[Antipsychotic-drug-induced hyperprolactinemia: physiopathology, clinical features and guidance]. / Hyperprolactinémies induites par les antipsychotiques : physiopathologie, clinique et surveillance.

BACKGROUND: Hyperprolactinemia is a frequent but neglected adverse effect observed in patients treated with antipsychotic-drugs. In this review, we summarize its physiopathogenetic mechanism, its clinical manifestations in men and women, and the way to manage it. LITERATURE FINDINGS: Prolactin is a hormone secreted by lactotroph cells in the anterior pituitary. Its synthesis and release are under the control of peptides, steroids and neurotransmitters. The main inhibitory regulation is made by dopamine, which binds dopamine receptors D2 on the membrane of lactotroph cells. Antipsychotic-drugs block these receptors and thus remove the inhibitory effect of dopamine on prolactin secretion. All antipsychotic-drugs block D2 receptors and all can induce hyperprolactinemia. Nonetheless, it seems that the faster the antipsychotic-drug dissociates from D2 receptors, the lesser the increase of prolactin in the plasma. Another way to explain hyperprolactinemia is the ability of antipsychotic-drugs to cross the blood-brain barrier. The role of their metabolites should also be considered. For these reasons, one can distinguish prolactin-raising (conventional neuroleptics, amisulpride, risperidone) and prolactin-sparing (clozapine, aripiprazole, olanzapine) antipsychotics. An English study showed that 18% of men and 47% of women treated with antipsychotics for severe mental illness had a prolactin level above the normal range. Hyperprolactinemia is in fact more frequent in women than in men. Sometimes it is asymptomatic, but the higher the prolactin level is, the more patients have clinical manifestations. Some symptoms are due to the hypogonadism caused by prolactin, which disturbs hypothalamic-pituitary axis function, and others are due to direct effects on target tissues. Consequently, patients can suffer from sexual dysfunction, infertility, amenorrhea, gynecomastia or galactorrhoea. Data suggest that these symptoms are common, but patients don't mention them spontaneously and clinicians underestimate their prevalence. In the long-term, hypogonadism involves a premature bone loss in men and women. Klibanski and colleagues showed that this loss is significant only in women with hyperprolactinemia associated with amenorrhea. That suggests that prolactin is not directly responsible for this clinical feature. Nevertheless, prolactin seems to be involved in the development of breast cancer, but its role is unclear for prostate cancer. DISCUSSION: Our review promotes a check-up before beginning a treatment with antipsychotic agents. First, a baseline prolactin level should be measured. It should also include the research on previous treatment with antipsychotic-drugs and the assessment of adverse effects suggestive of hyperprolactinemia. Questioning should finally look for any contra-indication to antipsychotics. Monitoring during antipsychotic treatment has been studied by a group of international experts in psychiatry, medicine, toxicology and pharmacy who made a critical review of clinical guidance on hyperprolactinemia. Experts notify that it is important to check whether patients have any sexual dysfunction, such as loss of libido or menstrual irregularity, and galactorrhoea. Prolactin level should also be controlled after three months of stable dose treatment, or if any clinical feature of hyperprolactinemia appears. If a patient prescribed antipsychotic-drugs has a confirmed prolactin level above the normal range, it is necessary to exclude other causes of hyperprolactinemia. If antipsychotics are really involved, the management should be adapted with the prolactin level and the patient him/herself. To summarize, clinicians can decrease the dose of the antipsychotic or switch to a prolactin-sparing drug. Oral contraceptives can be added whether to prevent pregnancy or to prevent bone loss and osteoporosis. Finally, experts recommend reserving dopamine agonists to treat antipsychotic-induced hyperprolactinemia in very exceptional circumstances as it can worsen the mental illness.

Immunomodulatory effects of therapeutic preparations of human albumin.

