Rufina Schuligoi

Increased content and transport of substance P and calcitonin gene-related peptide in sensory nerves innervating inflamed tissue: evidence for a regulatory function of nerve growth factor in vivo.

The responses of sensory neuropeptides during unilateral, Freunds adjuvant-induced, paw inflammation in the rat were examined. After five days of inflammation, the substance P and calcitonin gene-related peptide content in the sciatic nerve supplying the inflamed paw were increased by 60-75% when compared with the contralateral side. At this time-point, there was also a 30-40% increase in the substance P and calcitonin gene-related peptide content of the dorsal root ganglia (L4-L6), and a 40% increase in the calcitonin gene-related peptide content of the L4-L6 segments of the dorsal spinal cord on the inflammation side. In the dorsal root ganglia, calcitonin gene-related peptide content was also increased as early as 12 h and 48 h after induction of paw inflammation. On day 5 of inflammation, the axonal transport of both sensory neuropeptides towards the inflamed paw, as determined after sciatic nerve ligation, was also markedly increased as compared with the control side. Despite this increased transport, the amount of substance P and calcitonin gene-related peptide present in the inflamed paw itself was either reduced or remained unchanged from day 1 through to day 5 of inflammation pointing towards reduced storage and increased release of the peptides in the inflamed tissue. Nerve growth factor content was markedly increased in the sciatic nerve of the inflamed paw with a peak of +136% at time-point 24 h after induction of inflammation. When rats were systemically treated with anti-nerve growth factor serum, the increase in neuropeptide content in the sciatic nerve of the inflamed paw (day 5) was prevented. On the other hand, local injections of nerve growth factor for 5 days into a noninflamed paw were able to induce an increase in substance P and calcitonin gene-related peptide content in the supplying sciatic nerve. These findings point towards a regulatory function for nerve growth factor in vivo in the stimulation of sensory neuropeptide synthesis during prolonged inflammatory processes.

Effects of specific inhibition of cyclo-oxygenase-1 and cyclo-oxygenase-2 in the rat stomach with normal mucosa and after acid challenge.

Effects of the cyclo‐oxygenase (COX)‐1 inhibitor SC‐560 and the COX‐2 inhibitors rofecoxib and DFU were investigated in the normal stomach and after acid challenge. In healthy rats, neither SC‐560 nor rofecoxib (20 mg kg−1 each) given alone damaged the mucosa. Co‐treatment with SC‐560 and rofecoxib, however, induced severe lesions comparable to indomethacin (20 mg kg−1) whereas co‐administration of SC‐560 and DFU (20 mg kg−1 each) had no comparable ulcerogenic effect 5 h after dosing. SC‐560 (20 mg kg−1) inhibited gastric 6‐keto‐prostaglandin (PG) F1α by 86±5% and platelet thromboxane (TX) B2 formation by 89±4% comparable to indomethacin (20 mg kg−1). Rofecoxib (20 mg kg−1) did not inhibit gastric and platelet eicosanoids. Intragastric HCl elevated mucosal mRNA levels of COX‐2 but not COX‐1. Dexamethasone (2 mg kg−1) prevented the up‐regulation of COX‐2. After acid challenge, SC‐560 (5 and 20 mg kg−1) induced dose‐dependent injury. Rofecoxib (20 mg kg−1), DFU (5 mg kg−1) and dexamethasone (2 mg kg−1) given alone were not ulcerogenic but aggravated SC‐560‐induced damage. DFU augmented SC‐560 damage 1 but not 5 h after administration whereas rofecoxib increased injury after both treatment periods suggesting different time courses. Gastric injurious effects of rofecoxib and DFU correlated with inhibition of inflammatory PGE2. The findings show that in the normal stomach lesions only develop when both COX‐1 and COX‐2 are inhibited. In contrast, during acid challenge inhibition of COX‐1 renders the mucosa more vulnerable suggesting an important role of COX‐1 in mucosal defence in the presence of a potentially noxious agent. In this function COX‐1 is supported by COX‐2. In the face of pending injury, however, COX‐2 cannot maintain mucosal integrity when the activity of COX‐1 is suppressed.

Δ12-Prostaglandin J2, a Plasma Metabolite of Prostaglandin D2, Causes Eosinophil Mobilization from the Bone Marrow and Primes Eosinophils for Chemotaxis

PGD2, a major mast cell mediator, is a potent eosinophil chemoattractant and is thought to be involved in eosinophil recruitment to sites of allergic inflammation. In plasma, PGD2 is rapidly transformed into its major metabolite Δ12-PGJ2, the effect of which on eosinophil migration has not yet been characterized. In this study we found that Δ12-PGJ2 was a highly effective chemoattractant and inducer of respiratory burst in human eosinophils, with the same efficacy as PGD2, PGJ2, or 15-deoxy-Δ12,14-PGJ2. Moreover, pretreatment of eosinophils with Δ12-PGJ2 markedly enhanced the chemotactic response to eotaxin, and in this respect Δ12-PGJ2 was more effective than PGD2. Δ12-PGJ2-induced facilitation of eosinophil migration toward eotaxin was not altered by specific inhibitors of intracellular signaling pathways relevant to the chemotactic response, phosphatidylinositol 3-kinase (LY-294002), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (U-0126), or p38 mitogen-activated protein kinase (SB-202190). Desensitization studies using calcium flux suggested that Δ12-PGJ2 signaled through the same receptor, CRTH2, as PGD2. Finally, Δ12-PGJ2 was able to mobilize mature eosinophils from the bone marrow of the guinea pig isolated perfused hind limb. Given that Δ12-PGJ2 is present in the systemic circulation at relevant levels, a role for this PGD2 metabolite in eosinophil release from the bone marrow and in driving eosinophil recruitment to sites of inflammation appears conceivable.

