Inhibition of peripheral interleukin-1 beta-induced hyperalgesia by the intracerebroventricular administration of diclofenac and alpha-melanocyte-stimulating hormone

The present study was undertaken to investigate whether or not the endogeneous mechanisms in the brain can modulate the changes in nociception produced by peripherally-administered interleukin-1 beta (IL-1 beta) in rats. We administered diclofenac and alpha-melanocyte-stimulating hormone (alpha-MSH) into the lateral cerebroventricle (LCV) 10 min before the intraperitoneal (i.p.) injection of recombinant human IL-1 beta (rhIL-1 beta, 1 ng/kg-100 ng/kg) and then observed the changes in nociception using a hot-plate test. The i.p. injection of rhIL-1 beta (10 ng/kg and 100 ng/kg) reduced the paw-withdrawal latency without affecting the colonic temperature. The maximal reduction in the paw-withdrawal latency was observed 30 min after the i.p. injection of rhIL-1 beta at 100 ng/kg. The rhIL-1 beta (100 ng/kg)-induced hyperalgesia was inhibited by the LCV injection of both diclofenac (1 ng) and alpha-MSH (100 ng). The LCV injection of either diclofenac (1 ng) or alpha-MSH (100 ng) was found to have no effect on nociception by itself. These findings therefore suggest that the hyperalgesia induced by peripheral IL-1 beta can be modulated by a cyclooxygenase pathway of the arachidonate and alpha-MSH in the brain.     

Evidence that brain capillary endothelial cells can be infected with murine leukaemia retrovirus, LP-BM5

The effect of intraportal administrations of interleukin 1 beta (IL-1 beta) on the afferent activity of the hepatic branch of the vagus nerve was observed in urethane anesthetized rats. An intraportal injection of IL-1 beta in doses of 10 pg and 100 pg per animal (300-400 g body wt.) resulted in dose-dependent increase in the afferent activity, which lasted about 70-100 min. Further, intraportal injection of IL-1 beta (100 pg) induced reflex activation of efferent activity of the splenic (sympathetic) nerve and vagal thymic nerve. This reflex activation was not observed in hepatic vagotomized rat and after an administration of the same dose of IL-1 beta into the systemic vein in normal rat. The results suggest the existence of sensors for IL-1 beta which send information on IL-1 beta in the portal venous blood to the central nervous system through the hepatic branch of the vagus nerve and play some role in reflex regulation of immune function.     

Characterization of adenosine receptors evoking excitation of mesenteric afferents in the rat

We examined the effects of adenosine receptor agonists and antagonists on the discharge of mesenteric afferent nerves supplying the jejunum in pentobarbitone sodium-anaesthetized rats. Adenosine (0.03-10 mg kg(-1), i.v.), NECA (0.3-300 microg kg(-1), i.v.) and the A1 receptor agonist, GR79236 (0.3-1000 microg kg(-1), i.v.), each induced dose-dependent increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. The A1 receptor antagonist, DPCPX (3 mg kg(-1), i.v.), antagonized all the effects of GR79236 but only the haemodynamic effects of adenosine and NECA. The A2A receptor antagonist, ZM241385 (3 mg kg(-1), i.v.), antagonized the hypotensive effect of NECA but none of the effects of GR79236. The A2A receptor agonist, CGS21680 (0.3-300 microg kg(-1), i.v.), and the A3 receptor agonist, IB-MECA (0.3-300 microg kg(-1), i.v.), each induced only a dose-dependent hypotension. Subsequent administration of adenosine (3 mg kg(-1), i.v.) induced increases in afferent nerve activity and intrajejunal pressure and bradycardia. ZM241385 (3 mg kg(-1), i.v.) antagonized the hypotensive effect of CGS21680 but not the effects of adenosine. Bethanechol (300 microg kg(-1), i.v.) evoked increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. However, adenosine (3 mg kg(-1), i.v.) evoked greater increases in afferent nerve activity than bethanechol despite inducing smaller increases in intrajejunal pressure. In summary, A1 and A2B and/or A2B-like receptors evoke adenosine-induced increases in mesenteric afferent nerve activity and intrajejunal pressure in the anaesthetized rat. Furthermore, elevations in intrajejunal pressure do not wholly account for adenosine-evoked excitation of mesenteric afferent nerves.     