BACKGROUND AND OBJECTIVES: Albumin is the most abundant protein in plasma and is considered to be immunologically inert. However, we recently observed that therapeutic human albumin preparations, used as protein control in studies involving high doses of IVIg, modulated the MHC II-restricted activation of antigen-specific T cells. In the present work, we characterized this effect in more details. MATERIALS AND METHODS: An in vitro antigen presentation assay using mouse cells was used to evaluate the effect of therapeutic human albumin preparations on the activation of ovalbumin-specific T cells. Flow cytometry and quantitative real-time PCR were used to monitor the expression of genes involved in this process. RESULTS: Therapeutic human albumin preparations increased T cell activation in a dose-dependent manner. The effect was explained by an increase in the expression of MHC II and of two other genes (CIITA and H2-M) involved in antigen presentation by murine monocytic cells. Similarly, the expression of HLA-DR on the surface of human monocytic cells was increased following incubation with therapeutic human albumin preparations. CONCLUSION: Altogether, these results reveal a possible physiological role of albumin in immunological processes, leading to an increased ability of antigen presenting cells to trigger T cell activation. This immunomodulatory effect needs to be considered, at least in studies in which albumin is used as a presumably inert control protein.

[Cardiological monitoring of antipsychotic-treated patients: evaluation and evolution of a hospital protocol]. / Surveillance cardiologique des patients traités par neuroleptiques : évaluation et évolution d'un protocole hospitalier.

INTRODUCTION: Following the Agence Française de Sécurité Sanitaire des Produits de Santé (Afssaps) (French Health Authority) recommendations in 2001, which impose the rigorous follow-up (electrocardiogram [ECG] and ionogram) of patients treated with antipsychotics (AP), a monitoring protocol was elaborated and set up in the Caen psychiatric hospital in April 2002. Protocol evaluation compared with fixed aims was performed after two years' follow-up. AIM OF THE STUDY: This protocol had to answer a triple aim: better identification of patients at risk, ensure in-treatment monitoring, be simple and adapted to daily practice. INCLUSION CRITERIA: A systematic admission check-up (S0) which includes cardiological and biological controls and after one and six months' treatment control (S1 and S6) were recommended. The major risk factors (RF) researched were long QT interval, bradycardia and hypokaliemia. RESULTS: The initial monitoring was conducted in 601 patients (that only corresponded to 17% of hospital admission active files during the considered period). Means delays before obtaining an ECG were three times those obtained existing biological check-ups (11 days versus three days after date of admission). Systematic and integrated characterisation controls on admission were not respected. We noted that two-third of patients admitted during this period were hospitalized for only five days, although the mean time to obtain an ECG is of 11 days. This delay (approximately one week) between ECG and biological check-up is not compatible with a complete patient RF evaluation. Respectively, 83 and 68 patients were controlled under treatment at S1 and S6. Only half of the patients were controlled at the one-month (S1) and 16% at the six-months' theoretical dates (S6). These delays are inappropriate, notably with regard to the mean time of hospitalisation (15-17 days). The incidence of major RF was higher in treated (71%) than in non-treated patients. Major RF presence at S0 was not systematically associated with an AP treatment contraindication. The excessive delay before the first ECG could partially explain why this initial check-up was not able to detect a pretreatment contraindication. On the other hand, AP treated patients who presented at least one major RF at S0 were more frequently monitored at S1 than patients who did not (26% versus 13%, p=0.05). Among the 168 patients treated with AP or other drugs prolonging the QT interval at risk, 33 had at least one follow-up. This risk population was not better controlled than the initial cohort. DISCUSSION: Protocol evaluation is essential to improve its interest and feasibility. If systematic characterisation is simple, its application in practice is very difficult. The second version of the protocol presented here proposes to substitute the systematic ECG characterisation with the classification on admission of patients in "risk groups" that will condition the subsequent monitoring. Risk groups are identified into two RF types: those which are not related to AP treatment and those which are therapeutic attitude is adapted according to initial QTcorrigé (QTc), its progression between S0 and S (seven days) and kaliemia.

Genomewide identification of genes under directional selection: gene transcription Q(ST) scan in diverging Atlantic salmon subpopulations.

