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A local immuno-endocrine interaction may mediate rat adrenal glucocorticoid response to bacterial endotoxins
PII s0024-3205(98)00140-4
ELSEVIER
LifeSciences,Vol.62. No. 19.pp. 1783-1787, 19913 copyright0 1998ElsevierscienceInc. Printedin the USA. All tightsreserved 00%3205/98-$19.00 t .OO
A LOCAL IMMUNO-ENDOCRINE INTERACTION MAY MEDIATE RAT ADRENAL GLUCOCORTICOID RESPONSE TO BACTERIAL ENDOTOXINS Giuseppina Mazzocchi, Giuseppe Gottardo and Gastone G. Nussdorfer Department
of Anatomy, University of Padua, I-3 5 12 1 Padua, Italy
(Received in final form February 6, 1998)
Summary The effects of the bacterial endotoxin lipopolysaccharide (LPS) and interleukin (IL)-lp on corticosterone secretion has been studied in IWO by employing the technique of in situ perfUsion of the isolated rat left adrenal gland. Both LPS and IL- 1p dose-dependently raised corticosterone output, the response peacking at 60 and 90 min, respectively. IL-l receptor antagonist dose-dependently reversed the effect of LPS and IL-lb. The IL-l p converting enzyme (ICE) inhibitor AcWAD-CMK annulled the adrenal response to LPS, but did not affect that to ILlp. Collectively, these findings provide evidence that LPS, by enhancing adrenal production of IL- 1p, is able to evoke a sizable glucocorticoid response in the rat, thereby suggesting that local immuno-endocrine interactions may be operative in the adrenal gland of this species. Key Words: lipopolysaccharide, interleukin-lp, steroidogenesis, adrenals
involving the Compelling evidence indicates that immuno-neuroendocrine interactions, hypothalamo-pituitary CRI-VACTH system, play a crucial role in the modulation of adrenal secretory responses to inflammatory stresses (for review, see l-4). In recent years many findings have accumulated suggesting the existence, at least in the rat, of immune-endocrine interactions also at the adrenal level. Adrenal glands express interleukin (IL)-1 (5,6), and IL-l has been found to enhance in vitro production of corticosterone by adrenocortical cells (7-10). There are studies which show that lipopolysaccharide (LPS) raises IL-l immunoreactivity in rat adrenals (1 l), and LPS administration has been shown to evoke a sizable corticosterone response in hypophysectomized rats (12). The recent availability of purified IL- 1 receptor antagonist (IL- 1ra) and specific inhibitors of IL-l p converting enzyme (ICE) prompted us to check this possibility by employing the technique of in situ perfUsion of the isolated rat adrenal gland (13). In fact, this technique allows for the delivery of the chemicals directly to the adrenal gland and the study of their effects in vivo without any possible interference with other systemic regulatory mechanisms (e.g. the hypothalamo-pituitary axis).
Correspondence: Dr. G.G. Nussdorfer, Department of Anatomy, Ma Gabelli 65, I-35 121 Padova Italy
1x34
Interleukins
and Adrenals
Vol. 62, No. 19, 1998
Materials and methods Adult male Wistar rats (260 i 30 g BW) were obtained from Charles-River (Como, Italy) IL-ID and IL-lra were purchased from R & D Systems (Minneapolis, MN), the specific irreversible inhibitor of ICE AC-YVAD-CMK (ICE-I) (14) from Neosystem Laboratoires (Strasbourg, France), medium 199 from Difco (Detroit, MI), and LPS (from E.schevrchiu co//, serotype 0111.84, -10,000 U endotoxin/mg), human serum albumin (HSA) and other laboratory reagents from Sigma Chemical Co (St. Louis, MO) The rats were anesthesized with Nembutal, and their left adrenal gland was perfUsed /rl .sttu, as previously detailed (15) Briefly, perfusion medium was introduced via a cannula inserted in the celiac artery into an isolated segment of aorta from which the adrenal arteries arise, after flowing through the adrenal gland, medium was collected by a cannula inserted in