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YARWOOD H, NOURSHARGH S, BRAIN S, et al., 1993, EFFECT OF DEXAMETHASONE ON NEUTROPHIL ACCUMULATION AND EDEMA FORMATION IN RABBIT SKIN - AN INVESTIGATION OF SITE OF ACTION, BRITISH JOURNAL OF PHARMACOLOGY, Vol: 108, Pages: 959-966, ISSN: 0007-1188
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- Citations: 28
Weg VB, Williams TJ, Lobb RR, et al., 1993, A monoclonal antibody recognizing very late activation antigen-4 inhibits eosinophil accumulation in vivo., Journal of Experimental Medicine, Vol: 177, Pages: 561-566, ISSN: 1540-9538
Using an in vivo test system, the role of the beta 1 integrin very late activation antigen-4 (VLA-4) in eosinophil accumulation in allergic and nonallergic inflammatory reactions was investigated. Eosinophil infiltration and edema formation were measured as the local accumulation of intravenously injected 111In-labeled eosinophils and 125I-human serum albumin. The inflammatory reactions investigated were a passive cutaneous anaphylaxis (PCA) reaction and responses elicited by intradermal soluble inflammatory mediators (platelet-activating factor, leukotriene B4, C5a des Arg), arachidonic acid, and zymosan particles. The in vitro pretreatment of 111In-eosinophils with the anti-VLA-4 monoclonal antibody (mAb) HP1/2, which crossreacts with guinea pig eosinophils, suppressed eosinophil accumulation in all the inflammatory reactions investigated. Eosinophil accumulation was inhibited to the same extent when mAb HP1/2 was administered intravenously. It is interesting that HP1/2 had no effect on stimulated edema formation. These results suggest a role for VLA-4 in eosinophil accumulation in vivo and indicate a dissociation between the inflammatory events of eosinophil accumulation and edema formation.
Brady AJB, Warren JB, Poole-Wilson PA, et al., 1993, Nitric oxide attenuates cardiac myocyte contraction, American Journal of Physiology - Heart and Circulatory Physiology, Vol: 265, ISSN: 0002-9513
Cardiac muscle fibers have microvessels in close proximity, the distance from the nearest capillary being no greater than 8 μm. We performed experiments on isolated, electrically stimulated, contracting guinea pig cardiac myocytes to test whether NO from endothelium or nitrovasodilators or directly superfused in solution might affect myocyte contractility. In endothelium-myocyte coculture experiments, 10-7 M bradykinin reduced myocyte shortening by 11 ± 3.5%. This effect was abolished in the presence of N(G)-nitro-L-arginine methyl ester and was unaffected by indomethacin. Sodium nitroprusside, but not organic nitrovasodilators, reduced myocyte contraction amplitude by 23% at 3 x 10-5 M. This effect was reversed by methylene blue. Superfusion with NO solution had an effect similar to sodium nitroprusside, as did exposure to 8-bromoguanosine 3',5'-cyclic monophosphate. Thus the present study shows that cardiac myocyte contraction is attenuated by NO, which appears to act via production of guanosine 3',5'- cyclic monophosphate within the myocytes. Because cardiac myocytes in vivo are in such close proximity to endothelium, the effects of endothelial products on cardiac myocyte contractility may be important in myocardial function.
Collins PD, Connolly DT, Williams TJ, 1993, Characterization of the increase in vascular permeability induced by vascular permeability factor in vivo, British Journal of Pharmacology, Vol: 109, Pages: 195-199, ISSN: 0007-1188
Vascular permeability factor (VPF) is a protein secreted from a variety of human and rodent tumour and normal tissue cells. In addition to mediating angiogenesis and endothelial cell growth, VPF has been reported to be a potent mediator of increased microvascular permeability in vivo. In this study we have investigated these permeability changes in vivo using a quantitative model of local plasma leakage in rabbit skin. Our results reveal that VPF is a potent mediator of plasma leakage which, in the rabbit, depends on a synergistic interaction with arteriolar vasodilators such as prostaglandin E2. The requirement for an exogenous vasodilator further suggests that VPF does not act to increase blood flow in this model. We show that this response does not require the presence of circulating neutrophils and in this respect is similar to direct‐action permeability increasing mediators such as histamine and bradykinin. Similarly, the time course of plasma leakage induced by VPF resembles that of direct‐action mediators, where the greatest response occurs over the first 30 min. In contrast, the neutrophil‐dependent plasma leakage induced by the active component of zymosan‐activated plasma, C5ades arg, was maintained at a similar level over 2.5 h. Further, using mediator antagonists and enzyme inhibitors we demonstrate that the mechanism of action of VPF is not via activation of histamine, kinin, or platelet‐activating factor pathways. 1993 British Pharmacological Society
Hellewell PG, Jose PJ, Williams TJ, 1992, Inflammatory mechanisms in the passive cutaneous anaphylactic reaction in the rabbit: evidence that novel mediators are involved., Br J Pharmacol, Vol: 107, Pages: 1163-1172, ISSN: 0007-1188
