Andrew Roland Mackenzie

ICE-PROTEASE INHIBITORS BLOCK MURINE LIVER INJURY AND APOPTOSIS CAUSED BY CD95 OR BY TNF-ALPHA

The two apoptosis receptors of mammalian cells, i.e. the 55 kDa TNF receptor (TNF-R1) and CD95 (Fas/APO1) are activated independently of each other, however, their signaling involves a variety of ICE-related proteases [I]. We used a cell-permeable inhibitor of ICE-like protease activity to examine in vivo whether post-receptor signaling of TNF and CD95 are fully independent processes. Mice pretreated with the inhibitor, Z-VAD-fluoromethylketone (FMK) were dose-dependently protected from liver injury caused by CD95 activation as determined by plasma alanine aminotransferase and also from hepatocyte apoptosis assessed by DNA fragmentation (ID50 = 0.1 mg/kg). A dose of 10 mg/kg protected mice also from liver injury induced by TNF-alpha. Similar results were found when apoptosis was initiated via TNF-alpha or via CD95 in primary murine hepatocytes (IC50 = 1.5 nM) or in various human cell lines. In addition to prevention, an arrest of cell death by Z-VAD-FMK was demonstrated in vivo and in vitro after stimulation of apoptosis receptors. These findings show in vitro and in vivo in mammals that CD95 and the TNF-alpha receptor share a distal proteolytic apoptosis signal.

Increased Mature Interleukin-1β (IL-1β) Secretion from THP-1 Cells Induced by Nigericin Is a Result of Activation of p45 IL-1β-converting Enzyme Processing

Perregaux and Gabel (Perregaux, D., and Gabel, C. A. (1994) J. Biol. Chem. 269, 15195–15203) reported that potassium depletion of lipopolysaccharide-stimulated mouse macrophages induced by the potassium ionophore, nigericin, leads to the rapid release of mature interleukin-1β (IL-1β). We have now shown a similar phenomenon in lipopolysaccharide-stimulated human monocytic leukemia THP-1 cells. Rapid secretion of mature, 17-kDa IL-1β occurred, in the presence of nigericin (4–16 μm). No effects on the release of tumor necrosis factor-α, IL-6, or proIL-1β were seen. Addition of the irreversible interleukin-1β-converting enzyme (ICE) inhibitor, Z-Val-Ala-Asp-dichlorobenzoate, or a radicicol analog, inhibited nigericin-induced mature IL-1β release and activation of p45 ICE precursor. The radicicol analog itself did not inhibit ICE, but markedly, and very rapidly depleted intracellular levels of 31-kDa proIL-1β. By contrast, dexamethasone, cycloheximide, and the Na+/H+ antiporter inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, had no effect on nigericin-induced release of IL-1β. We have therefore shown conclusively, for the first time, that nigericin-induced release of IL-1β is dependent upon activation of p45 ICE processing. So far, the mechanism by which reduced intracellular potassium ion concentration triggers p45 ICE processing is not known, but further investigation in this area could lead to the discovery of novel molecular targets whereby control of IL-1β production might be effected.

Different effects of cyclosporin a on bone remodeling in young and adult rats

Reported effects of cyclosporin A (Sandimmun, CsA) on bone have been both contradictory and controversial. Thus, stimulation of new bone formation as well as increased mineral and matrix resorption have been observed. To investigate the response of basal mineral and matrix turnover to CsA treatment at different stages of skeletal development, comparative experiments were conducted in young growing female rats and in adults. Fifty-six young animals (study A) and 40 adults (study B) received orally either the carrier substance or 5, 15, and 30 mg/kg CsA for 30 days. The following parameters were measured: (a) total skeletal mineral content by dual energy X-ray absorptiometry (DEXA) on days 1 and 30; (b) tibial trabecular volume at day 30; (c) serum osteocalcin at 5-day intervals; (d) urinary deoxypyridinoline (Dpd) excretion (days 1, 15, and 30); and (e) plasma levels of CsA. Results can be summarized as follows: in young rats (study A), total skeletal mineral was not modified by the 5- and 15-mg/kg doses of CsA, whereas 30 mg/kg induced a significant decrease (-15%, p < 0.01). This parameter was not significantly modified in adult animals (study B) subjected to the same doses. The administration of 5 mg/kg CsA did not alter tibial trabecular volume in young rats, but 15 and 30 mg/kg significantly lowered this parameter (-16.3%, p < 0.02, and -42%, p < 0.001, respectively). In adult rats, tibial trabecular volume remained unchanged with the exception of the group receiving 30 mg/kg which exhibited significantly lower values (-28%, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin A. Mode of Action and Effects on Bone and Joint Tissues

Cyclosporin A is an established immunomodulatory agent with an increasing number of clinical applications. Although its precise mechanisms of action remain elusive, one of the most important known properties of CyA is its ability to inhibit the production of cytokines involved in the regulation of T-cell activation. In particular, CyA inhibits de novo synthesis of interleukin 2(IL-2), the major cytokine involved in T-cell proliferation, as well as other cytokines, probably at the level of gene transcription, as shown by the suppression of mRNA levels in activated T-cells. Although the major actions of CyA are on T-cells, there is some evidence for possible direct effects on other cell types e.g. B-cells, macrophages and, from our own work, on bone and cartilage cells. Cyclosporin A is thought to enter cells and to bind to cyclophilins, which are members of a family of high-affinity cyclosporin A-binding proteins, now known as immunophilins. The binding of cyclosporins to such proteins appears to be closely linked to the immunosuppressive action of cyclosporins. The immunophilins possess enzyme activity, ie. peptidyl-prolyl cis-trans isomerase, also known as rotamase, which can regulate protein folding, and may therefore alter the functional state of many cell proteins. Cyclosporin A blocks peptidyl-prolyl cis-trans isomerase activity but it is not clear whether this plays a part in its selective inhibition of cytokine-gene transcription. Moreover, the ubiquitous presence of cyclophilins and immunophilins raises the question of why cyclosporin A has its apparent major effects only on T-cells. Recent proposals regarding the intracellular mode of action of CyA suggest that it interacts with cyclophilin and other regulatory proteins including calmodulin and calcineurin, which is a serine/threonine phosphatase, and thereby affects the functional state of key regulators of gene transcription in its target cells. The effects of CyA on T-cells and directly or indirectly on connective tissue cells, including bone, cartilage and synovial cells, which all can produce a range of cytokines, are of interest in relation to the tissue changes that occur in inflammatory diseases, such as rheumatoid arthritis. Thus, for example, cyclosporin A inhibits in vitro the bone resorbing activity of interleukin 1, 1,25-dihydroxy-vitamin D3, parathyroid hormone and prostaglandin E2 by apparently non-T-cell effects, while in vivo protects against bone and cartilage loss in adjuvant arthritis. More needs to be known about the direct and indirect modulation of cytokine production by cyclosporin A in connective tissues, in order to understand its potential value in clinical disorders.

Graded Doses of Recombinant Interleukin-1β Induce Generalized Osteopenia in Rats without Altering Skeletal Growth and Joint Integrity

Background: Whereas a primary role of interleukin-1β (IL-1β) in local bone remodelling and articular inflammation has been well established, the effect of prolonged systemic administration of this cytokine on total skeletal Ca, somatic growth and joint tissue has not yet been investigated. Methods: Five groups of 14 rats each, aged 7–8 weeks, had miniosmotic pumps (Alzet 200 µl) implanted and primed to release 100, 200, 500, 1,000 and 2,000 ng/kg/24 h of human recombinant IL-1β (rIL-1β) daily for 14 days. On days 0 and 14 total skeletal mineral content (BMC) was assessed by means of X-ray absorptiometry and vertebral and tibial growth was measured by computer-assisted radiometry. On the same days, blood was drawn and analyzed for rat growth hormone (rGH), insulin-like growth factor (IGF-1), and osteocalcin. Also 24-hour urine was collected for d-pyridinoline (dpd) determinations. Hind- and forepaw diameter as a parameter of joint inflammation was assessed using a micrometric calliper. Subsequently the animals were sacrificed and one tibia dissected for measurement of trabecular volume by computerized histomorphometry. Results: BMC decreased in a dose-dependent manner reaching significance at 1,000 and 2,000 ng/kg (p < 0.03 and 0.04) in close correlation with tibial trabecular volumes (r = 0.84; p < 0.02). Normal vertebral and tibial growth was recorded at all dosages. There was no evidence of joint involvement. Blood rGH and IGF-1 remained normal as did osteocalcin, the latter reflecting lack of osteoblast activation. In contrast dpd increased in a dose-dependent manner indicating enhanced bone matrix turnover. Conclusion: It is concluded that graded infusions of supraphysiological doses of rIL-1β capable of inducing osteopenia did not affect skeletal growth in the absence of articular reaction. This is in contrast with the experience recorded in experimental arthritis in which growth retardation, in addition to osteopenia, may be caused by factors other than circulating IL-1β.

Long-Term Systemic Administration of Human Recombinant Interleukin-1β Induces a Dose-Dependent Fall in Circulating Parathyroid Hormone in Rats

The synergism/antagonism between interleukin (IL)-1β and parathyroid hormone (PTH) has been the subject of in vitro and in vivo work, but a possible direct action of the cytokine on PTH release has not been reported. We have investigated the effect of a continuous infusion of human recombinant IL-1β (rIL-1β) on circulating PTH during a 14-day period in 7-week-old female rats. This time interval was chosen in order to exclude initial hypercalcemia and to enable data collection under steady-state conditions. Five groups of 20 animals each had miniosmotic pumps (Alzet 2002, 200 µl) implanted subcutaneously and primed to release either distilled water (controls) or 100, 500, 1,000 and 2,000 ng/24 h of rIL-1β. Blood was drawn on days 1 and 14 for PTH, corticosterone and Ca2+ determinations. Adequate biological activity of the infused rIL-1β was supported by elevated rectal temperature records and significant elevations of plasma corticosterone on day 14. The 100-ng dose had no effect but 500–2,000 ng rIL-1β/24 h significantly reduced plasma PTH in a dose-dependent manner down to 54% of basal value (20.4 ± 1.1 vs. 15.3 ± 1.4 pg/ml for 500 ng, p < 0.005; 20.5 ± 1.3 vs 12.3 ± 1.1 for 1,000 ng, p < 0.001, and 19.5 ± 2.0 vs. 10.6 ± 1.1 pg/ml for 2,000 ng, p < 0.0008). Despite these findings, no differences in blood Ca2+ could be detected between treated animals and controls. The following conclusions can be inferred from the foregoing: Systemic administration of rIL-1β to rats induced a dose-dependent fall in circulating PTH without altering calcemia, calling into question the biological relevance of the former finding. Although the recorded PTH depression may indeed not have been severe enough to cause hypocalcemia, it can be hypothesized that osteoclast activation by rIL-1β would enhance bone mineral release into the pool compensating for depressed PTH activity.

