Calcium-dependent PAF-stimulated generation of reactive oxygen species in a human keratinocyte cell line.

During inflammation and other pathological states, the lipid mediator platelet-activating factor (PAF) and reactive oxygen species (ROS) are both generated. We have been investigating the effect of exogenous PAF on ROS formation in the human keratinocyte cell line (HaCaT). ROS production, measured using luminol-enhanced chemiluminescence (CL), proved to be rapid, transient, PAF receptor-mediated, and totally dependent on an increase in intracellular Ca2+ ([Ca2+]i) and on the presence of extracellular Ca2+. Repeated administration of PAF resulted in refractoriness to the agonist in terms of both capacities to increase [Ca2+]i and generate ROS. The cells, however, continued to respond fully to other stimulants (bradykinin, epidermal growth factor, thapsigargin). The PAF-induced increases in [Ca2+]i (monitored using the fluorescent probe Fluo-3) were also rapid and transient and paralleled those of ROS generation. Relatively specific inhibitors of potential ROS-producing systems were administered in an attempt to characterize the ROS producing system(s). Inhibitors of xanthine oxidase, phospholipase A2, lipoxygenase, cyclooxygenase and NO synthase did not interfere with PAF evoked ROS. The flavoprotein inhibitor diphenyleneiodonium and the mitochondrial cytochrome oxidase inhibitor KCN, prevented generation of ROS, making NAD(P)H a candidate for the electron source of the ROS and the mitochondria a potential major site of formation.

Nature of seasonal variation in development of acute tolerance to morphine.

Morphine inhibits contractions of coaxially stimulated guinea pig ileum. Acute tolerance to this effect occurs on continuous perfusion with morphine for 1.5 h. This is associated with an increase in the sensitivity of the tissue to acetylcholine. Acute tolerance does not always develop in spite of identical treatment, but shows a distinct seasonal incidence. It is significantly higher in the summer (May--Nov.) than winter (Dec.--April). This seasonal variation in development of tolerance does not appear to be related to changes in diet, environmental light or temperature, or to the sensitivity of the ileum to morphine. In the winter, the acetylcholine release from stimulated ilea is lower than in the summer. In addition to inhibiting acetylcholine release, morphine also has a non-specific blocking effect in the winter. This postjunctional effect could prevent the development of supersensitivity to acetylcholine during continuous morphine treatment so that acute tolerance cannot occur.

Induction of acute synovitis in the rat by human interferon.

Intra-articular injection of human interferon (alpha, beta or gamma) into rat knee joints induced increases in knee diameter, synovial effusion and synovial membrane weight. An inflammatory cell infiltrate was also noted in synovial membranes. Hydrocortisone diminished the inflammatory response when injected together with interferon. Mouse beta interferon did not produce synovitis in rat knees. These results further support the hypothesis that interferon may play a role in the pathogenesis of human arthritis.

Interferon triggers experimental synovitis and may potentiate auto-immune disease in humans.

From these data it appears that IFN is capable of stimulating prostaglandin E and hyaluronic acid production by human synovial fibroblasts in vitro and of initiating an inflammatory reaction in animal joints. In chronic arthritis its production may result from persisting viral or other antigenic stimulation. IFN may enhance the immune response and mediate the inflammatory process in the joint. Its role in the pathogenesis of rheumatic and various other autoimmune diseases is undergoing further study.

Crosstalk between elevation of [Ca2+]i, reactive oxygen species generation and phospholipase A2 stimulation in a human keratinocyte cell line.

The aim of the study was to explore the possible interrelationship between reactive oxygen species (ROS) formation and cPLA2 activation and the mediator role that [Ca2+]i may play in these processes in the human keratinocyte cell line, HaCaT. HaCaT cells can be invoked to transiently produce ROS by epidermal growth factor (EGF), thapsigargin (TPG) and the Ca(2+)-ionophore, A23187. These 3 agonists transiently increase [Ca2+]i with characteristic kinetics and magnitude. TPG and A23187 each activates on its own [3H]AA release from prelabeled cells, whereas EGF on its own has no effect on [3H]AA release. However, EGF augments [3H]AA release invoked by TPG or A23187 several fold. EGF activates MAP kinase cascades in HaCaT cells, leads to ROS formation and induces relatively small (1.6 fold) elevation in [Ca2+]i, whereas A23187 and TPG lead to a substantial elevation in [Ca2+]i (2.5 to 5 fold) and to ROS formation. Both have a minor effect on MAP kinase activation. The synergism in PLA2 activation by EGF and TPG or A23187, and the sensitivity of [3H]AA release to N-acetylcysteine (NAC) and dithiothreitol (DTT) (potent reducing agents) or to DPI (an inhibitor of FAD-dependent oxidases) lead to the suggestion that ROS formation, elevation of [Ca2+]i and PLA2 activation are causally related. Since we show that elevation of [Ca2+]i is a prerequisite for both ROS and PLA2 activation, it is possible that these processes contribute to the toxicity (apoptosis) exerted by chronic elevation of [Ca2+]i.

Nitric oxide: a mediator in anaphylactic shock in guinea-pigs.

In this study we show that the pathophysiology of anaphylaxis includes generation of nitric oxide (NO), a very powerful, short-acting vasodilator. Guinea-pigs sensitized to ovalbumin were treated with 200 microgram/kg diphenylene iodonium (DPI), and NO synthase inhibitor, prior to antigen challenge. Mortality following the challenge fell from 71 to 39% (p < 0.001, n = 59). In the Langendorff preparation perfused isolated hearts from sensitized guinea-pigs were challenged to initiate cardiac anaphylaxis. The coronary flow rate (CFR), a direct reflection of coronary arterial resistance, was reduced by antigen challenge to 56 +/- 4% (n = 16) of the basal rate. DPI (2 micrograms/ml) intensified the antigen-induced fall in CFR to 13 +/- 3% of control (p < 0.005, n = 5), and the false substrate for NO, L-N-methylarginine, to 37 +/- 3% (p < 0.05, n = 4). Sodium nitroprusside (SNP), a NO generator, raised the basal CFR by 46% (from 11.2 +/- 1.7 ml/min to 16.3 +/- 1.9 ml/min) and blunted the antigen-induced fall in CFR. Paradoxically, DPI, which can inhibit flavoprotein enzymes other than NO synthase, potentiated the vasodilator effect of SNP, raising the basal CFR by 116%. Together these results strongly indicate that the vasodilator NO is generated in anaphylaxis. However, whereas in the heart it may function as a counterweight to the vasospasm of the coronary arteries, in the intact animal it appears to be a major contributor to the potentially lethal hypotension of anaphylactic shock.

Attenuation of anaphylactic shock and related mortality in guinea-pigs after administration of a potent protein kinase inhibitor, K252a.

We examined whether protein kinases have a role in the expression of anaphylactic shock (AS). Guinea-pigs sensitized to ovalbumin were administered i.p. saline (control) or 10 micrograms/kg K252a, a potent protein kinase inhibitor, 30 min before challenge. The development of AS and mortality was observed for the next 2 h. In the K252a-treated group the incidence of AS fell to 53% from 100%, the maximum intensity was 62% less than the control, and mortality dropped to 16% from 50% of the animals. We suggest that protein kinases are involved in the expression of AS, and that inhibitors of these enzymes may protect against the symptoms of AS and allergy.

Magnesium protects against anaphylactic shock and cardiac myolysis in guinea-pigs.

The pathophysiological responses to immune stress (IS) include activation of several processes which are dependent on cytosolic Ca2+ elevation. Magnesium frequently acts as a natural Ca2+ antagonist. In this study we have observed that Mg2+ can protect guinea-pigs against IS. Antigen-sensitized guinea-pigs, which had been fed a magnesium-deficient diet, were given a single dose (15 mg) of MgCl2 intraperitoneally 1 h before antigen challenge. The development of anaphylactic shock (AS) was observed during the next 2 h, and the hearts were subsequently examined histologically for signs of cardiac myolysis (CM). Magnesium (i) reduced the incidence of CM from 40% to 10% (p < 0.05); (ii) reduced the incidence of AS from 61% to 35% (p < 0.05); (iii) attenuated the severity of the AS; and (iv) lowered mortality from 39% in the control to 19% in the Mg(2+)-treated group (p = 0.1). Serum and tissue total [Mg2+] were not affected by the administration of MgCl2. Also, the serum and heart Mg2+ levels were the same whether or not the guinea-pigs developed AS or CM. In cell culture we demonstrated that by elevating the [Mg2+] in the medium bathing sensitized rat basophilic leukemia (RBL) cells, the increase in cytosolic [Ca2+] subsequent to antigen challenge was reduced from 174 +/- 23.28% (1 mM) to 82.74 +/- 13.22% (3 mM). We conclude that a single treatment with Mg2+ can considerably diminish damage induced by immune stress, probably by its altering the Ca2+: Mg2+ ratio. Since the physiological reaction to different types of stress is similar, Mg2+ could prove beneficial in preventing stress-induced shock in general. Studies examining the mechanisms by which Mg2+ exerts its effects thus provide a scientific basis for the current clinical use of Mg2+ in acute myocardial infarction (AMI) and asthma.

Generation of reactive oxygen species in a human keratinocyte cell line: role of calcium.

In the human keratinocyte cell line HaCaT, reactive oxygen species (ROS) were generated in a dose- and time-dependent manner in response to epidermal growth factor (EGF), bradykinin, thapsigargin, and the Ca(2+)-ionophore A23187, agonists that interact with different primary cell targets. ROS formation was assessed by both chemiluminescence- and fluorescence-based methods. The ROS evoked by EGF and bradykinin decayed within 8 and 4 min, respectively, this transient effect resulting probably from down-regulation of the specific agonist receptors or dissipation of the secondary signals. In contrast, the response to thapsigargin and A23187 was sustained for at least 15 min. Extracellular Ca2+ and a rise in intracellular Ca2+ concentration ([Ca2+]i) proved essential for ROS production. Chelation by BAPTA suppressed ROS formation. Direct measurement of [Ca2+]i using fura fluorescence revealed that EGF and bradykinin evoked a modest, transient [Ca2+]i elevation of less than twofold, whereas with thapsigargin and A23187 there was a sustained two- to fourfold elevation. For each agonist, the kinetics of the rise and decay of [Ca2+]i were similar to those of ROS. The enzyme(s) involved in ROS formation were inhibited by diphenyleneiodonium, indicating dependence on FAD. Our results suggest a close link between ROS and changes in [Ca2+]i generated by growth factors and hormones. This is a particularly interesting connection because elevation of ROS and/ or [Ca2+]i has been linked to cell proliferation, differentiation, and apoptosis.

Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils.

Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca2+ concentration ([Ca2+]i) and induces production of leukotriene C4 (LTC4). This model was used to examine the role of Ca2+ in LTC4 formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2 x 10(-7) M), selectively prevented the stimulatory effect of the antigen on LTC4 production whereas the response to calcium ionophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 micrograms/ml) did not overcome the inhibitory action of HC. An elevated [Ca2+]i is known to be essential for the activation of both 5-lipoxygenase and phospholipase A2. The stimulatory effect of the antigen on LTC4 production was abolished when the cells were incubated in Ca2+-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca2+. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca2+ concentration of 150 microM and 40 microM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca2+]i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of 45Ca2+ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca2+]i, and this may be associated with the inhibitory action of HC on LTC4 formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.

Lipoxygenase inhibitor and colchicine as anti-arthritic agents in the rat.

The non-steroidal anti-inflammatory agents do not have identical activities on the various pathways of arachidonic acid metabolism. The purpose of this study is to examine and compare the activities of colchicine, an anti-arthritic agent, indomethacin, a known prostaglandin synthesis inhibitor and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, in an experimental model of arthritis. Acute arthritis of the knee was induced in rats by injection of lipopolysaccharide (LPS) into the joints. Arthritis was characterized by an increase in joint diameter (18%), increased synovial weight (34%) and an increase in synovial prostaglandin E (PGE) production (56%). While administration of all of the agents examined abolished LPS-induced joint diameter and synovial weight increase, only indomethacin reduced increased PGE content. NDGA and colchicine had no inhibitory effect on LPS-induced PGE production, and moreover they actually stimulated PGE production when compared to control values. It is concluded that: Among the mediators of the inflammatory process are factors sensitive to colchicine and NDGA which are not PGs. Lipoxygenase products of arachidonic acid including leukotrienes may have an important role in inflammation. Leukotrienes and prostaglandins may act in concert in mediating the inflammatory process.

Immunological challenge with virus initiates leukotriene C4 production in the heart and induces cardiomyolysis in guinea pigs.

Cardiac myolysis was observed in guinea pigs sensitized with vesicular stomatitis virus (VSV), following challenge with this antigen. The phenomenon developed within 1 h of challenge, appearing as islands in the myocardium. The speed and focal nature of the damage point to obstruction of blood flow as a cause of the myolysis. The myolysis was not a toxic effect of the virus itself, but probably a consequence of cardiac anaphylaxis. It occurred only after challenge, and was abolished in 71% of the animals by pretreatment with a mixture of the lipoxygenase-cyclooxygenase inhibitor, BW755C and H1 histamine receptor antagonist, diphenhydramine. Treatment with BW755C alone before challenge prevented myolysis from developing in 46% of the animals. Challenge in vitro with VSV to the perfused, spontaneously beating, sensitized isolated guinea pig heart increased sulfidopeptide-leukotriene (LTC4, LTD4, LTE4) production from undetectable levels (0.5 ng LTD4-equivalent/heart/15' to 13 ng LTD4-equivalent/heart/15'. At the same time, there were derangements in cardiac rate, contractility and coronary outflow typical of cardiac anaphylaxis. The reduction in coronary outflow rate during cardiac anaphylaxis is due largely to the powerful vasoconstrictor effect of LT, as well as perhaps platelet-activating-factor. Thus it is speculated that there is a causal relationship between LT release, vasoconstriction, ischemia and myolysis in the heart, following VSV challenge to sensitized guinea pigs.

Magnesium-deficient diet aggravates anaphylactic shock and promotes cardiac myolysis in guinea pigs.

Actively sensitized guinea pigs were rendered Mg(2+)-deficient for 2-3 weeks and then subjected to immune stress. No differences could be seen between treated and control groups prior to immune challenge. 1-2 h after antigen challenge, 95% of the Mg(2+)-deficient animals were observed to be anaphylactic, i.e. apathetic, dyspneic, and they had a rapid pulse rate. Only 4% of the control animals showed signs of anaphylaxis. Serum magnesium concentration, [Mg2+], fell from 1.32 +/- 0.07 mM in control guinea pigs to 0.56 +/- 0.04 mM in those fed an Mg(2+)-deficient diet. Cardiomyolysis (CM) developed in 19% of the anaphylactic animals and in 3% of the controls. We conclude that Mg(2+)-deficient animals continue to function, provided conditions are normal, but they are unable to withstand stress. The heart, however, appears to be better equipped to defend itself against Mg2+ deficiency and low serum [Mg2+], a supposition supported by the fact that heart [Mg2+] is not significantly reduced in hypomagnesemic guinea pigs (0.864 +/- 0.021 microgram/mg dry weight in control, and 0.834 +/- 0.062 microgram/mg dry weight in magnesium-deficient animals). The data indicate that hypomagnesemia heightens the intensity of the immune response, thereby exacerbating both anaphylactic shock (AS) and CM. A normal serum [Mg2+] would thus seem essential for protection against immune stress.