The influence of the lymph node on the protein concentration of efferent lymph leaving the node

1. Experiments have been performed in sheep to determine the contribution of lymph formed within a lymph node to the total protein output in lymph leaving the node. 2. The lymphatic duct leaving the popliteal lymph node was cannulated and the protein and lymphocyte output in efferent lymph determined. The afferent lymph flow to the popliteal node was then diverted and lymph formed only within the lymph node collected from the efferent cannula. It appeared from the results that the popliteal lymph node forms lymph at the rate of approximately 1 ml. per hour and may contribute 30-50% of the protein output observed in efferent lymph. 3. The importance of lymph formation within the lymph node varied between nodes found in different regions of the body. This was due in part to the different protein concentrations in the afferent lymph to the different nodes. 4. A positive correlation was found between the protein and lymphocyte concentrations in efferent lymph from the popliteal lymph node in seven out of eleven sheep and in lymph formed within the popliteal lymph node in two out of three sheep. It is suggested that this relationship may be due to an increased transfer of plasma proteins through the post-capillary venules in the lymph node accompanying the continual traffic of lymphocytes across the wall of these vessels. The results indicated that the protein transfer across the post-capillary venules was not an indiscriminate transfer of plasma per se but a selective transport from the blood plasma compartment based on molecular size.     

The effect of central angiotensin II receptor blockade on interleukin-1beta- and prostaglandin E-induced fevers in rats: possible involvement of brain angiotensin II receptor in fever induction

We investigated the role of the brain angiotensin II (Ang II) receptor subtypes AT1 and AT2 in the development of fever induced in freely moving rats by administration of interleukin-1beta (IL-1beta) or prostaglandin E2 (PGE2). Intraperitoneal (i.p.) injection of IL-1beta (2 microg/kg) induced a marked fever of rapid onset. Intracerebroventricular (i.c.v.) administration, immediately before IL-1beta injection, of a selective AT2 receptor antagonist, CGP42112A (5 or 20 microg), reduced the fever in a dose-related manner. Rats given an i.c.v. injection of PGE2 (200 ng) developed a monophasic fever response that was attenuated by i.c.v. treatment with CGP42112A (10 or 20 microg) in a dose-related manner. The IL-1beta (2 microg/kg i.p.)- and PGE2 (200 ng i.c.v.)-induced fevers were unchanged by the selective AT1 receptor antagonist losartan (60 microg i.c.v.). Treatment with exogenous Ang II (100 ng i.c.v.), which itself had no effect on resting body temperature, resulted in an enhancement of the PGE2 (50 ng i.c.v.)-induced fever. The administration of CGP42112A (2 and 5 microg) into the rostral hypothalamus (preoptic/anterior hypothalamic region) reduced fevers induced by IL-1beta (2 microg/kg i.p.) or intrahypothalamic (i.h.) PGE2 (100 ng). Moreover, i.h. injection of Ang II (25 ng) augmented the PGE2 (25 ng i.h.)-induced fever. Finally, the i.h. administration, 15 min before i.h. PGE2 (100 ng), of the angiotensin-converting enzyme (ACE) inhibitor lisinopril (5 and 10 microg) attenuated the PGE2-induced fever. These results suggest that brain AT2 receptors contribute to the induction of such febrile responses in rats.     

Tracheal vascular dilatation elicited by vagal nerve stimulation in rats

We investigated the effects of the electrical stimulation of a unilateral cervical vagal nerve on the blood flow in the trachea using laser Doppler flowmetry in urethane anesthetized Wistar King rats. Stimulation for 30 s at 1, 2, 5, 10, 20 or 50 Hz with 10 V intensity caused an increase in tracheal blood flow (TBF) in a frequency-dependent manner; the effects were most dominant with the 10-Hz stimulation among the six frequencies used. The increased responses of TBF with the muscarinic receptor antagonist atropine (1.0 mg/kg, i.v.) were significantly reduced when compared with those without atropine at 5 Hz stimulation (123.3 +/- 11.9% vs. 180.1 +/- 24.5%). This shows the existence of vasodilation due to a cholinergic mechanism. The increased responses of TBF after the ganglion blocking agent hexamethonium (20 mg/kg) i.v. administration were significantly reduced when compared with those without hexamethonium at 1, 2 Hz stimulation (1 Hz: 18.9 +/- 2.7% vs. 35.4 +/- 4.7%, 2 Hz: 40.5 +/- 8.9% vs. 58.8 +/- 6.7%); this shows the existence of vasodilation due to a non-cholinergic parasympathetic efferent mechanism which itself appears to be due to the release of neuropeptides such as VIP and PHI. The increased responses after hexamethonium administration were augmented probably because of the enhanced release of other neuropeptides like SP and CGRP especially at 10 Hz and 20 Hz stimulation. These findings suggest that the mechanism of vasodilation by the activity in the vagal fibers in the trachea of the rat has cholinergic and non-cholinergic efferent components and a non-cholinergic afferent component. In rats, the afferent component may play an important role in controlling tracheal vascular changes.     

