Expression of CD39 by human peripheral blood CD4+ CD25+ T cells denotes a regulatory memory phenotype.

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4+ Foxp3+ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood-derived human CD4+ CD25+ CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4+ CD39+ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4+ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4+ CD25+ CD39+ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell-populations to allow tracking of these in health and disease, as in renal allograft rejection.

Leucocytoclastic and renal vasculitis in a patient with autoimmune pancreatitis: new associations.

Autoimmune pancreatitis (AIP) is an uncommon condition which comprises diffuse or discrete pancreatic enlargement and irregular pancreatic duct strictures of autoimmune origin leading to pain or obstructive jaundice associated with extra-pancreatic manifestations. It is characterized by an elevated IgG, especially IgG4, level. We illustrate the first described case of a patient with AIP in association with leucocytoclastic and renal vasculitis.

Inhibition of stimulated bone resorption in vitro by TIMP-1 and TIMP-2.

Recombinant human TIMP-1 and TIMP-2 (tissue inhibitors of metalloproteinases) inhibited bone resorption induced by either parathyroid hormone or 1,25-dihydroxyvitamin D3 in cultured neonatal mouse calvariae. The inhibition was reversible, dose-dependent and complete at 1 microgram/ml inhibitor concentration. TIMP-2 was more potent than TIMP-1. TIMP-1 and TIMP-2 also inhibited basal bone resorption. Neither metalloproteinase inhibitor affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of beta-glucuronidase or the spontaneous release of lactate dehydrogenase. These results suggest that endogenous TIMPs play a central role in regulating both physiological and pathological bone resorption.

The purification and partial characterization of bone resorptive polypeptides from bovine bone matrix.

Matrix proteins were extracted from bovine cortical bone with EDTA/Tris-HCl under non-dissociative conditions at neutral pH. Four distinct bone resorptive proteins with molecular masses of 14, 25, 29 and 40 kDa were purified and partially characterized using an in vitro neonatal mouse calvarial assay and a growth factor assay using BALB/c/3T3 cells. The 14 kDa protein was purified by anion exchange chromatography (Mono Q) and gel filtration (Superdex 75HR) using FPLC (fast protein liquid chromatography); this factor stimulated the proliferation of MCF-7 human breast cancer cells, a bioassay which is specific for the insulin-like growth factors (IGFs). The 25, 29 and 40 kDa proteins were purified by sequential chromatography as follows: anion-exchange (Mono Q), heparin-Sepharose, hydroxyapatite, concanavalin A-Sepharose, phenyl-Superose, reversed phase high performance liquid chromatography (HPLC) and sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The 25 kDa protein was identified as TGF-beta by its inhibitory effect on the proliferation of mink lung cells. The 40 kDa protein enhanced the formation of multinucleate tartrate-resistant acid phosphatase positive cells in a murine bone marrow differentiation assay, but was without effect in an isolated osteoclast assay and had no growth factor activity; this protein is likely to be a colony stimulating factor. The 29 kDa protein was also without growth factor activity; it was, however, able to stimulate bone resorption in the isolated osteoclast assay, suggesting a direct action in osteoclast function. The 29 and 40 kDa proteins may be osteoblast gene products that have been sequestrated by the bone matrix in a similar fashion to TGF-beta and the IGFs. This is the first report of proteins isolated from bone matrix which directly stimulate osteoclast differentiation and activity.

Multiple extracellular signals promote osteoblast survival and apoptosis.