1. We have examined the mechanisms of local oedema formation in the passive cutaneous anaphylactic (PCA) reaction in the rabbit. 2. IgE-containing antiserum was injected i.d. and allowed to sensitize skin sites for periods up to 240 h. Antigen (bovine gamma globulin) was injected i.d. or i.v. and local oedema formation assessed by the accumulation of i.v. injected 125I-labelled rabbit serum albumin. Potential inhibitors were mixed with antigen prior to i.d. injection or were administered i.v. 3. Maximum oedema formation was observed when a sensitization period of 48-72 h was used. Oedema formation in the PCA reaction was of short duration with a t 1/2 of approximately 15 min. No evidence of late oedema formation (up to 6 h) was found. 4. Local oedema formation in the PCA was reduced by indomethacin suggesting that vasodilator, oedema-potentiating prostaglandins were released. However, it was likely that other vasodilators were also generated. 5. Antihistamines were poor inhibitors of oedema formation as were PAF antagonists, a 5-lipoxygenase inhibitor, a kallikrein inhibitor, a bradykinin antagonist and anti-C5a antibody. 6. Local oedema formation in the PCA was partially reduced by neutrophil depletion and colchicine suggesting that neutrophil-dependent mediators were involved. 7. Exudate fluid from anaphylactic reactions in the rabbit peritoneal cavity contained permeability-increasing activity when injected into rabbit skin. This activity is now being characterized. 8. A vasodilator prostaglandin appears to be released in the rabbit PCA reaction but none of the established permeability-increasing mediators appears to be involved. Thus, there may be novel inflammatory mediators generated in this reaction which may have relevance for human allergic skin diseases.
von Uexküll C, Nourshargh S, Williams TJ, 1992, Comparative responses of human and rabbit interleukin-1 in vivo: effect of a recombinant interleukin-1 receptor antagonist., Immunology, Vol: 77, Pages: 483-487, ISSN: 0019-2805
The ability of recombinant human and rabbit interleukin-1 alpha (IL-1 alpha) in inducing inflammatory responses in rabbit skin were compared. Intradermal (i.d.) injections of recombinant human IL-1 alpha and recombinant rabbit IL-1 alpha induced intense accumulation of 111In-labelled neutrophils which was dependent on the dose of the cytokines administered. Both forms of IL-1 alpha induced very small levels of plasma protein leakage. Co-injection of the cytokines with the mRNA synthesis inhibitor actinomycin D (Act D) attenuated the number of neutrophils accumulating in response to both human and rabbit forms of IL-1 alpha. Local injection of a recombinant human IL-1 receptor antagonist (IL-1Ra) caused a dose-dependent inhibition of local inflammatory responses initiated by human and rabbit IL-1 alpha s well as rabbit IL-1 beta indicating the species cross-reactivity of the antagonist. IL-1Ra was selective for IL-1 in rabbit skin, as responses induced by C5ades Arg and formyl-methionyl-leucyl-phenylalanine (FMLP) were not inhibited. IL-1Ra significantly inhibited the IL-1-induced neutrophil accumulation only when co-injected with the cytokine. The local administration of the antagonist 30 min after rabbit IL-1 alpha failed to inhibit the inflammatory response. These results suggest that the in vivo events leading to the accumulation of neutrophils in response to IL-1 alpha are rapidly initiated.
Lad N, Williams TJ, Booth RF, 1992, Neutrophil infiltration into the ischaemic/reperfused rabbit isolated myocardium: effect of PF-5901 and cycloheximide., Eur J Pharmacol, Vol: 223, Pages: 163-171, ISSN: 0014-2999
The Langendorff-perfused rabbit heart preparation has been used to study the interaction of isolated rabbit neutrophils with regionally ischaemic myocardium. Short durations of regional ischaemia (10-60 min) and subsequent reperfusion (30 min) of the hearts with neutrophils resulted in a significant time-dependent accumulation of neutrophils (as assessed by myeloperoxidase activity) in the area at risk. Pre-activation of neutrophils with zymosan-activated serum prior to their infusion into the myocardium potentiated neutrophil accumulation in the area at risk. Pretreatment of the myocardium with a lipoxygenase inhibitor, PF-5901 (10 microM), or a de novo protein synthesis inhibitor, cycloheximide (10 microM), significantly reduced the accumulation of neutrophils in the ischaemic/reperfused myocardium. In contrast, pretreatment of neutrophils with cycloheximide (10 microM, for 15 min) prior to their infusion had no significant effect on neutrophil accumulation in the area at risk. The cyclooxygenase inhibitor, indomethacin (10 microM), had no effect on neutrophil accumulation in the area at risk following ischaemia and reperfusion. These results suggest the involvement of de novo protein synthesis and the lipoxygenase products in the infiltration of neutrophils following ischaemia and reperfusion in vitro.
