Biomedical subjects

R J Flower

Publications and source records attributed to R J Flower.

At least 127 records · Page 7Linked to original sources

PubMed1993

Modulation of IL-1-induced neutrophil migration by dexamethasone and lipocortin 1.

IL-1 is a pro-inflammatory cytokine which controls many features of the immune and inflammatory response. When injected into a mouse 6-day-old air-pouch, human rIL-1 (1 to 100 ng) induced in a dose-dependent fashion a migration of PMN that could be reliably assessed 4 h after injection. Both IL-1 alpha and IL-1 beta were active in this model. The effect of the cytokine was inhibited by local administration of actinomycin D (1 to 10 micrograms), alpha-melanocyte-stimulating hormone (200 micrograms), and a mAb recognizing IL-1R type I (10 micrograms). Indomethacin (1 mg/kg), an inhibitor of cyclo-oxygenase, and BW4AC (2 mg/kg), a selective lipoxygenase inhibitor, were without effect but moderate inhibition was seen with the platelet-activating factor antagonist WEB2086 (1 to 10 mg/kg). The glucocorticoid dexamethasone (0.015 to 1.5 mg/kg) potently inhibited the elicitation of neutrophils induced by IL-1 when given systemically 2 h before the cytokine. The steroid-induced anti-inflammatory protein lipocortin 1 (LC1) also produced a dose-dependent inhibition of PMN migration into the pouch with an ED50 of approximately 0.15 to 0.21 mg/kg. The denatured protein was without effect. Passive immunization of mice with a polyclonal sheep antiserum or a mAb raised against LC1 abolished the inhibitory action of dexamethasone whereas preimmune serum or control IgG were without significant effect. These findings provide further evidence that LC1 is involved in the anti-inflammatory action of glucocorticosteroids and suggest that this protein may act as an endogenous regulator of IL-1 action.

Animals↗

PubMed1993

Effect of chronic capsaicin and guanethidine treatment on skin blood flow of the rat.

Using a non-invasive laser Doppler velocimetry technique to measure skin blood flow in the rat skin, we have shown that chronic treatment with capsaicin inhibited the vasodilator response to a non-pathological, local heat (44 degrees C, 20 min duration) stimulus. However, sympathectomy, accomplished with chronic treatment of guanethidine, had no effect on this heat-induced vasodilation. Topical steroids have also been shown to inhibit this response thus, since the heat-dilator response appears from these results to be brought about by the release of sensory neuropeptides, the sensory neuron may be a target for topical steroid action.

Animals↗

PubMed1993

Pharmacology of interleukin-1-induced neutrophil migration.

Neutrophil (PMN) accumulation induced by interleukin-1 beta (IL-1 beta, 5-20 ng) into the mouse air pouch was inhibited in a dose-dependent manner (2-200 micrograms) by concomitant injection of IL-1 receptor antagonist (IL-1RA). Similarly, co-administration of the neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) resulted in a reduction of the number of migrated PMN but only at the highest dose tested (200 micrograms). Although IL-1RA does not select between the two types of receptors so far described for IL-1, the effectiveness of alpha-MSH suggests that this property of the cytokine may occur through IL-1 type I receptor. This observation was confirmed by using a specific monoclonal antibody (mAb) raised against this receptor type, and which strongly inhibited (87%) IL-1-induced PMN recruitment.

Animals↗

PubMed1993

Development of specific antibody and in vivo response to antigen in different rat strains: effect of dexamethasone and importance of endogenous corticosteroids.

Endogenous glucocorticoids undoubtedly play a role in the control of immune responses: their contribution to inter-strain variation is unknown. The development of specific IgG and IgE was measured following inoculation with ovalbumin in Lewis, Fischer, Wistar and Brown Norway rats. The Lewis gives a smaller IgG and IgE response than the other strains and the response in vivo to antigen injected into the paw correlates with the titre of specific antibody. Treatment with the steroid receptor antagonist RU486 (mifepristone) following inoculation reveals that in the Lewis, and to a lesser extent in the Brown Norway, the development of a specific IgG response is limited by endogenous corticosteroids. The IgG response in different strains is differently sensitive to treatment with the synthetic glucocorticoid dexamethasone, the Lewis being particularly resistant. The importance of control by endogenous corticosteroids should not be overlooked in contributing to strain differences in immune response.

