Effects of human recombinant tumor necrosis factor-alpha and interleukin 1 on the synthesis of glycosaminoglycan and DNA in cultured rat costal chondrocytes

We examined effects of human rTNF alpha on the synthesis of glycosaminoglycan and DNA in cultured rat costal chondrocytes. The effects of human recombinant IL-1 alpha and IL-1 beta were also given attention. rTNF alpha, as well as rIL-1 alpha and rIL-1 beta, decreased the incorporation of [35S]sulfate into glycosaminoglycan to about 10% of the levels in the control. The half-maximal doses of rTNF alpha, rIL-1 alpha or rIL-1 beta required for the suppression of glycosaminoglycan synthesis (by rTNF alpha, rIL-1 alpha, and rIL-1 beta) were 2 ng/ml, 30 ng/ml, or 5 ng/ml, respectively. rTNF alpha stimulated incorporation of [3H]thymidine in the chondrocytes in a dose- and time-dependent manner. DNA synthesis was increased to about threefold over the control cultures in the presence of 1 microgram/ml rTNF alpha for 72 hr. The stimulatory effect of rTNF alpha on DNA synthesis was observed in both subconfluent and confluent cultures, whereas rIL-1 alpha and rIL-1 beta had no stimulatory activity on DNA synthesis. The addition of rTNF alpha to the cultures of chondrocytes stimulated DNA synthesis, even in medium containing no fetal calf serum. The fetal calf serum acted synergistically with rTNF alpha in increasing DNA synthesis. We propose that both TNF and IL-1 may be involved in inflammatory diseases of cartilage, and that TNF alpha, but not IL-1, may have some physiologic growth factor function for chondrocytes.     

Cytokine activation of vascular endothelium. Effects on tissue-type plasminogen activator and type 1 plasminogen activator inhibitor

Regulation of the fibrinolytic system of cultured human umbilical vein endothelial cells (HUVECs) by recombinant interleukin 1 beta (rIL-1 beta) and tumor necrosis factor alpha (rTNF alpha) was investigated. Functional and immunologic assays indicated that both cytokines decreased HUVEC tissue-type plasminogen activator (tPA) and increased type 1 plasminogen activator inhibitor (PAI-1) in a dose- and time-dependent manner. Maximal effects (50% decrease in tPA antigen; 300-400% increase in PAI-1 activity) were achieved with 2.5 units/ml rIL-1 beta and 200 units/ml rTNF alpha. Combinations of rIL-1 beta and rTNF alpha were not additive at these maximal concentrations. After a 24-h pretreatment with rIL-1 beta, HUVECs secreted tPA at one-quarter of the rate of control cells and released PAI-1 at a rate that was 5-fold higher than controls. Neither the basal rate of PAI-1 release nor the increased rate of release of PAI-1 in response to rIL-1 beta was affected by subsequently treating the cells with secretagogues (e.g. phorbol myristate acetate) suggesting that PAI-1 is not contained within a rapidly releasable, intracellular storage pool. Northern blot analysis using a PAI-1 cDNA probe indicated that the cytokines increased the steady-state levels of the 3.2- and 2.3-kb PAI-1 mRNA species, but with a preferential increase in the larger mRNA form. The fact that both rIL-1 beta and rTNF alpha act in a similar manner strengthens the hypothesis that the local development of inflammatory/immune processes could reduce endothelial fibrinolytic activity.     

Interleukin 1 and tumor necrosis factor synergistically stimulate prostaglandin synthesis and phospholipase A2 release from rat renal mesangial cells

Treatment of rat glomerular mesangial cells with recombinant human interleukin 1 alpha (rIL-1 alpha), recombinant human interleukin 1 beta (rIL-1 beta) or recombinant human tumor necrosis factor (rTNF) induces prostaglandin E2 (PGE2) synthesis and the release of a phospholipase A2 (PLA2) activity. rIL-1 beta is significantly more potent than rIL-1 alpha or rTNF in stimulating PGE2 as well as PLA2 release from mesangial cells. When given together, rTNF interacts in a synergistic fashion with rIL-1 alpha and rIL-1 beta to enhance both, PGE2 synthesis and PLA2 release. The released PLA2 has a neutral pH optimum and is calcium-dependent. Pretreatment of cells with actinomycin D or cycloheximide inhibits basal and cytokine-stimulated PGE2 and PLA2 release.     