Programed cell death (PCD) or apoptosis is a naturally occurring cell suicide pathway induced in a variety of cell types. In many cases, PCD apparently arises as a result of competition for limiting amounts of survival signals. In this study, we have investigated the potential role of growth factors (GF), cytokines, and osteotropic hormones on osteoblast survival in vitro. Our results indicate that in the absence of any of these factors, osteoblasts rapidly undergo PCD, as determined by cell morphology, mitochondrial function, and nuclei fragmentation. Osteoblast survival was promoted by insulin-like growth factor I (IGF-I), IGF-II, insulin, and basic fibroblast growth factor (bFGF). Platelet-derived growth factor had no effect on osteoblast survival, but this GF potentiated the survival-promoting effects of IGF-I, IGF-II, and insulin. A similar effect occurred when bFGF was added in combination with either of the IGFs or insulin. The effects of the IGFs were blocked by alphaIR-3, an antibody to the type I IGF receptor, whereas the effects of insulin were only partially blocked. This antibody blocked the potentiating effects of platelet-derived growth factor on IGF-I-mediated osteoblast survival, but only partially blocked those of bFGF. Although a 100% survival of osteoblasts was seen in the presence of 2% FCS, the highest level attained by any of the above GF combinations was approximately 75%. The monocyte-derived factor, tumor necrosis factor-alpha (TNF alpha) was the only agent that enhanced PCD in this study. These results suggest that osteoblast survival is promoted by those GFs sequestrated in bone matrix and that the type I, but not the type II, IGF receptor is involved in the response. Our data also indicate that other unidentified GFs or components of the extracellular matrix may be involved in promoting osteoblast survival and that TNF alpha may abrogate their effects in vivo. We propose that these GFs may be released from bone matrix during phases of bone resorption and promote osteoblast survival, thereby playing an important role in bone remodeling, and that PCD induced by TNF alpha may contribute to the bone loss in inflammatory bone disease.

Osteoblasts mediate insulin-like growth factor-I and -II stimulation of osteoclast formation and function.

Insulin-like growth factor-I (IGF-I) and IGF-II have powerful, well defined effects on osteoblastic cells, stimulating their proliferation and inducing collagen synthesis, but the role of IGF-I and -II in modulating osteoclast differentiation and activity remains unclear. We first examined the bone-resorptive effects of IGF-I and IGF-II by assessing 45Ca2+ release from neonatal mouse calvarial bones. Both IGFs dose dependently stimulated bone resorption, with an EC50 of 8 x 10(-9) M for IGF-I and 2 x 10(-8) M for IGF-II. We then tested the effects of the IGFs on bone resorption by rat isolated osteoclasts cultured on ivory slices. Neither IGF-I nor IGF-II stimulated isolated osteoclast activity. However, in the presence of either primary mouse osteoblasts or human osteosarcoma MG 63 cells, both IGFs enhanced osteoclast resorptive activity, with an EC50 of 5 x 10(-10) M for IGF-I and 10(-9) M for IGF-II. Stimulation was not mediated by BALB/c/3T3 cells, a nonosteoblastic cell line. The effects of the IGFs were blocked by alpha IR-3, an antibody to the type I IGF receptor, but not by beta-galactosidase, a lysosomal enzyme that competes with IGF-II for the type II IGF receptor. We then examined the effects of the IGFs on the formation of osteoclast-like multinucleate cells (MNCs) in mouse bone marrow cultures. IGF-I and -II dose dependently increased the number of tartrate-resistant acid phosphatase (TRAP)-positive MNCs, although their effects were less than that of 1,25-dihydroxyvitamin D3 (a hormone that induces osteoclast differentiation). No TRAP-positive MNCs appeared in the absence of these hormones. Like authentic osteoclasts, the TRAP-positive MNCs formed in response to IGF-I and -II bound [125I]salmon calcitonin. When mouse bone marrow cells were cultured on ivory slices in the presence of either IGF-I or IGF-II for 10 days, numerous resorption lacunae were formed. beta-Galactosidase had no effect on IGF-mediated osteoclast formation. These results are strong evidence that both IGF-I and IGF-II stimulate bone resorption in vitro by enhancing osteoclast formation and function. Our data also suggest that the IGFs act through the intermediary of osteoblastic cells to stimulate osteoclast activity and that the type I, but not the type II, IGF receptor is involved in their responses. We propose that the local production of IGF-I and IGF-II may modulate both osteoblast-osteoclast interactions and osteoclast formation and play an important role in bone remodeling.

