Mice deficient in IL-1beta manifest impaired contact hypersensitivity to trinitrochlorobenzone.

Mice rendered deficient in IL-1 beta by gene targeting in embryonic stem cells develop and grow normally in a protected laboratory environment. Endotoxin-stimulated peritoneal macrophages from IL-1beta-deficient mice showed normal synthesis and cellular release of IL-1alpha after treatment with 5 mM ATP demonstrating that IL-1beta is not necessary for expression and release of the IL-1alpha isoform. Mice deficient in IL-1beta showed unaltered sensitivity to endotoxic shock, with or without pretreatment with D-galactosamine. In contrast, IL-1beta-deficient mice showed defective contact hypersensitivity responses to topically applied trinitrochlorobenzene (TNCB). This defect could be overcome either by application of very high doses of sensitizing antigen, or by local intradermal injection of recombinant IL-1beta immediately before antigen application. These data demonstrate an essential role for IL-1beta in contact hypersensitivity and suggest that IL-1beta acts early during the sensitization phase of response. They suggest an important role for IL-1beta in initiation of the host of response at the epidermal barrier.

Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin.

Mice rendered deficient in lymphotoxin (LT) by gene targeting in embryonic stem cells have no morphologically detectable lymph nodes or Peyer's patches, although development of the thymus appears normal. Within the white pulp of the spleen, there is failure of normal segregation of B and T cells. Spleen and peripheral blood contain CD4+CD8- and CD4-CD8+ T cells in a normal ratio, and both T cells subsets have an apparently normal lytic function. Lymphocytes positive for immunoglobulin M are present in increased numbers in both the spleen and peripheral blood. These data suggest an essential role for LT in the normal development of peripheral lymphoid organs.

Detection of fos protein during osteogenesis by monoclonal antibodies.

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

Inhibition by verapamil of neutrophil responses to formylmethionylleucylphenylalanine and phorbol myristate acetate. Mechanisms involving Ca2+ changes, cyclic AMP and protein kinase C.

Verapamil inhibits in human neutrophils the respiratory burst, the secretion and the change of transmembrane potential induced by formylmethionylleucylphenylalanine, a Ca2+-dependent stimulus, and by phorbol myristate acetate, a Ca2+-independent stimulus. Besides the blocking of Ca2+ channels, many mechanisms are responsible for the inhibition of neutrophil responses. In fact, verapamil (i) increases the intracellular cAMP concentration, potentiates the cAMP response induced by the chemotactic peptide and induces the appearance of a cAMP response also when the stimulant is phorbol myristate acetate; (ii) causes a decrease of Ca2+ association to cell membranes, so depleting the pools of exchangeable Ca2+ and depressing the 'Ca2+ response' in terms of rise in [Ca2+]i monitored with Quin 2 and of rapid mobilization from cell membranes monitored by chlorotetracycline fluorescence change; (iii) inhibits the Ca2+-activated phospholipid-dependent protein kinase C. The data, discussed in relation to the biochemical mechanisms of the stimulus-response coupling, are compatible with the hypothesis of an involvement of the activation of protein kinase C as key step in the sequence of transduction events for the induction of many neutrophil functions.

Intensity and kinetics of the respiratory burst of human neutrophils in relation to receptor occupancy and rate of occupation by formylmethionylleucylphenylalanine.

Studies on the relationship between the binding of fMet-Leu-Phe and the respiratory response in human neutrophils have been carried out under two different conditions of stimulus presentation, i.e., instantaneously and over a period of time. The main findings are as follows (1) Under the first condition the activation of the respiratory response reaches the maximum value very quickly, when the receptor occupancy is less than 20% that at equilibrium. After reaching this maximal value, the activated respiration progressively decreases, while the specific binding of the stimulant continues until equilibrium. (2) Under the second condition, i.e., when the stimulus to neutrophils is presented over a time of 1, 2 or 4 min, the respiratory response (and also the secretory one) is depressed or absent, and the initial rate of the binding (initial Vass) is lower, but the maximal values of the receptor occupancy at equilibrium and of the rate of receptor occupation (maximal Vass) are similar and only slightly lower than those reached under the condition of instantaneous presentation of the stimulus. (3) This form of desensitization is specific for fMet-Leu-Phe and does not consist of the inactivation of the target (NADPH oxidase), since neutrophils desensitized by the slow presentation of the peptide are able to respond to a second challenge with other stimulants. These results indicate that: (1) the efficacy of the stimulus-receptor complexes is short-lived; (2) the intensity of the respiratory response is dependent on the rate of reaching a threshold of binding; (3) when this initial rate is slow, owing to the slow presentation of the stimulus, a specific desensitization takes place, indicating the existence of a molecular mechanism, linked in some way to the initial rate of binding, that modulates the capacity of the stimulus-receptor complexes to transduce signals for cell responses. The physiological role of this type of desensitization is discussed.

Independence with respect to Ca2+ changes of the neutrophil respiratory and secretory response to exogenous phospholipase C and possible involvement of diacylglycerol and protein kinase C.

Exogenous phospholipase C induces in human neutrophils the activation of a respiratory burst, measured as O2 consumption and O-2 production, and of secretion of specific granules, measured as release of vitamin B-12 binding protein. The secretory response is minimal and follows the onset of the respiratory response. Studies carried out using cells prelabeled with [3H]glycerol and 32P on the molecular mechanism of the stimulations demonstrate that the effects are dependent on the formation of diacylglycerol by hydrolysis of different classes of glycerophospholipids. They are, however, independent of the activation of a 'phosphoinositide turnover' as occurs in cells stimulated with fMet-Leu-Phe. Furthermore, the respiratory and secretory responses to exogenous phospholipase C are not associated with modifications of cytosolic Ca2+ concentration, measured with the Quin-2 method, and of the release of bound Ca2+, measured with the membrane probe, chlorotetracycline. Apart from a quantitative difference, mostly regarding the ratio of the intensity of the respiratory and secretory responses, the effects caused by exogenous phospholipase C are qualitatively similar to those induced by phorbol myristate acetate and are probably linked to an involvement of protein kinase C, activated by diacylglycerol liberated in the plasma membrane.

Relationship between the binding of N-formylmethionylleucylphenylalanine and the respiratory response in human neutrophils.

The results presented in this paper demonstrate that the chemotactic peptide N-formylmethionylleucylphenylalanine (f-Met-Leu-Phe) is rapidly inactivated by the products of the respiration of human neutrophils stimulated by the peptide itself. The process of inactivation is impeded by the addition of inhibitors of myeloperoxidase (KCN, NaN3), of catalase, of methionine but not by the addition of superoxide dismutase, indicating that the mechanism of inactivation is the oxidation of methionine residue by myeloperoxidase-H2O2-halide system. The oxidation of the peptide causes the rapid cessation of the respiratory burst, since the sulfoxide derivative loses its ability to bind the specific receptors of neutrophil surface and, hence, its biological activity. The comparison between the time course of the binding of f-Met-Leu-[3H]Phe to the specific receptors and the rate of the respiratory response of neutrophils in the presence and in the absence of the process of peptide oxidation was used to investigate the mechanism of the activation of the respiratory burst by the peptide-receptor complexes. In conditions where the inactivation of the stimulatory agent takes place the stimulated respiration slows down and resumes the resting state shortly after the cessation of the binding, although a substantial amount of the peptide remains bound to the specific receptors. In conditions where the degradation of the peptide does not occur the binding of the peptide and the respiratory burst continue for a longer period of time, but the rate of the respiration, calculated in terms of the instantaneous velocity (Vist), is not correlated to the amount of the ligand bound to the membrane receptors measured at various times, indicating that a summation of the effects of the ligand-receptor complexes does not occur as they form. These findings demonstrate, as far as the respiratory response is concerned, that the biological activity of the peptide-receptor complexes is short-lived and that continuous de-novo receptor occupancy is necessary for the maintenance of the activated respiration.

Studies on stimulus-response coupling in human neutrophils. II. Relationships between the effects of changes of external ionic composition on the properties of N-formylmethionylleucylphenylalanine receptors and on the respiratory and secretory responses.