Adrenal Cortex Hormones↗

PubMed1993

Lipocortin 1 (annexin 1) immunoreactivity in the cervical spinal cord of Lewis rats with acute experimental allergic encephalomyelitis.

Spontaneous recovery from acute experimental allergic encephalomyelitis (EAE) by the Lewis rat is probably mediated by endogenous corticosteroids. It has been proposed that the anti-inflammatory actions of the glucocorticoids may be effected via the induction of mediator proteins termed lipocortins and recently we have demonstrated increased levels of lipocortin 1 in the central nervous system (CNS) of EAE-diseased rats (Bolton C., A-J. Elderfield and R.J. Flower (1990), J. Neuroimmunol. 29: 173-181). In this study, utilizing antisera raised against recombinant human lipocortin 1, immunohistochemistry and light microscopy have been used to determine the distribution of the protein in the cervical spinal cord of Lewis rats during EAE. In normal animals lipocortin 1 immunoreactivity was localized predominantly in the walls of larger blood vessels and to a lesser extent capillaries. The same staining pattern was found in adjuvant-inoculated controls. In sections from EAE-inoculated animals there was no change during the induction phase, but with the onset of clinical symptoms and the appearance of inflammatory infiltrates in the CNS, a marked increase in lipocortin 1 immunostaining was observed. This additional staining was due to widespread immunoreactivity of the lesions, was maximal at the height of disease and decreased following recovery and lesion regression. Within the lesions the vast majority of infiltrating lymphocytes and macrophages were positive for lipocortin 1, including some very heavily stained macrophage-like cells. Measurement of corticosterone in the sera of these animals showed that changes in lipocortin 1 immunostaining in the CNS during EAE closely parallel serum corticosterone levels.

Acute Disease↗

PubMed1993

Topical betamethasone-17-valerate inhibits heat-induced vasodilatation in man.

Topical betamethasone-17-valerate inhibits the vasodilatation response to local non-pathological heating of rat skin. We have shown that this effect can also be demonstrated in man. Topical betamethasone-17-valerate significantly inhibited the increase in human forearm cutaneous blood flow in response to heat of 44 degrees C, as measured by laser-Doppler velocimetry. This suggests that the effects of topical steroids upon skin blood flow in the rat and in man are similar, and supports the use of the animal model as a paradigm for studying human skin blood flow, and changes in response to anti-inflammatory steroids.

Administration, Topical↗

PubMed1993

Effects of lipocortin 1 and dexamethasone on the secretion of corticotrophin-releasing factors in the rat: in vitro and in vivo studies.

Lipocortin 1 (LC1: also called annexin 1) was first described as a putative second messenger protein for the anti-inflammatory steroids in peripheral tissues. In the present study, in vitro and in vivo methods were used to examine its potential role within the hypothalamus as a mediator of the regulatory actions of the glucocorticoids on the hypothalamo-pituitary-adrenocortical axis of the rat. In the in vitro studies, the effects of human recombinant LC1 (hu-r-LC1) on the concomitant release of the two major corticotrophin-releasing factors (CRF-41 and arginine vasopressin, AVP) from isolated hypothalami removed from chronically adrenalectomized rats were compared with those of dexamethasone in the presence and absence of appropriate secretagogues, namely phospholipase A2 (PLA2), interleukin-6 (IL-6) and a non-specific depolarizing agent, K+ (56 mM). The spontaneous release of CRF-41 in vitro was unaffected by either hu-r-LC1 (5 to 100 ng/ml) or dexamethasone (1 microM). Both compounds however reduced the release of the neuropeptide evoked by IL-6 (5 ng/ml) but failed to modify the secretory responses to PLA2 (25 U/ml) or K+ (56 mM). Dexamethasone (1 microM) had no effect on the basal release of AVP but effectively blocked the secretion of the peptide induced by either IL-6 (10 ng/ml) or PLA2 (25 U/ml). In complete contrast, hu-r-LC1 (5 to 100 ng/ml) stimulated the release of AVP and potentiated the secretory responses to IL-6 (10 ng/ml) and PLA2 (25 U/ml) but not to K+ (56 mM). The hypothalamic responses to PLA2 stimulation (25 U/ml) were associated with significant (P < 0.01) increases in prostaglandin E2 release which, in some instances, were potentiated by hu-r-LC1 (5 to 20 ng/ml). In vivo, administration of histamine (0.6 mg/100 g body wt, ip) produced significant (P < 0.01) increases in the serum corticosterone concentration and in the hypothalamic LC1 content. Neither hu-r-LC1 (0.6 to 1.2 micrograms) nor a polyclonal anti-LC1 antibody (3 microliters, diluted 1:200), injected intracerebroventricularly (icv), influenced either the resting serum corticosterone concentration or the hypersecretion of the steroid evoked by histamine stress. A lower dose of the recombinant protein (0.3 micrograms icv) also failed to alter basal corticosterone release but, in contrast to the higher doses, potentiated the pituitary-adrenocortical responses to histamine. The results suggest that LC1 may contribute to some aspects of peptide release in the hypothalamus but that its actions are not necessarily related to those of the glucocorticoids.