Local intestinal immune responses to infections with Trichinella spiralis. Real-time, continuous assay of cytokines in the intestinal (afferent) and efferent thoracic duct lymph of rats

Levels of IL-4, IL-5, TNF-alpha, and IFN-gamma were quantitated in the intestinal (afferent) and efferent thoracic duct lymph of rats during the course (0 to 289 h) of an infection with Trichinella spiralis. Intestinal lymph was collected by cannulating thoracic ducts of mesenteric lymphadenectomized animals. These studies showed that cytokines typical of a Th2 type (IL-4 and IL-5) and a Th1 type (IFN-gamma) were simultaneously detected in the intestinal lymph during the first 8 days after infection. Worm expulsion (day 11 to 12) was associated with increased levels of IL-4 and IL-5 in the intestinal lymph. IL-5 levels rose as early as 15 to 20 h and remained elevated throughout the infection. IL-4 activity appeared in intestinal lymph 60 h after infection and reached peak levels during worm expulsion. Despite the predominantly Th2 nature of cytokine response, IFN-gamma levels showed several cycles of high and low production during the course of infection. A comparison of cytokine levels between intestinal and efferent lymph values showed no significant differences in IL-4 or IL-5 levels suggesting no contribution by the mesenteric node to efferent lymph. However, IFN-gamma and TNF-alpha levels were lower in efferent lymph compared with intestinal lymph suggesting mesenteric node consumption. Adoptive transfer experiments showed that protective CD4+CD45RC- cells primed the gut for a more rapid TH2-type response that was faster than in a primary infection. In contrast, adoptive transfer of CD4+CD45RC+ cells primed the gut for a more rapid Th1-type(IFN-gamma) response. These studies demonstrate a novel method for measuring real-time changes in cytokine levels in the gut during the course of an active infection.     

Effects of water deprivation and angiotensin II intracerebroventricular administration on brain nitric oxide synthase activity

Intracranial administration of L-arginine causes a reduction of the water intake induced by water deprivation or by intracerebroventricular (i.c.v.) injection of angiotensin II (angiotensin II), through the release of nitric oxide (NO) in the central nervous system. We studied the effects of i.c.v. angiotensin II (120 ng/rat) in association with i.c.v. L-arginine (2.5-10 microg/rat) on blood pressure. We also studied the effects of both peripheral and central angiotensin II injection (1.5-6 mg kg(-1) i.p. and 30-120 ng rat(-1) i.c.v., respectively) on NO synthase activity in the cortex, diencephalon and brainstem, after water deprivation (24 h), conditions producing activation of the renin-angiotensin system. L-arginine dose dependently antagonized the increase in blood pressure induced by i.c.v. angiotensin II (P < 0.001). Peripheral administration of angiotensin II produced a dose-dependent reduction of NO synthase activity in the brainstem and cortex (P < 0.001), but not in the diencephalon. Water deprivation produced similar effects on brain NO synthase activity. Angiotensin II i.c.v. injection caused NO synthase activity reduction in all brain regions studied (P < 0.001). Our findings suggest that NO and angiotensin II could play opposite roles in brain regulation of blood pressure and drinking behaviour.     

Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation

The order of potency of tachykinin (TK) receptor agonists suggests that TK NK-1 receptors mediate their inhibitory effect on water intake induced by intracerebroventricular (i.c.v.) injection of angiotensin II (AngII) in rats. The present study was aimed at further evaluating which TK receptor subtype mediates the effect, using selective antagonists for the TK receptor subtypes. Pulse i.c.v. injection of the TK agonist neuropeptide gamma (NP gamma), 31-250 ng/rat, markedly inhibited AngII-induced water intake. The i.c.v. injection of the NK-1 receptor antagonist SR14033, 0.5 microgram/rat, significantly reduced, while 1 microgram/rat completely abolished the inhibitory effect of NP gamma, 125 ng/rat. The selective NK-2 receptor antagonist SR48968 and the selective NK-3 receptor antagonist R820 were devoid of any effect up to the i.c.v. dose of 2 micrograms/rat. On the other hand, i.c.v. injection of SR140333, 1 microgram/rat, did not increase drinking induced by i.c.v. injection of AngII, 0.1-10 ng/rat, and did not increase drinking in water sated or water deprived rats. The results of the present study confirm that central TKergic mechanisms inhibit AngII-induced drinking in rats, and provide further evidence that TK NK-1 receptors mediate the effect. Failure of i.c.v. injected SR 140333 to increase AngII-induced drinking, as well as water intake in sated or deprived rats suggests that brain NK-1 receptor mechanisms apparently do not exert a tonic control on AngII-induced drinking and, in general, on water intake in rats. From a pharmacological point of view, the inhibitory effect of TKs on the dipsogenic action of AngII can represent a functional test for activity at central NK-1 receptors in rats.     