Brady AJ, Williams FM, Williams TJ, 1992, Inflammatory injury in myocardial ischaemia., Clin Sci (Lond), Vol: 83, Pages: 511-518, ISSN: 0143-5221
Williams TJ, Hellewell PG, 1992, Endothelial cell biology. Adhesion molecules involved in the microvascular inflammatory response., Am Rev Respir Dis, Vol: 146, Pages: S45-S50, ISSN: 0003-0805
Accumulation of leukocytes in tissues is essential for effective host defense. To fulfill this role the cell must interact with and penetrate the vessel wall and migrate in the tissue. It is now clear that cell adhesion molecules play a crucial role in orchestrating these processes. A number of families of such adhesion molecules that mediate the interaction of circulating leukocytes and vascular endothelial cells have been identified. These include the leukocyte integrins, the selectins, members of the immunoglobulin family, and certain carbohydrates. Studies in vitro have elucidated which of these adhesion molecules are important in the interaction of different leukocyte classes with the endothelium under both basal and stimulated conditions. With the aid of monoclonal antibodies, the role that these molecules play in the interaction of inflammatory cells in the microvasculature in vivo is being assessed. Studies to date have demonstrated the key role of cell adhesion molecules in inflammation.
Rossi AG, Norman KE, Donigi-Gale D, et al., 1992, The role of complement, platelet-activating factor and leukotriene B4 in a reversed passive Arthus reaction., Br J Pharmacol, Vol: 107, Pages: 44-49, ISSN: 0007-1188
1. The mechanisms underlying oedema formation induced in a reversed passive Arthus (RPA) reaction and, for comparison, in response to zymosan in rabbit skin were investigated. 2. Oedema formation at skin sites was quantified by the accumulation of intravenously-injected 125I-labelled human serum albumin. 3. Recombinant soluble complement receptor type 1 (sCR1), administered locally in rabbit skin, suppressed oedema formation induced in the RPA reaction and by zymosan. 4. The platelet-activating factor (PAF) antagonists, WEB 2086 and PF10040 administered locally, inhibited oedema formation induced in the RPA reaction and by PAF but not by zymosan. 5. A locally administered leukotriene B4 (LTB4) antagonist, LY-255283, inhibited oedema formation induced by LTB4 but did not inhibit oedema responses to PAF, zymosan or the RPA reaction. 6. The results demonstrate a role for complement in oedema formation in both the RPA reaction and in response to zymosan. An important contribution by PAF is indicated in the RPA reaction but not in response to zymosan whereas no evidence was obtained to suggest a role for LTB4 in either inflammatory response.
Warren JB, Coughlan ML, Williams TJ, 1992, Endotoxin-induced vasodilatation in anaesthetized rat skin involves nitric oxide and prostaglandin synthesis., Br J Pharmacol, Vol: 106, Pages: 953-957, ISSN: 0007-1188
1. The effect of intradermally injected endotoxin on skin blood flow was investigated in anaesthetized male Wistar rats in vivo. 2. Local skin blood flow changes were measured hourly for 6 h in the shaved dorsal skin with a laser-Doppler flow probe and compared to changes in control sites which had been injected with 100 microliters of phosphate-buffered saline. By 3 h, skin blood flow increased above basal by 129 +/- 27% and 186 +/- 29% with 1 and 10 micrograms of endotoxin respectively. Blood flow remained significantly elevated at 6 h, the corresponding figures being 129 +/- 24% and 154 +/- 31% (P less than 0.05, n = 6 rats, mean +/- s.e.mean). 3. In further experiments, the response to 3 micrograms of endotoxin was measured at 4 h and treatment with a cyclo-oxygenase inhibitor, nitric oxide synthase inhibitors or a topical steroid all significantly inhibited this response (P less than 0.05 in each case, n = 6 rats in each group with duplicate sites in each animal). 4. Indomethacin 3 x 10(-9) mol per site injected 3.5 h after injection of endotoxin suppressed the mean 4 h response to endotoxin by 78%; NG-nitro-L-arginine methyl ester (L-NAME) 10(-7) mol per site suppressed the response by 95%; NG-monomethyl-L-arginine (L-NMMA) 10(-7) mol per site suppressed the response by 50%; whereas the D-isomer of NG-monomethyl-arginine 10(-7) mol per site had no significant effect.5. Topical application of the corticosteroid, betamethasone 17-valerate (1% solution) 18 h before injection of endotoxin inhibited the mean 4 h response to endotoxin by 66% and the 6 h response by 48%.6. In the same model, the vasodilator response to arachidonic acid was inhibited by both indomethacin and nitric oxide synthase inhibitors (P<0.05 in each case).7. These data suggest that the microcirculatory vasodilator response to endotoxin and arachidonic acid injected locally involves both nitric oxide synthase and cyclo-oxygenase in this in vivo model.