Species differences in human and rat islet sensitivity to human cytokines. Monoclonal anti-interleukin-1 (IL-1) influences on direct and indirect IL-1-mediated islet effects

Species differences in sensitivity to human recombinant cytokines were observed when human or rat islets were co-cultured with human recombinant cytokines for 6 days. Suppression of both human and rat islet insulin secretion resulted from co-culture with recombinant interleukin-1 alpha (rIL-1 alpha) or interleukin-1 beta (rIL-1 beta); however, direct rIL-1 alpha and rIL-1 beta cytotoxicity was seen with rat islets but not with human islets. Human islet insulin secretion was also suppressed during co-culture with recombinant tumor necrosis factor (rTNF) or interferon (rIFN), but not with lymphotoxin (rLT) or rIL-6; rat islet insulin secretion was not suppressed by any of these cytokines. No direct cytotoxic effects resulted from co-culture of human islets with rLT, rTNF, rIFN, or rIL-6; rLT was slightly cytotoxic for rat islets. Human islet cytotoxic synergy occurred between rLT and rIL-1 alpha, rIL-1 beta, or rIFN; synergy in suppression of human islet insulin secretion occurred between rLT and rIL-1 beta, and between rIFN and rTNF. Pretreatment of rIL-1 with monoclonal antibody (mAb) specific for non-crossreactive epitopes on rIL-1 alpha (H43 and H12) or rIL-1 beta (H34 and H21) prevented islet cytotoxic synergy between rIL-1 alpha or rIL-1 beta, respectively, and rLT. Although all four mAb's neutralize the thymocyte and fibroblast stimulatory activities of rIL-1 alpha or rIL-1 beta, mAb H21 does not neutralize rIL-1 beta activity against rat islets. Implications for cytokine-mediated islet cytotoxicity and suppression of insulin secretion are discussed.     

Human recombinant interleukin-1 beta- and tumor necrosis factor alpha-mediated suppression of heparin-like compounds on cultured porcine aortic endothelial cells

Cytokines are known to tip the balance of the coagulant-anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface-associated heparin-like compounds have not been examined yet. Incorporation of [35S]sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin-1 beta (rIL-1 beta) or tumor necrosis factor alpha (rTNF alpha) in a dose- and time-dependent manner with little effect on cell number, protein content, and [3H]leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rIL-1 beta or 5 ng/ml of rTNF alpha for 12-24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S-heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I-labeled antithrombin III to the endothelial cell surface was reduced to 40-60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor (heparin-like) activity was partially diminished in cytokine-treated endothelial cells. Thus, cytokine-mediated suppression of heparin-like substance on endothelial cells appears to be another cytokine-inducible endothelial effects affecting coagulation.     

Trace levels of bacterial lipopolysaccharide prevent interferon-gamma or tumor necrosis factor-alpha from enhancing mouse peritoneal macrophage respiratory burst capacity

Preexposure of resident mouse peritoneal macrophages for 1 hr to traces of bacterial lipopolysaccharide (LPS) (less than or equal to 1 ng/ml) rendered the cells refractory to activation by recombinant interferon-gamma (rIFN gamma) or recombinant tumor necrosis factor-alpha (rTNF alpha), as evaluated by release of H2O2 upon stimulation with phorbol myristate acetate. Inhibition persisted for at least 4 days. Fifty percent inhibition of activation mediated by rIFN gamma followed 1 hr exposure to 10 pg/ml LPS. Fifty percent inhibition of activation mediated by rTNF alpha was achieved with 1 hr exposure to 1 pg/ml LPS. Such low levels LPS exposures (concentration X time) are far below those reported for many other actions of LPS on host cells. Inhibition was partially prevented by the cyclooxygenase inhibitors indomethacin, ibuprofen, and acetylsalicylic acid. Exogenous prostaglandins PGE1 and PGE2, and the 3',5'-cyclic adenosine monophosphate analog dibutyryl cyclic adenosine monophosphate (cAMP), mimicked the inhibitory effect of LPS in a dose-dependent manner, consistent with the hypothesis that formation of endogenous cyclooxygenase products in response to LPS may elevate intracellular cAMP and that the latter may mediate the observed inhibition. In addition, neutralizing antibody against IFN alpha and IFN beta selectively prevented LPS inhibition of activation mediated by rIFN gamma, but not by rTNF alpha. This suggests that IFN alpha and/or IFN beta induced by LPS also contributed to inhibition of activation by rIFN gamma. Thus, release of LPS may afford microorganisms a means by which to interfere with immunologically mediated enhancement of the respiratory burst-dependent antimicrobial capacity of macrophages.     

Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) is induced in rabbit articular chondrocytes by cotreatment with interleukin 1 beta and a protein kinase C activator.

The synthesis of an 88-kDa gelatinolytic enzyme, identified as a zymogen of matrix metalloproteinase (proMMP)-9, was induced in the primary culture of rabbit articular chondrocytes by cotreatment with recombinant interleukin 1 beta (rIL-1 beta) and the protein kinase C (PKC) agonists, phorbol 12,13-dibutyrate (PDBu) or mezerein. Negligible 88-kDa gelatinolytic activity was produced by unstimulated cells or cells treated with a PKC activator alone at concentrations up to 100 ng/ml, and only a modest induction occurred with rIL-1 beta alone at concentrations of 1-100 ng/ml. However, when these cells were treated with a PKC activator in the presence of IL-1 beta (1 ng/ml), induction was striking, with enzymic activity detectable at a concentration as low as 1 ng/ml of mezerein or 10 ng/ml of PDBu. Rabbit chondrocytes in culture constitutively produced the zymogen of MMP-2 (proMMP-2) and its production was not altered by treatment with IL-1 beta or PKC agonists alone or in combination. Recombinant tumor necrosis factor alpha (rTNF alpha) did not substitute for IL-1 beta in inducing proMMP-9 in the presence of PKC activators, nor was the combination of IL-1 beta or TNF alpha alone effective. These data indicate that rabbit articular chondrocytes have a potential to synthesize and secrete proMMP-9 under certain biological and pathological conditions but that the expression of proMMP-9 is differently regulated from that of other MMPs.     

Transforming growth factor-beta inhibits Leydig cell steroidogenesis in primary culture

The effects of transforming growth factor (TGF) on Leydig cell steroidogenesis in primary culture were investigated. Basal testosterone levels were 3.7 +/- 0.54 ng/ml (mean +/- SE, N = 7). In the presence of hCG (10 ng/ml), testosterone levels increased to 22.77 +/- 3.05 ng/ml. TGF-beta caused a dose dependent inhibition of hCG-stimulated testosterone formation but without effects on basal levels. TGF-beta also inhibited 8-bromo cyclic AMP-induced testosterone formation and hCG-stimulated cyclic AMP formation. In contrast, TGF-alpha had no effect on either basal or hCG-stimulated testosterone formation and did not modify the inhibitory effect of TGF-beta. Present study indicates that TGF-beta can modulate Leydig cell steroidogenesis.     

Interleukin-1 alpha as a potent inhibitor of gonadotropin action in porcine Leydig cells: site(s) of action.

The effects of interleukin on testicular steroidogenesis have been studied in several laboratories, most often by using cultured rat Leydig cells. Several reports have indicated that interleukin-1 beta (IL-1 beta), but not interleukin-1 alpha (IL-1 alpha), exert a potent effect on gonadotropin action in rat Leydig cells. By using cultured porcine Leydig cells as a model, we found that IL-1 alpha (and to a lesser extent IL-1 beta), contrary to previous reports, is a potent inhibitor of LH/hCG steroidogenic action; and we further localized the steroidogenic biochemical step(s) affected by IL-1 alpha. IL-1 alpha inhibited hCG-induced testosterone secretion (about 67%) in a dose- and time-dependent manner. Half maximal and maximal effects were obtained with 4 U/ml (approximately 0.4 ng/ml, 0.3 x 10(-10) M) and 20 U/ml (approximately 2 ng/ml, 1.4 x 10(-10) M) of IL-1 alpha, respectively. The inhibitory effect of IL-1 alpha on gonadotropin action was detected at 6 h and was maximal after 24 h of treatment with the cytokine. The IL-1 alpha inhibitory effect was more potent than that of IL-1 beta: the maximal inhibitory effect of IL-1 beta was obtained with 400 U/ml. Subsequent investigations indicated that IL-1 alpha inhibited different biochemical steps involved in gonadotropin-induced testicular steroidogenesis. In this context, although IL-1 alpha appears to inhibit Leydig cell membrane functions (through a decrease in LH/hCG binding and gonadotropin-induced cAMP production), the antigonadotropin action of the cytokine is probably exerted predominantly at a step(s) located beyond cAMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant interleukin-1 beta inhibits elastin formation by a neonatal rat lung fibroblast subtype