Evolutionary genomics has benefited from methods that allow identifying evolutionarily important genomic regions on a genomewide scale, including genome scans and QTL mapping. Recently, genomewide scanning by means of microarrays has permitted assessing gene transcription differences among species or populations. However, the identification of differentially transcribed genes does not in itself suffice to measure the role of selection in driving evolutionary changes in gene transcription. Here, we propose and apply a "transcriptome scan" approach to investigating the role of selection in shaping differential profiles of gene transcription among populations. We compared the genomewide transcription levels between two Atlantic salmon subpopulations that have been diverging for only six generations. Following assessment of normality and unimodality on a gene-per-gene basis, the additive genetic basis of gene transcription was estimated using the animal model. Gene transcription h(2) estimates were significant for 1044 (16%) of all detected cDNA clones. In an approach analogous to that of genome scans, we used the distribution of the Q(ST) values estimated from intra- and intersubpopulation additive genetic components of the transcription profiles to identify 16 outlier genes (average Q(ST) estimate = 0.11) whose transcription levels are likely to have evolved under the influence of directional selection within six generations only. Overall, this study contributes both empirically and methodologically to the quantitative genetic exploration of gene transcription data.

Activation of human neutrophils in vitro and dieldrin-induced neutrophilic inflammation in vivo.

Many chemicals of environmental concern are known to alter the immune system and are considered toxic molecules because they affect immune cell functions. Inflammation related to environmental chemical exposure, however, is poorly documented, except that from air pollutants. In this study, we found that the organochlorine insecticide dieldrin could not alter the ability of human neutrophils to phagocytose opsonized sheep red blood cells at nonnecrotic concentrations (0.1, 1, 10, and 50 microM). However, dieldrin was found to increase human neutrophil superoxide production, RNA synthesis, and proinflammatory cytokine interleukin-8 production. The normal apoptotic rate of neutrophils evaluated by both cytology and flow cytometry (CD-16 staining) was not altered by dieldrin treatments, and this was correlated with its inability to inhibit spreading of neutrophils onto glass. Using the murine air pouch model, we found that dieldrin induces a neutrophilic inflammation. Taken together, these results demonstrated that dieldrin is a proinflammatory contaminant. To our knowledge, this is the first report establishing that dieldrin is a contaminant exhibiting proinflammatory properties. In addition, it is the first time that the murine air pouch model has been successfully used to confirm that a chemical of environmental concern can induce an inflammatory response in vivo.

Activation of human neutrophils by technical toxaphene.

Toxaphene is a persistent organic pollutant (POP) known to be composed of numerous congeners. Toxaphene technical mixture applied as a pesticide consists of over 800 congeners. Among these, T(2) and T(12) are the two environmentally prevalent forms found in humans. Although toxaphene is known to exert some toxic effects, including potential proinflammatory properties, little is known concerning its action on cells of the human immune system, especially neutrophils. In the present study, we found that toxaphene was not necrotic for human neutrophils incubated for up to 24 h with concentrations ranging from 0.1 to 50 microg/ml. Toxaphene was found to induce neutrophil superoxide production (O(-)(2)) in a concentration-dependent manner. The potency and the kinetics of toxaphene-induced O(-)(2) by neutrophils were found to be similar to that of the classical neutrophil agonists phorbol 12-myristate 13-acetate (PMA). Furthermore, the use of various transduction signal inhibitors (genistein, pertussis toxin, staurosporine, H-7, and HA-1077), suggests that, as for PMA, toxaphene mediates its effect primarily via PKCs and, to a lesser extend, via tyrosine kinases. In this respect, staurosporine, H-7, and genistein were found to inhibit toxaphene- and PMA-induced O(-)(2) production by 52, 72, and 31% and by 63, 62, and 23%, respectively. Toxaphene was also found to significantly enhance neutrophil phagocytosis of opsonized sheep red blood cells and to induce neutrophil apoptosis. The induction of neutrophil apoptosis was paralleled with a decrease in CD16 expression. T(2) and T(12), the two prevalent congeners found in humans, were also found to significantly increase the O(-)(2) production in neutrophils at a concentration of 5 microg/ml. We conclude that neutrophils are important targets for toxaphene, as this POP can activate O(-)(2) production by a PKC- and tyrosine kinase-dependent mechanism, induce phagocytosis, and accelerate the apoptotic rate. This is the first study that focuses on toxaphene/human neutrophil interactions.

Quality of life and home enteral tube feeding: a French prospective study in patients with head and neck or oesophageal cancer.