Henriques MG, Rae GA, Cordeiro RS, et al., 1992, Endothelin-1 inhibits PAF-induced paw oedema and pleurisy in the mouse., Br J Pharmacol, Vol: 106, Pages: 579-582, ISSN: 0007-1188
1. The current study analyses the effects of endothelin-1 (ET-1) on paw oedema and pleurisy induced by platelet activating factor (PAF) and other inflammatory agents in the mouse. 2. Combined subplantar injection of ET-1 (0.5 pmol/paw) did not modify oedema caused by histamine (1 to 100 mumol/paw), 5-hydroxytryptamine (1 to 100 mumol/paw) or bradykinin (1 to 100 nmol/paw) but markedly inhibited the response to PAF (0.95 to 3.8 nmol/paw). The selective action of ET-1 against PAF-induced (1.9 nmol/paw) oedema was dose-dependent, reaching a maximum at 0.5 pmol/paw and lasted up to 2 h. 3. ET-1 (0.5 pmol/paw) also inhibited paw oedema (3-4 h) caused by zymosan (500 micrograms/paw). In contrast, it did not modify either the early (1-4 h) or late (48-72 h) phases of the oedematogenic response to carrageenin (300 micrograms/paw), when given either together with or 24 h after the carrageenin. 4. Intrathoracic injection of PAF (1.9 nmol/cavity) induced pleurisy characterized by an increase in pleural exudate volume, and in accumulation of Evans Blue which was maximal at 30 min and lasted up to 4 h. When injected together with PAF, ET-1 (0.5 pmol/cavity) virtually abolished PAF-induced pleurisy. 5. It is concluded that ET-1 is a potent inhibitor of PAF-induced inflammation in the mouse. Its mechanism of anti-inflammatory action in this species, in contrast to what has been found in other species, does not appear to derive from its potent vasoconstrictor properties as ET-1, at the doses used, failed to affect oedematogenic responses to other inflammatory mediators.
Warren JB, Larkin SW, Coughlan M, et al., 1992, Pituitary adenylate cyclase activating polypeptide is a potent vasodilator and oedema potentiator in rabbit skin in vivo., Br J Pharmacol, Vol: 106, Pages: 331-334, ISSN: 0007-1188
1. The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on microvascular blood flow and plasma protein leakage were investigated in rabbit skin in vivo. 2. Intradermal injection of PACAP38, the 38 amino acid form of the peptide, caused a dose-dependent increase in blood flow measured by a 133Xe clearance technique. An equivalent increase in blood flow was induced by 10(-12) mol per site of PACAP38, 10(-12) mol per site of human alpha-calcitonin gene-related peptide (CGRP) and 10(-10) mol per site of vasoactive intestinal polypeptide (VIP). 3. The vasodilator activity of PACAP38 was not significantly different from that of the 27 amino acid form of the peptide, PACAP27, when measured with a laser Doppler flow meter, causing a 104 +/- 14% compared with 110 +/- 18% increase above basal blood flow at 10(-12) mol per site respectively. 4. At 10(-12) mol per site the effect of PACAP38 was longer lasting than that of CGRP. Blood flow remained significantly increased above control at 2 h with PACAP38 (P less than 0.05) whereas blood flow after intradermal CGRP had returned to control values by this time. 5. PACAP38 injected alone had no significant effect on microvascular leakage of 125I-labelled albumin. However, PACAP38 significantly potentiated bradykinin-induced oedema where it was approximately 100 fold more potent than VIP. 6. Oedema potentiation induced by PACAP38 was not inhibited by indomethacin at a dose which did inhibit potentiation of bradykinin-induced oedema by arachidonic acid.7. PACAP38 is at least as potent as other peptides which have been postulated to be involved in the inflammatory response when tested in rabbit skin in vivo. PACAP may contribute to both the hyperaemia and oedema components of inflammation.
Buckley TL, Brain SD, Jose PJ, et al., 1992, The partial inhibition of inflammatory responses induced by capsaicin using the Fab fragment of a selective calcitonin gene-related peptide antiserum in rabbit skin., Neuroscience, Vol: 48, Pages: 963-968, ISSN: 0306-4522
The effect of an anti-calcitonin gene-related peptide (CGRP) antibody on responses induced by the sensory C-fibre neuropeptide, CGRP, and capsaicin, which selectively activates C-fibre nerves, was investigated in rabbit skin. Test agents and antibody were injected intradermally. Local blood flow changes were measured by 133Xenon clearance and oedema formation by [125I]albumin accumulation. Preinjection intradermally with the Fab fragment of a goat anti-human alpha CGRP antibody selectively inhibited increased blood flow induced by CGRP (3 x 10(-12) mol/site) and caused a partial, but significant inhibition of increased blood flow induced by capsaicin (3 x 10(-7) mol/site). Oedema induced by histamine and bradykinin was potentiated by vasodilator doses of CGRP and capsaicin. These potentiating effects were significantly inhibited by pretreatment with anti-CGRP Fab. The Fab fragment was more potent in inhibiting capsaicin-induced responses than the parent IgG. These results suggest that capsaicin releases vasodilator quantities of CGRP in rabbit skin.