the renal vein Once perfusion had begun the animal was killed, and thereafter was used only as a platform for the adrenal preparation. PerfUsion medium [medium 199 and Krebs-Ringer bicarbonate buffer (2 1, vol/vol), containing 0 2% glucose and 5 mg/ml HSA] was gassed with 95% 02 and 5% COZ, maintained at 37”C, and delivered at a constant rate of 2 ml/10 min After an initial equilibration period of 20 min, three IO-min samples were collected, then chemicals dissolved in 200 ~1 of medium were injected in the arterial perfUsion cannula, and further nine or twelve IO-min samples were collected The following chemicals were injected (i) LPS (from 0 5 to 2 0 ctg), (ii) IL- I f3 (from 1 to 1000 pg), (iii) LPS (1 5 pg) or IL-ID (100 pg) alone and in the presence of IL-lra (from 10 to 1000 pg) or ICE-I (from 10 to 1000 pg) Steroid hormones were extracted from the perfUsion medium, and corticosterone was purified by HPLC, as previously described (16) Corticosterone concentration in the IO-min samples was measured by RIA, as described earlier (17) Sensitivity, 25 pg/ml; in&a- and interassay variations, 7% and 9%, respectively. For each rat the rate of corticosterone output was calculated as the average of three consecutive IO-min collection periods (30-min samples) before (baseline) and after the injection of the chemicals In the dose-response studies, corticosterone output was the average of the first three 30-min post-injection samples, while in the time-response studies it was measured separately in four 30-min post-injection samples. For each rat, corticosterone response was expressed as percent change from the respective baseline value. For each experimental point, 5 rat adrenals were perfUsed and data were calculated as means i SEM. The statistical comparison of results was done by ANOVA, followed by the Duncan’s multiple range test Results The bolus administration of both LPS and IL- 10 raised corticosterone release by perfUsed adrenals in a dose-dependent manner, maximal effective doses being 1 5 pg and 100 pg, respectively (Fig 1) Corticosterone response to I 5 pg LPS was significant at 60 min and peacked at 90 min, while that to 100 pg IL-ID was already high at 30 min and reached its maximum at 60 min (Fig. 2) ILlra and ICE-I dose-dependently counteracted the LPS (1 5 pg)-evoked corticosterone response, and annulled it at a dose of 1 ng (Fig 3A). The secretory response to IL-I p ( 100 pg) was reversed by IL-lra, but not affected by ICE-I (Fig. 3B)
Vol. 62, No. 19, 1998
Interleukins
and Adrenals
corticosterone
output(%)
*
* 400-
*
. .
. *
*. J *
300-
’ 200-
1oo-
I
*
P
LPS (at) or IL-lb (pg:
I
0
I
I
0.5
1 .o
1.5
1
IO
100
,
2.0 0 10000
Fig. 1. Dose-related effects of LPS (0) and IL-1 p (0) l on corticosterone output from I?? situ perked rat adrenal glands. Data are means rt SEM (n = 5). *P
corticosterone
*
output (%) 400
2oc
ioa time after LPS or IL-I p injection (min
0
30
60
I
90
’ 120
Fig. 2. Time-related effects of the maximal effective dose of LPS (0) and IL-l 0 (@) on corticosterone output from in situ perfksed rat adrenal glands. Data are means k SEM (n = 5). *P
1785
1786
Interleukins
and Adrenals
Vol. 62. No. 19, 1998
400.
300.
200.
1 oo-
A
I B
C
I 10
I 100
IL-lra (WI lB I 1000
B
C
10
8 100
ICE-I(w) I 1000
Fig 3 Dose-related effects of IL-lra (panel A) and ICE-I (panel B) on the raise in corticosterone output from 111SIW perfused rat adrenal glands, evoked by the maximal effective dose of LPS (0) and IL-ID (a) (control groups) Data are means * SEM (n = 5) *P
Vol. 62, No. 19, 1998
Interleukins
and Adrenals
1787
glucocorticoid response to mild infective stresses, overcoming the difficulties related to the fact that blood-borne bacterial endotoxins must cross the blood-brain barrier to penetrate in the hypothalamus and activate CRH/ACTH release. References 1. 2. 3. 4. 5.
R-C. GAILLARD, Trends Endocrinol. Metab. 5 303-309 (1994). N.C. VAMVAKOPOULOS and G.P. CHROUSOS, Endocr. Rev. s 409-420 (1994) B.L. SPANGELO and W.C. GOROSPE, Front. Neuroendocrinol. j$ l-22 (1995). H.O. BESEDOVSKY and A. DEL REY, Endocr. Rev. 11 64-102 (1996). M.G. DE SIMONI, R. DEL BO, A. DE LUIGI, S. SIMARD and G FORLONI, Endocrinology 136 897-902 (1995). 6. M. SCHULTZBERG, S. TINGSBORG, S. NOBEL, J. LUNDKVIST, S. SVENSON, A SIMONCSITZ and T. BARTFAI, J. Interf Cytokine Res. B 721-729 (1995). 7. P.G. ANDREIS, G. NERI, A.S. BELLONI, G. MAZZOCCHI, A. KASPRZAK and G G NUSSDORFER, Endocrinology 129 53-57 (1991). 8. A.R. GWOSDOW, N.A. O’CONNELL, J.A. SPENCER M.S.A. Km R.K. AGARWAL, H.H.BODE and A.B. ABOU-SAMRA, Am. J. Physiol. 263 E461-E466 (1992). 9. N.A. O’CONNELL, A. KUMAR, K. CHATZIPANTELI, A. MOHAN, R.K. AGARWAL, C HEAD, S.R. BORNSTEIN, A.B. ABOU-SAMRA and A.R. GWOSDOW, Endocrinology 135 460-467 (1994). 10. M.J.M. VAN DER MEER, A.R.M.M. HERMUS, G.J. PESMAN and C.G.J. SWEEP, Cytokine 8 238247 (1996). 11. C. ANDERSSON, S.B. SVENSON, S. VAN DEVENTER A. CERAMI AND T. BARTFAI, Neuroscience 47 481-485 (1992). 12. G. MAZZOCCHI, S. ROCCO, L.K. MALENDOWICZ, P. REBUFFAT and G.G NUSSDORFER, Endocr. Res. U 525-536 (1995). 13. G.P. VINSON, J.P. HINS0NandP.W. RAVEN, Cell. Biochem. Funct. 3 235-253 (1985). 14. M. ENARI, H. HUG AND S. NAGATA, Nature 375 78-81 (1995). 15. G. MAZZOCCHI, G.P. ROSSI, P. REBUFFAT, L.K. MALENDOWICZ, A. MARKOWSKA and G.G. NUSSDORFER, Endocrinology 138 2333-2337 (1997). 16. G. NERI, L.K. MALENDOWICZ, P.G. ANDREIS and G.G. NUSSDORFER, Endocrinology 133_ 511-514 (1993). 17. L.K. MALENDOWICZ, G.G. NUSSDORFER A. MARKOWSKA K.W. NOWAK and T TORLINSKI, Neuropeptides 24 l-4 (1993). 18. CA. DINARELLO, Adv. Pharmacol. 2i 21-51 (1994). 19. J.L. BANKERS-FULBRIGHT, K.R. KALLI and D.J. MC KEAN, Life Sci 59 61-83 (1996). 20. D.K. MILLER, J. MYERSON and J.W. BECKER J. Cell Biochem. 64 2-10 (1997)
ELSEVIER
LifeSciences,Vol.62. No. 19.pp. 1783-1787, 19913 copyright0 1998ElsevierscienceInc. Printedin the USA. All tightsreserved 00%3205/98-$19.00 t .OO
A LOCAL IMMUNO-ENDOCRINE INTERACTION MAY MEDIATE RAT ADRENAL GLUCOCORTICOID RESPONSE TO BACTERIAL ENDOTOXINS Giuseppina Mazzocchi, Giuseppe Gottardo and Gastone G. Nussdorfer Department
of Anatomy, University of Padua, I-3 5 12 1 Padua, Italy
(Received in final form February 6, 1998)
Summary The effects of the bacterial endotoxin lipopolysaccharide (LPS) and interleukin (IL)-lp on corticosterone secretion has been studied in IWO by employing the technique of in situ perfUsion of the isolated rat left adrenal gland. Both LPS and IL- 1p dose-dependently raised corticosterone output, the response peacking at 60 and 90 min, respectively. IL-l receptor antagonist dose-dependently reversed the effect of LPS and IL-lb. The IL-l p converting enzyme (ICE) inhibitor AcWAD-CMK annulled the adrenal response to LPS, but did not affect that to ILlp. Collectively, these findings provide evidence that LPS, by enhancing adrenal production of IL- 1p, is able to evoke a sizable glucocorticoid response in the rat, thereby suggesting that local immuno-endocrine interactions may be operative in the adrenal gland of this species. Key Words: lipopolysaccharide, interleukin-lp, steroidogenesis, adrenals
involving the Compelling evidence indicates that immuno-neuroendocrine interactions, hypothalamo-pituitary CRI-VACTH system, play a crucial role in the modulation of adrenal secretory responses to inflammatory stresses (for review, see l-4). In recent years many findings have accumulated suggesting the existence, at least in the rat, of immune-endocrine interactions also at the adrenal level. Adrenal glands express interleukin (IL)-1 (5,6), and IL-l has been found to enhance in vitro production of corticosterone by adrenocortical cells (7-10). There are studies which show that lipopolysaccharide (LPS) raises IL-l immunoreactivity in rat adrenals (1 l), and LPS administration has been shown to evoke a sizable corticosterone response in hypophysectomized rats (12). The recent availability of purified IL- 1 receptor antagonist (IL- 1ra) and specific inhibitors of IL-l p converting enzyme (ICE) prompted us to check this possibility by employing the technique of in situ perfUsion of the isolated rat adrenal gland (13). In fact, this technique allows for the delivery of the chemicals directly to the adrenal gland and the study of their effects in vivo without any possible interference with other systemic regulatory mechanisms (e.g. the hypothalamo-pituitary axis).
Correspondence: Dr. G.G. Nussdorfer, Department of Anatomy, Ma Gabelli 65, I-35 121 Padova Italy
1x34
Interleukins
and Adrenals
Vol. 62, No. 19, 1998
Materials and methods Adult male Wistar rats (260 i 30 g BW) were obtained from Charles-River (Como, Italy) IL-ID and IL-lra were purchased from R & D Systems (Minneapolis, MN), the specific irreversible inhibitor of ICE AC-YVAD-CMK (ICE-I) (14) from Neosystem Laboratoires (Strasbourg, France), medium 199 from Difco (Detroit, MI), and LPS (from E.schevrchiu co//, serotype 0111.84, -10,000 U endotoxin/mg), human serum albumin (HSA) and other laboratory reagents from Sigma Chemical Co (St. Louis, MO) The rats were anesthesized with Nembutal, and their left adrenal gland was perfUsed /rl .sttu, as previously detailed (15) Briefly, perfusion medium was introduced via a cannula inserted in the celiac artery into an isolated segment of aorta from which the adrenal arteries arise, after flowing through the adrenal gland, medium was collected by a cannula inserted in the renal vein Once perfusion had begun the animal was killed, and thereafter was used only as a platform for the adrenal preparation. PerfUsion medium [medium 199 and Krebs-Ringer bicarbonate buffer (2 1, vol/vol), containing 0 2% glucose and 5 mg/ml HSA] was gassed with 95% 02 and 5% COZ, maintained at 37”C, and delivered at a constant rate of 2 ml/10 min After an initial equilibration period of 20 min, three IO-min samples were collected, then chemicals dissolved in 200 ~1 of medium were injected in the arterial perfUsion cannula, and further nine or twelve IO-min samples were collected The following chemicals were injected (i) LPS (from 0 5 to 2 0 ctg), (ii) IL- I f3 (from 1 to 1000 pg), (iii) LPS (1 5 pg) or IL-ID (100 pg) alone and in the presence of IL-lra (from 10 to 1000 pg) or ICE-I (from 10 to 1000 pg) Steroid hormones were extracted from the perfUsion medium, and corticosterone was purified by HPLC, as previously described (16) Corticosterone concentration in the IO-min samples was measured by RIA, as described earlier (17) Sensitivity, 25 pg/ml; in&a- and interassay variations, 7% and 9%, respectively. For each rat the rate of corticosterone output was calculated as the average of three consecutive IO-min collection periods (30-min samples) before (baseline) and after the injection of the chemicals In the dose-response studies, corticosterone output was the average of the first three 30-min post-injection samples, while in the time-response studies it was measured separately in four 30-min post-injection samples. For each rat, corticosterone response was expressed as percent change from the respective baseline value. For each experimental point, 5 rat adrenals were perfUsed and data were calculated as means i SEM. The statistical comparison of results was done by ANOVA, followed by the Duncan’s multiple range test Results The bolus administration of both LPS and IL- 10 raised corticosterone release by perfUsed adrenals in a dose-dependent manner, maximal effective doses being 1 5 pg and 100 pg, respectively (Fig 1) Corticosterone response to I 5 pg LPS was significant at 60 min and peacked at 90 min, while that to 100 pg IL-ID was already high at 30 min and reached its maximum at 60 min (Fig. 2) ILlra and ICE-I dose-dependently counteracted the LPS (1 5 pg)-evoked corticosterone response, and annulled it at a dose of 1 ng (Fig 3A). The secretory response to IL-I p ( 100 pg) was reversed by IL-lra, but not affected by ICE-I (Fig. 3B)
Vol. 62, No. 19, 1998
Interleukins
and Adrenals
corticosterone
output(%)
*
* 400-
*
. .