Adrenalectomy↗

PubMed1993

Investigations into the mechanism of vasoconstrictor action of the topical steroid betamethasone-17-valerate in the rat.

1. The effect of topical betamethasone upon skin blood flow was investigated in the rat. Two types of vasodilator stimuli were used; local heating to the surface of the skin and intradermal application of inflammatory agents. Blood flow was measured by laser doppler velocimetry. 2. Topical betamethasone-17-valerate (1 g with an 18 h pretreatment) significantly inhibited the heat-induced vasodilatation in the rat skin, as also did systemically administered betamethasone (1 mg kg-1, 3 h pretreatment). 3. Angiotensin converting enzyme (ACE) inhibitors (captopril, 5 mg kg-1 and enalapril, 1 mg kg-1, 30 min pretreatments) were the only drugs out of several different types of systemically administered inhibitors and antagonists that were tested which also inhibited the heat-induced vasodilatation. Aprotinin (100,000 KIU kg-1, 5 min pretreatment) a serine protease inhibitor, significantly potentiated the heat-induced response. 4. Bradykinin (50 nmol per site), des-Arg9-bradykinin (5 nmol per site), substance P (0.1 nmol per site) and capsaicin (1 mumol per site) induced an increase in skin blood flow. 5. Topical betamethasone treatment resulted in a significant inhibition of the vasodilator response to des-Arg9-bradykinin, whereas captopril treatment inhibited the responses to substance P, capsaicin, bradykinin and des-Arg9-bradykinin. 6. Intradermal application of captopril (10-100 micrograms) also caused a dose-dependent inhibition of the heat-induced vasodilatation. 7. These results suggest that topical betamethasone may be acting in a manner similar to that of the ACE inhibitors to produce an inhibition of the flow responses in the skin and that this effect may be brought about by interfering with the action of vasodilator peptide(s) or protein(s).

Administration, Cutaneous↗

PubMed1993

Anti-inflammatory actions of an N-terminal peptide from human lipocortin 1.

An acetylated polypeptide corresponding to residues 2-26 of human lipocortin 1 was synthesized and the anti-inflammatory activity assessed in three models of acute inflammation in rat and mouse. In the carrageenin rat paw oedema test, the peptide produced a maximal inhibition of approximately 41% at the 3 h time point with a 10 micrograms dose. When rat paw oedema was induced by the injection of venom phospholipase A2, the peptide produced a significant inhibition (31%) at the top dose of 20 micrograms per paw. In the mouse air-pouch model, systemic treatment with the peptide produced a dramatic reduction in cytokine-induced leukocyte migration with an ID50 of approximately 40 micrograms per mouse. The N-terminal peptide 2-26 shares the actions of lipocortin 1 in these acute models of inflammation.

Amino Acid Sequence↗

PubMed1993

The local anti-inflammatory action of dexamethasone in the rat carrageenin oedema model is reversed by an antiserum to lipocortin 1.