Experimental arthritis: Effect on growth parameters and total skeletal calcium

Aim of the study was to investigate the possible mechanisms leading to stunted growth and osteoporosis in experimental arthritis. Fourty-two female rats of 7-8 weeks of age were randomly assigned to three groups of 14 animals each: (a) controls; (b) adjuvant-inoculated (AA); and (c) adjuvant-inoculated rats receiving 10 mg cyclosporin A (CsA) orally for 30 days. Biological parameters studied were: hindpaw swelling; vertebral length progression expressed as Delta increments between days 1 and 30 as a parameter of skeletal growth, and estimation of total skeletal mineral content by dual energy X-ray absorptiometry (n=10 each group) on day 30. Endocrine parameters measured were pulsatile release of growth hormone (rGH) on day 30 following jugular cannulation and measurement of insulin-like growth factor (IGF-1) in pooled plasma from rGH profiles. Results can be summarized as follows: Untreated AA rats exhibited local signs of inflammation in comparison with controls (hindpaw diameter 8.1-8.9 mm vs. 5.3-5.6 mm in controls). Treatment with CsA normalized this parameter (4.9-5.6 mm). Vertebral growth was significantly retarded in AA rats in comparison with controls (214+/-32 vs. 473+/-33 microm; p<0.001). Administration of CsA normalized vertebral size increment with a clear tendency to overgrowth (523+/-43 microm, n.s.). There was also a marked reduction in total skeletal mineral content in diseased (AA) rats as compared to controls (5.8+/-0.1 vs. 7.5+/-0.1g [OH-apatite]; p<0.001), and a moderate but significant increment above controls in the group receiving CsA (8.0+/-0.1 vs. 7.5+/-0.1g [OH-appatite]; p<0.04). Integrated rGH profiles exhibited a significant fall in arthritic rats and were completely restored to normal under CsA treatment. A trend toward higher rGH values was observed in the latter group (2908+/-554 in AA vs. 8317+/-1492 ng/ml/240 min in controls; p<0.001, and 10940+/-222 ng/ml/240 min, n.s. in the CsA group). There was a good correlation between skeletal growth and rGH pulsatility (r=0.81; p<0.001). IGF-1 followed a similar pattern (630+/-44 in AA vs. 752+/-30 ng/ml in controls; p<0.04, and 769+/-59 ng/ml in the CsA group, n.s. vs. controls). Thus, a clear tendency to skeletal overgrowth following treatment was observed in agreement with the hormonal data. It can therefore be concluded that, in experimental arthritis, attenuated GH-spiking and reduced circulating IGF-1 appear to be causally related to growth retardation, probably mimicking signs and symptoms observed in juvenile arthritis. Therapy with CsA is followed by normalization of hormonal and biological parameters accompanied by a catch up phenomenon in skeletal growth which is also observed clinically in juvenile arthritis. Generalized osteopenia is a prominent feature seemingly connected with the growth abnormalities as they parallel each other during the evolution of the disease and respond equally to therapy.

Radiometrical, hormonal and biological correlates of skeletal growth in the female rat from birth to senescence

OBJECTIVE We investigated the skeletal growth profile of female rats from birth to senescence (100weeks) on the basis of sequential radiometrical, hormonal and biochemical parameters. DESIGN Weaning rats entered the study which was divided into two sections: a) sequential measurements of vertebral and tibial growths and bone mineral density (BMD), estimation of mineral content of the entire skeleton (BMC) and chemical analysis of vertebral Ca; and b) determination of basal and pulsatile growth hormone (rGH), insulin-like growth hormone (IGF-I), estradiol (E2), parathyroid hormone (PTH), osteocalcin (OC) and urinary d-pyridinoline (dp) throughout the experimental period. RESULTS Vertebral and tibial growths ceased at week 25 whereas BMD and BMC as well as total vertebral Ca exhibited a peak bone mass at week 40. rGH pulsatile profiles were significantly higher in younger animals coinciding with the period of active growth and IGF-I peaked at 7weeks, slowly declining thereafter and stabilizing after week 60. OC and dp closely paralleled IGF-I coinciding with the period of enhanced skeletal growth, remaining thereafter in the low range indicative of reduced bone turnover. E2 increased during reproductive life but the lower values subsequently recorded were still in the physiological range, strongly suggesting a protective role of this steroid on bone remodeling. PTH followed a similar profile to E2, but the significance of this after completion of growth remains unclear. CONCLUSIONS Mechanisms governing skeletal growth in the female rat appear similar to those in humans. Bone progression and attainment of peak bone mass are under simultaneous control of rGH, IGF-I and calciotropic hormones and are modulated by E2. This steroid seems to protect the skeleton from resorption before senescence whereas the role of PTH in this context remains uncertain.