Tibial slope in the posterolateral quadrant with and without ACL injury.

INTRODUCTION: An increased tibial slope is a risk factor for rupture of the anterior cruciate ligament. In addition, a tibial bone bruise or posterior lateral impression associated with slope changes also poses chronic ligamentous instability of the knee joint associated with an anterior cruciate ligament (ACL) injury. In the majority of cases, the slope is measured in one plane X-ray in the lateral view. However, this does not sufficient represent the complex anatomy of the tibial plateau and especially for the posterolateral quadrant. Normal values from a "healthy" population are necessary to understand if stability of the knee joint is negatively affected by an increasing slope in the posterolateral area. Until now there are no data about the physiological slope in the posterolateral quadrant of the tibial plateau. MATERIALS AND METHODS: In 116 MRI scans of patients without ligamentous lesions and 116 MRI scans with an ACL rupture, tibial slope was retrospectively determined using the method described by Hudek et al. Measurements were made in the postero-latero-lateral (PLL) and postero-latero-central (PLC) segments using the 10-segment classification. In both segments, the osseous as well as the cartilaginous slope was measured. Measurements were performed by two independent surgeons. RESULTS: In the group without ligamentous injury the mean bony PLL slope was 5.8° ± 4.8° and the cartilaginous PLL slope was 6.7° ± 4.8°. In the PLC segment the mean bony slope was 6.6° ± 5.0° and the cartilaginous slope was 9.4° ± 5.7°. In the cohort with ACL rupture, the bony and cartilaginous slope in both PLL and PCL were significantly higher (P < 0.001) than in the group without ACL injury (bony PLL 9.8° ± 4.8°, cartilage PLL 10.4° ± 4.7°, bony PLC 10.3° ± 4.8°, cartilage PLL 12.8° ± 4.3°). Measurements were performed independently by two experienced surgeons. There were good inter- (CI 87-98.7%) and good intraobserver (CI 85.8-99.6%) reliability. CONCLUSION: The bony and the cartilaginous slope in the posterolateral quadrant of the tibial plateau are different but not independent. Patients with an anterior cruciate ligament injury have a significantly steeper slope in the posterolateral quadrant compared to a healthy group. Our data indicate that this anatomic feature might be a risk factor for a primary ACL injury which has not been described yet. LEVEL OF EVIDENCE: III.

Analysis of low-dose estrogen on callus BMD as measured by pQCT in postmenopausal women.

BACKGROUND: Osteoporosis affects elderly patients of both sexes. It is characterized by an increased fracture risk due to defective remodeling of the bone microarchitecture. It affects in particular postmenopausal women due to their decreased levels of estrogen. Preclinical studies with animals demonstrated that loss of estrogen had a negative effect on bone healing and that increasing the estrogen level led to a better bone healing. We asked whether increasing the estrogen level in menopausal patients has a beneficial effect on bone mineral density (BMD) during callus formation after a bone fracture. METHODS: To investigate whether estrogen has a beneficial effect on callus BMD of postmenopausal patients, we performed a prospective double-blinded randomized study with 76 patients suffering from distal radius fractures. A total of 31 patients (71.13 years ±11.99) were treated with estrogen and 45 patients (75.62 years ±10.47) served as untreated controls. Calculated bone density as well as cortical bone density were determined by peripheral quantitative computed tomography (pQCT) prior to and 6 weeks after the surgery. Comparative measurements were performed at the fractured site and at the corresponding position of the non-fractured arm. RESULTS: We found that unlike with preclinical models, bone fracture healing of human patients was not improved in response to estrogen treatment. Furthermore, we observed no dependence between age-dependent bone tissue loss and constant callus formation in the patients. CONCLUSIONS: Transdermally applied estrogen to postmenopausal women, which results in estrogen levels similar to the systemic level of premenopausal women, has no significant beneficial effect on callus BMD as measured by pQCT, as recently shown in preclinical animal models. TRIAL REGISTRATION: Low dose estrogen has no significant effect on bone fracture healing measured by pQCT in postmenopausal women, DRKS00019858 . Registered 25th November 2019 - Retrospectively registered. Trial registration number DRKS00019858 .

[Cement augmentation on the spine : Biomechanical considerations]. / Zementaugmentation an der Wirbelsäule : Was ist biomechanisch zu beachten?