Release of somatostatin and its role in the mediation of the anti-inflammatory effect induced by antidromic stimulation of sensory fibres of rat sciatic nerve

1. The effect of antidromic stimulation of the sensory fibres of the sciatic nerve on inflammatory plasma extravasation in various tissues and on cutaneous vasodilatation elicited in distant parts of the body was investigated in rats pretreated with guanethidine (8 mg kg(-1), i.p.) and pipecuronium (200 microg kg(-1), i.v.). 2. Antidromic sciatic nerve stimulation with C-fibre strength (20 V, 0.5 ms) at 5 Hz for 5 min elicited neurogenic inflammation in the innervated area and inhibited by 50.3 +/- 4.67% the development of a subsequent plasma extravasation in response to similar stimulation of the contralateral sciatic nerve. Stimulation at 0.5 Hz for 1 h also evoked local plasma extravasation and inhibited the carrageenin-induced (1%, 100 microl s.c.) cutaneous inflammation by 38.5 +/- 10.0% in the contralateral paw. Excitation at 0.1 Hz for 4 h elicited no local plasma extravasation in the stimulated hindleg but still reduced the carrageenin-induced oedema by 52.1 +/- 9.7% in the paw on the contralateral side. 3. Plasma extravasation in the knee joint in response to carrageenin (2%, 200 microl intra-articular injection) was diminished by 46.1 +/- 12.69% and 40.9 +/- 4.93% when the sciatic nerve was stimulated in the contralateral leg at 0.5 Hz for 1 h or 0.1 Hz for 4 h, respectively. 4. Stimulation of the peripheral stump of the left vagal nerve (20 V, 1 ms, 8 Hz, 10 min) elicited plasma extravasation in the trachea, oesophagus and mediastinal connective tissue in rats pretreated with atropine (2 mg kg(-1), i.v.), guanethidine (8 mg kg(-1), i.p.) and pipecuronium (200 microg kg(-1), i.v.). These responses were inhibited by 37.8 +/- 5.1%, 49.7 +/- 9.9% and 37.6 +/- 4.2%, respectively by antidromic sciatic nerve excitation (5 Hz, 5 min) applied 5 min earlier. 5. Pretreatment with polyclonal somatostatin antiserum (0.5 ml/rat, i.v.) or the selective somatostatin depleting agent cysteamine (280 mg kg(-1), s.c.) prevented the anti-inflammatory effect of sciatic nerve stimulation (5 Hz, 5 min) on a subsequent neurogenic plasma extravasation of the contralateral paw skin. The inhibitory effect of antidromic sciatic nerve excitation on plasma extravasation in response to vagal nerve stimulation was also prevented by somatostatin antiserum pretreatment. 6. Cutaneous blood flow assessment by laser Doppler flowmetry indicated that antidromic vasodilatation induced by sciatic nerve stimulation was not inhibited by excitation of the sciatic nerve of the contralateral leg (1 Hz, 30 min) or by somatostatin (10 microg/rat, i.v.) injection. 7. Plasma levels of somatostatin increased more than 4 fold after stimulation of both sciatic nerves (5 Hz, 5 min) but the stimulus-evoked increase was not observed in cysteamine (280 mg kg(-1), s.c.) pretreated rats. 8. These results suggest that somatostatin released from the activated sensory nerve terminals mediates the systemic anti-inflammatory effect evoked by stimulating the peripheral stump of the sciatic nerve.     

Inhibition of IL-1 induced tissue factor (TF) synthesis and procoagulant activity (PCA) in purified human monocytes by IL-4, IL-10 and IL-13