The cellular actions of interleukin-11 on bone resorption in vitro.

The pleiotropic cytokine interleukin-11 (IL-11) stimulates osteoclast formation in vitro, but it is not known whether it influences other steps in the bone-resorptive cascade. Using a variety of in vitro model systems for studying bone resorption we have investigated the effects of IL-11 on 1) osteoclast formation, fusion, migration, and activity; and 2) osteoblast-mediated osteoid degradation. The involvement of matrix metalloproteinases (MMPs) and products of arachidonic acid metabolism in IL-11-mediated resorption were also assessed. We first examined the bone-resorptive effects of IL-11 by assessing 45Ca release from neonatal mouse calvarial bones. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10(-10) M. The kinetics of IL-11-mediated 45Ca release demonstrated that it was without effect for the first 48 h of culture, but by 96 h, it stimulated 45Ca release to the same level as that produced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (a hormone that stimulates osteoclast formation and activity). IL-11 also produced a dose-dependent increase in osteoblast-mediated type I collagen degradation with a maximum of 58.0 +/- 6.2% at 5 x 10(-9) M; this effect of IL-11 was less than that produced by 1,25-(OH)2D3 (76.5 +/- 7.1%) and was prevented by an inhibitor of MMPs, but not those blocking arachidonic acid metabolism. We then tested the effects of IL-11 on isolated mouse osteoclasts cultured on ivory slices in the presence and absence of primary mouse osteoblasts. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts. We then examined the effects of IL-11 on the formation of osteoclast-like multinucleate cells in mouse bone marrow cultures and the resorptive activity of such cultures using ivory as a substrate. IL-11 dose-dependently increased 1) the number of tartrate-resistant acid phosphatase-positive osteoclast-like multinucleate cells and 2) the surface area of lacunar resorption, although the effects were less than that of 1,25-(OH)2D3. The effect of IL-11 on bone marrow lacunar resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. These results provide strong evidence that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating osteoclast migration and activity. Our data also suggest that the stimulatory effects of IL-11 involve both MMPs and products of arachidonic acid metabolism.

DDAVP stimulates prostacyclin production.

Des-amino-D-arginine vasopressin (DDAVP) stimulates the release of factor VIII and plasminogen activator from the vascular endothelium. An infusion of exogenous factor VIII given to haemophiliacs causes an increase in platelet activation. This activation does not occur after stimulating a rise in the patient's own factor VIII level caused by DDAVP infusion. We hypothesised therefore that DDAVP could also cause the endothelial release of prostacyclin (PGI2), a potent anti-platelet agent which would counteract the aggregating effect of factor VIII. To examine this possibility we studied the effect of DDAVP on prostacyclin release, as measured by its stable metabolite 6-oxo-PGF1 alpha, in vitro and in vivo. Rabbit aortic rings were incubated with different concentrations of DDAVP using saline as control. The supernatant was assayed for 6-oxo-PGF1 alpha by radioimmunoassay. All concentrations of DDAVP gave a significant release of 6-oxo-PGF1 alpha. Vasopressin was much less potent. When DDAVP was infused into haemophilic patients there was a significant increase in circulating 6-oxo-PGF1 alpha levels immediately after the infusion. The facial flushing observed as a side-effect of DDAVP could therefore be prostacyclin-mediated. We confirmed this by abolishing the DDAVP induced flushing seen in normal subjects by prior treatment with aspirin which inhibits PGI2 formation.

Autocrine signals promote osteoblast survival in culture.