Yellon DM, Iliodromitis E, Latchman DS, et al., 1992, Whole body heat stress fails to limit infarct size in the reperfused rabbit heart., Cardiovasc Res, Vol: 26, Pages: 342-346, ISSN: 0008-6363
OBJECTIVE: It has recently been shown that induction of heat stress proteins by whole body heat stress confers myocardial protection in the isolated in vitro rat and rabbit heart. This study extends the above studies by examining the effects of stress protein synthesis on the limitation of infarct size in the in vivo rabbit heart model. METHODS: 30 male New Zealand white rabbits were used. Six rabbits were used for measurement of heat stress protein; 10 were used for infarct size determination in a heat stress group (HS); 14 were used for infarct size determination in a control group. There were 10 exclusions. Under anaesthesia, body temperature was raised to 42 degrees C for 15 min in the HS group. Following 24 hours of recovery rabbits were reanaesthetised and the hearts subjected to a 45 min period of regional ischaemia followed by 3 h reperfusion. The risk zone was defined with fluorescent particles and the infarct area determined by tetrazolium staining. Western blotting showed an increase in the 72 KD heat stress protein in hearts in the HS group. RESULTS: Infarct size as a percent of risk area was 61.4 (SEM 6.4)% (n = 14) in control hearts and 71.8(7.3)% (n = 10) in the HS hearts. These results were not statistically significant. CONCLUSIONS: No protective effect of heat stress could be seen when infarct size was used as the end point. Either the protection seen in earlier studies using the Krebs perfused isolated heart model does not accurately reflect protection against myocardial infarction, or heat stress itself may induce injurious factors in the blood which will negate any direct protective effect to the myocardium in this model.
Walls AF, Brain SD, Desai A, et al., 1992, Human mast cell tryptase attenuates the vasodilator activity of calcitonin gene-related peptide., Biochem Pharmacol, Vol: 43, Pages: 1243-1248, ISSN: 0006-2952
Calcitonin gene-related peptide (CGRP) is localized in and released from sensory nerves. It is a potent and long acting vasodilator which has been suggested to play a role in the control of blood flow. Using HPLC and trichloroacetic acid precipitation techniques, we have examined the ability of human mast cell lysates and a purified preparation of mast cell tryptase to degrade CGRP. We found that CGRP is effectively cleaved by tryptase (Km = 6.8 x 10(-6) mol/L at 37 degrees). Enzymatic activity was inhibited by antipain, leupeptin, N-alpha-p-tosyl-L-lysine chloromethyl ketone, benzamidine or aprotinin, but not by soybean trypsin inhibitor or N-tosyl-L-phenylalanine chloromethyl ketone. The degradation of CGRP by lysates of purified skin mast cells showed a similar pattern of inhibition suggesting that tryptase may be the major enzyme involved. The activity of tryptase was not affected by the presence of heparin. Incubation of CGRP with tryptase resulted in a loss of its vasodilator activity as observed by intravital microscopy of the hamster cheek pouch microvasculature. CGRP preincubated with tryptase failed to relax arterioles when added topically. It is suggested that the catalysis of CGRP by tryptase could represent an important means by which the activity of this neuropeptide is regulated in vivo.
Sciberras DG, Brain SD, Williams TJ, 1992, The inflammatory effects of CGRP and substance P in human skin, ISSN: 0306-5251
Nourshargh S, Perkins JA, Showell HJ, et al., 1992, A comparative study of the neutrophil stimulatory activity in vitro and pro-inflammatory properties in vivo of 72 amino acid and 77 amino acid IL-8., J Immunol, Vol: 148, Pages: 106-111, ISSN: 0022-1767
IL-8, a potent neutrophil-activating protein, can be produced by many cell types including monocytes, lymphocytes, fibroblasts, neutrophils, and endothelial cells. Depending on the cell source, the N-terminal amino acid sequence of IL-8 displays heterogeneity that has been shown to confer differences in its neutrophil stimulatory activity in vitro. Despite these observations the relative potency of different IL-8 molecules in vivo is unknown. To address this question we have investigated the biologic activity of the two predominant forms of IL-8, the 72 and the 77 amino acid proteins, in vitro and in vivo. In vitro, human rIL-8(72) and human rIL-8(77) dose dependently induced adherence of rabbit peritoneal neutrophils and human neutrophils to laminin-coated plates and elevated cytoplasmic levels of Ca2+ ([Ca2+]i) in fura-2 loaded neutrophils. In these in vitro assays human rIL-8(72) was more potent than human rIL-8(77) while inducing comparable responses to human rC5a. With respect to enhancing [Ca2+]i, neutrophils desensitized to human rIL-8(72) failed to respond to human rIL-8(77). However, neutrophils fully desensitized to human rIL-8(77) could exhibit a partial response to human rIL-8(72). Further, human rIL-8(72) was approximately 10-fold more effective than human rIL-8(77) in displacing human [125I]rIL-8(72) from rabbit peritoneal neutrophils in a receptor-binding assay. In vivo, intradermally administered human rIL-8(72) and human rIL-8(77) induced 111In-neutrophil accumulation and edema formation in rabbit skin. In contrast to the in vitro studies, the two forms of IL-8 gave identical responses in vivo although they were less potent than human rC5a. Our results demonstrate that, in vitro, human rIL-8(72) is more potent than human rIL-8(77) in stimulating neutrophils. It may be that IL-8)72) has a greater affinity and/or efficacy for the neutrophil IL-8 cell-surface receptors. One possibility for the observation that both forms of IL-8 are equipotent in induc
Weg VB, Watson ML, Cordeiro RS, et al., 1991, Histamine, leukotriene D4 and platelet-activating factor in guinea pig passive cutaneous anaphylaxis., Eur J Pharmacol, Vol: 204, Pages: 157-163, ISSN: 0014-2999
The involvement of histamine, leukotriene D4 (LTD4) and platelet-activating factor (PAF) in cutaneous anaphylaxis was investigated in a guinea pig model. When given alone, the H1 receptor antagonist chlorpheniramine, the LTD4/E4 antagonist LY171883 and the PAF antagonist WEB2086 were unable to inhibit increased microvascular plasma protein leakage in passive cutaneous anaphylaxis (PCA) reactions, as monitored by the extravasation of intravenously injected 125I-albumin. Furthermore the H2 receptor antagonist cimetidine and the serotonin antagonist methysergide were unable to reduce PCA responses when given alone or in combination with chlorpheniramine. In marked contrast, combinations of antagonists were able to reduce plasma leakage significantly. A combination of chlorpheniramine, LY171883 and WEB2086 virtually abolished plasma leakage during the PCA response, but did not influence the plasma protein leakage induced by intradermal injection of bradykinin. These results demonstrate that these allergic reactions involve several mediators and that the inability of an individual mediator antagonist to reduce responses does not necessarily rule out a role for that mediator.