BACKGROUND: Vertebral compression fractures are the most common osteoporotic fractures. Since the introduction of vertebroplasty and screw augmentation, the management of osteoporotic fractures has changed significantly. AIMS: The biomechanical characteristics of the risk of adjacent fractures and novel treatment modalities for osteoporotic vertebral fractures, including pure cement augmentation by vertebroplasty, and cement augmentation of screws for posterior instrumentation, are explored. MATERIALS AND METHODS: Eighteen human osteoporotic lumbar spines (L1-5) adjacent to vertebral bodies after vertebroplasty were tested in a servo-hydraulic machine. As augmentation compounds we used standard cement and a modified low-strength cement. Different anchoring pedicle screws were tested with and without cement augmentation in another cohort of human specimens with a simple pull-out test and a fatigue test that better reflects physiological conditions. RESULTS: Cement augmentation in the osteoporotic spine leads to greater biomechanical stability. However, change in vertebral stiffness resulted in alterations with the risk of adjacent fractures. By using a less firm cement compound, the risk of adjacent fractures is significantly reduced. Both screw augmentation techniques resulted in a significant increase in the withdrawal force compared with the group without cement. Augmentation using perforated screws showed the highest stability in the fatigue test. DISCUSSION AND CONCLUSION: The augmentation of cement leads to a significant change in the biomechanical properties. Differences in the stability of adjacent vertebral bodies increase the risk of adjacent fractures, which could be mitigated by a modified cement compound with reduced strength. Screws that were specifically designed for cement application displayed greatest stability in the fatigue test.

Calcium homeostasis influences radiological fracture healing in postmenopausal women.

INTRODUCTION: Recent studies suggest that calcium and 25-[OH]-cholecalciferol represent substantial co-factors in fracture healing. However, there still seems to be no sustainable consensus regarding the influence on fracture healing patterns. In this study, the influence of calcium and vitamin D levels on fracture callus formation was prospectively analysed using pQCT scan. METHODS: 94 postmenopausal females with distal radius fractures and consecutive surgery were included. Calcium, 25-[OH]-cholecalciferol, parathyroid hormone and bone-specific alkaline phosphatase levels were obtained prior surgical treatment and after 6 weeks. A pQCT scan was performed on both sites. Bone mineral density and fracture callus area were determined after detecting the outer border contour at a threshold of 280 mg/ccm. Patients received daily supplements of 1000 mg calcium and 880 IU 25-[OH]-cholecalciferol. RESULTS: Mean 25-[OH]-cholecalciferol level was 19.61 ± 21.87 ng/ml, mean parathyroid hormone level was 52.6 ± 58.9 ng/l and mean Ca level was 2.23 ± 0.35 mmol/l. After 6 weeks of supplementation a significant increase of calcium (p < 0.001) and 25-[OH]-cholecalciferol (p < 0.001), and a significant decrease of parathyroid hormone (p < 0.001) levels were observed. Sixth week follow-up fracture callus area correlated significantly with postoperative normal range calcium levels on the fractured site (p = 0.006). Bone mineral density correlated with age (p < 0.001), but not with calcium and 25-[OH]-cholecalciferol levels after 6 weeks. All fractures presented timely adequate callus formation. CONCLUSION: Calcium and parathyroid hormone serum levels influence fracture callus area interpreted as fracture callus formation patterns. Calcium levels within physiological range accounted for highest fracture callus area. Therefore, a balanced calcium homeostasis is required for appropriate callus formation.

Pupil evaluation in addition to Glasgow Coma Scale components in prediction of traumatic brain injury and mortality.

