Lipopolysaccharide Administration In Rats Research Articles

Lipopolysaccharide impairs neurodevelopment and induces changes in astroglial reactivity, antioxidant defenses and bioenergetics in the cerebral cortex of neonatal rats.

Neonates have an immature immune system, which increases their vulnerability to infectious agents and inflammatory insults. The administration of the immunostimulatory agent lipopolysaccharide (LPS) has been shown to induce the expression of pro-inflammatory cytokines and cause behavior alterations in rodents at different ages. However, the effects of LPS administration during the neonatal period and its consequences during immune system maturation remain to be elucidated. We showed here that a single intraperitoneal administration of LPS in rats on postnatal day (PND) 7 caused early and variable alterations in TNF-α, S100B and GFAP levels in the cerebral cortex, CSF and serum of the animals, indicating long-term induction of neuroinflammation and astroglial reactivity. However, on PND 21, only GFAP levels were increased by LPS. Additionally, LPS induced oxidative stress and altered energy metabolism enzymes in the cerebral cortex on PND 21, and caused neurodevelopment impairment over time. These data suggest that neuroinflammation induction during the neonatal period induces glial reactivity, oxidative stress and bioenergetic disruption that may lead to neurodevelopment impairment and cognitive deficit in adult life.

Beneficial effects of selenium against the behavioral consequences of lipopolysaccharide administration in rats

ObjectiveThe effects of selenium on behavioral consequences of lipopolysaccharide (LPS) injection in rats was assessed. Materials and methodsThe animals were randomized into the following four groups (n = 8 in each): (1) control group, (2) LPS group (that received 1 mg/kg of LPS two hours before conducting the behavioral tests), and (3 and 4) LPS- Sel 100 and LPS- Sel 200 groups (that were treated with selenium 100 or 200 μg/kg before LPS). Daily administration of selenium started three days before beginning the behavioral tests. Open- field test (OFT), elevated plus maze test (EPM) and forced swimming test (FST) were done. The brains and blood were then collected to examine oxidative stress parameters and determine white blood cell (WBC) count. ResultsLPS increased immobility and decreased active and climbing time in FST. In EPM, LPS decreased the time spent in the open arms but increased the time spent in the closed arms. In OFT, LPS decreased the time spent in the central zone compared to the control group. High-dose selenium improved performances of the rats in behavioral tests. Also, LPS injection increased malondialdehyde (MDA) while decreased thiol, superoxide dismutase (SOD) and catalase (CAT). Selenium protected brain tissues from oxidative damage. The number of total WBCs was significantly increased in the LPS group compared to the control group. Both doses of selenium induced a significant reduction in total WBC count. ConclusionIt was demonstrated that selenium improved behavioral consequences of LPS that were mediated by neuro-inflammatory mediators. These effects were accompanied by improved oxidative status in the brain tissues and reduced WBCs count.

Prior Ablation of the Splanchnic Sympathetic Nerves Increases Plasma Inflammatory Cytokine Levels and Suppresses Bacteremia in Response to Systemically Administered E. coli in Sheep

The splanchnic sympathetic nerves exert a powerful anti‐inflammatory action in response to systemic administration of lipopolysaccharide in rats (Martelli et al. J. Physiol. 592: 1677–1686, 2014). However, it is not known how this endogenous anti‐inflammatory influence of the splanchnic nerve affects the body’s ability to fight infection. The aim of this study was to determine if and how the endogenous reflex activation of the splanchnic anti‐inflammatory pathway affects blood cytokine levels and the disease course of bacteremia after intravenous infusion of live E. coli into a conscious large animal (sheep). One month prior to infection, the greater splanchnic nerve was cut bilaterally (SplancX, n = 8) or sham surgery (Sham, n = 7) performed in anesthetized sheep (isoflurane/O2). After 1 month, these conscious sheep were infused with live E. coli (2.8 × 109 CFU in 30 min) into a jugular vein. Blood samples were obtained at 1.5, 3, 6, 24 and 48 h after bacterial injection. As expected, i.v. infusion of E. coli caused systemic bacteremia with high blood counts of E. coli [3.02 ± 0.64 Log10(CFU/ml + 1), mean ± sem] in 5 of 7 sham sheep. By contrast, no significant bacteremia was observed in any of the 8 SplancX sheep (0.18 ± 0.18 Log10[CFU/ml + 1], p < 0.01, c.f. Sham sheep). Plasma levels of tumour necrosis factor‐α peaked after 1.5 h, and were significantly greater in SplancX vs Sham sheep (2.4 ± 0.6 vs 1.5 ± 0.2 ng/ml, p < 0.05) at this time. Plasma levels of another pro‐inflammatory cytokine, interleukin‐6, peaked at 6h and were higher in SplancX vs Sham sheep (7.4 ± 0.6 vs 2.4 ± 0.6 ng/ml, p < 0.01). Although plasma levels of the anti‐inflammatory cytokine interleukin‐10 increased in both groups, they were lower in SplancX vs Sham sheep from 1.5 h to 24 h (2.9 ± 0.4 vs 6.7 ± 1.1 ng/ml at 24 h, p < 0.01). All plasma cytokine levels measured had returned to baseline by 48 h. Plasma cortisol levels were higher (p<0.01) in SplancX sheep only at 6 h (298 ± 29 vs 138 ± 37 ng/ml), while the fever was less (p < 0.05) in SplancX at 6 h (40.3 ± 0.3 vs 40.8 ± 0.3°C). The greater pro‐inflammatory response in splanchnic denervated animals most likely contributes to their enhanced ability to clear bacteremia. The present findings demonstrate the powerful consequences of neural control of inflammation in the context of an acute systemic infection.Support or Funding InformationSupported by the National Heart Foundation of Australia