Jose PJ, Collins PD, Perkins JA, et al., 1991, Identification of a second neutrophil-chemoattractant cytokine generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to melanoma-growth-stimulatory activity., Biochem J, Vol: 278 ( Pt 2), Pages: 493-497, ISSN: 0264-6021
The intraperitoneal injection of zymosan in the rabbit results in the generation of an inflammatory exudate containing oedema-forming and chemoattractant activities. Previous studies demonstrated the early appearance of the complement fragment C5a, followed by the generation of two mediators related to the cytokine interleukin-8 that were separable by cation-exchange h.p.l.c. N-Terminal amino acid sequencing identified one of these mediators as rabbit interleukin-8. This paper describes the purification of the second cytokine by cation-exchange, gel-filtration and reversed-phase h.p.l.c. The purified material had both oedema-forming and chemoattractant activity when assayed in rabbit skin in vivo. On SDS/PAGE a single 6-8 kDa band was observed and N-terminal amino acid sequencing of the reduced and alkylated protein positively identified 36 amino acids. This sequence revealed the rabbit homologue of melanoma-growth-stimulatory activity. The identification of these two cytokines in vivo will provide an opportunity to investigate the importance of their co-release in the inflammatory process.
Seale JP, Nourshargh S, Hellewell PG, et al., 1991, Mechanism of action of platelet activating factor in the pulmonary circulation: an investigation using a novel isotopic system in rabbit isolated lung., Br J Pharmacol, Vol: 104, Pages: 251-257, ISSN: 0007-1188
1. Rabbit isolated lungs were perfused via the pulmonary artery with Tyrode solution containing 4.5% Ficoll and 0.1% bovine serum albumin at a constant rate of 20 ml min-1. Lung perfusate was drawn for alternating 5 min periods from two reservoirs, one containing 125I-albumin and the other unlabelled albumin to wash out the intravascular label. Microvascular 125I-albumin leakage was determined from the count remaining at the end of the washout phase with an external gamma scintillation probe. In addition, perfusion pressure was monitored continuously. Each experiment comprised 6 cycles over a total period of 60 min. 2. Infusion of platelet activating factor (PAF, 3 nmol min-1 for 10 min) resulted in microvascular 125I-albumin leakage, whereas lyso-PAF was without effect. During PAF infusions there was also an increase in perfusion pressure. Both the permeability and pressor effects of PAF were inhibited by the PAF antagonist L-652731. 3. Infusion of the thromboxane analogue U-46619 (0.6 nmol min-1 for 10 min) caused an increase in perfusion pressure but protein accumulation was not significantly different from that observed with control infusions. 4. Bolus injections of PAF (1 nmol) caused increases in perfusion pressure which were reduced by indomethacin, dazmegrel and BW 755C. Bolus injections of PAF, repeated at 30 min intervals caused reproducible pressor responses; however, repeated injections at 60 min intervals resulted in augmented responses. This augmentation did not occur in the presence of indomethacin. 5. Retrograde perfusion of PAF via the pulmonary vein induced increased perfusion pressure and microvascular 125I-albumin leakage. The observed increase in leakage when compared with forward perfusion suggests that PAF produces predominantly arteriolar constriction i.e. proximal to the site of leakage during forward perfusion. 6. These results indicate that PAF is a vasoconstrictor in the rabbit pulmonary circulation and augmented responses occur with repe
Buckley TL, Brain SD, Rampart M, et al., 1991, Time-dependent synergistic interactions between the vasodilator neuropeptide, calcitonin gene-related peptide (CGRP) and mediators of inflammation., Br J Pharmacol, Vol: 103, Pages: 1515-1519, ISSN: 0007-1188
1. The action of the long lasting neuropeptide vasodilator, calcitonin gene-related peptide (CGRP), in potentiating oedema formation and neutrophil accumulation was investigated in the dorsal skin of the rabbit, in vivo. Combinations of agents under test were administered by intradermal (i.d.) injection. Oedema formation and neutrophil accumulation were then measured by quantitative radiolabel techniques. 2. CGRP (1 x 10(-11) mol per site) potentiated neutrophil accumulation induced by zymosan activated plasma, (used as a source of C5a des Arg), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and leukotriene B4 (LTB4). In contrast CGRP did not induce neutrophil accumulation when injected alone. 3. Oedema formation induced by a series of chemically distinct mediators of increased microvascular permeability; bradykinin, platelet activating factor (PAF), FMLP and zymosan-activated plasma; measured 0-30 min after i.d. injection was potentiated by CGRP (1 x 10(-11) mol per site). 4. Oedema formation induced by zymosan activated plasma and FMLP but not bradykinin and PAF, was also significantly potentiated by CGRP when oedema was measured 30-60 min after i.d. injection. The potentiation of oedema induced by zymosan activated plasma measured 30-60 min after i.d. injection was not observed in the presence of the shorter acting prostanoid vasodilator prostacyclin (PGI2, 3 x 10(-10) mol per site). 5. These results suggest that CGRP, as a consequence of its sustained vasodilator activity could have prolonged potentiating effects on neutrophil accumulation and oedema formation in inflammatory conditions.