. *
*. J *
300-
’ 200-
1oo-
I
*
P
LPS (at) or IL-lb (pg:
I
0
I
I
0.5
1 .o
1.5
1
IO
100
,
2.0 0 10000
Fig. 1. Dose-related effects of LPS (0) and IL-1 p (0) l on corticosterone output from I?? situ perked rat adrenal glands. Data are means rt SEM (n = 5). *P
corticosterone
*
output (%) 400
2oc
ioa time after LPS or IL-I p injection (min
0
30
60
I
90
’ 120
Fig. 2. Time-related effects of the maximal effective dose of LPS (0) and IL-l 0 (@) on corticosterone output from in situ perfksed rat adrenal glands. Data are means k SEM (n = 5). *P
1785
1786
Interleukins
and Adrenals
Vol. 62. No. 19, 1998
400.
300.
200.
1 oo-
A
I B
C
I 10
I 100
IL-lra (WI lB I 1000
B
C
10
8 100
ICE-I(w) I 1000
Fig 3 Dose-related effects of IL-lra (panel A) and ICE-I (panel B) on the raise in corticosterone output from 111SIW perfused rat adrenal glands, evoked by the maximal effective dose of LPS (0) and IL-ID (a) (control groups) Data are means * SEM (n = 5) *P
Vol. 62, No. 19, 1998
Interleukins
and Adrenals
1787
glucocorticoid response to mild infective stresses, overcoming the difficulties related to the fact that blood-borne bacterial endotoxins must cross the blood-brain barrier to penetrate in the hypothalamus and activate CRH/ACTH release. References 1. 2. 3. 4. 5.
R-C. GAILLARD, Trends Endocrinol. Metab. 5 303-309 (1994). N.C. VAMVAKOPOULOS and G.P. CHROUSOS, Endocr. Rev. s 409-420 (1994) B.L. SPANGELO and W.C. GOROSPE, Front. Neuroendocrinol. j$ l-22 (1995). H.O. BESEDOVSKY and A. DEL REY, Endocr. Rev. 11 64-102 (1996). M.G. DE SIMONI, R. DEL BO, A. DE LUIGI, S. SIMARD and G FORLONI, Endocrinology 136 897-902 (1995). 6. M. SCHULTZBERG, S. TINGSBORG, S. NOBEL, J. LUNDKVIST, S. SVENSON, A SIMONCSITZ and T. BARTFAI, J. Interf Cytokine Res. B 721-729 (1995). 7. P.G. ANDREIS, G. NERI, A.S. BELLONI, G. MAZZOCCHI, A. KASPRZAK and G G NUSSDORFER, Endocrinology 129 53-57 (1991). 8. A.R. GWOSDOW, N.A. O’CONNELL, J.A. SPENCER M.S.A. Km R.K. AGARWAL, H.H.BODE and A.B. ABOU-SAMRA, Am. J. Physiol. 263 E461-E466 (1992). 9. N.A. O’CONNELL, A. KUMAR, K. CHATZIPANTELI, A. MOHAN, R.K. AGARWAL, C HEAD, S.R. BORNSTEIN, A.B. ABOU-SAMRA and A.R. GWOSDOW, Endocrinology 135 460-467 (1994). 10. M.J.M. VAN DER MEER, A.R.M.M. HERMUS, G.J. PESMAN and C.G.J. SWEEP, Cytokine 8 238247 (1996). 11. C. ANDERSSON, S.B. SVENSON, S. VAN DEVENTER A. CERAMI AND T. BARTFAI, Neuroscience 47 481-485 (1992). 12. G. MAZZOCCHI, S. ROCCO, L.K. MALENDOWICZ, P. REBUFFAT and G.G NUSSDORFER, Endocr. Res. U 525-536 (1995). 13. G.P. VINSON, J.P. HINS0NandP.W. RAVEN, Cell. Biochem. Funct. 3 235-253 (1985). 14. M. ENARI, H. HUG AND S. NAGATA, Nature 375 78-81 (1995). 15. G. MAZZOCCHI, G.P. ROSSI, P. REBUFFAT, L.K. MALENDOWICZ, A. MARKOWSKA and G.G. NUSSDORFER, Endocrinology 138 2333-2337 (1997). 16. G. NERI, L.K. MALENDOWICZ, P.G. ANDREIS and G.G. NUSSDORFER, Endocrinology 133_ 511-514 (1993). 17. L.K. MALENDOWICZ, G.G. NUSSDORFER A. MARKOWSKA K.W. NOWAK and T TORLINSKI, Neuropeptides 24 l-4 (1993). 18. CA. DINARELLO, Adv. Pharmacol. 2i 21-51 (1994). 19. J.L. BANKERS-FULBRIGHT, K.R. KALLI and D.J. MC KEAN, Life Sci 59 61-83 (1996). 20. D.K. MILLER, J. MYERSON and J.W. BECKER J. Cell Biochem. 64 2-10 (1997)