Studies were carried out on the mechanism responsible for the enhancement of the respiratory and secretory responses to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) exhibited by human neutrophils suspended in Na+-free, high-K+ buffered solution. The results demonstrate that: (a) the variation of Na+ concentration in the suspending solution induces in human neutrophils a marked modification of the recognition apparatus for the chemotactic peptide fMet-Leu-Phe, the lack of or low concentration of this ion increasing the number of the receptors and their specific affinity for the ligand; (b) the greater respiratory burst and secretion induced by fMet-Leu-Phe in human neutrophils suspended in Na+-free, high-K+ medium are due to the increased formation of receptor-ligand complexes at the cell membrane; (c) the greater respiratory response is partially due also to a higher efficiency of these receptor-ligand complexes. The molecular mechanism by which Na+ exerts a regulative role on the properties of the recognition apparatus for the chemotactic peptide and its possible significance are discussed.

Cyclic AMP inhibition of phosphoinositide turnover in human neutrophils.

The effect of increased intracellular levels of cyclic AMP on phosphoinositide metabolism was studied in human neutrophils stimulated with fMet-Leu-Phe. Intracellular cyclic AMP was raised by preincubation either with dibutyryl cyclic AMP and theophylline or with prostaglandin E1. Concentrations of dibutyryl cyclic AMP and theophylline fully inhibitory for the metabolic responses inhibited phosphoinositide breakdown and phosphatidic acid formation to a large extent. The accumulation of the water-soluble inositol phosphates was also measured. In agreement with the data obtained on the phospholipids, inositol phosphate generation was found to be severely, though not completely, reduced. Treatment with dibutyryl cyclic AMP and theophylline also inhibited resynthesis of membrane inositol lipids. Treatment with prostaglandin E1 had a similar, though less, marked effect on inositol lipid turnover, which was parallel with a smaller inhibition of metabolic responses. We therefore suggest that the elevation of intracellular cyclic AMP mainly affects neutrophil responses by inhibiting the phosphoinositide cycle.

Mechanism of desensitization of neutrophil response to N-formylmethionylleucylphenylalanine by slow rate of receptor occupancy. Studies on changes in Ca2+ concentration and phosphatidylinositol turnover.

Previous studies on the regulation of responses of neutrophils to fMet-Leu-Phe have demonstrated the relevance of the role of the rate of occupation of the receptors by the stimulant. When this rate is decreased by presenting the peptide to neutrophils over a period of time by means of an infusion pump, the activation of the respiratory burst and of the secretion is greatly depressed or is absent. This paper deals with further investigations on the mechanisms of this desensitization, which previous results have shown to consist of an uncoupling between the ligand-receptor complexes and the target for cell responses, caused by the deceleration of the initial rate of occupation of the receptors. The data presented here demonstrate that this desensitization is not linked to the formation of a negative intermediate such as cAMP, but is associated with: (i) a depression of the rate and magnitude of the phosphatidylinositol response (activation of phosphatidylinositol turnover measured as modification of incorporation of [32P]Pi and [3H]glycerol into phosphatidylinositol and phosphatidic acid); (ii) a deceleration of the rate of the release of bound Ca2+, without a decrease in the total quantity of Ca2+ liberated (measured as fluorescence changes of chlorotetracycline treated neutrophils); (iii) a slower rise of cytosolic free Ca2+ concentration [Ca2+]i, without a decrease in the magnitude of the final increase of [Ca2+]i (monitored with Quin 2). These findings, which are discussed in relation to the recent hypotheses on the transduction reactions of receptor-mediated stimuli for neutrophil responses, are consistent with a mechanism of desensitization involving decreased production of diacylglycerol by the hydrolysis of phosphatidylinositol and deficient activation of Ca2+-phospholipid-dependent protein kinase C.

Quantification of vitamin D receptor mRNA by competitive polymerase chain reaction in PBMC: lack of correspondence with common allelic variants.