Atorvastatin suppresses vascular hypersensitivity and remodeling induced by transient adventitial administration of lipopolysaccharide in rats.

The phenotypic transition of vascular smooth muscle cells (VSMCs) from a contractile to a proliferative state markedly affects the pathophysiology of cardiovascular diseases. The adventitial inflammation can promote neointimal formation and vascular remodeling. We used direct administration of lipopolysaccharide (LPS) into the periphery of the carotid artery to investigate the influence of transient adventitial inflammation on vascular remodeling and its potential mechanism. Male 15-week-old Wistar rats were randomly assigned to four groups with six rats in each group. The rats of groups I and II were administered distilled water, and group III and IV were treated with fasudil and atorvastatin respectively. All treatments were given daily for 11 days. On day 8, the adventitia in group I was injected with 5 µL sterile saline, and the group II-IV were injected with 5 µL sterilized LPS. The carotid blood flow and femoral blood pressure were measured in vivo, and the thickness of vascular intima and middle layer was measured in vitro. Serum interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were determined using enzyme-linked immunosorbent assay (ELISA) assay. And the Rho-associated coiled-coil-containing protein kinase 2 (ROCK2), myosin phosphatase target subunit 1 (MYPT1), myosin light chain (MLC), myocardin, SM-α actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected by western blot. The comparisons were made by one-way analysis of variance with Bonferroni's post hoc test. A value of P<0.05 was considered to represent a statistically significant difference. Transient adventitial inflammation induced by LPS caused no obvious change in basal blood flow, but did lead to vascular hypersensitivity to serotonin. Morphological examinations revealed that the medial layer was the only domain affected, and showed VSMC proliferation and rearrangement. LPS increased serum IL-6 and TNFα contents, ROCK2 expression and activity, and caused changes in the expression levels of some stereotypical VSMC genes. Similar to the Rho-kinase inhibitor fasudil, atorvastatin completely restored the morphological alterations, even increased blood flow. Our study confirms the beneficial effect of atorvastatin on the vascular system in terms of morphology and function.

S76. Omega-3 Fatty Acid Deficiency Augments C-Reactive Protein Biosynthesis in Response to Repeated Lipopolysaccharide Administration in Rats

The pathoetiology of major depressive disorder (MDD) is associated with elevated biomarkers of systemic inflammation, including C-reactive protein (CRP), and lower red blood cell (RBC) omega-3 polyunsaturated fatty acid (n-3 PUFA) levels. The present study investigated the effects of dietary-induced alterations in n-3 PUFA biostatus on basal and stimulated CRP levels in rats.