We have studied the survival requirements of osteoblasts to test the hypothesis that osteoblasts undergo programmed cell death (PCD) or apoptosis unless they are continuously signalled by other cells not to do so. Osteoblasts survived for 6 days in culture at high cell density in the absence of other cell types, serum or exogenous proteins, but they died with the morphological features of apoptosis in these conditions at low cell density. Osteoblast survival was enhanced during the first 2 days of culture by the addition of the sulphydryl compound, cysteine to the culture medium which was converted intracellularly to the antioxidant glutathione. Catalase, an enzyme decomposing hydrogen peroxide, also protected the cells, whereas superoxide dismutase had no effect. Therefore, osteoblasts in culture are sensitive to toxic compounds derived from molecular oxygen, i.e. hydroxyl radicals or hydrogen peroxide spontaneously generated in CMRL medium containing ascorbate and ferrous ions. Conditioned medium from high density cultures prevented osteoblast apoptosis in low density cultures, as long as antioxidants were also present. The enhancing effect of conditioned medium on osteoblast survival was prevented by neutralizing antibodies to insulin-like growth factor-I (IGF-I) and IGF-II but not by antibodies to either platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF). These results suggest that in addition to regulating cell growth and differentiation, IGF-I and IGF-II also function as survival factors for osteoblasts. Our data also indicate that antioxidants are required for osteoblast survival and that they enhance growth factor mediated osteoblast survival.

The effects of serine proteinase inhibitors on bone resorption in vitro.

The aims of this study were to identify the role and sites of action of serine proteinases (SPs) in bone resorption, a process which involves a cascade of events, the central step of which is the removal of bone matrix by osteoclasts (OCs). This resorbing activity, however, is also determined by recruitment of new OCs to future resorption sites and removal of the osteoid layer by osteoblasts (OBs), which enables OCs to gain access to the underlying mineralized bone. The resorption systems we have studied consisted of (i) neonatal calvarial explants, (ii) isolated OCs cultured on ivory slices, (iii) mouse OBs cultured on either radiolabelled type I collagen films or bone-like matrix, (iv) bone marrow cultures to assess OC formation and (v) 17-day-old fetal mouse metatarsal bone rudiments to assess OC migration and fusion. Two separate SP inhibitors, aprotinin and alpha(2)-antiplasmin dose-dependently inhibited (45)Ca release from neonatal calvarial explants: aprotinin (10(-6) M) was the most effective SP inhibitor, producing a maximum inhibitory effect of 55.9%. Neither of the SP inhibitors influenced either OC formation or OC resorptive activity. In contrast, each SP inhibitor dose-dependently inhibited OB-mediated degradation of both type I collagen fibrils and non-mineralized bone matrix. In 17-day-old metatarsal explants aprotinin produced a 55% reduction in the migration of OCs from the periosteum to the mineralized matrix after 3 days in culture but after 6 days in culture aprotinin was without effect on OC migration. Primary mouse osteoblasts expressed mRNA for urokinase type plasminogen activator (uPA), tIssue type plasminogen activator (tPA), the type I receptor for uPA, plasminogen activator inhibitor types I and II and the broad spectrum serine proteinase inhibitor, protease nexin I. In situ hybridization demonstrated expression of tPA and uPA in osteoclasts disaggregated from 6-day-old mouse long bones. We propose that the regulation of these various enzyme systems within bone tIssue determines the sites where bone resorption will be initiated.

Effect of poly DL-lactide--co-glycolide implants and xenogeneic bone matrix-derived growth factors on calvarial bone repair in the rabbit.

Polymer implant discs composed of 50:50 poly DL-lactide-co-glycolide (molecular weight about 9000) were used to repair 5 mm calvarial defects in 2 kg rabbits and osseous repair compared to spontaneous healing (control). After 4 weeks the implants had undergone substantial degradation with little evidence of residual polymer. The extent to which the defects had been replaced by bone showed individual variation. In some animals a layer of bone with normal cancellous architecture had bridged the defect, but at no time was bone observed in intimate contact with the polymer matrix, suggesting that the material had acted as a tissue spacer rather than an osteoconductive substrate. Non-osseous tissue consisted of a highly vascular fibrous connective tissue containing variable numbers of inflammatory cells. In some sites numerous macrophages and multinucleate giant cells were observed, the majority of which were shown by immunocytochemistry to be MHC class II-positive. Histomorphometric analysis demonstrated no statistically significant difference in osseous repair between control and polymer implant groups after 1, 2 or 3 months. Incorporation of bone matrix proteins extracted from bovine cortical bone into the discs, however, provoked a cellular and humoral immune response which had a significant inhibitory effect on osseous repair. These data suggest, first, that while synthetic polymers have potential as bone graft substitutes, improvements in their performance in vivo are needed and, second, it is advisable to use allogeneic proteins in rabbit models of bone regeneration.