Exposure of monocytes to pro-inflammatory cytokines or lipopolysaccharide (LPS) may induce synthesis and expression of tissue factor (TF). In this paper we have focused on the induction of TF-activity in human monocytes by the pro-inflammatory cytokines recombinant human interleukin 1 (rhIL-1 alpha) (rhIL-1 beta) (rhIL-6) and human tumour necrosis factor alpha (rhTNF-alpha), measured as procoagulant activity (PCA) in a microtitre plate-based clot assay. In addition we have studied the modulation of IL-1 alpha/beta induced TF-mRNA and PCA by rhIL-4, rhIL-10 and rhIL13. IL-1 alpha and IL-1 beta induced a concentration dependent increase in TF-activity. Neither IL-6 nor TNF-alpha gave rise to procoagulant activity at the concentrations tested (0.2-20 ng/ml). IL-4, IL-10 and IL-13, all effectively diminished IL-1 alpha/beta induced PCA, shown at the protein- and at the mRNA-level, while cell viability was unaffected. These results add to the previously demonstrated role of IL-4 and IL-10 as inhibitors of LPS-induced TF-activity, showing that these anti-inflammatory cytokines are not specific for LPS-activation but interfere with other stimulating substances such as IL-1, which may be involved in diseases where LPS is not present.     

Relaxing actions of corticotropin-releasing factor on rat resistance arteries

1. Although it well established that corticotropin-releasing factor (CRF) injected i.v. can cause hypotension and vasodilatation, there is no in vitro evidence that CRF acts as a vasodilator. We have therefore tested the hypothesis that the hypotensive effect of i.v. CRF is due to a direct vasodilator action by carrying out experiments in vitro on rat resistance arteries (i.d. 150-300 microns). 2. Initial in vivo experiments confirmed that CRF (1.5 nmol.kg-1) injected i.v. caused hypotension in rats, this being partially antagonized by the CRF analogue CRF9-41. 3. For the in vitro experiments, vessels were taken from the mesenteric, cerebral and femoral vascular beds, and mounted as ring preparations in an isometric myograph. The vessels were pre-contracted with one of 3 agonists (prostaglandin F2 alpha, arginine vasopressin or noradrenaline) or with a high-potassium solution (K+). 4. With maximal concentrations of the agonists, CRF caused relaxation of mesenteric and cerebral vessels with 10 nM, and near complete relaxation with 100 nM. Femoral vessels pre-constricted with agonists and all vessels pre-constricted with K+ were less affected by CRF. In the mesenteric vessels, with sub-maximal levels of pre-constriction, CRF caused substantial relaxation at 1 nM and could cause complete relaxation at 10 nM. 5. The relaxant effect of CRF on contractions of mesenteric vessels was antagonized by 100 nM CRF9-41. Neither tetraethyl ammonium (30 mM) nor glibenclamide (3 microM) antagonized the relaxant effect of CRF. 6. The relaxant effect of CRF on mesenteric small arteries was found to be unaffected by removal of the endothelium. 7. The results indicate that CRF causes an endothelial-independent vasodilatation of rat resistance arteries under in vitro conditions at concentrations which are consistent with this being an important cause of the hypotension observed with i.v. injection of CRF.     

Disposition kinetics of cobalt mesoporphyrin in mouse, rat, monkey and dog

1. Radiometric and UV analyses indicated > 95% unchanged cobalt mesoporphyrin (CoMP) in plasma after i.v. or i.m. administration. Blood clearance of CoMP is < 2% of hepatic blood flow in mouse and rat, and < 0.5% of hepatic blood flow in monkey and dog. CoMP elimination t1/2 ranged from 3.1 to 9.9 days in animals after i.v. administration. 2. CoMP is highly (> 99.5%) bound to plasma proteins, but has low affinity for blood cells (Kp < 0.15). The volume of CoMP distribution (Vss < 0.91/kg) is reflective of a distribution to total body water following i.v. administration to mouse, rat, monkey and dog. 3. [14C]CoMP reached highest levels in rat tissue between 1 and 4 days following i.m. injection. Liver, kidney cortex, lymph node, adrenal and spleen demonstrated greatest uptake of radiolabel. Concentration in tissues was readily detectable at 60 days post-dose. 4. CoMP was slowly absorbed after i.m. administration showing dose-dependent pharmacokinetics. The major route of radiolabel elimination was faecal excretion (54% of dose) in rat after an i.m. dose of [14C]CoMP. Approximately 1% of the 14C dose was recovered in the urine over 7 days post-dose. 5. As a polar metalloporphyrin, CoMP has low clearance, restricted tissue distribution and long elimination t1/2 in the laboratory animals.     