It has been recently claimed that polymorphism for the vitamin D receptor (VDR) influences several aspects of calcium and bone metabolism. To evaluate the physiologic plausibility of these claims, we compared the abundance of the VDR mRNA in peripheral blood mononuclear cells (PBMCs) between different VDR genotypes using a quantitative reverse transcribed polymerase chain reaction-based method. The method is based on the coamplification of VDR cDNA and an internal standard consisting of known concentrations of a human VDR CDNA mutated at a BglII restriction site; the interassay coefficient of variation is 11%. To validate the method, we made use of earlier receptor binding studies indicating that normal human monocytes and activated, but not resting, lymphocytes expressed the VDR. The concentration of the VDR mRNA was 10(-8) to 10(-7) g/g of total RNA in cell-sorted monocytes and in in vitro activated lymphocytes, but only 10(-12) g/g of total mRNA in resting lymphocytes, establishing that the VDR mRNA determined by our method in PBMCs is due to constitutive expression in monocytes. Following an initial genotype screening of 85 normal volunteers by polymerase chain reaction or restriction fragment length polymorphism analysis, 14 individuals with the Bb genotype, 12 with the bb genotype, and 12 with the BB genotype were selected. The concentration of the VDR mRNA, corrected for the number of monocytes, was similar among the three genotype groups, as were the other variables examined: serum calcitriol, serum osteocalcin, and vertebral and hip bone density. We conclude that VDR polymorphism does not affect the abundance of the VDR mRNA.

Increased basal and thrombin-induced free calcium in platelets of essential hypertensive patients.

Intracellular free calcium, [Ca2+]i, was studied in platelets of essential hypertensive subjects and normotensive controls under basal conditions and after stimulation with epinephrine, norepinephrine, angiotensin II, ouabain, and thrombin, using the fluorescent calcium indicator quin 2. Basal [Ca2+]i was significantly higher in hypertensive subjects (n = 32) than in normotensive controls (n = 30; 167.4 +/- 5.0 vs 143.2 +/- 3.1 nmol/L; p less than 0.001). Epinephrine, norepinephrine, angiotensin II, and ouabain had no effect on platelet calcium, whereas thrombin induced a dose-dependent increase in [Ca2+]i in both the presence and absence of extracellular calcium. This [Ca2+]i increase in the presence of extracellular calcium, which depends mainly on calcium influx, was significantly higher (p less than 0.05) in platelets of hypertensive subjects at all thrombin concentrations (ranging from 0.025-0.1 U/ml), while the [Ca2+]i increase in the absence of extracellular calcium, which depends only on release from intracellular stores, was similar in hypertensive subjects and controls. These results suggest that, in essential hypertension, there is not only increased platelet resting [Ca2+]i but also an increase in agonist-mediated calcium influx, which appears to indicate a cell membrane abnormality in the platelets of subjects with essential hypertension.

Relationships between phosphoinositide metabolism, Ca2+ changes and respiratory burst in formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils. The breakdown of phosphoinositides is not involved in the rise of cytosolic free Ca2+.

The relationships between the changes of cellular Ca2+, the activation of phosphoinositide turnover and the functional responses induced by the stimulus-receptor interactions in neutrophils are matter of controversy. By measuring the concentration dependency of different formyl-leucyl-methionyl-phenylalanine (FMLP)-induced changes, the following values of ED50 were found: 1.6 and 0.8 nM for the rise in [Ca2+]i monitored with Quin-2, in the presence and absence of exogenous Ca2+, respectively; 20 nM for the activation of phosphoinositide metabolism, monitored as change in the 32Pi of phosphatidate; 14 nM for membrane-bound Ca2+ mobilization, monitored with chlorotetracycline (CTC); 34 nM for 45Ca2+ influx and 32 nM for the respiratory burst. Furthermore, low dose of FMLP causes an increase in [Ca2+]i in absence of activation of breakdown of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-biphosphate monitored as changes in [3H]glycerol radioactivity. The results clearly demonstrate that the increase in [Ca2+]i, due to the release from intracellular stores, is not caused by the breakdown of phosphatidylinositides. On the other hand, the data of the similarity of ED50 are compatible with an involvement of phosphoinositide response in the release of membrane bound Ca2+, monitored with CTC, and in the 45Ca influx and in the respiratory burst.