Faccioli LH, Nourshargh S, Moqbel R, et al., 1991, The accumulation of 111In-eosinophils induced by inflammatory mediators, in vivo., Immunology, Vol: 73, Pages: 222-227, ISSN: 0019-2805
Eosinophils are implicated in the pathogenesis of a variety of allergic inflammatory diseases such as asthma. Several substances have been shown to be chemotactic for eosinophils in vitro, but the inflammatory mediators involved in the accumulation of eosinophils in vivo are as yet unidentified. In this study we have developed a system to measure the accumulation of 111In-eosinophils in guinea-pig skin in vivo. Horse serum-induced guinea-pig peritoneal eosinophils were radiolabelled with 111In and injected intravenously into recipient animals. 125I-albumin was also injected intravenously in order to measure local oedema formation simultaneously. A range of putative mediators was injected intradermally and responses measured for up to 2 hr. Of the mediators tested, guinea-pig C5a des Arg in zymosan-activated plasma was the most active. Recombinant human C5a (rHC5a) was also highly active, but less than the guinea-pig material. C5a des Arg in maximally activated plasma induced a 1500% increase in eosinophil accumulation, while rHC5a (10(-10) mol dose) induced a 600% increase. Platelet-activating factor (PAF) and leukotriene B4 (LTB4) were also tested for comparison. With respect to 111In-eosinophil accumulation, the order of potency of the mediators tested was as follows: guinea-pig C5a des Arg greater than LTB4 greater than PAF. In contrast, the order of potency of the mediators with respect to oedema formation was: PAF greater than guinea-pig C5a des Arg greater than LTB4. The techniques described will facilitate analysis of the mechanisms involved in eosinophil accumulation in defined inflammatory reactions.
Buckley TL, Brain SD, Collins PD, et al., 1991, Inflammatory edema induced by interactions between IL-1 and the neuropeptide calcitonin gene-related peptide., J Immunol, Vol: 146, Pages: 3424-3430, ISSN: 0022-1767
The neuropeptide calcitonin gene-related peptide (CGRP) is a potent vasodilator with a long duration of action. CGRP is widely distributed and is present in perivascular nerves of tissues that include skin and the synovium. In this study we have investigated the possibility that CGRP can modulate the inflammatory actions of the cytokine IL-1 by using an inflammatory model in rabbit skin. The intradermal injection of IL-1 (1.4 x 10(-14) mol/site) alone stimulated little edema formation. However, when IL-1 was injected with CGRP (10(-11) mol/site), a highly significant edema was observed, and neutrophil accumulation induced by IL-1 was potentiated. These results suggest that the action of IL-1 as a potent mediator of increased microvascular permeability is only observed when skin blood flow is increased in this model. This was confirmed by experiments in which PGE2 (3 x 10(-9) mol/site) at a dose with a similar duration of vasodilator action as CGRP (10(-11) mol/site) also potentiated edema induced by IL-1. Further experiments investigated the mechanism by which IL-1 increased microvascular permeability. Edema induced by IL-1 was dependent on de novo protein synthesis and the presence of circulating neutrophils. However, selective platelet-activating factor and histamine H1 antagonists had no inhibitory effect on this response. Thus it appears that when a microvascular bed is dilated by the long-lasting vasodilator CGRP, edema induced by IL-1 is clearly observed. These results highlight a potentially important synergistic interaction between cytokines and neuropeptides in inflammation.