A prospective study was conducted to evaluate the impact of home enteral tube feeding on quality of life in 39 consecutive patients treated for head and neck or oesophageal cancer at the Centre François Baclesse in Caen, France. Patients were taken as their own controls. Quality of life was evaluated using the EORTC QLQ-C30 core questionnaire, and the EORTC H&N35 and OES24 specific questionnaires. The feeding technique tolerance was evaluated using a questionnaire specifically developed for this study. Two evaluations were made, the first a week after hospital discharge (n = 39) and the second 3 weeks later (n = 30). Overall, the global health status/quality of life scale score slightly improved; among symptoms, scale scores that significantly improved (P < 0.05) concerned constipation, coughing, social functioning and body image/sexuality. The physical feeding technique tolerance was acceptable while the technique was psychologically less tolerated with two-thirds of the patients longing to have the tube removed. One third of the patients was also uncomfortable about their body image. Home enteral tube feeding was responsible for not visiting family or close relations in 15% of patients, and not going out in public in 23%. We conclude that home enteral tube feeding is a physically well accepted technique although a substantial proportion of patients may experience psychosocial distress.

Metabolic engineering.

Metabolic engineering is the science that combines systematic analysis of metabolic and other pathways with molecular biological techniques to improve cellular properties by designing and implementing rational genetic modifications. As such, metabolic engineering deals with the measurement of metabolic fluxes and elucidation of their control as determinants of metabolic function and cell physiology. A novel aspect of metabolic engineering is that it departs from the traditional reductionist paradigm of cellular metabolism, taking instead a holistic view. In this sense, metabolic engineering is well suited as a framework for the analysis of genome-wide differential gene expression data, in combination with data on protein content and in vivo metabolic fluxes. The insights of the integrated view of metabolism generated by metabolic engineering will have profound implications in biotechnological applications, as well as in devising rational strategies for target selection for screening candidate drugs or designing gene therapies. In this article we review basic concepts of metabolic engineering and provide examples of applications in the production of primary and secondary metabolites, improving cellular properties, and biomedical engineering.

The effects of local anesthetics on postoperative pain.

This study was performed to determine if intraoperative local anesthesia improved control of postoperative pain after inguinal herniorrhaphy and to compare the effects of two commonly used local anesthetics on pain management. The Gate Control Theory of Pain formed the theoretical basis for this study. A retrospective nonexperimental study in an ex post facto design was used. Data were collected from 1990 through 1997 on 120 patient charts. The use of local anesthetic intraoperatively significantly decreased patients' lengths of stay postoperatively (P = 0.00) and need for postoperative narcotics (P = 0.00). Bupivacaine was found to be superior to lidocaine in decreasing the need for postoperative narcotic analgesia. Researchers concluded that many patients would benefit from intraoperative injection of local anesthesia. This information can affect patient care outcomes through decreasing recovery time, reducing postoperative pain, and reducing health care costs.

Granulocyte-macrophage colony-stimulating factor enhances EBV-induced synthesis of chemotactic factors in human neutrophils.

We have recently demonstrated that EBV binds to human neutrophils and stimulates a wide range of activities, including homeotypic aggregation, total RNA synthesis, and expression of the chemokines IL-8 and macrophage inflammatory protein-1alpha (MIP-1alpha). Neutrophil function is also known to be modulated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have therefore investigated the modulation of EBV-induced activation of human neutrophils by GM-CSF. Treatment of neutrophils with GM-CSF before EBV activation enhanced the production of both MIP-1alpha and IL-8. The IL-8 produced under these conditions was biologically active as determined in the calcium mobilization assay. GM-CSF was also found to increase the ability of EBV to prime neutrophils for increased leukotriene B4 (LTB4) synthesis. Prior treatment of GM-CSF with neutralizing Abs inhibited these effects. GM-CSF also increased the specific binding of FITC-EBV to the neutrophil surface, as evaluated by fluorocytometry. Local production of GM-CSF in tissues invaded by EBV could therefore serve to potentiate a host defense mechanism directed toward the destruction of the infectious virus via increased production of chemotactic factors. Since both IL-8 and MIP-1alpha are reported to be chemoattractants in vitro for T cells and T and B cells, respectively, the ability of EBV to induce their production by neutrophils may enhance its ability to infect B and T lymphocytes via increased recruitment to sites of infection.

Epstein-Barr virus induces GM-CSF synthesis by monocytes: effect on EBV-induced IL-1 and IL-1 receptor antagonist production in neutrophils.