BACKGROUND: Early diagnosis and prediction of traumatic brain injury (TBI) is essential for determining treatment strategies and allocating resources. This study evaluated the predictive accuracy of Glasgow Coma Scale (GCS) verbal, motor and eye components alone, or in addition to pupil size and reactivity, for TBI. METHODS: A retrospective cohort analysis of data from 51 425 severely injured patients registered in the Trauma Registry of the German Society for Trauma Surgery from 1993 to 2009 was undertaken. Only directly admitted patients alive on admission and with complete data on GCS, pupil size and pupil reactivity were included. The unadjusted predictive roles of GCS components and pupil parameters, alone or in combination, were modelled using area under the receiver operating characteristic (AUROC) curve analyses and multivariable logistic regression regarding presence of TBI and death. RESULTS: Some 24 115 patients fulfilled the study inclusion criteria. Best accuracy for outcome prediction was found for pupil reactivity (AUROC 0.770, 95 per cent confidence interval 0.761 to 0.779) and GCS motor component (AUROC 0.797, 0.788 to 0.805), with less accuracy for GCS eye and verbal components. The combination of pupil reactivity and GCS motor component (AUROC 0.822, 0.814 to 0.830) outmatched the predictive accuracy of GCS alone (AUROC 0.808, 0.800 to 0.815). Pupil reactivity and size were significantly correlated (r(s) = 0.56, P < 0.001). Patients displaying both unequal pupils and fixed pupils were most likely to have TBI (95.1 per cent of 283 patients). Good outcome (Glasgow Outcome Scale score 4 or more) was documented for only 1929 patients (8.0 per cent) showing fixed and bilateral dilated pupils. CONCLUSION: The best predictive accuracy for presence of TBI was obtained using the GCS components. Pupil reactivity together with the GCS motor component performed best in predicting death.

Apoptotic effects on B-cell chronic lymphocytic leukemia (B-CLL) cells of heterocyclic compounds isolated from Guttiferaes.

A series of 10 heterocyclic compounds purified from Allanblackia were tested on two B cell lines, ESKOL and EHEB, and on cells from B-CLL patients. Several molecules inhibited the proliferation of both cell lines and promoted apoptosis of B-CLL cells through different mechanisms, some of them elicited a dissipation of the mitochondrial transmembrane potential, other triggered caspase-3 activation and cleavage of the inducible nitric oxide synthase. Blood mononuclear cells and B-lymphocytes from healthy donors appeared less sensitive than B-CLL cells. These results indicate that these molecules may be of interest in the development of new therapies for B-CLL.

Hyperforin, a new lead compound against the progression of cancer and leukemia?

Extracts of the plant St John's wort, Hyperforin perforatum L., have been used for centuries in traditional medicine, notably for the treatment of depression. One of their main lipophilic components, a natural prenylated phloroglucinol termed hyperforin (HF), has been identified as the major molecule responsible for the antidepressant effects of this plant. Within the last few years, a number of studies have demonstrated that HF displays, in addition, several other biological properties of potential pharmacological interest. They include an antibacterial capacity and inhibitory effects on inflammatory mediators. It is worth noting that HF also promotes apoptosis of various cancer cells from solid tumors and hematological malignancies, including B-cell chronic lymphocytic leukemia. In addition, HF inhibits the capacity of migration and invasion of different tumor cells, as well as exhibiting antiangiogenic effects. Altogether, these properties qualify HF as a lead structure for the development of new therapeutic molecules in the treatment of various diseases, including some malignant tumors.

Hyperforin inhibits MMP-9 secretion by B-CLL cells and microtubule formation by endothelial cells.

We previously reported that hyperforin (HF), a natural phloroglucinol purified from Saint John's wort, can induce the apoptosis of leukemic cells from patients with B-cell lymphocytic leukemia (B-CLL) ex vivo. We show here that treatment of cultured B-CLL patients' cells with HF results in a marked inhibition of their capacity to secrete matrix metalloproteinase-9, an essential component in neo-angiogenesis through degradation of the extracellular matrix process. The phloroglucinol acts by decreasing the production of the latent 92 kDa pro-enzyme. The inhibitory effect of HF is associated with a decrease in VEGF release by the leukemic cells. Moreover, HF is found to prevent the formation of microtubules by human bone marrow endothelial cells cultured on Matrigel, evidencing its capacity to inhibit vessel formation. Our results show the antiangiogenesis activity of HF and strengthen its potential interest in the therapy of B-CLL.

Pro-apoptotic properties of hyperforin in leukemic cells from patients with B-cell chronic lymphocytic leukemia.