Ultrastructural morphology of granule release from juxtaglomerular myoepithelioid and peripolar cells.

The morphology of granule release from juxtaglomerular myoepithelioid and peripolar cells has been examined. Renin release occurs from myoepithelioid granules by an unusual form of exocytosis via deep channel-like invaginations of the plasma membrane. Such release occurs towards the juxtaglomerular interstitium as well as towards the arteriolar lumen. Peripolar cell granule exocytosis occurs directly into the urinary space in sodium-depleted sheep, tentatively supporting the speculation that it may be the source of an intrarenal sodium-retaining hormone.

Macrophages in renal injury.

There is good evidence from experimental studies that glomerular macrophages are important in acute renal injury and an increasing acceptance that they also play a role in chronic glomerular injury by stimulating mesangial cell proliferation and glomerulosclerosis. However, it is now evident that the contribution of macrophages and T cells within the interstitium must be taken into account. Indeed, if it is proved that progressive renal injury occurs via interstitial DTH mechanisms, regardless of the nature of the initial glomerular insult, then such mechanisms may provide a suitable target for the development of novel therapeutic strategies.

A case of granulomatous dermatitis: cutaneous leishmaniasis.

A case is reported of a 75-year-old woman, with a past clinical history of granuloma annulare, who developed groups of papulonodular skin lesions on the trunk and face six weeks after returning from a trip to the Mediterranean. The initial biopsy showed a granulomatous dermatitis which was considered consistent with the sarcoidal variant of granuloma annulare, and the lesions were treated with topical and intralesional steroid. A second biopsy performed four months later revealed large numbers of histiocytes containing diagnostic Leishman bodies. It is not clear whether the first biopsy was from a chronic lesion and the second from an acute lesion, or whether local steroid treatment enhanced proliferation of organisms and made a definitive diagnosis possible on the second biopsy.

Tubulointerstitial nephritis following high-dose ifosfamide in three breast cancer patients.

We report three cases of women with breast cancer who developed renal impairment following treatment with high-dose ifosfamide. All the women underwent renal biopsy, which demonstrated severe interstitial damage with tubular changes by light and electron microscopy. Although reversible acute tubular dysfunction is well recognised with ifosfamide therapy, the long-term outcome of ifosfamide-induced renal injury remains unclear. The results of the current study suggest that ifosfamide can cause severe irreversible renal tubulointerstitial injury and should be used with caution even when there is initially normal renal function.

Ultrastructural changes in the sheep renal juxtaglomerular apparatus in response to sodium depletion or loading.

The ultrastructural changes in various components of the renal juxtaglomerular apparatus have been examined in sheep subjected to sodium depletion or loading. In normal sheep, juxtaglomerular arteriolar myoepithelioid cell granulation was relatively sparse whereas peripolar cell granulation was prominent. Sheep subjected to rapid sodium depletion in response to parotid cannula drainage showed features indicating increased activity of the renin-angiotensin system with a rise in plasma renin concentration, an increase in the juxtaglomerular index and morphological evidence of increased renin granule production in arteriolar myoepithelioid cells. In contrast, in sheep subjected to dietary sodium loading, there was evidence of decreased activity of the renin-angiotensin system with a fall in plasma renin concentration and relatively poor myoepithelioid cell granulation. In sodium depleted sheep, juxtaglomerular peripolar cells showed increased synthetic activity and marked heterogeneity of granule density; in sodium loaded sheep, peripolar cells showed no consistent changes. A major new finding was the detection of exocytotic release of granule material from peripolar cells into the urinary space during sodium depletion, confirming the secretory nature of such cells and supporting the concept that they may play a role in sodium homeostasis.