Neutrophils play an important role in the control of viral infections by releasing a variety of potent agents. We previously demonstrated that Epstein-Barr virus (EBV) binds to human neutrophils and stimulates cytokine synthesis including interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1Ra). Since neutrophil functions are known to be modulated by the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), we therefore investigated the cellular source of GM-CSF synthesis following treatment of leukocytes with EBV and the effect of GM-CSF on the production of IL-1, IL-1Ra, and superoxide by EBV-treated neutrophils. In enriched-cell populations, only monocytes were found to produce GM-CSF in response to EBV, which was maximal after 12 h of incubation. The results obtained with UV-irradiated particles or EBV neutralized with monoclonal antibody 72A1 suggest that contact between the cell and the gp350 of the viral envelope is sufficient to induce the release of GM-CSF. On the other hand, GM-CSF differentially upregulated EBV-induced IL-1 and IL-1Ra production by neutrophils. Pretreatment of neutrophils with GM-CSF prior to EBV activation synergistically enhanced the production of IL-1 alpha and IL-1 beta, but only marginally affected IL-1Ra synthesis. In addition, GM-CSF was also found to synergistically enhance the superoxide production by neutrophils in response to EBV. Molecular analysis showed that GM-CSF did not alter the IL-1 beta and IL-1Ra mRNA synthesis induced by EBV, suggesting that GM-CSF could act at a posttranslational level. Local production of GM-CSF by monocytes in tissues invaded by EBV could serve to potentiate the host defense mechanisms directed toward the destruction of the infectious virus.

EBV induces the production and release of IL-8 and macrophage inflammatory protein-1 alpha in human neutrophils.

As the first line of defense in the immune system, neutrophils may release a variety of potent agents upon exposure to infectious agents. In this study we have investigated the ability of human neutrophils to produce chemotactic cytokines, or chemokine in response to EBV. Exposure of neutrophils to EBV led to an increase in accumulation of mRNA for IL-8 and macrophage inflammatory protein-1alpha (MIP-1alpha). EBV stimulated a time-dependent production of immunoreactive IL-8 and MIP-1alpha by neutrophils. The ability of EBV to stimulate the synthesis of IL-8 and MIP-1alpha protein was reflected by both an accumulation of the protein in the intracellular compartment as well as increased secretion. A variety of control studies support the idea that infectious EBV is not required for induction of chemokine gene expression; however, the response is dependent on the interaction between the glycoprotein gp350 of the viral envelope and the neutrophil surface. Since both IL-8 and MIP-1alpha are reported to be chemoattractants in vitro for T cells and for T and B cells, respectively, the ability of EBV to induce their production by neutrophils may enhance the ability of this virus to infect B and T lymphocytes via increased recruitment to sites of infection.

Rapid DNA purification for Hal gene PCR diagnosis in porcine tissues and extension to other meat species.

Two different halothane (Hal) gene polymerase chain reaction (PCR) tests were applied to genomic DNA extracted from porcine blood, semen, muscle and fat tissues by a rapid and simple Chelex-100 based method. One of the PCR procedure is designed from the ryanodine receptor coding sequence to produce a 81 base pair (bp) fragment, while the other is designed from pig intron sequences to produce a 659 bp fragment. Oligonucleotide primers derived from the coding sequence were also used for other meat species. Amplification products obtained from porcine, bovine, ovine, equine and deer genomic DNA were successfully digested with Hha I restriction enzyme to produce the same electrophoretic pattern as in the normal homozygous (NN) pig. No PCR products could be amplified from chicken and turkey DNA.

The IL-1 and IL-1 receptor antagonist (IL-1Ra) response of human neutrophils to EBV stimulation. Preponderance of IL-Ra detection.