The effects of the hyperforin (HF), a natural phloroglucinol purified from Hypericum perforatum, were investigated ex vivo on leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). HF was found to promote apoptosis of B-CLL cells, as shown by time- and dose-dependent stimulation of phosphatidylserine externalization and DNA fragmentation, by disruption of the mitochondrial transmembrane potential, caspase-3 activation and cleavage of the caspase substrate PARP-1. Moreover, HF-induced downregulation of Bcl-2 and Mcl-1, two antiapoptotic proteins that control mitochondrial permeability. HF also downregulated two proteins which are overexpressed by B-CLL patients' cells, the cell cycle inhibitor p27kip1 through caspase-dependent cleavage into a p23 form, and the nitric oxid (NO) synthase of type 2 (inducible NO synthase). This latter was accompanied by reduction in the production of NO known to be antiapoptotic in B-CLL cells. Preventing effects of the general caspase inhibitor z-VAD-fmk indicated that HF-promoted apoptosis of B-CLL cells was mostly caspase dependent. Furthermore, normal B lymphocytes purified from healthy donors appeared less sensitive to HF-induced apoptosis than B-CLL cells. These results indicate that HF may be of interest in the development of new therapies for B-CLL based on the induction of apoptosis and combination with cell cycle-dependent antitumor drugs.

Evidence for splenic suppressor cells in C3H/He, T-cell-deprived C3H/He, and nude mice bearing a 3-methylcholanthrene-induced fibrosarcoma.

Suppressor cells were demonstrated in the spleens of C3H/He mice carrying 3-methylcholantrene-induced fibrosarcomas. These cells inhibited the in vitro reactivity of normal lymphocytes to T- and B-cell mitogens. They disappeared within a few days after the tumor was surgically removed. Pretreatment of spleen cells (ScC) from tumor-bearing (TB) mice with either iron and a nagnet, antiserum against Thy 1.2 antigen plus complement, or antiserum against immunoglobulin plus complement demonstrated that the suppressor cells were adherent, non-T-cells bearing immunoglobulin at their surfaces. The suppressive effect could still be demonstrated by addition of SpC from TB mice 24 or 48 hours after phytohemagglutinin stimulation of normal SpC, SpC from TB C3H/He mice inhibited mitogen-induced stimulation of both C3H/He and DBA/2 lymphocytes. In T-cell-deprived TB C3H/He mice, suppressor cells were also observed and had the same characteristics as those in non-T-cell-deprived mice. In nude mice, however, although suppressor cells were active, they were not adherent and did not bear immunoglobulin at their surfaces. The existence of these suppressor cells may be one reason why the immune system of TB animals is unable to reject the tumor.

Immunity to a 3-methylcholanthrene-induced fibrosarcoma in the C57BL/6 mouse: in vivo analysis by the adoptive tumor neutralization test.

Analyses of the adoptive tumor neutralization test (modified Winn test) in C57BL/6 mice made immune to a 3-methylcholantheene-induced fibrosarcoma showed that the reaction was mediated by a thymus-derived lymphocyte, it was tumor-specific, and the resistance of the immunized donor mouse to the challenge was strongly correlated with the protection of the recipient mouse. Proliferation of immune cells and close contact between tumor cells and immune T-cells were required. The hypothesis of a participation of the recipient in the reaction was considered because of the lack of adoptive protection of pangenic nude mice.

Mechanisms involved in the pro- and anti-apoptotic role of NO in human leukemia.

Nitric oxide (NO) exerts contrasting effects on apoptosis, depending on its concentration, flux and cell type. In some situations, NO activates the transduction pathways leading to apoptosis, whereas in other cases NO protects cells against spontaneous or induced apoptosis. The redox state of the cells appears to be a crucial parameter for the determination of the ultimate action of NO on cell multiplication and survival. Apoptosis is mostly associated with the delivery of NO by chemical donors and with myelomonocytic cells, whereas antiapoptotic effects seem to be related to the endogenous production of NO by NO synthases and is observed more frequently in cells of the B lymphocyte lineage. Pro-apoptotic effects are often observed when NO reacts with superoxide to produce the highly toxic peroxynitrite. Through the induction of damages to DNA, NO stimulates the expression of enzymes and transcription factors involved in DNA repair and modulation of apoptosis, such as the tumor suppressor p53. The latter molecule transactivates the expression of pro-apoptotic genes, such as bax, and that of the cyclin-dependent kinase inhibitor p21, whereas it down-regulates the expression of the anti-apoptotic protein bcl-2. On the other hand, NO inactivates caspases through oxidation and S-nitrosylation of the active cystein, providing an efficient means to block apoptosis. Other protective effects of NO on apoptosis rely on the stimulation of cGMP-dependent protein kinase (PKG), modulation of the members of the bcl-2/bax family that control the mitochondrial pore transition permeability, induction of the heat shock protein HSP 70 and interaction with the ceramide pathway. A defect in the apoptotic process contributes to the accumulation of tumoral cells in leukemia, notably in B-CLL. A better knowledge of the targets of NO would provide efficient means to control cell apoptosis, and hence would possibly lead to the development of new therapeutic approaches for diseases where an alteration of apoptosis is involved.