1. A local pre-injection of 1 micrograms dexamethasone sodium phosphate strongly inhibited (> 60% inhibition at 3 h; P < 0.001 at all time points) the development of carrageenin-induced paw oedema in the rat induced by a subplantar injection of 0.1 ml, 2% carrageenin. 2. Coinjection of a polyclonal rabbit antiserum raised against human 1-188 recombinant lipocortin 1, which also recognised the rat protein, reversed the inhibitory action of dexamethasone (P < 0.05 at 4 h and 5 h). At the highest volume used (40 microliters) control antisera were without any effect. 3. These data further support the concept that lipocortin 1 is involved in the anti-inflammatory mechanism of action of the glucocorticoids.

Animals↗

PubMed1993

Glucocorticoid-and non-glucocorticoid induction of lipocortins (annexins) 1 and 2 in rat peritoneal leucocytes in vivo.

1. We have studied the occurrence, distribution and disposition of lipocortins (annexins) 1, 2 and 5 in mixed peritoneal leucocytes obtained from rats in which glucocorticoid levels were altered by adrenalectomy, administration of the glucocorticoid antagonist, RU486, or by injection of dexamethasone or hydrocortisone, as well as from rats in which the peritoneal cells were elicited by inflammatory stimuli. 2. In cells obtained from untreated rats with an intact adrenal cortex, lipocortins 1, 2 and 5 were readily detectable: the majority of each of the proteins was apparently located intracellularly with much smaller amounts in the membrane. Lipocortin 1 and to a lesser extent lipocortin 5 were also seen in a Ca(2+)-dependent association with the external plasma membrane. Following administration of RU486 (2 x 20 mg kg-1) the amounts of lipocortin 1 and 2 in cells were greatly reduced. Conversely, injection of hydrocortisone (1 mg kg-1) or dexamethasone (0.08 mg kg-1) caused an increase in the amount of lipocortin 1 and 2 in peritoneal cells within 30 min. Lipocortin 5 was unchanged by any manipulation of glucocorticoid levels. 3. Lipocortins 1 and 2 were elevated in both intracellular and membrane-associated fractions of macrophages elicited by intraperitoneal injection in inflammogens. This phenomenon also occurred in adrenalectomized animals. 4. Our data indicate that glucocorticoids control the synthesis of some members of the lipocortin family in rat mixed peritoneal cells but also suggest the existence of a separate system for controlling the generation of this protein. The significance of these observations is considered in relation to the mechanism of glucocorticoid hormone action on eicosanoid production.

Adrenalectomy↗

PubMed1993

Serum corticosterone, interleukin-1 and tumour necrosis factor in rat experimental endotoxaemia: comparison between Lewis and Wistar strains.

1. Circulating corticosterone, interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF alpha) activities in serum of Lewis and Wistar rats were measured following injection of lipopolysaccharide (LPS). IL-1 was measured as 'lymphocyte activation factor' (LAF) activity following precipitation of inhibitory activity with polyethylene glycol. TNF alpha activity was measured as cytotoxic activity. 2. Compared to the Wistar, the Lewis rat had higher circulating LAF and TNF activities following LPS, and release of both cytokines was prolonged in this strain. 3. Corticosterone increases in response to LPS were less in the Lewis than in the Wistar rat following the initial peak at 1 h; basal corticosterone was lower in the Lewis rat. 4. Adrenalectomized Lewis rats had even greater amounts of circulating LAF and TNF activities following LPS than did intact animals; the effect of adrenalectomy was not however mimicked by acute treatment with the steroid receptor antagonist, RU486, suggesting that endogenous corticosteroids did not acutely control cytokine release. 5. Although in vivo administration of anti-murine IL-1 alpha antiserum significantly lowered LAF activity of serum, circulating corticosterone in response to LPS was not affected. Similarly, treatment with anti-murine TNF alpha monoclonal antibody (mAb) abrogated TNF activity without affecting corticosterone, suggesting that other mediators may be responsible for corticosterone release following LPS. 6. This 'overproduction' of inflammatory cytokines together with lower circulating corticosterone may contribute to the susceptibility of the Lewis rat to diseases such as adjuvant arthritis or experimental allergic encephalomyelitis.