Evaluation of Olive Leaf Extract Effects on Functional and Structural Changes of Liver Tissue Following Lipopolysaccharide Administration in Rats

Evaluation of Olive Leaf Extract Effects on Functional and Structural Changes of Liver Tissue Following Lipopolysaccharide Administration in Rats

P.1.f.014 - Central and peripheral effects of lipopolysaccharide administration in rats

P.1.f.014 - Central and peripheral effects of lipopolysaccharide administration in rats

LPS-induced renal inflammation is prevented by (−)‐epicatechin in rats

This work investigated the capacity of (−)-epicatechin to prevent the renal damage induced by LPS administration in rats. Male Sprague Dawley rats were fed for 4 days a diet without or with supplementation with (−)-epicatechin (80mg/kg BW/d), and subsequently i.p. injected with lipopolysaccharide (LPS). Six hours after injection, LPS-treated rats exhibited increased plasma creatinine and urea levels as indicators of impaired renal function. The renal cortex of the LPS-treated rats showed: i) increased expression of inflammatory molecules (TNF-α, iNOS and IL-6); ii) activation of several steps of NF-κB pathway; iii) overexpression of TLR4, and iv) higher superoxide anion production and lipid peroxidation index in association with increased levels of gp91phox and p47phox (NOX2) and NOX4. Pretreatment with dietary (−)-epicatechin prevented the adverse effects of LPS challenge essentially by inhibiting TLR4 upregulation and NOX activation and the consequent downstream events, e.g. NF-kB activation.

Systematic Analysis of the Cytokine and Anhedonia Response to Peripheral Lipopolysaccharide Administration in Rats.

Inflammatory processes may cause depression in subsets of vulnerable individuals. Inflammation-associated behavioral changes are commonly modelled in rodents by administration of bacterial lipopolysaccharide (LPS). However, the time frame in which immune activation and depressive-like behavior occur is not very clear. In this study, we showed that systemic administration of LPS robustly increased circulating levels of corticosterone, leptin, pro- and anti-inflammatory cytokines, and chemokines. Serum concentrations of most analytes peaked within the first 6 h after LPS injection and returned to baseline values by 24 h. Chemokine levels, however, remained elevated for up to 96 h. Using an optimized sucrose preference test (SPT) we showed that sickness behavior was present from 2 to 24 h. LPS-induced anhedonia, as measured by decreased sucrose preference, lasted up to 96 h. To mimic the human situation, where depression develops after chronic inflammation, rats were preexposed to repeated LPS administration or subchronic restraint stress and subsequently challenged with LPS. While these procedures did not increase the duration of anhedonia, our results do indicate that inflammation may cause depressive symptoms such as anhedonia. Using our SPT protocol, more elaborate rodent models can be developed to study the mechanisms underlying inflammation-associated depression in humans.

Combined in utero hypoxia-ischemia and lipopolysaccharide administration in rats induces chorioamnionitis and a fetal inflammatory response syndrome

IntroductionPreterm birth is a major cause of infant morbidity and long-term disability, and is associated with numerous central nervous system (CNS) deficits. Infants exposed to intrauterine inflammation, specifically chorioamnionitis, are at risk for very early preterm birth and neurological complications including cerebral palsy, epilepsy, and behavioral and cognitive deficits. However, placenta-brain axis abnormalities and their relationship to subsequent permanent CNS injury remain poorly defined. MethodsIntrauterine injury was induced in rats on embryonic day 18 (E18) by transient systemic hypoxia-ischemia (TSHI) and intra-amniotic lipopolysaccharide (LPS) injection. Placenta, brain and serum were collected from E19 to postnatal day 0 (P0). Histology, TUNEL staining, western blot and multiplex immunoassays were used to quantify placental and brain abnormalities, and fetal serum cytokine levels. ResultsPrenatal TSHI + LPS caused acute and subacute placental injury hallmarked by inflammatory infiltrate, edema, hemorrhage and cell death along with placental increases in IL-1β and TNFα. TSHI + LPS increased a diverse array of circulating inflammatory proteins including IL-1β, TNFα, IL-6, IL-10, IL-4, IFNγ and CXCL1, both immediately after TSHI + LPS and in live born pups. CNS inflammation was characterized by increased CXCL1. DiscussionPrenatal TSHI + LPS in rats induces placental injury and inflammation histologically consistent with chorioamnionitis, concomitant with elevated serum and CNS pro-inflammatory cytokines. This model accurately recapitulates key pathophysiological processes observed in extremely preterm infants including placental, fetal, and CNS inflammation. Further investigation into the mechanism of CNS injury following chorioamnionitis and the placental-brain axis will guide the use of future interventions.