Autologous peripheral blood stem cell transplantation and adoptive immunotherapy with activated natural killer cells in the immediate posttransplant period

Relapse after high-dose chemotherapy supported by peripheral blood stem cell transplantation (HDC-PBSCT) is the main cause of therapeutic failure in patients with lymphoma and breast cancer. Adoptive immunotherapy with activated natural killer (A-NK) cells and interleukin 2 might eliminate surviving residual tumor without adding to toxicity. Eleven patients with relapsed lymphoma and one with metastatic breast cancer were entered on a pilot clinical trial of HDC-PBSCT followed on day 2 after transplant by infusion of cultured autologous A-NK cells. Simultaneously, recombinant human interleukin 2 (rhIL-2) was initiated as a 4-day continuous i.v. infusion at 2 x 10(6) IU/m2/day, referred to as high-dose rhIL-2. Therapy with high-dose rhIL-2 was followed by a 90-day continuous i. v. infusion at 3 x 10(5) IU/m2/day, referred to as low-dose rhIL-2. All patients engrafted and nine completed treatment. Posttransplant days to a neutrophil count of 500/microliter and to a platelet count of 50,000/microliter were similar to comparable patients treated with HDC-PBSCT alone. Generation of A-NK cells for therapy was feasible in all patients except the three patients with Hodgkin's disease, whose cells did not proliferate in culture. Overall toxicity associated with early posttransplant transfer of A-NK cells and interleukin 2 did not differ from that observed with peripheral blood stem cell transplantation alone in comparable patients. There was early amplification of natural killer cell activity in the peripheral blood of four patients that appeared to result from the transfused A-NK cells. Adoptive transfer of A-NK cells and rhIL-2 during the pancytopenic phase after HDC-PBSCT was feasible and well tolerated, did not adversely affect engraftment, and resulted in amplified natural killer activity in the peripheral blood during the immediate posttransplantation period.     

Pancreas carcinoma

1. To assess in vivo chemotactic activity of tumour necrosis factor (TNF), interleukin-1 (IL-1), IL-8, and cytokine-induced neutrophil chemoattractant (CINC), we injected these cytokines into the pleural cavity of rats. 2. CINC (0.1-1 microgram) and recombinant human IL-8 (rhIL-8, 0.2-5 micrograms) caused neutrophil infiltration into the rat pleural cavity in a dose-dependent fashion, peaking at 3 h. The number of leukocytes in the peripheral blood did not change significantly. 3. RhTNF alpha and rhIL-1 alpha also induced neutrophil accumulation. The dose response curves of rhTNF alpha (0.67 ng-6.7 micrograms) and rhIL-1 alpha (0.45 ng-4.5 micrograms) at 3 h were bell shaped. On the other hand, unlike CINC and rhIL-8, rhTNF alpha and rhIL-1 alpha caused transient marked leukopenia at 3 h in a simple dose-dependent fashion. 4. Concomitant injection of actinomycin D dose-dependently and completely at 10 micrograms inhibited neutrophil infiltration induced by rhTNF alpha (0.67 microgram) and rhIL-1 alpha (0.45 microgram) at 3 h. However, that induced by CINC or rhIL-8 was not affected by actinomycin D. 5. Peaking at 1 h, CINC production in the pleural cavity was found after intrapleural injection of rhTNF alpha (0.67 microgram) or rhIL-1 alpha (0.45 microgram), but not after that of rhIL-8 (5 micrograms). The CINC production induced by rhTNF alpha or rhIL-1 alpha and the neutrophil infiltration was suppressed by concomitant injection of actinomycin D (1 and 10 micrograms). 6. These results indicate that CINC and IL-8 themselves are direct chemoattractants for neutrophils, whereas TNF and IL-1 induce neutrophil infiltration indirectly via newly synthesized mRNA for chemotactic protein including CINC, which may be involved in neutrophil emigration at local inflammatory sites in rats.     

Age-related changes in localization of injected radiolabelled lymphocytes in the lymph nodes of antigen-stimulated mice

The localization of i.v. injected syngeneic lymph node cells, radiolabelled with 51Cr or 75Se-L-selenomethionine, was studied in male CBA/H mice aged between 3 and 30 months. The following results were obtained. (1) Localization of cells from young adult donors was greater in the s.c. lymph nodes of old than of young recipients, the main increase being between 15 and 17 months of age. Increases in lymph node weight and DNA-synthesis were also seen at this time; but the rise in cell localization was significant even when calculated per unit of tissue weight. Splenic localization either declined slightly with age or, like the liver, showed no significant change. (2) Local antigenic stimulation by a single injection of sheep erythrocytes into one front footpad, 24 hr before lymph node cell injection, resulted in increased localization in the regional lymph nodes of 3-17 month old, but rarely of 24-30 month old mice. (3) No consistent differences in localization were observed between lymph node cells from 4-month and 25-month old donors. Both age-related and antigen-related increases in cell localization were at least partly attributable to an enhanced rate of entry of lymphocytes from the blood to the lymph nodes. Although the changes underlying the decline in antigen-related localization of cells in old recipients have still to be clarified, it is probable that the defective immune responses of old mice result partly from this decline.     