Adrenal Cortex Hormones↗

PubMed1993

Modulation of the hypothalamo-pituitary-adrenocortical responses to cytokines in the rat by lipocortin 1 and glucocorticoids: a role for lipocortin 1 in the feedback inhibition of CRF-41 release?

Our recent studies indicate that lipocortin 1 (LC1), a putative second messenger protein for the anti-inflammatory steroids in peripheral tissues, may also contribute to the regulatory actions of the glucocorticoids on the hypothalamo-pituitary-adrenal axis. In the present study we have used in vitro and in vivo models to compare the effects of adrenalectomy, LC1 and dexamethasone on the cytokine-induced secretion of the 41-amino acid corticotrophin releasing factor (CRF-41) and arginine vasopressin (AVP) by the rat hypothalamus. In addition, western blot analysis was used to examine the influence of dexamethasone on the expression of LC1 in the hypothalamus. In vitro, interleukins- (IL-) 1 alpha (100 and 200 pg/ml), 1 beta (0.5 and 1.0 ng/ml) and 8 (0.25-1.0 ng/ml) readily initiated the release of CRF-41 and AVP from hypothalami removed from intact rats. IL-6 (10 and 20 ng/ml) was also an effective CRF-41 secretagogue but, unlike the other interleukins tested, it was ineffective with regard to AVP. Adrenalectomy 7-14 days prior to autopsy increased significantly (p < 0.01) the magnitude of the CRF-41 responses to IL-1 alpha, IL-1 beta and IL-6 but not to IL-8. In contrast however, while hypothalamic tissue from adrenalectomized rats, unlike that from intact animals, responded to IL-6 (5-20 ng/ml) with a pronounced hypersecretion of AVP, the AVP responses to IL-1 alpha and IL-1 beta were largely unaffected by adrenalectomy as too were those to IL-8. The marked increases in CRF-41 and AVP release from hypothalami from adrenalectomized rats initiated in vitro by IL1 alpha, IL-1 beta, IL-6 and IL-8 were readily overcome by preincubation of the tissue with dexamethasone (10(-7) M). In addition, the steroid caused 'externalization' of two species of immunoreactive (ir-) LC1 (37 and 58 kDa) by the hypothalamic cells but failed to influence the total LC1 content of the tissue. The inhibitory effects of dexamethasone on the cytokine-induced release of CRF-41 in vitro were mimicked by LC1 (10 ng/ml) which alone had no effect on the basal release of the peptide. However, unlike dexamethasone, LC1 failed to influence the concomitant release of AVP from the hypothalamic tissue elicited by IL1 alpha, IL-1 beta or IL-8 and potentiated that evoked by IL-6.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Cortex↗

PubMed1993

Endogenous tachykinins play a role in IL-1-induced neutrophil accumulation: involvement of NK-1 receptors.

Pretreatment of mice with capsaicin resulted in approximately 40% inhibition of the polymorphonuclear leucocyte (PMN) influx elicited by interleukin-1 (IL-1) injected into a murine air-pouch. This inhibition was mimicked by two selective antagonists of neurokinin-1 (NK-1) tachykinin (TK) receptors, i.e. CP-96,345 and RP-67,580, but not by the inactive enantiomer RP-68,651. A selective NK-2 antagonist, SR-48,968, was inactive. The natural peptide, substance P (SP), and a selective NK-1 agonist, (Sar9)SP, induced PMN infiltration into the murine air-pouch, whereas a selective NK-2 agonist, (beta Ala8)NK-A(4-10), was ineffective. Moreover, SP-induced PMN accumulation was prevented by co-administration of RP-67,580 and CP-96,345, but not by RP-68,651. These findings suggest that the release of an endogenous TK, possibly SP, may occur following IL-1 injection in vivo, indicating a contributory role for neuropeptides in the PMN migration elicited by this cytokine. The action of selective agonists and antagonists suggests the involvement of NK-1 receptors.

Animals↗

PubMed1992

Evidence for the presence and location of annexins in human platelets.