Peripheral bacterial endotoxin administration triggers both memory consolidation and reconsolidation deficits in mice

Peripherally administered inflammatory stimuli, such as lipopolysaccharide (LPS), induce the synthesis and release of proinflammatory cytokines and chemokines in the periphery and the central nervous system, and trigger a variety of neurobiological responses. Indeed, prior reports indicate that peripheral LPS administration in rats disrupts contextual fear memory consolidation processes, potentially due to elevated cytokine expression. We used a similar, but partially olfaction-based, contextual fear conditioning paradigm to examine the effects of LPS on memory consolidation and reconsolidation in mice. Additionally, interleukin-1β (IL-1β), brain-derived neurotrophic factor (BDNF), and zinc finger (Zif)-268 mRNA expression in the hippocampus and the cortex, along with peripheral cytokines and chemokines, were assessed. As hypothesized, LPS administered immediately or 2 h, but not 12 h, post-training impaired memory consolidation processes that support the storage of the conditioned contextual fear memory. Additionally, as hypothesized, LPS administered immediately following the fear memory trace reactivation session impaired memory reconsolidation processes. Four hours post-injection, both central cytokine and peripheral cytokine and chemokine levels were heightened in LPS-treated animals, with a simultaneous decrease in BDNF, but not Zif-268, mRNA. Collectively, these data reinforce prior work showing LPS- and cytokine-related effects on memory consolidation, and extend this work to memory reconsolidation.

Capillary Defects and Exaggerated Inflammatory Response in the Airways of EphA2-Deficient Mice

Both Eph receptors and ephrin ligands have been implicated in blood vessel and neuronal development. Recent studies suggested that EphA2 inhibition reduces tumor angiogenesis, but its role in blood vessel development and inflammation is unclear. We examined these issues using either airways of pathogen-free, EphA2-deficient mice at various ages or EphA2-deficient mice whose airways were inflamed by either Mycoplasma pulmonis infection or ovalbumin sensitization and challenge. EphA2-deficient mice had fewer capillaries, a greater number of endothelial sprouts, and greater capillary diameters than age-matched, wild-type control mice. Moreover, capillaries in EphA2-deficient mice had significantly less pericyte coverage, suggesting abnormal interactions between endothelial cells and pericytes. These differences were apparent in early postnatal life but decreased during progression into adulthood. In inflamed airways, significantly more angiogenesis and lymphangiogenesis, a greater number of infiltrating leukocytes, and higher expression levels of inflammatory cytokine mRNA were present in EphA2-deficient mice after M. pulmonis infection. Additionally, in allergic airway inflammation with ovalbumin sensitization and challenge, a greater number of lymphatic sprouts and infiltrating leukocytes, higher mRNA expression levels of TH2 cytokines and chemokines related to allergic airway inflammation, and enhanced airway hyper-responsiveness were present in EphA2-deficient mice. We conclude that defective pericyte coverage causes capillary defects, abundant endothelial sprouts, and thick capillary diameters in EphA2-deficient mice, indicating that these animals have exaggerated responses to airway inflammation.

Multiple pathogenic factor-induced complications of cirrhosis in rats: A new model of hepatopulmonary syndrome with intestinal endotoxemia

To develop and characterize a practical model of Hepatopulmonary syndrome (HPS) in rats. The experimental animals were randomized into five feeding groups: (1) control (fed standard diet), (2) control plus intraperitoneal injection with lipopolysaccharide (LPS), (3) cirrhosis (fed a diet of maize flour, lard, cholesterol, and alcohol plus subcutaneously injection with carbon tetrachloride (CCl(4)) oil solution), (4) cirrhosis plus LPS, and (5) cirrhosis plus glycine and LPS. The blood, liver and lung tissues of rats were sampled for analysis and characterization. Technetium 99m-labeled macroaggregated albumin (Tc99m-MAA) was used to test the dilatation of pulmonary microvasculature. Typical cirrhosis and subsequent hepato-pulmonary syndrome was observed in the cirrhosis groups after an 8 wk feeding period. In rats with cirrhosis, there were a decreased PaO(2) and PaCO(2) in arterial blood, markedly decreased arterial O(2) content, a significantly increased alveolar to arterial oxygen gradient, an increased number of bacterial translocated within mesenteric lymph node, a significant higher level of LPS and tumor necrosis factor-alpha (TNF-alpha) in plasma, and a significant greater ratio of Tc99m-MAA brain-over-lung radioactivity. After LPS administration in rats with cirrhosis, various pathological parameters got worse and pulmonary edema formed. The predisposition of glycine antagonized the effects of LPS and significantly alleviated various pathological alterations. The results suggest that: (1) a characteristic rat model of HPS can be non-invasively induced by multiple pathogenic factors including high fat diet, alcohol, cholesterol and CCl(4); (2) this model can be used for study of hepatopulmonary syndrome and is clinically relevant; and (3) intestinal endotoxemia (IETM) and its accompanying cytokines, such as TNF-alpha, exert a crucial role in the pathogenesis of HPS in this model.