Interleukin-1 beta induced transient diabetes mellitus in rats. A model of the initial events in the pathogenesis of insulin-dependent diabetes mellitus?

When aiming at preventing IDDM in man, knowledge of the molecular mechanisms leading to beta cell destruction may facilitate identification of new possible intervention modalities. A model of IDDM pathogenesis in man suggests that cytokines, and IL-1 in particular, are of major importance in the initial events (Nerup et al 1994) (Fig. 1). In vitro rat experiments demonstrated that rhIL-1 beta inhibits beta cell function and induces beta cell death both in isolated islets of Langerhans and in the isolated perfused pancreatic gland. With the long term goal of identifying new modalities capable of preventing IDDM in man, the aim af this review was to investigate the effects of rhIL-1 beta on beta-cell function and viability in normal rats. This review discussed 1) the pharmacokinetics of IL-1 beta in rats as the basis for choice of route of administration and dose of rhIL-1 beta, 2) the effects and molecular mechanisms of IL-1 beta on temperature and food intake used as control parameters for successful injection of rhIL-1 beta in rats, 3) the effects of one or more injection of IL-1 beta on rat beta cell function, 4) the molecular mechanisms leading to IL-1 beta induced beta cell inhibition in vivo, and some possible intervention modalities based on the molecular mechanisms, 5) the effects of IL-1 beta on spontaneous diabetes mellitus in DP BB rats, and 6) the effects and molecular mechanisms of IL-1 beta induced inhibition of thyroid epithelial cell function and aggravated thyroiditis in DP BB rats, compared to the effects of IL-1 beta on rat beta cell function. Finally, this review discussed the effects of IL-1 beta on human beta cells in vitro, and the clinical relevance of these experiments, with special reference to a clinical trial with the aim of preventing IDDM in man. The pharmacokinetic studies suggested that IL-1 beta is distributed according to a two-compartment model with a first-order elimination. Interleukin-1 beta reached all the investigated organs in the rats, was accumulated in kidneys and was excreted in the urine. The data suggested that IL-1 beta also accumulated in the islets of Langerhans. After injection of 4.0 micrograms/kg pathophysiologically relevant concentrations of rhIL-1 beta were reached and intact rhIL-1 beta persisted for up to 5 hrs in plasma. Peripheral injections of IL-1 beta dose-dependently induced fever and anorexia in rats, probably via induction of PGE2 in the brain or in peripheral tissues thereafter passing the blood-brain barrier. Nitric oxide produced by cNOS seems to be a molecular mediator of IL-1 beta induced fever but not of anorexia. Fever and anorexia are well described effects of IL-1 beta in rats, and are as such usefull control parameters of the absorption and biological activity of IL-1 beta after peripheral injection. Injections of rhIL-1 beta to normal, non-diabetes prone rats induced initial beta cell stimulation followed by inhibition, in accordance with in vitro data. Furthermore, induction of peripheral insulin resistance coincided with beta cell inhibition after one daily injection for 5 days, leading to a transient diabetes mellitus-like state, characterized by hyperglycemia and hypoinsulinemia. At this time point, electron-microscopy did not demonstrate beta cell destruction. However, IL-1 beta induced intercellularly edema and microvillous processes on the beta cells, which might be early evidence of apoptosis. The diabetes mellitus-like state was not aggravated if the daily injections were continued beyond 5 days. Daily injections of rhIL-1 beta for 2 to 4 weeks induced formation of blocking IL-1 beta-antibodies in normal rats. Hence, injections exceeding 2 weeks should only be performed using species homologous IL-1 beta. The molecular mechanism of IL-1 beta induced beta cell inhibition in rats in vivo as in vitro, are likely to involve binding of IL-1 beta to the IL-1RtI, since the IL-1RtII is considered to be a decoy receptor. (ABSTRACT TRUNCATED)     

Different receptor mechanisms mediate the effects of endotoxin and interleukin-1 on female sexual behavior