In this study the identity of annexins in human platelets has been determined together with their ability to be released by agents which induce platelet degranulation. The presence of proteins cross-reacting to antibodies against annexins I and V was detected in human platelets. However, the study provided evidence that these annexins are not located on the surface of the plasma membrane in a Ca++ dependent manner. Moreover, activation of platelets with several agents which induced platelet degranulation did not cause release of annexins I or V as determined by both immunoblotting and ELISA.

Annexin A1↗

PubMed1992

Lipocortin 1 mediates dexamethasone-induced growth arrest of the A549 lung adenocarcinoma cell line.

The synthetic glucocorticoid dexamethasone (1 microM to 1 pM) strongly (maximum greater than 80%) inhibits proliferation of the A549 human lung adenocarcinoma line (EC50 greater than 1 nM) and leads to the appearance, or a further increase (approximately 3-fold) in the expression on the cell surface, of the calcium and phospholipid binding protein lipocortin (annexin) 1. Both these effects, which are shared by hydrocortisone (1 microM) but not by progesterone or aldosterone (1 microM), are inhibited by the antiglucocorticoids RU38486 and RU43044 (1 microM). The nonsteroidal antiinflammatory drugs indomethacin (1 microM) and naproxen (10 microM) and human recombinant lipocortin 1 (0.05-5.0 micrograms/ml) also produce growth arrest in this cell line. During proliferation A549 cells spontaneously release prostaglandin E2 [10-20 ng (28-57 pmol) per ml per 5-day period] into the growth medium. In concentrations that cause growth-arrest, dexamethasone, indomethacin, and lipocortin 1 abolish the generation of this eicosanoid by A549 cells. Prostaglandin E2 itself (0.01-1 pM) stimulates cell growth and partially reverses (approximately 50%) the inhibition of growth caused by dexamethasone and indomethacin. Addition of the neutralizing anti-lipocortin 1 monoclonal antibody 1A (5 micrograms/ml), but not the nonneutralizing anti-lipocortin monoclonal antibody 1B, substantially reversed (greater than 80%) the inhibitory activity of dexamethasone on both growth and prostaglandin E2 synthesis. The generation of prostaglandin E2 by A549 cells seems to be an important regulator of cell proliferation in vitro and the dexamethasone-induced suppression of proliferation in this model is attributable to eicosanoid inhibition caused by lipocortin 1.

Adenocarcinoma↗

PubMed1992

Peptide 204-212 of lipocortin 5 inhibits the generation of a prostacyclin-like factor from rat aorta preparations in vitro.

The release of an endogenous prostacyclin-like factor (PLF) from rat thoracic aorta rings was evaluated through inhibition of platelet ADP-induced aggregation and assessed with a micro-method using 96-multiwell plates. Aggregation was assessed in an ELISA reader by measuring changes of optical density at 620 nm in each well. The generation of endogenous PLF by aortic rings was time-dependent and was inhibited by indomethacin (3 microM). ADP-induced aggregation was inhibited by iloprost in a dose-dependent manner. Incubation of rat aorta rings with peptide 204-212 of human lipocortin 5 (1-100 micrograms/ml) resulted in a dose-dependent inhibition of PLF release with a maximal inhibition of 90-95%. The effect of peptide 204-212 was reversible. A control peptide (amino-acids 104-112 of lipocortin 5) was without any significant effect. Peptide 204-212 (100 micrograms/ml) did not modify PLF release from rat aorta preparations challenged with arachidonic acid (10(-6)-10(-4) M).

Amino Acid Sequence↗

PubMed1992

Lipocortins (annexins) 1, 2, 4 and 5 are increased in the central nervous system in multiple sclerosis.

Western blotting and densitometry have been used to investigate the lipocortin content of post-mortem central nervous system (CNS) tissue samples from multiple sclerosis (MS) patients and normal controls. Lipocortins 1, 2, 4 and 5 were all detected in normal control grey and white matter. In white matter samples from MS patients these lipocortins were found to be significantly increased, a further elevation in lipocortin content was observed in MS plaque tissue. The implications of these findings with respect to the role of these proteins in inflammatory CNS disease and a possible mechanism of steroid action in the therapy of MS are discussed.

Adult↗