Verapamil inhibits platelet aggregation by a calcium-independent mechanism.

We studied the inhibitory effects of the calcium channel blocker verapamil both on platelet aggregation and intracellular calcium [Ca2+]i in platelets loaded with a fluorescent indicator (quin 2). The inhibitory effects of verapamil on the platelet aggregation response to both thrombin and ionomycin were seen to be clearly dissociated from the verapamil-induced inhibition of the [Ca2+]i increase produced by these agonists. Verapamil-induced inhibition of platelet aggregation was also obtained when using the "calcium-independent" agonist phorbol-myristate acetate (PMA). It may be deduced that a calcium-independent mechanism plays a role in verapamil-induced inhibition of platelet aggregation. We postulate that this mechanism may operate via a protein-kinase C pathway.

Increased cytosolic calcium in cystic fibrosis neutrophils effect on stimulus-secretion coupling.

A disorder of calcium homeostasis has been related to the pathogenesis of Cystic Fibrosis (CF). The Authors have studied the relationship between the cytosolic free calcium concentration ([Ca2+]i), the amount of Ca2+ released from endogenous stores and the secretory response in CF neutrophils. Significantly elevated resting [Ca2+]i and depressed Ca2+ release induced by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) is present in CF neutrophils. In the absence of exogenous Ca2+ the secretory response of CF neutrophils after a weak stimulus such as Cytochalasin B (CB) is greater than in normal neutrophils, while a depressed secretion of azurophilic granules is evident in CF neutrophils stimulated by CB + FMLP. The data confirm the hypothesis of an altered Ca2+ homeostasis in CF cells. Cystic Fibrosis (CF), an autosomal recessive exocrinopathy, is characterized by secretory abnormalities and ion transport dysfunctions (for review see 1,2). Since intracellular Ca2+ seems to play a role in stimulus-secretion coupling and ion movements, several aspects of Ca2+ homeostasis have been investigated in CF. The total Ca2+ content has been reported to be increased in fibroblast cultures and in lymphocytes (3,4,5) and mitochondrial Ca2+ uptake was found elevated in fibroblast cultures (6). An elevated free cytosolic calcium concentration ([Ca2+]i) has been recently reported in buccal epithelial cells (7), while normal concentration has been found in lymphocytes and Epstein Barr virus transformed lymphoblasts (5,8). The present paper shows the results of a study in human neutrophils, a cell whose several functions such as secretion, movement and respiratory burst are in some way regulated by Ca2+. The data report that in neutrophils of CF patients the resting [Ca2+]i is higher and the secretory response is partly modified.

Mechanisms and functions of the oxygen radicals producing respiration of phagocytes.

Respiratory burst is due to the activation of a membrane bound NADPH oxidase induced by perturbation of the plasma membrane during phagocytosis or following interaction between the cell surface and a number of environmental stimuli. It refers to the increase in the non-mitochondrial O2 consumption with a concomitant production of different reactive species (superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen ...). The effects of the respiratory burst depend on the intensity and combination of the different actions which are defensive, toxic, activatory and modulatory of the inflammatory process.

Cyclic AMP inhibition of fMet-Leu-Phe-dependent metabolic responses in human neutrophils is not due to its effects on cytosolic Ca2+.

Cyclic AMP powerfully inhibits the fMet-Leu-Phe-dependent respiratory burst and exocytosis of azurophilic and specific granules without affecting Ca2+ release from intracellular stores. The elevation of [Ca2+]i induced by fMet-Leu-Phe is short-lived in cyclic AMP-treated cells and similar to that of untreated cells stimulated in the absence of external Ca2+. Nevertheless, in these latter cells fMet-Leu-Phe induces metabolic activation. We therefore suggest that the inhibitory action of cyclic AMP on neutrophil responses is not due to its effects on [Ca2+]i homoeostasis.

Spontaneous insertion of an IS2 element into the promoter region of the lac operon.

A spontaneous mutation in pUC18 has revealed the insertion of a chromosomal insertion sequence (IS)2 element into the promoter region of the lac operon. The IS2 insertion site, at the pentanucleotide sequence TCGAG, is unlike previously described junctional sequences.