Intraplantar injection of nerve growth factor into the rat hind paw: local edema and effects on thermal nociceptive threshold

&NA; Nerve growth factor (NGF) is known to produce hyperalgesia as well as to stimulate synthesis of neuropeptides in dorsal root ganglia (DRG). In the present study, we wanted to determine the effects of local NGF administration and assess to which extent mast cell‐dependent factors are mediating NGF responses. Rats received 1 daily unilateral intraplantar injection for 3 days. Local edema (days 1–3), changes in thermal nociceptive threshold (days 1–4), and the content of calcitonin gene‐related peptide (CGRP) and substance P (SP) in the sciatic nerve (day 4), were determined. NGF injection caused edema which was absent in rats pretreated with compound Symbol as well as in rats treated neonatally with capsaicin (‘capsaicin denervation’). NGF‐induced edema was not reduced by the neurokinin‐1 receptor antagonist SR140333, but attenuated by the CGRP receptor antagonist CGRP[8–37]. Symbol. No caption available On each day, NGF injection caused a decrease in thermal nociceptive threshold which lasted for less than 3 h. Capsaicin denervation, but not treatment with indomethacin, abolished NGF‐induced thermal hyperalgesia. Treatment with compound Symbol attenuated hyperalgesia produced by the first, but not by subsequent, NGF injections. Symbol. No caption available On day 4, 24 h after the last of 3 NGF injections, thermal nociceptive threshold was not different from control values, but at that time, CGRP and SP were elevated in the sciatic nerve. We suggest therefore that NGF‐induced local edema was caused by mast cell‐derived vasoactive compounds which act together with afferent neuron‐derived CGRP to increase vascular permeability. NGF‐induced thermal hyperalgesia most likely was caused by an increased sensitivity of peripheral endings of capsaicin sensitive afferents. This effect of NGF was not mediated by products of the cyclooxygenase pathway, and was also observed in mast cell‐depleted rats.

Selective cyclo‐oxygenase‐2 inhibitors aggravate ischaemia‐reperfusion injury in the rat stomach

Effects of indomethacin, the selective cyclo‐oxygenase (COX)‐2 inhibitors NS‐398 and DFU, and dexamethasone on gastric damage induced by 30 min ischaemia followed by 60 min reperfusion (I‐R) were investigated in rats. Modulation of gastric levels of COX‐1 and COX‐2 mRNA by I‐R was evaluated using Northern blot and reverse transcription‐polymerase chain reaction. I‐R‐induced gastric damage was dose‐dependently aggravated by administration of indomethacin (1–10 mg kg−1), NS‐398 (0.4–4 mg kg−1) or DFU (0.02–2 mg kg−1) as assessed macroscopically and histologically. Likewise, administration of dexamethasone (1 mg kg−1) significantly increased I‐R damage. Low doses of 16,16‐dimethyl‐prostaglandin(PG)E2, that did not protect against ethanol‐induced mucosal damage, reversed the effects of the selective COX‐2 inhibitors, indomethacin and dexamethasone. I‐R had no effect on gastric COX‐1 mRNA levels but increased COX‐2 mRNA levels in a time‐dependent manner. Dexamethasone inhibited the I‐R‐induced expression of COX‐2 mRNA. I‐R was not associated with a measurable increase in gastric mucosal formation of 6‐keto‐PGF1α and PGE2. PG formation was substantially inhibited by indomethacin (10 mg kg−1) but was not significantly reduced by NS‐398 (4 mg kg−1), DFU (2 mg kg−1) or dexamethasone (1 mg kg−1). The findings indicate that selective COX‐2 inhibitors and dexamethasone markedly enhance gastric damage induced by I‐R. Thus, whereas COX‐2 has no essential role in the maintenance of gastric mucosal integrity under basal conditions, COX‐2 is rapidly induced in a pro‐ulcerogenic setting and contributes to mucosal defence by minimizing injury. This suggests that in certain situations selective COX‐2 inhibitors may have gastrotoxic effects.