The present study investigated the effect of EBV on gene expression and protein synthesis of IL-1 and its natural IL-1R antagonist (IL-1Ra) in human peripheral blood neutrophils. EBV induced a rapid accumulation of IL-1 and IL-lRa mRNA in neutrophils that was associated with the later appearance of considerable amounts of IL-1alpha, IL-1beta, and IL-Ra proteins. Approximately 3200 and 610 times more IL-Ra than IL-1alpha a or IL-1beta, respectively, was secreted by neutrophils in response to EBV. The effect induced by EBV cannot reflect an overall metabolic activity of neutrophils, since EBV failed to induce granulocyte-macrophage OF synthesis. Heat-inactivated virus was unable to stimulate cytokine synthesis, whereas UV-irradiated virus retained the full IL-1- and IL-1Ra-inducing potential of the native particle. Furthermore, pretreatment of cells with cycloheximide or phosphonoacetic acid did not abrogate the effect of EBV, suggesting that EBV does not penetrate the cell, but that a virion's structural molecule is required to induce such an effect. In this respect, neutralization of the viral particles with the mAb 72A1, which is known to react with glycoprotein gp350 of the viral envelope, inhibits the production of IL-1 and IL-1Ra, suggesting that gp350 could be involved in this process. Thus, the elevated levels of IL-1Ra detected for EBV-stimulated neutrophils might be part of a mechanism used by the virus to evade the immune system.

Paradoxical effects of colchicine on the activation of human neutrophilis by chemotactic factors and inflammatory microcrystal.

Neutrophil activation by chemotactic factors and by inflammatory microcrystals is accompanied by increases in protein tyrosine phosphorylation and by the activation of the NADPH oxidase. The addition of colchicine inhibited both responses induced by triclinic monosodium urate or calcium pyrophosphate crystals. On the other hand, colchicine enhanced the tyrosine phosphorylation of specific protein in neutrophils stimulated by chemotactic factor and augmented the production of superoxide anions induced by these same agonists. The effects of colchicine were shared by other anti-microtubule agents (nocodazole and vinblastine) but not by its inactive analogue beta-lumicolchicine, trimethylcolchicinic acid, indomethacin, or phenylbutazone. Furthermore, the (enhancing as well as inhibitory) effects of colchicine on tyrosine phosphorylation and superoxide anion production were reversed by taxol. Finally, in human cytoplasts colchicine again inhibited microcrystal-stimulated tyrosine phosphorylation but did not change chemotactic factor-stimulated phosphorylation. These data strongly support the hypothesis that microtubule-related mechanisms are involved in the modulation of the tyrosine phosphorylation response in human neutrophils, and suggest that a relationship may exist between the augmentation of tyrosine phosphorylation and of the stimulation of the NADPH oxidase induced by chemotactic factors.

Tyrosine phosphorylation in activated human neutrophils. Comparison of the effects of different classes of agonists and identification of the signaling pathways involved.

The tyrosine phosphorylation responses initiated in human neutrophils by soluble and particulate agonists were characterized. Chemotactic factors, hematopoietic growth factors, and inflammatory microcrystals stimulated in a time- and concentration-dependent manner the tyrosine phosphorylation of distinct patterns of substrates: pp120, pp85, pp70, and pp60 in the case of chemotactic factors; pp155, pp130, pp120, pp85, pp60, and pp40 in the case of granulocyte macrophage-CSF; and pp130, pp120, pp70, and pp60 in the case of monosodium urate (MSU) crystals. Several of the single bands on one-dimensional blots (including pp40, pp70, and pp120) could be resolved into multiple spots on two-dimensional gels. The responses of several other chemotactic factors resembled those of FMLP. Cytokineplasts retained the capacity to respond to FMLP, granulocyte-macrophage-CSF, or MSU crystals with a stimulation of tyrosine phosphorylation, and contained the major substrates detected in intact neutrophils. Several unrelated tyrosine kinase inhibitors (herbimycin A, genistein, and erbstatin) strongly diminished the tyrosine phosphorylation response to chemotactic factors. Pertussis toxin abrogated the tyrosine phosphorylation response to FMLP, whereas protein kinase C (Ro 21-8220, chelerithryn) inhibitors were without effect. Chelation of intracellular calcium attenuated the tyrosine phosphorylation response to FMLP. These results indicate that G proteins play a crucial role in the coupling of chemotactic factor receptors to tyrosine phosphorylation and that this coupling occurs in parallel to that of phospholipase C. These results also underline the complexity of the transduction pathways implicated in the initiation of tyrosine phosphorylation.

Crystal-induced neutrophil activation. V. Differential production of biologically active IL-1 and IL-1 receptor antagonist.