Collins PD, Jose PJ, Williams TJ, 1991, The sequential generation of neutrophil chemoattractant proteins in acute inflammation in the rabbit in vivo. Relationship between C5a and proteins with the characteristics of IL-8/neutrophil-activating protein 1., J Immunol, Vol: 146, Pages: 677-684, ISSN: 0022-1767
An in vivo experimental peritonitis model was investigated in the rabbit using zymosan as the inflammatory stimulus. After an i.p. injection of zymosan, exudate was removed at intervals and tested in the back skin of assay rabbits. Assay rabbits received i.v. injections of 125I-albumin and 111In-neutrophils, and the local accumulation of each label was measured in response to intradermal injections of exudate samples mixed with a potentiating dose of PGE2. When peritoneal exudate samples were tested in the presence of a specific anti-C5a antibody, virtually all the edema-inducing and neutrophil chemoattractant activity was abolished in samples taken up to 2 h after the zymosan injection. Later samples, however, contained increasing levels of a non-C5a component. In C5a-depleted 6-h exudate two peaks of inflammatory activity were separated using cation exchange HPLC. Evidence is presented that C5a itself is unable to stimulate the production of these activities. Both peaks of activity appear related to IL-8/NAP-1 as they inhibited the binding of 125I-IL-8/NAP-1 to human neutrophils.
Williams TJ, Das A, von Uexkull C, et al., 1991, Neutrophils in asthma., Ann N Y Acad Sci, Vol: 629, Pages: 73-81, ISSN: 0077-8923
Watson ML, Williams TJ, 1991, Measurement of cell accumulation., Agents Actions Suppl, Vol: 34, Pages: 429-438, ISSN: 0379-0363
Beaubien BC, Collins PD, Jose PJ, et al., 1990, A novel neutrophil chemoattractant generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to interleukin 8., Biochem J, Vol: 271, Pages: 797-801, ISSN: 0264-6021
An inflammatory reaction was induced in vivo by injection of zymosan into the peritoneal cavity of the rabbit. The inflammatory exudate was found to contain oedema-inducing and neutrophil chemoattractant activity when assayed in rabbit skin in vivo, using 125I-albumin and 111In-neutrophils. This activity was additional to that of complement fragment C5a, which was removed by an affinity gel. Two chemoattractants were isolated by cation-exchange, gel-filtration and reversed-phase h.p.l.c. One of these, which ran as a single band of 6-8 kDa on SDS/PAGE, was subjected to N-terminal sequence analysis without reduction and alkylation of cysteine residues. Positive identification of 28 of the first 31 amino acids revealed a rabbit homologue of interleukin-8 (75% sequence identity with human interleukin-8). The demonstration of interleukin-8 as a major neutrophil chemoattractant in an inflammatory reaction in vivo provides the basis for further investigations into the role of this cytokine in the inflammatory process.
Nourshargh S, Williams TJ, 1990, Evidence that a receptor-operated event on the neutrophil mediates neutrophil accumulation in vivo. Pretreatment of 111In-neutrophils with pertussis toxin in vitro inhibits their accumulation in vivo., J Immunol, Vol: 145, Pages: 2633-2638, ISSN: 0022-1767
The role of neutrophil chemoattractant receptors in neutrophil stimulation in vitro is well established, however, the precise mechanisms underlying local neutrophil accumulation at inflammatory sites in vivo have not been defined. A fundamental question that remains open is whether chemoattractants act on the endothelial cell or the neutrophil to initiate the process of neutrophil migration in vivo. To address this question we have investigated whether neutrophil accumulation in vivo can occur if chemoattractant receptor occupancy is uncoupled from neutrophil stimulation. For this purpose we have used pertussis toxin (PT) as the pharmacologic tool. We have investigated the effect of in vitro pretreatment of rabbit neutrophils with PT on their responses in vitro and on their accumulation in vivo. Pretreatment of rabbit neutrophils with PT inhibited FMLP- and C5a-, but not PMA- induced increases in CD18 expression, neutrophil adherence, and degranulation in vitro. This pretreatment procedure with PT inhibited the accumulation of radiolabeled neutrophils in vivo in response to intradermally injected FMLP, C5a, C5a des Arg, leukotriene B4, IL-8, and zymosan in rabbit skin. Further, in contrast to the in vitro results, PT inhibited the PMA-induced 111In-neutrophil accumulation in vivo. Interestingly, pretreatment of neutrophils with PT also inhibited accumulation in response to intradermally injected IL-1, despite the reports that IL-1 lacks neutrophil chemoattractant activity in vitro. Although the experimental techniques used cannot distinguish the different stages of neutrophil migration involved, these results suggest that the accumulation of neutrophils induced by local extravascular chemoattractants in vivo depends on a pertussis toxin-sensitive receptor operated event on the neutrophil itself. Further, PMA and IL-1 may release secondary chemoattractants in vivo.
JOSE PJ, MOSS IK, MAINI RN, et al., 1990, MEASUREMENT OF THE CHEMOTACTIC COMPLEMENT FRAGMENT C5A IN RHEUMATOID SYNOVIAL-FLUIDS BY RADIOIMMUNOASSAY - ROLE OF C5A IN THE ACUTE INFLAMMATORY PHASE, ANNALS OF THE RHEUMATIC DISEASES, Vol: 49, Pages: 747-752, ISSN: 0003-4967
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