Gastric Acid-Evoked c-fos Messenger RNA Expression in Rat Brainstem Is Signaled by Capsaicin-Resistant Vagal Afferents

BACKGROUND & AIMS Gastric acid is known to contribute to ulcer pain, but the mechanisms of gastric chemonociception are poorly understood. This study set out to investigate the pathways and mechanisms by which gastric acid challenge is signaled to the brain. METHODS Neuronal excitation in the rat brainstem and spinal cord after intragastric administration of HCl (0.35-0.7 mol/L) was examined by in situ hybridization autoradiography for the immediate early gene c-fos. RESULTS Gastric acid challenge did not induce c-fos transcription in the spinal cord but caused many neurons in the nucleus tractus solitarii and area postrema to express c-fos messenger RNA (mRNA). The HCl concentration-dependent excitation of medullary neurons was in part associated with behavioral manifestations of pain but not directly related to the acid-induced injury and contraction of the stomach. Subdiaphragmatic vagotomy suppressed the c-fos mRNA response to intragastric acid, and morphine inhibited it in a naloxone-reversible manner, whereas pretreatment of rats with capsaicin was without effect. CONCLUSIONS Gastric acid challenge is signaled to the brainstem, but not the spinal cord, through vagal afferents that are sensitive to acid but resistant to capsaicin. It is hypothesized that the gastric acid-induced c-fos transcription in the brainstem is related to gastric chemonociception.

CRTH2 and D-type prostanoid receptor antagonists as novel therapeutic agents for inflammatory diseases.

Accumulation of type 2 T helper (Th2) lymphocytes and eosinophils is a hallmark of bronchial asthma and other allergic diseases, and it is believed that these cells play a crucial pathogenic role in allergic inflammation. Thus, Th2 cells and eosinophils are currently considered a major therapeutic target in allergic diseases and asthma. However, drugs that selectively target the accumulation and activation of Th2 cells and eosinophils in tissues are unavailable so far. Prostaglandin (PG)D2 is a key mediator in various inflammatory diseases including allergy and asthma. It is generated by activated mast cells after allergen exposure and subsequently orchestrates the recruitment of inflammatory cells to the tissue. PGD2 induces the chemotaxis of Th2 cells, basophils and eosinophils, stimulates cytokine release from these cells and prolongs their survival, and might hence indirectly promote IgE production. PGD2 mediates its biologic functions via 2 distinct G protein-coupled receptors, D-type prostanoid receptor (DP), and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). DP and CRTH2 receptors are currently being considered as highly promising therapeutic targets for combating allergic diseases and asthma. Here, we revisit the roles of PGD2 receptors in the regulation of eosinophil and Th2 cell function and the efforts towards developing candidate compounds for clinical evaluation.

Stimulation by nerve growth factor of neuropeptide synthesis in the adult rat in vivo: bilateral response to unilateral intraplantar injections

Unilateral intraplantar injections (1/day for 3 days) of 4 mu g nerve growth factor (NGF) into the rat hindpaw increased the expression of prepro-tachykinin (PPT)- and prepro-calcitonin gene-related peptide (ppCGRP)-mRNA in bilateral L5 dorsal root ganglia (DRGs). This was accompanied by an increase of CGRP-like immunoreactivity in the ipsi- and contralateral sciatic nerve but by no detectable change of CGRP-IR in other afferents. NGF injections into the skin of one ear or into the plantar side of one forepaw increased CGRP-IR in the respective afferents (trigeminal ganglion, or nerves arising from the brachial plexus, respectively), but had no effect on sciatic CGRP-IR. This suggests that the NGF-induced symmetrical increase of CGRP synthesis in L5 DRGs was not caused by systemic action of NGF, which, therefore, may be a useful tool to further investigate mechanisms which are responsible for contralateral effects of unilateral inflammation.

Protein Carbamylation Renders High-Density Lipoprotein Dysfunctional

Carbamylation of proteins through reactive cyanate has been demonstrated to predict an increased cardiovascular risk. Cyanate is formed in vivo by breakdown of urea and at sites of inflammation by the phagocyte protein myeloperoxidase. Because myeloperoxidase (MPO) associates with high-density lipoprotein (HDL) in human atherosclerotic intima, we examined in the present study whether cyanate specifically targets HDL. Mass spectrometry analysis revealed that protein carbamylation is a major posttranslational modification of HDL. The carbamyllysine content of lesion-derived HDL was more than 20-fold higher in comparison with 3-chlorotyrosine levels, a specific oxidation product of MPO. Notably, the carbamyllysine content of lesion-derived HDL was five- to eightfold higher when compared with lesion-derived low-density lipoprotein (LDL) or total lesion protein and increased with lesion severity. The carbamyllysine content of HDL, but not of LDL, correlated with levels of 3-chlorotyrosine, suggesting that MPO mediated carbamylation in the vessel wall. Remarkably, one carbamyllysine residue per HDL-associated apolipoprotein A-I was sufficient to induce cholesterol accumulation and lipid-droplet formation in macrophages through a pathway requiring the HDL-receptor scavenger receptor class B, type I. The present results raise the possibility that HDL carbamylation contributes to foam cell formation in atherosclerotic lesions.

Prostaglandin E2 Inhibits Eosinophil Trafficking through E-Prostanoid 2 Receptors

The accumulation of eosinophils in lung tissue is a hallmark of asthma, and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E2 exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE2 potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD2, and C5a. Activated monocytes similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a cyclooxygenase inhibitor. The selective E-prostanoid (EP) 2 receptor agonist butaprost mimicked the inhibitory effect of PGE2 on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE2, inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE2 on eosinophil migration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases.