation of macrophages Five rats/experiment were killed by cervical dislocation and the testes removed and perfused with phosphate-buffered saline (without calcium and magnesium, PBS) to remove contaminating blood cells. The testes were gently decapsulated without cutting seminiferous tubules and then digested in a solution of collagenase (Sigma Type I, 100 units/ml in 50 ml PBS). The digestion took place at 34°C in a shaking water bath at 120 cycles/min for 20 min in a plastic Erlenmeyer 250 ml flask (Coming Glass Works, Coming, NY). At the end of the digestion, the flask was inverted several times until the tubules loosened but remained attached at the rete testis. The flask was then placed on ice for 2 min to allow the tubules to settle. The supernatant, containing the crude interstitial cells, was then centrifuged at 400 x g for 10 min at 4 ° C. The cells were suspended in PBS acclimated to 34°C and plated into 60-mm Coming Petri dishes (Coming Glass Works, Corning, NY). The dishes were placed on a glass plate (commonly used for cooling an electrophoresis gel) through which water was circulated to maintain the surface temperature at 34°C. After 4 min, the unattached cells were removed by vigorous washings with PBS. Three milliliters of complete medium was then added (Dulbecco's modified Eagle's medium, containing 2.2 g/1 sodium bicarbonate, 10 mM HEPES, 10% horse serum (donor herd), 100 units/ml penicillin and 100/~g/ml streptomycin, (all from Sigma Chemical Co., St. Louis, MO). The pH of this medium was strictly controlled between 7.0 and 7.4 at all times. Preliminary studies indicated that testicular macrophages are seriously influenced by extremes of pH. Macrophages prepared by this technique are greater than 95°/~ positive for Fc receptor, secrete superoxide anion and lysozyme and phagocytose and kill pathogenic bacteria (Hutson, 1988; Wei et al., 1988). The cells were then treated for 24 h with 100 ng/ml oFSH-17 (N1DDK), 100 ng/ml testosterone (Sigma Chemical Co., St. Louis, MO) or 1 /~g/ml LPS (Sigma Chemical Co., St. Louis, MO) for 24 h at 34°C in a humidified atmosphere containing 95% air and 5% CO2. The medium was then filtered (0.2 #m) and either stored frozen at -16°C or tested immediately in the TNFot bioassay as described below.
Isolation of Sertoli cells and peritubular cells Sertoli cells and peritubular cells were isolated from testes of six 20-23day-old Kyoto pups per experiment as previously described (Tung et al.,
66
1984). Briefly, the testes were decapsulated and sequentially digested in 0.25% trypsin, 0.1% collagenase and 0.1% hyaluronidase (Sigma Chemical Co., St. Louis, MO). The Sertoli cell preparation was taken from the final hyaluronidase digestion while the peritubular cells were obtained in the collagenase supernatant. The Sertoli cells were plated into 60-mm Petri dishes (Corning Glass Works, Corning, NY) and cultured in 3 ml complete medium without serum but with and without 100 ng/ml oFSH-23. The peritubular cells were plated similarly except that 10% defined bovine calf serum (Hyclone, Logan, UT) was added. Peritubular cells were treated with or without 100 ng/ml testosterone (Sigma Chemical Co., St. Louis, MO). Sertoli cells and peritubular cells were maintained in culture as described above for testicular macrophages.
Isolation of Leydig cells Leydig cells were isolated from testes of 4 adult rats/experiment as previously described (Hutson, 1988). Briefly, the testes were digested in 50 ml of collagenase (100 units/ml) in PBS and the cells in the supernatant fractionated on Percoll density gradients. Leydig cells found in the most dense band (those located just above band of red cells at a density of 1.09-1.12) were used in these studies. Preparations obtained by this technique are approximately 80-85% positive for 3/3-steroid dehydrogenase activity. The cells were plated into 35-mm Petri dishes (Primaria, Falcon Plastics, Oxnard, CA) in 2 ml complete medium with or without 100 ng/ml o-LH. Cells were maintained in culture as described above. TNFa assay This assay was performed as previously described based on the known sensitivity of L929 cells to TNFo~ (Baarsch et al., 1991). L929 cells (American Type Culture Collection, Rockville, MD) were plated in 96-well plates (Corning Glass Works, Corning, NY) on the morning of the assay at a concentration of 10 000 cells/well in 200/A complete medium. At the beginning of the assay, the medium was replaced with control medium (Dulbecco's modified Eagle's medium plus 1% horse serum (Sigma Chemical Co., St. Louis, MO, donor herd), 2.2 g/1 sodium bicarbonate, 10 mM HEPES, 100 units/ml penicillin and 100 t~g/ml streptomycin and 3 /~g/ml actinomycin D (Sigma Chemical Co., St. Louis, MO) plus various dilutions of medium previously exposed to testicular macrophages, Sertoli cells, peritubular cells or Leydig cells. Test substances (FSH, LH, testosterone and LPS) and control medium that had not been exposed to cells were also tested individually. Medium from macrophages was also incubated with rabbit polyclonal anti-murine recombinant TNFot (10 #g/ml) or preimmune IgG (10 #g/ml) (Endogen, Boston, MA) for 48 h at 4°C prior to testing in the assay.
67
The L929 cells were then incubated in the presence of dilutions of the conditioned media or test substances at 37°C for 20-24 h. MTT (3-(4,5dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) (Sigma Chemical Co., St. Louis, MO) was then added at a concentration of 2.5 mg/ml PBS and the cells incubated an additional 1 h at 37°C in a humidified atmosphere of 100% air. MTT is a water-soluble yellow tetrazolium salt that is converted by mitochondrial dehydrogenases only by living cells to a water-insoluble blue precipitate. The MTT was then removed and the blue precipitate solubilized with ethanol. Cell viability was then assessed spectrophotometrically at -4550. The percentage viable cells was calculated by comparison with cells treated only with control medium. Cell viability was also assessed microscopically by determining the percentage of cells containing blue reaction product. Statistical methods The means of the various treatment groups were compared for significant differences (95% confidence level) using a computer program for analysis of variance and Scheffe's F-test (StatView SE+, Abacus Concepts, Inc. Berkeley, CA). Results
It was found that conditioned medium from testicular macrophages had TNFot activity and induced optimal lysis of L292 cells at 12-24 h depending upon the density of the macrophage cultures (Fig. 1). The L929 cells exposed 100
80 o~
60
~ ~
40
~'~
20
~g
0
,
HOURS Fig. 1. Testicular macrophage conditioned medium was diluted 1:]0 and added to L929 cells for various times. The ceils were then j~ubat~d with M l " r and the percentage of viable (blue) cells determined
microscopically.
68
to macrophage-conditioned medium changed from firmly-attached slightly flattened fibroblastic cells to poorly-attached rounded cells over this time (Fig. 2). Therefore, the assay was subsequently terminated between 18 and 20 h. It was found in these pilot studies that viable cell number correlated well with absorbance at A550 as previously reported (Baarsch et al., 1991). Therefore, we used results from the spectrophotometric assay to illustrate subsequent studies. The lytic activity of testicular macrophage-conditioned medium was not significantly stimulated by addition of FSH or testosterone, while LPS had a very slight but statistically significant influence (Fig. 2). These factors had no influence on viability of L929 cells when tested alone as vehicle controls. Lytic activity of medium from macrophages was significantly reduced by addition of rabbit anti-mouse recombinant TNFot but not by the addition of preimmune IgG (Fig. 3). Preliminary studies also indicated that a monoclonal antibody to recombinant human TNFot was unable to neutralize the lyric activity of the rat TNFot activity from testicular macrophages in accordance with known species specificity (data not illustrated). Conditioned medium from Sertoli cells, Leydig cells and peritubular cells did not contain T N F a activity (Fig. 4). Conversely, conditioned medium from both peritubular cells and Leydig cells stimulated growth of L929 cells in the presence of actinomycin D (Fig. 4). The addition of testosterone or LH to peritubular cells or Leydig cells, respectively, had no influence on proliferation of L929 cells.
100 o
g
75-
o 50-
25
0 o
P, M a c r o p h a g e Conditioned M e d i u m
Fig. 2. This graph illustrates the effects of various test substances on the production of TNFc~ by testicular macrophages treated with 1 #g/ml LPS, 100 ng/ml testosterone (T), or 100 ng/ml follicle-stimulating hormone (FSH). LPS, FSH and T, in the absence of macrophage conditioned medium, were also tested (3 bars to the far right o f the graph). Values represent the mean ± S.E.M. of four culture dishes• Asterisks indicate a statistical significant difference from the control at P >_ 0.05.
69 125" 100 ¢j
1 1111 1111
75
50
25 0
~
~
.~..c2.
~: 4: 4-: T . . ~.
Preimmune IgG
Anti-TNF alpha
i
o
i
I
Macrophage Conditioned Medium Fig. 3. The neutralizing influence of 10 t~g/ml a n t i - T N F or preimmune I g G on TNFc~ activity. Values are the mean ± S.E.M. (n = 4). Asterisks indicate a statistical significant difference from the control at P _> 0.05.
Discussion Three important parameters are necessary to establish that a single factor such as TNFo~ is an important factor in a complex mixture of factors such as conditioned medium from macrophages. First, addition of the factor by
175
-
150 m
o
125
~o o~
0 ,:.: ,:.: .~.
o
~H I
i
Sertoli cells
"~ ".: .. I
I
--
•
I
.~. J
LH I
Peritubular cells
,:.: ~ I
T I
...~.
I
I
Leydig cells
Fig. 4. The cellular specificity of TNFc~ secretion by various testicular cell types. Sertoli cells, peritubular cells and Leydig cells were cultured for 24 h and the medium assayed for T N F a activity. Values are the mean ± S.E.M. (n = 4). Asterisks indicate a statistical significant difference from the control at P _ 0.05.
70
itself should produce a response similar to the response observed in the conditioned medium. Secondly, it should be demonstrated that the putative factor is present within the conditioned medium. And finally, removal or neutralization of the factor from the conditioned medium should reduce the activity of the medium. The first criterion, that purified TNFc~ has an influence on Leydig cells, has been established previously (Calkins et al., 1990; Warren et al., 1990; Mauduit et al., 1991). The results of the present study satisfy the second criterion, that the conditioned medium in fact contains TNFo~. The final criterion, to demonstrate a change in the activity of conditioned medium by removal of TNFo~ remains to be completed. It was surprising to find that LPS had little influence on the secretion of TNFot since endotoxin has been shown to have a stimulatory action on macrophages from other tissues (Carswell et al., 1975; Francis et al., 1991). It is possible that the testicular macrophage is artificially activated by the isolation procedure even though care was taken to be sure the solutions used in isolating and culturing the macrophages were sterile and low in endotoxin. However, collagenase or other unknown factors could have 'activated' these cells. It was also interesting to learn that FSH had no activity on TNFo~ secretion since FSH has been shown to bind to specific receptors on testicular macrophages and elicit changes in amino acid incorporation into acid precipitable material (Yee and Hutson, 1985b,c). The rationale for testing the effects of testosterone on the secretion of TNFot was based on the possibility that a feedback loop may exist between macrophages and Leydig cells. That is, the secretion of TNFc~ by macrophages might have altered testosterone production by Leydig cells which would in return modify macrophage activity. While such a loop was not found for TNFot and testosterone, other factors may be involved. TNFot exists in two forms; one is associated with the cytoplasmic membrane and the other is soluble (Kriegler et al., 1988). The present findings tested only the TNFc~ that was released into the culture medium. It is tempting to speculate that the bound form is also involved in influencing Leydig cells in vivo since the testicular macrophage and Leydig cell are so closely associated in situ. The presence of the membrane digitations between these two cells (Christensen et al., 1969; Hutson, 1992) would be a strategic site for such a mechanism to exist. The secretion of TNFot appeared to be specific to macrophages in that Sertoli cells, peritubular cells and Leydig cell did not secrete TNFot. However, it should be pointed out that the Sertoli cells and peritubular cells were isolated from immature animals while the macrophages were isolated from adult animals. It is yet to be determined if the secretion of TNFot is agedependent in any of the cell types tested. The unexpected observation that peritubular cells and Leydig cells produce a factor or factors that increase the
71
number of L929 cells is intriguing. It may be possible that this activity is due to the production of one or several growth factors by these cells. It is interesting that Sertoli cells lacked similar activity since they have been shown to produce a factor capable of stimulating the proliferation of other cell lines (Feig et al., 1980). The physiological significance of TNFot secretion by testicular macrophages remains to be established. While LH is generally thought to be the primary hormone involved in regulating testosterone production by Leydig cells, it should be noted that LH activity can be drastically reduced in culture in the presence of TNFo~ (Mauduit et al., 1991). Thus only when the entire spectrum of autocrine, paracrine and endocrine regulatory mechanisms is understood, can conclusions concerning relative importance of any one signal be appreciated. However, the present studies together with previous findings indicating that Leydig cells respond to TNFc~ (Calkins et al., 1990; Mauduit et al., 1991) provide additional information supporting the hypothesis that TNFot from testicular macrophages plays a role in Leydig cell regulation.
Acknowledgements The pituitary hormones were generous gifts from the NIDDK National Pituitary Program. This work was supported by a grant from the NICHD (HD26733).
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72 Francis, G., Newman, R., Betz, J. and Poth, M. (1991) Macrophage conditioned media but not tumor necrosis factor inhibits in vitro testosterone production by rat Leydig cell enriched cultures. Abstract presented at the 73 annual meeting of the Endocrine Society, June 19-22, p. 52. Hutson, J.C. (1988) Leydig cells do not have Fc receptors. J. Androl. 10, 159-165. Hutson, J.C. (! 990) Changes in the concentration and size of testicular macrophages during development. Biol. Reprod. 43, 885-890. Hutson, J.C. (1992) Development of interdigitations between Leydig cells and macrophages. Cell Tissue Res. 267, 385-389. Hutson, J.C. and Stocco, D.M. (1989) Comparison of cellular and secreted proteins of macrophages from the testis and peritoneum on two-dimensional polyacrylamide gels. Regional Immunol. 2, 249-253. Kriegler, M., Perez, C., DeFay, K., Albert, I. and Lu, S.D. (1988) A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: Ramifications for the complex physiology of TNF. Cell 53, 45-53. Miller, S.C., Bowman, B.M. and Rowland, H.G. (1983) Structure, cytochemistry, endocytic activity and immunoglobulin (Fc) receptors of rat testicular interstitial-tissue macrophages. Am. J. Anat. 168, 1-13. Miller, S.C., Bowman, B.M. and Roberts, L.K. (1984) Identification and characterization of mononuclear phagocytes isolated from rat testicular interstitial tissues. J. Leuk. Biol. 36, 679-687. Mauduit, C., Hartman, D.J., Chauvin, M.A., Revol, A., Morera, A.M. and Benahmed, M. (1991) Tumor necrosis factor t~ inhibits gonadotropin action in cultured porcine Leydig cells: Site(s) of action. Endocrinology 129, 2933-2940. Raburn, D.J., Coquelin, A. and Hutson, J.C. (1991) Human chorionic gonadotropin increases the concentration of macrophages in neonatal rat testis. Biol. Reprod. 45, 172-177. Sinha, A.A., Erickson, A.W. and Seal, U.S. (1977) Fine structure of Leydig cells in crabeater, leopard and ross seals. J. Reprod. Fertil. 49, 51-54. Tung, P.S., Skinner, M.K. and Fritz, I.B. (1984) Fibronectin synthesis is a marker for peritubular cell contaminants in Sertoli cell-enriched cultures. Biol. Reprod. 30, 199-211. Warren, D.W., Pasupuleti, V., Lu, Y., Platler, B.W. and Horton, R. (1990) Tumor necrosis factor and interleukin-I stimulate testosterone secretion in adult male rat Leydig cells in vitro. J. Androl. 11, 3530-360. Wei, R.Q., Yee, J.B., Straus, D.C. and Hutson, J.C. (1988) Bactericidal activity of testicular macrophages. Biol. Reprod. 38, 830-835. Wing, T.Y. and Lin, H.S. (1977) The fine structure of testicular interstitial cells in the adult golden hamster with special reference to seasonal changes. Cell Tissue Res. 183, 385-393. Yee, J.B. and Hutson, J.C. (1985a) Effects of testicular macrophage-conditioned medium on Leydig cells in culture. Endocrinology 116, 2682-2684. Yee, J.B. and Hutson, J.C. (1985b) Biochemical consequences of FSH binding to testicular macrophages. Biol. Reprod. 32, 872-879. Yee, J.B. and Hutson, J.C. (1985c) In vivo effects of FSH on testicular macrophages. Biol. Reprod. 32, 880-883.