Production of matrix metalloproteinase-9 in early stage B-CLL: suppression by interferons.

Besides vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs) play critical roles in angiogenesis, tumor invasion and metastasis. Increased angiogenesis is observed in chronic B lymphocytic leukemia (B-CLL) and published data reported VEGF and bFGF production in this disease. The purpose of this study was to investigate MMP expression in early stage B-CLL. Elevated MMP-9 concentrations were detected by ELISA in the sera of B-CLL patients (median level 250 ng/ml) compared with healthy donors (67 ng/ml) (P < 0.0001), and immunostaining with antibodies against MMP-9 and B cell antigens (CD19, CD23) substantiated the presence of MMP-9 in tumoral B lymphocytes. By using RT-PCR, ELISA and zymography experiments, we confirmed that B-CLL cells expressed and released the pro-form of MMP-9 with Mr 92 kDa (158-1300 pg/ml/10(6) cells/48 h), p-aminophenylmercuric acetate generating a 82 kDa active form. In contrast, the production of MMP-9 by normal counterpart B cells was significantly low (28-169 pg/ml/10(6)cells/48 h). Moreover, B-CLL culture supernatants contained bFGF (median levels 17 pg/ml/10(6) cells/48 h), VEGF (1.4 pg/ml/10(6) cells/48 h) and TNF-alpha (0.2 pg/ml/10(6) cells/48 h). TNF-alpha and VEGF antibodies blocked MMP-9 at the mRNA and protein levels. Interferons (IFNs) type I or type II repressed MMP-9 gelatinolytic activity in a dose and time dependency, and this was reflected by a parallel inhibition of MMP-9 mRNA and protein. IFNs however did not affect the production of bFGF, VEGF and TNF-alpha. Together, our data show that B-CLL lymphocytes synthesize MMP-9 and emphasize the specific inhibitory actions of IFNs on its expression.

Proliferative response of hairy cells to B cell growth factor (BCGF): in vivo inhibition by interferon-alpha and in vitro effects of interferon-alpha, -beta, and -gamma.

Hairy cell leukemia (HCL) is a pre-plasma B cell tumor which responds to interferon (IFN)-alpha therapy. In vitro, B cell growth factor (BCGF) can induce proliferation of hairy cells. We have investigated the effect of in vitro and in vivo treatments with different recombinant IFN on the capacity of hairy cells to proliferate in response to human BCGF. In vitro treatment of leukemic cells from HCL patients with recombinant IFN-alpha-2 (5/5 cases) or IFN-beta (4/5 cases) resulted in a marked inhibition of the BCGF-dependent response. This suppressive effect was obtained with IFN concentrations of 1000, 100 IU/ml, and even occasionally 10 IU/ml. In contrast, no such inhibition was observed with IFN-gamma, despite the presence of specific IFN-gamma receptors on hairy cells at densities similar to receptors for IFN-alpha/beta. The IFN-alpha-induced suppression of the proliferative response of hairy cells to BCGF was also observed in vivo in two patients within 6-12 hr after administration of single doses of IFN-alpha. When hairy cells were maintained in culture for 1 week, they recovered their capacity to be stimulated by BCGF. This reversion was also shown in vivo in hairy cells isolated 1 week after IFN administration. Since in vivo growth of hairy cells could possibly result from the autocrine secretion of BCGF, we propose that the therapeutic effect of IFN-alpha on HCL may be due in part to an inhibition of such autocrine loop.

Flavopiridol downregulates the expression of both the inducible NO synthase and p27(kip1) in malignant cells from B-cell chronic lymphocytic leukemia.

Flavopiridol, an inhibitor of cyclin-dependent kinases and other protein kinases, induces in vitro apoptosis of malignant cells from B-cell chronic lymphocytic leukemia (B-CLL). Previously, we reported that nitric oxide (NO), produced by an inducible NO synthase (iNOS), spontaneously expressed by the B-CLL cells, contributed to their deficiency in apoptosis. In the present work, we show that ex vivo treatment of leukemic cells from B-CLL patients with flavopiridol results in the inhibition of iNOS expression, as determined by immunofluorescence and Western blotting, and in a marked inhibition of NO production measured in situ with a specific fluorescent probe (DAF-2 DA). These effects are accompanied by membrane, mitochondrial and nuclear events of apoptosis. Flavopiridol exposure also results in the stimulation of caspase 3 activity and in caspase-dependent cleavage of p27(kip1), a negative regulator of the cell cycle, which is overexpressed in B-CLL. Thus, flavopiridol is capable of downregulating both iNOS and p27(kip1) expression in B-CLL cells. Furthermore, flavopiridol-promoted apoptosis is partly reverted by an NO donor, suggesting that inhibition of the NO pathway could participate in the apoptotic effects of flavopiridol on the leukemic cells.

Expression of a functional inducible nitric oxide synthase in hairy cell leukaemia and ESKOL cell line.

The expression of nitric oxide synthase (NOS) isoforms was investigated in the established ESKOL hairy cell line and in leukemic cells of patients with hairy cell leukemia (HCL). By reverse transcription-polymerase chain reaction (RT-PCR), these cells were found to spontaneously express inducible NOS (iNOS)-specific mRNA, but not endothelial constitutive NOS (ecNOS) mRNA. The iNOS protein was detected by immunofluorescence in the cytoplasm of permeabilized leukemic cells and ESKOL cells, using different anti-iNOS monoclonal antibodies. A protein of 135 kDa was identified by Western blotting in ESKOL and HCL lysates, confirming the presence of an iNOS in these cells. Cytosolic homogenates displayed NOS catalytic activity, as measured by the conversion of 14C-labelled L-arginine into 14C L-citrulline and by detection in situ using the DAF-2DA (diaminofluorescein diacetate) NO-sensitive fluorescent probe. Ligation of CD23 (low affinity IgE receptor) was found to increase iNOS expression in ESKOL and conversely to decrease the percentage of cells undergoing apoptosis, as measured by the percentage of cells expressing annexin V. These results indicate that, as in chronic B cell lymphocytic leukemia cells (B-CLL) a functional iNOS is expressed constitutively in hairy cells that contributes to protecting these tumoral cells from apoptosis.

Expression of the B8.7 antigen on hairy cells and relation with the LMW-BCGF response.

Hairy cells are classified as B cell tumors at a preplasma cell stage of differentiation and are believed to represent cells undergoing a switch process. These cells are stimulated in vitro to DNA synthesis and multiplication in the presence of the lymphokine LMW-BCGF. We have tested the level of expression on these cells of the newly described B8.7 activation marker which has been reported to be associated with the capacity of various B cells to respond to LMW-BCGF. The presence of this marker has been readily detected on the hairy cells of 10 of the 12 patients tested in this study; interestingly, for one of the negative cases, the tumor cells were unable to proliferate in response to LMW-BCGF. As on normal B cells, a marked inhibition of the LMW-BCGF dependent response could be achieved in the presence of a monoclonal anti-B8.7 antibody, sustaining the proposal that the B8.7 molecule is involved in the signaling pathway of this growth factor. IFN-alpha is highly efficient in the therapy of hairy cell leukemia (HCL), and we confirm in the present study that IFN-alpha also inhibits the LMW-BCGF dependent proliferation of hairy cells in vitro. In addition, we show that this inhibition is independent of a significant modulation of the B8.7 antigen, a molecule putatively associated with the LMW-BCGF receptor.

Heterogeneous spontaneous and interleukin-4-induced nitric oxide production by human monocytes.

The generation of nitric oxide by human monocytes has long been a subject of controversy because of the difficulty of rationalizing this production. In this work we evaluated the capacity of human monocytes to produce nitric oxide (NO) as measured by nitrite (NO2-) release. Resting unstimulated monocytes (2 x 10(6) cells/ml) were found to produce significant amounts of NO2- after 8 to 12 days in culture. This production appeared to be highly heterogeneous. Indeed, approximately, 75% of monocytes from the different donors produced up to 10 microM NO2- and were considered low producers; the last 25% produced higher amounts of NO2- (from 10 to 110 microM) and were considered high producers. In any case the spontaneous production of NO2- by monocytes was overcome in the presence of 1 mM N omega-monomethyl-L-arginine (LNMMA). This inhibitory effect was reversed in the presence of an excess of L-arginine (5 mM), indicating that this process is effectively dependent on L-arginine metabolism. Because interleukin-4 (IL-4) is considered an important NO-regulatory cytokine, its regulatory effect on this spontaneous production of NO was also evaluated. In the presence of a defined dose of IL-4 (1 to 100 ng/ml) the spontaneous production of the high-producing population of monocytes was abrogated, whereas IL-4 stimulated the production by the low-producing population of monocytes, which was suppressed in the presence of LNMMA. The present data indicate that NO production by human monocytes is heterogeneous and that IL-4 can be a potent inducer or inhibitor of this production, suggesting a variability in the activation state of these cells.

Ligation of CD23 activates soluble guanylate cyclase in human monocytes via an L-arginine-dependent mechanism.

Transduction through Fc epsilon R2/CD23 was analyzed in normal human monocytes using immunoglobulin E (IgE)-anti-IgE immune complexes (IgE ICs) and monoclonal antibodies (mAbs) to CD23. Anti-CD23 mAb and IgE IC triggered a time-dependent increase in cGMP and cAMP in interleukin-4-preincubated (CD23+) but not in unstimulated (CD23-) monocytes. Maximal cGMP and cAMP accumulations were observed 10 and 20 min, respectively, after the onset of CD23 ligation. The increase in cGMP was inhibited with N omega-monomethyl-L-arginine (L-NMMA), which also partially affected cAMP accumulation. Addition of an anti-CD23 mAb Fab fragment inhibited the IgE IC- and the anti-CD23 mAb-induced cGMP and cAMP accumulation, confirming the engagement of CD23. In addition, IgE IC and anti-CD23 mAb induced, at least in some donors, a production of nitrite that was inhibited in the presence of L-NMMA. Taken together, these findings suggest a possible involvement of the nitric oxide synthase pathway in IgE IC-mediated activation of CD23+ monocytes.

Beta 2-adrenoceptor agonists regulate the IL-4-induced phenotypical changes and IgE-dependent functions in normal human monocytes.

The beta 2-adrenoceptor agonists salbutamol and fenoterol were tested for their regulatory effects on human monocyte phenotype and functions, either alone or in combination with interleukin-4 (IL-4). These drugs enhanced in a dose-dependent manner the IL-4-induced membrane and mRNA expression of the low-affinity receptor for immunoglobulin E (IgE) (CD23), as well as the release of its soluble form, sCD23. Salbutamol and fenoterol alone elicited expression of the monomorphic beta 2-chain (CD18) of the leukocyte functional antigen (LFA1) family. This effect appeared to be restricted to CD11b (CR3) and CD11c (gp 150-95), because CD11a (LFA-1 alpha chain) was not modified. beta 2-Adrenoceptor stimulation was also found to potentiate the effect of IL-4 on CD11b, CD11c, and CD18 expression. In contrast, these agents alone did not alter the level of major histocompatibility complex class II and CD14 antigens or modify their respective up- and down-regulation by IL-4. Ligation of CD23 on IL-4-preincubated (CD23+) monocytes with IgE/anti-IgE immune complexes induced the release of free radicals nitric oxide and of the proinflammatory mediators IL-6 and thromboxane B2 (TxB2). Addition of salbutamol, inactive alone, potentiated the generation of superoxide anion and of nitric oxide generation, as well as the production of IL-6 and TxB2 triggered by CD23 ligation. These results indicate that beta 2-adrenoceptor stimulation potentiates in vitro the IL-4-induced phenotypical and functional changes on monocytes and suggest that such an interaction could occur in IgE-dependent immune reactions.