The effect of recombinant interleukin-1 beta (rIL-1 beta) on elastin accumulation by lipid-laden interstitial cells (LIC) derived from neonatal rat lung was examined. The LIC, a fibroblast subtype, synthesized large amounts of elastin which was deposited into the extracellular matrix. This elastin was alkali-resistant and had an amino acid composition typical of adult rat elastin. Treatment of lipid-laden interstitial cell cultures with rIL-1 beta at 100 pg/ml caused a dramatic decrease in elastin accumulation as assessed by hot alkali treatment and transmission electron micrographs of the cell cultures. Tropoelastin formation was selectively decreased by rIL-1 beta relative to other proteins. Steady state levels of elastin mRNA were slightly decreased by rIL-1 beta at 5 pg/ml and markedly decreased by rIL-1 beta at 50 pg/ml or greater. The addition of indomethacin had no effect on rIL-1 beta-induced decreases in elastin mRNA levels. Inhibiting protein synthesis with cycloheximide blocked the effect of rIL-1 beta on elastin mRNA levels. The level of alpha 1(I) collagen mRNA was decreased by rIL-1 beta, but only at concentrations higher than that needed to induce a decrease in elastin mRNA. These data indicate that rIL-1 beta decreased steady state levels for elastin mRNA and elastin accumulation and can selectively regulate the accumulation of elastin and collagen.     

Effect of lutropin and cycloheximide on lutropin receptors and cyclic AMP production in Leydig tumour cells in vitro.

A system to study lutropin-induced desensitization of tumour Leydig cells in vitro has been investigated. Tumour Leydig cells were purified on a Percoll gradient and then incubated for 30 min with lutropin (0-1000ng/ml). The cells were then washed and incubated in suspension media at 32 degrees C. 125I-labelled human choriogonadotropin binding and basal and lutropin-stimulated cyclic AMP production were determined at various times. Initially the cells showed a dose-dependent decrease in human choriogonadotropin binding (1.18 and 0.13fmol/10(6) cells respectively) followed by an increase at 1 h (2.32 and 0.87fmol/10(6) cells respectively). Human choriogonadotropin binding remained elevated in the cells pre-incubated without lutropin, whereas the cells pre-incubated with lutropin showed a dose-dependent decrease over the next 10 h (2.20-0.18fmol/10(6) cells respectively). Basal production of cyclic AMP initially reflected the pre-incubation conditions (1.17-21.19ng/10(6) cells per h for 0-1000ng of lutropin/ml respectively). However, by 1 h there was a marked rise in basal cyclic AMP production which returned to the initial lower values by 4 h. At all time intervals studied, lutropin-induced cyclic AMP production showed a decrease that was proportional to lutropin concentration in the pre-incubated media. The decreases in human choriogonadotropin binding produced by pre-incubations with lutropin (100ng/ml) was partially inhibited by the presence of cycloheximide in the pre-incubation media and totally prevented by the continuous presence of cycloheximide. These results demonstrate that desensitization of tumour Leydig cells occurs after exposure to lutropin in vitro. This desensitization involves both a loss of plasma membrane receptors for lutropin and lutropin-stimulated adenylate cyclase. These events can be prevented by cycloheximide and are therefore probably dependent on protein synthesis.     

The aging Leydig cell: 1. Testosterone and adenosine 3',5'-monophosphate responses to gonadotropin stimulation in rats

Plasma testosterone levels before and after a single injection of hCG were significantly lower in 24-month old rats than 60--90 day old animals (p less than 0.001). Even with repeated hCG administration for three weeks, plasma testosterone levels of old rats could not be restored to levels present in unstimulated young rats. In response to in vitro LH and 8-bromo-cyclic AMP stimulation, purified young Leydig cells produced significantly higher amounts of testosterone than Leydig cells from old rats. Maximal testosterone formation of the young Leydig cells in response to LH was 42.0 +/- 6.88 ng/10(6) cells, while cells from old rats produced only 16.8 +/- 3.69 ng/10(6) cells (p less than 0.01). However, the dose of LH at which one half maximal response (ED50) occurred was 0.1 mIU/ml for young Leydig cells and 0.05 mIU/ml for old Leydig cells. Basal and 1.0 mIU LH-stimulated cyclic AMP formation were comparable in both groups, but cyclic AMP formation in response to 10 mIU of LH was significantly less in the old rats (p less than 0.05). Present results demonstrate impaired steroidogenic capacity of old rats both in vivo and in vitro. Decreased testosterone response in old rats most likely is the consequence of understimulation of Leydig cells by gonadotropin; however, there appear to be additional intrinsic defects in old Leydig cells.     