Activation of the immune system by lipopolysaccharide (LPS) produces physiological, neuroendocrine and behavioral effects, some of which are mediated by cytokine production. We have previously shown that the cytokine interleukin-1 (IL-1) inhibits sexual behavior in female, but not male rats, while producing a comparable suppression of locomotion in both sexes. The present study examined the effects of LPS on sexual behavior and locomotion of male and female rats, and the involvement of IL-1 receptors in mediating the effects of IL-1 and LPS on females' behavior. Peripheral (i.p.) administration of LPS (50 or 250 microg/kg) significantly decreased sexual behavior in females, up to 6 h after administration, while it had no effect on male sexual behavior. However, locomotor activity, measured in the open-field test, was similarly reduced by LPS in both males and females. Pretreatment with the IL-1 receptor antagonist (IL-1ra) either i.p. (10 mg/kg) or intracerebroventricularly (i.c.v.) (50 microg/rat) did not prevent the inhibition of female sexual behavior and locomotion induced by either i.p. (50 microg/kg) or i.c.v. (200 or 400 ng/rat) administration of LPS, respectively. However, identical doses of IL-1ra significantly reversed the effects of IL-1beta, administered either i.p. (5 microg/kg) or i.c.v. (50 ng/rat), respectively. These results demonstrate that both LPS and IL-1beta produce marked inhibition of sexual behavior in female, but not in male rats. However, IL-1 receptors are not required for the effects of LPS on sexual behavior in female rats.     

Further evidence that the tachykinin PG-KII is a potent agonist at central NK-3, but not NK-1, receptors

Intracerebroventricular (i.c.v.) injection of tachykinins (TKs) inhibits ethanol intake and angiotensin II-induced water intake; the effects are apparently mediated by NK-3 and NK-1 receptors, respectively. The present study evaluated the effect of the TK PG-KII, a novel kassinin-like peptide isolated from the skin of the Australian frog Pseudophryne güntheri, in these in vivo tests for central activity. PG-KII, given by i.c.v. injection, potently inhibited alcohol intake in genetically selected alcohol-preferring rats, being about 3 times more potent than the selective NK-3 receptor agonist NH2-SENK. The dose of 100 ng/rat, that markedly inhibited ethanol intake, did not inhibit food intake and prandial drinking in food deprived rats, providing evidence that the effect of PG-KII on ethanol intake is behaviorally selective. The effect on ethanol intake was inhibited by i.c.v. injection of the NK-3 receptor antagonist R820, but was not modified by the NK-1 receptor antagonist SR 140333. PG-KII inhibited drinking induced by angiotensin II only at doses of 300 or 1000 ng/rat, being about 5 times less potent than the selective NK-1 receptor agonist [Sar9, Met(O2)11] substance P. These doses of PG-KII produced also marked increase in competing behaviors, such as grooming and locomotion. The dose of 1000 ng/rat evoked a general inhibition of the ingestive behavior, reducing also food intake. The i.c.v. injection of the NK-1 receptor antagonist SR 140,333 only slightly inhibited the effect of PG-KII on angiotensin II-induced drinking, while it markedly reduced that of [Sar9, Met(O2)11] substance P. These findings, in accordance with those of previous studies, indicate that PG-KII is endowed with marked activity at central NK-3 receptors, and low activity at NK-1 receptors.     

Studies on the lymphocytes of sheep. III. Destination of lymph-borne immunoblasts in relation to their tissue of origin

Lymph-borne immunoblasts were labeled in vitro with 125I[]iodo-deoxy-uridine, washed and returned by intravenous injection to the sheep from which they had been collected. Twenty h later the sheep were killed and the distribution of the immunoblasts was determined by assaying the radioactivity in various organs. Immunoblasts from the efferent lymph of peripheral somatic lymph nodes (PSLN) went mainly to the spleen, lungs and other PSLN, while immunoblasts from intestinal lymph went mainly to the small gut. This ability of intestinal immunoblasts to home to the gut was demonstrated also in the sterile environment of fetuses in utero; apparently the migratory behavior of immunoblasts, like that of small lymphocytes, is not primarily "antigen-driven". A technique was devised for the collection of peripheral (i.e. afferent to the mesenteric node) intestinal lymph which was found to contain 10-20 times the numbers of small lymphocytes that occur in the peripheral lymph from other tissues. Immunoblasts from peripheral intestinal lymph also homed to the gut. The immunoglobulin content of immunoblasts was studied by making detergent extracts of lymph cells, by applying immuno-peroxidase techniques to cell films and by investigating the incorporation of 14C-labeled amino acids into immunoglobulins by immunoblasts in vitro. Immunoblasts from both somatic and intestinal lymph contained and made IgG and IgM, but many intestinal immunoblasts contained and made IgA. It is not known whether this immunoglobulin mediates the extravasation of immunoblasts into the gut. Nonetheless, there is compelling evidence that there are two major migratory pathways for lymphoid cells; one through the gut-associated lymphoid tissue and the other through the somatic-splenic lymphoid tissues.