Neutrophils produce IL-1 when stimulated by monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals. Neutrophils also generate the IL-1R antagonist (IL-1Ra), especially when incubated with granulocyte-macrophage CSF (GM-CSF) or TNF-alpha. We studied the simultaneous expression of IL-1 and IL-1Ra by GM-CSF- or TNF-alpha-treated neutrophils activated by MSU or CPPD. Neutrophils incubated with GM-CSF or TNF-alpha produced approximately 300 or 200 times more IL-1Ra than IL-1, respectively. Suboptimal concentrations of MSU or CPPD induced low amounts of IL-1 without affecting IL-1Ra. Interaction of GM-CSF- and TNF-alpha-treated neutrophils with MSU or CPPD up-regulated IL-1 while simultaneously down-regulating IL-1Ra. As a result, the bioactivity of IL-1 secreted was enhanced. Synergistic increases of IL-1 (but not IL-1Ra) mRNA levels were noted in GM-CSF- or TNF-alpha-treated neutrophils exposed to CPPD. Treatment of neutrophils with colchicine before incubation with GM-CSF or TNF alpha, inhibited crystal-induced IL-1 by 50 to 55%, but failed to significantly affect IL-1Ra. The IL-1Ra to IL-1 ratio was significantly increased by 185 to 220%. These results demonstrate that IL-1 and IL-1Ra production by human neutrophils are differentially regulated, that the combined presence of GM-CSF or TNF-alpha and microcrystals favor the production of biologically active IL-1 over that of IL-1Ra, and that colchicine selectively inhibits IL-1 without affecting IL-1Ra production.

Crystal-induced neutrophil activation. IV. Specific inhibition of tyrosine phosphorylation by colchicine.

We recently demonstrated that pathologically relevant inflammatory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, potently stimulate a characteristic protein tyrosine phosphorylation pattern in human neutrophils that differed from that observed in response to other soluble or particulate agonists. In this study, the effects of colchicine on protein tyrosine phosphorylation induced by MSU and CPPD crystals in human blood neutrophils were investigated. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that colchicine dose-dependently inhibited the tyrosine phosphorylation of all the proteins phosphorylated in response to MSU and CPPD crystals. Other microtubule-disruptive agents such as vinblastine, nocodazole, and colcemid also inhibited crystal-induced protein tyrosine phosphorylation while lumicolchicine and trimethylcolchicinic acid were without effect. Indomethacin and phenylbutazone were similarly without effect on microcrystal-induced tyrosine phosphorylation. Colchicine, as well as the other active alkaloids, failed to inhibit the protein tyrosine phosphorylation elicited by FMLP, C5a, leukotriene B4, and unopsonized zymosan. Overall, these results demonstrate that colchicine specifically and significantly inhibits the protein tyrosine phosphorylation induced by MSU and CPPD crystals and suggest that its effects are associated, at least in part, with its interaction with microtubules. Furthermore, the use of microtubule-disrupting drugs demonstrate that the mechanisms implicated in the induction of protein tyrosine phosphorylation by microcrystals differed from those involved in response to other soluble or particulate agonists.

Inhibition of tyrosine phosphorylation by wortmannin in human neutrophils. Dissociation from its inhibitory effects on phospholipase D.

BACKGROUND: Recent studies have indicated that the regulation of the activation of human neutrophils depends on tyrosine phosphorylation and on phospholipase D. Furthermore, a tentative causal relationship between these two signalling pathways has been indirectly implied derived through the use of inhibitors of tyrosine kinases. The fungal metabolite, wortmannin is at present the only compound known to inhibit the receptor-mediated activation of phospholipase D in human neutrophils. Its mechanism of action is presently unknown. EXPERIMENTAL DESIGN: The ability of peripheral blood neutrophils to respond to various agonists with an increase in activity of phospholipase D and an enhancement of tyrosine phosphorylation in the absence or presence of wortmannin was monitored. RESULTS: Wortmannin was found to inhibit the stimulation of tyrosine phosphorylation by fMet-Leu-Phe, and by the inflammatory microcrystals monosodium urate and calcium pyrophosphate dihydrate. This effect of wortmannin was not secondary to inhibition of phospholipase D as U73122, a previously described phospholipase C inhibitor, was also found to inhibit phospholipase D without affecting tyrosine phosphorylation. CONCLUSIONS: The results make it likely that one of the earliest sites of action of wortmannin in human neutrophils is at the level of tyrosine phosphorylation which then exerts a modulatory influence on the activation of phospholipase D.