Structural and functional studies of the adrenal cortex and renal juxtaglomerular apparatus in pregnant sheep subjected to sodium depletion or loading.

The morphological changes in the adrenal cortex and renal juxtaglomerular apparatus have been examined in pregnant sheep subjected to sodium depletion or loading. These changes were similar to those found in non-pregnant sheep in response to sodium depletion or loading, with the additional finding of interstitial edema in the zona glomerulosa of sheep with twin fetuses. As in non-pregnant sheep, sodium depletion resulted in evidence of juxtaglomerular peripolar cell granule release. The major finding was that sodium depletion in sheep with twin fetuses triggered the onset of ovine toxemia or "twin lamb disease".

Ultrastructural localisation of CD44 in the rat lung in experimental Goodpasture's syndrome.

Although CD44 is known to be involved in a wide array of cell to cell and cell to matrix interactions, its role in immune-mediated disease is not well understood. Therefore, using immunogold electron microscopy we have determined the precise localisation of CD44 in the rat lung in experimental Goodpasture's (GP) syndrome, a model of immune-mediated pulmonary disease. In normal rat lung CD44 was present on the surface of alveolar macrophages but was not detectable on endothelium. In GP syndrome there was strong CD44 expression on all infiltrating inflammatory leucocytes, both adherent to endothelium and within the alveolar spaces and interstitium. However the most striking finding was the progressively strong antibody staining for CD44 on pulmonary endothelium of alveolar capillaries and larger vessels over the 21 days of GP syndrome. In situ hybridisation confirmed that the endothelial CD44 staining was due to local protein synthesis. All epithelial cell surfaces, including bronchial epithelium and type I and II alveolar epithelial cells, were negative in normal rat lung and GP syndrome. De novo CD44 expression by endothelial cells during the progression of GP syndrome may contribute to leucocyte recruitment and cell-mediated lung injury.

Ceramide-induced cell death/survival in murine osteoblasts.

Programmed cell death (PCD) or apoptosis is a naturally occurring cell suicide pathway induced in a variety of cell types. We determined whether ceramide treatment contributes to reduced cell viability and increased PCD in primary osteoblasts and the signalling pathways that are involved. Cell viability was determined by the 3-(4,5-dimethyl-thiozol-2-yl)-2,5-diphenyl tetrazolium bromide assay. We found that C(2)-ceramide (or=2 x 10(-6) M significantly reduced osteoblast viability in a dose- and time-dependent manner. The effect of ceramide on cell viability was specific since C(2)-dihydroceramide had no effect. Increasing intracellular ceramide levels with either sphingomyelinase (SMase) or an inhibitor of ceramide metabolism also increased osteoblast apoptosis. Ceramide-induced PCD in osteoblasts was determined by nuclear appearance and DNA fragmentation. PCD was induced by both C(2)-ceramide and SMase. The ability of ceramide (5 x 10(-8) M) to promote osteoblast survival was prevented by a general protein kinase C (PKC) inhibitor and by a PKC zeta inhibitor, whilst osteoblast survival was enhanced in the presence of a protein phosphatase 1 (PP1) inhibitor. Phosphatidylinositol-3 kinase (PI3K) inhibitors had no effect on osteoblast survival. The ability of ceramide (5 x 10(-5) M) to induce apoptosis was prevented by the inhibitors of PP1 and PKC delta, whilst the general PKC and PI3K inhibitors had no effect on it. Our findings suggest that ceramide signals osteoblast survival and apoptosis through different intracellular pathways, and that alteration in the intracellular levels of ceramide may play an important role in bone remodelling.