Macrophages rapidly internalize their tumor necrosis factor receptors in response to bacterial lipopolysaccharide

The effect of bacterial lipopolysaccharide (LPS) on macrophage receptors for tumor necrosis factor/cachectin (TNF-R) was studied. At equilibrium, iodinated recombinant human TNF alpha (rTNF alpha) bound to 1100 +/- 200 sites/cell on macrophage-like RAW 264.7 cells with a Kd of 1.3 +/- 0.1 x 10(-9) M. Preexposure of RAW 264.7 cells to 10 ng/ml LPS for 1 h at 37 degrees C resulted in complete loss of cell surface TNF alpha binding sites. 50% loss ensued after 1 h with 0.6 ng/ml LPS, or after 15 min with 10 ng/ml LPS. Complete loss of TNF alpha binding sites occurred without change in numbers of complement receptor type 3. No decrease in TNF-R followed preexposure to LPS at 4 degrees C, nor could LPS displace 125I-rTNF alpha from its binding sites. Although TNF-R disappeared from the surface of intact macrophages following exposure to LPS, specific TNF alpha binding sites were unchanged in permeabilized macrophages, indicating that TNF-R were rapidly internalized. Conditioned media from LPS-treated RAW 264.7 cells induced 30% down-regulation of TNF-R on macrophages from LPS-hyporesponsive mice (C3H/HeJ), suggesting that a soluble macrophage product may be responsible for a minor portion of the LPS effect. Additional evidence against endogenous TNF alpha being the major cause of TNF-R internalization was the rapid onset of the effect of LPS on TNF-R compared to the reported onset of TNF alpha production, the relatively high concentrations of exogenous rTNF alpha required to mimic the effect of LPS, and the inability of TNF alpha-neutralizing antibody to block the effect of LPS. LPS-induced down-regulation of TNF-R was complete or nearly complete not only in RAW 264.7 cells, but also in primary macrophages of both human and murine origin, was less marked in human endothelial cells, and was absent in human granulocytes and melanoma cells and mouse L929 cells. Thus, in situ, macrophages and some other host cells may be resistant to the actions of TNF alpha produced during endotoxinemia, because such cells may internalize their TNF-R in response to LPS before TNF alpha is produced.     

Neurotensin regulation of TSH secretion in the rat

The ionophore A23187 (6.7 microM) increased the rates of formation of prostaglandins and cyclic AMP in suspensions of thioglycollate-elicited rat peritoneal macrophages. Both effects were inhibited by the calmodulin blocker trifluoperazine (50 microM) and the calcium channel blocker verapamil (500 microM). Inhibitors of phospholipase A2 and cyclo-oxygenase also blocked both actions of A23187. The stimulated prostaglandin formation was markedly reduced when the cells were preincubated with 8-bromo-cyclic AMP (1mM), dibutyryl cyclic AMP (1mM) or cholera toxin (500ng/ml). Addition of exogenous arachidonic acid (30 microM) alleviated this inhibition. We propose that the effect of A23187 on macrophages includes a 'self-limiting' mechanism whereby newly-synthesized prostaglandins can inhibit, via cyclic AMP, a step(s) prior to the transformation of arachidonic acid and thus modulate their own production.     

Tumor necrosis factor, alone or in combination with IL-2, but not IFN-gamma, is associated with macrophage killing of Mycobacterium avium complex

Organisms belonging to the Mycobacterium avium complex (MAC) are the most common bacterial pathogens in patients with AIDS but factors associated with the activation of cellular defense mechanisms against this atypical mycobacterium have not been defined. Peritoneal macrophages harvested from a chronic MAC infection in C57 black mice are able to kill approximately 86% of intracellular MAC in contrast to 0 to 20% killing by unstimulated human and mouse macrophages in vitro. The availability of human rTNF-alpha, rIFN-gamma, and rIL-2 permitted evaluation of the role of each of these lymphokines/monokines, alone or in combination, in activating macrophages in vitro to kill MAC. Human monocyte-derived macrophages were cultured in vitro, stimulated with rIL-2, rIFN-gamma, or rTNF, and then infected with MAC (serovars 1 and 8). Mouse peritoneal macrophages were harvested, cultured in vitro, and stimulated with rIFN-gamma. rTNF (10(4) U/ml) was associated with a modest increase of intracellular killing of MAC (58 +/- 5%) even when utilized 24 or 48 h after macrophage infection or when administered for 5 consecutive days after infection (78.1 +/- 4%). Both human and murine IFN-gamma were associated with increased intracellular growth of MAC (32 +/- 4% for murine and 38 +/- 3% for human macrophages). However, intracellular killing (53 +/- 6% compared with control) was observed after 6 days of treatment with IFN-gamma. This latter effect was fully blocked by anti-TNF antibody, whereas rIL-2 alone did not augment the intracellular killing of MAC by human macrophages. rTNF plus either rIFN-gamma or rIL-2 triggered significant increases in superoxide anion production, but subsequent MAC killing was no greater than with rTNF alone. Treatment of macrophages with 10 U/ml of rTNF followed by rIL-2 (200 U/ml) was associated with 68% of intracellular killing. TNF seems to be an important monokine, promoting activation of mycobactericidal mechanisms in human macrophages.     

Interaction of recombinant IL-1 and recombinant tumor necrosis factor in the induction of mouse acute phase proteins

Recombinant mouse and human IL-1 (alpha and beta forms), as well as rTNF-alpha when administered in vivo, induced the production of the mouse acute phase reactants: serum amyloid P-component (SAP), C3, and fibrinogen. The SAP response to all three rIL-1 proteins reached a maximum at a dose of 10(4) U/mouse, which corresponds to 1 to 10 micrograms of protein. The maximum in vivo response consisted of a 10-fold increase in SAP levels, a 2-fold increase in C3 levels, and a 3-fold increase in fibrinogen concentration. By contrast, rTNF-alpha induced a much smaller acute phase (AP) protein response (4-fold increase in SAP) when administered in vivo. Administration of a combination if rIL-1 and rTNF resulted in an AP response that was additive for SAP, synergistic for fibrinogen, but resulted in only the same amount of C3 induced by IL-1 alone. Both recombinant monokines induced new SAP synthesis by isolated hepatocytes in vitro with an optimal response occurring with either 1 U of rIL-1/ml per 2 x 10(5) hepatocytes or 10(-3) U/ml of rTNF. The hepatocyte response to IL-1 was of the same magnitude as the response of intact mice; however, the response to TNF was approximately 10(4) times more efficient in vitro. A mixture of the monokines induced an in vitro SAP response that was additive when suboptimal doses of rIL-1 were combined with optimal amounts of rTNF-alpha. Overall, the findings indicate that both monokines directly trigger hepatocyte synthesis of SAP and that their combined effect probably accounts for a substantial portion of the synthesis of these AP proteins in mice.     

The aging Leydig cell: 2. Two distinct populations of Leydig cells and the possible site of defective steroidogenesis

Using metrizamide gradient centrifugation two populations of Leydig cells were found in both 60-90 day-old and 24 month-old rats. Cells from both Band 2 (B2) and Band 3 (B3) responded to LH stimulation with increased cyclic AMP formation; however, only B3 cells produced significant amounts of testosterone. Cells from both B2 and B3 of the old rats synthesized less cyclic AMP and testosterone than cells from their younger counterparts. In response to LH stimulation, 0.01 - 1.0 mIU/ml, no appreciable difference of cyclic AMP formation could be detected between young and old Leydig cells. Maximal testosterone production occurred when 1 mIU/ml LH was used. Only when LH concentration was increased to 10 and 100 mIU/ml, did young Leydig cells produce significantly more cyclic AMP than old Leydig cells. After addition of 5X10(-7)M of pregnenolone or progesterone to the incubation medium, both young and old Leydig cells produced comparable amounts of testosterone. These results demonstrate no impairment of old rat Leydig cells to synthesize testosterone from pregnenolone and progesterone.     

Release of arachidonic acid and the effects of corticosteroids on steroidogenesis in rat testis Leydig cells.

The release of arachidonic acid by luteinizing hormone (LH) and the effects of inhibiting phospholipase A2 (PLA2) in vivo and in vitro on LH stimulated steroidogenesis in rat testis Leydig cells has been investigated. It was found that arachidonic acid is rapidly incorporated into phospholipids and is released within 1 min after addition of LH. The effects of treating adult rats with dexamethasone and human chorionic gonadotropin (hCG) in vivo on steroidogenesis and prostaglandin synthesis in Leydig cells isolated 6 h later were determined. It was found that hCG caused a marked increase in prostaglandin F2 alpha formation which was inhibited by treatment with dexamethasone. LH-stimulated testosterone production was inhibited in the hCG treated rats and dexamethasone caused a further decrease. Treatment with dexamethasone alone also caused a decrease in the response to LH. HCG, but not dexamethasone, had similar inhibitory effects on LH-stimulated cyclic AMP production. Similarly, the PLA2 inhibitors quinacrine, dexamethasone and corticosterone, added to the Leydig cells in vitro, inhibited LH-stimulated testosterone production but not cyclic AMP production. 11-Dehydrocorticosterone also inhibited LH-stimulated testosterone production, but higher concentrations were required to give 50% inhibition compared to corticosterone (50 and 25 microM, respectively). Ring A-reduced metabolites of corticosterone and progesterone were also found to inhibit LH-stimulated steroidogenesis. The results obtained in this and previous studies are consistent with the activation of PLA2, (either directly by LH and/or via cyclic AMP), which results in the release of arachidonic acid and the formation of leukotrienes, which stimulate steroidogenesis in the Leydig cell. This study also indicates that corticosteroids and their metabolites may exert inhibitory effects at other sites in the steroidogenic pathways, in addition to PLA2.     

Cytokine regulation of interleukin 6 production by human endothelial cells

The influence of recombinant (r) human tumor necrosis factor alpha (rTNF-alpha), r human interleukin 1 beta (rIL-1 beta), and r human interferon gamma (rIFN-gamma) on the production of interleukin 6 (IL-6) by human endothelial cells (HEC) was investigated. The addition of 1-100 U/ml of either rTNF-alpha or rIL-1 beta to cultures of HEC monolayers caused a dose-related increase in IL-6 production as detected after 24 hr of incubation. In contrast to rIL-1 beta and rTNF-alpha, the use of up to 1000 U/ml of rIFN-gamma caused only a moderate increase in IL-6 production. However, significantly greater quantities of IL-6 were produced by HEC monolayers subjected to 1000 U/ml of rIFN-gamma in combination with 1-100 U/ml of rTNF-alpha. Furthermore, the addition of graded concentrations of human transforming growth factor beta (TGF-beta) to cultures resulted in a dose-related inhibition of rIL-1 beta- and rTNF-alpha-induced IL-6 production by HEC. The results demonstrate that rIL-1 beta and rTNF-alpha share the ability to stimulate HEC for production of IL-6 and indicate that TGF-beta may act as an immunosuppressive agent, at least partially, through its ability to inhibit the action of TNF-alpha and IL-1 on endothelial cells.     

Effects of Cordyceps sinensis on testosterone production in normal mouse Leydig cells.

The stimulatory effect of Cordyceps sinensis (CS) on MA-10 mouse Leydig tumor cell steroidogenesis was previously demonstrated in our laboratory. In the present studies, we further determined the effect of CS on steroidogenesis in purified normal mouse Leydig cells. Different concentrations of CS (0.1-10 mg/ml) were added to Leydig cells without or with human chorionic gonadotropin (hCG) (50 ng/ml), and the steroid production was determined by radioimmunoassay (RIA). The results illustrated that CS stimulated normal mouse Leydig cell steroidogenesis in a dose-dependent relationship. CS at 3 mg/ml significantly stimulated testosterone production (p<0.05). Concerning the temporal relationship, CS at 3 mg/ml stimulated maximal testosterone production between 2 to 3 hr. Interestingly, hCG-stimulated testosterone productions were suppressed by CS in a dose-dependent relationship. CS also reduced dbcAMP-stimulated testosterone productions, which indicated that CS affected signal transduction pathway of steroidogenesis after the formation of cyclic AMP. Moreover, cycloheximide inhibited CS-treated mouse Leydig cell testosterone production, suggesting that new protein synthesis was required for CS-stimulated steroidogenesis.