A new method to detect systemic inflammatory response syndrome by continuous monitoring system: an experimental study in rats

A continuous monitoring system was designed for detection of systemic inflammatory response syndrome (SIRS) using spectrum analyser and local temperature mapping system with deep body thermometer. This method demonstrated significant increases of heart and respiratory rates and deep body temperature from normal levels within 120 minutes after lipopolysaccharide administration in rats. Our monitoring system appears promising for clinical detection of SIRS in the early stages to prevent multiple organ failure.

Subcutaneous and continuous administration of lipopolysaccharide increases serum levels of triglyceride and monocyte chemoattractant protein-1 in rats.

Previous studies have suggested an association between chronic infection and cardiovascular disease, and combined infection/inflammation is frequently related with hypertriglyceridemia, which may be a risk factor of cardiovascular disease. In fact, experimental transient or intermittent administration of lipopolysaccharide (LPS) is known to cause hypertriglyceridemia. We investigated the effects of subcutaneous and continuous administration of LPS in rats, which was considered to mimic chronic infection such as periodontal disease, on the serum levels of lipids [triglyceride (TG), total cholesterol (TC) and free fatty acid (FFA)] and cytokines [tumor necrosis factor-alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1)], all of which are thought to be etiological factors of atherosclerotic cardiovascular disease. Ten fasted Wistar rats were subcutaneously injected with LPS (500 micro g/100 g of body weight) (LPS-injected rats; n = 5) or saline (saline-injected rats; n = 5) as a model of transient infection. Further, mini-osmotic pumps containing the same concentration of LPS (LPS-implanted rats; n = 5) or saline (saline-implanted rats; n = 5) were subcutaneously implanted into an additional 10 rats as a model of chronic infection. After the injection or implantation, time-dependent changes in serum levels of TG, TC, FFA, TNF-alpha, MCP-1 and LPS were determined. LPS was detected in serum until 24 h in LPS-injected rats and until day 5 in LPS-implanted rats. Serum TG levels significantly increased from 6 to 24 h in LPS-injected rats and from day 5 to 14 in LPS-implanted rats. In both LPS-injected and LPS-implanted rats, serum FFA levels increased only slightly, whereas serum TC levels did not appreciably change. Both types of LPS administration increased serum TNF-alpha levels transiently and MCP-1 levels continuously. These findings suggest that chronic infection such as periodontal disease induces hypertriglyceridemia and increases serum MCP-1 levels in a manner that increases the risk of atherosclerotic cardiovascular disease.

Role of nitric oxide in lipopolysaccharide-induced release of vasopressin in rats

This study evaluated the role of nitric oxide (NO) in vasopressin (AVP) release induced by intravenous lipopolysaccharide (LPS) in rats previously treated with intracerebroventricular (i.c.v.) saline, l-NAME, l-arginine or sodium nitroprusside (SNP). In control rats given i.c.v. saline, l-NAME, l-arginine or SNP, AVP levels did not change from baseline. After LPS, plasma AVP increased, reaching a peak at 60 min, and returning to basal levels 4 h later in all i.c.v. pre-treated groups ( P<0.05). The LPS administration in rats previously treated with l-NAME induced higher AVP levels ( P<0.05) that remained elevated throughout the period of the experiment ( P<0.05). These findings confirm the inhibitory role of NO in AVP secretion induced by LPS.

Myeloperoxidase-associated tyrosine nitration after intratracheal administration of lipopolysaccharide in rats.

Previous studies have shown that lipopolysaccharide-induced inflammation in the lung results in tyrosine nitration. The objective of this study was to evaluate the contribution of myeloperoxidase and peroxynitrite pathway to the tyrosine nitration in lipopolysaccharide-administered lungs of rats that were otherwise untreated or leukocyte-depleted by cyclophosphamide or received inhaled nitric oxide (NO). The authors analyzed the immunoreactivity of inducible nitric oxide synthase (iNOS), nitrotyrosine (a product of the myeloperoxidase or peroxynitrite pathway), and chlorotyrosine (a byproduct of the myeloperoxidase pathway) by use of specific antibodies. The number of neutrophils in bronchoalveolar lavage fluid (BALF) and levels of myeloperoxidase activity in lung homogenates were also measured. Lipopolysaccharide enhanced the immunoreactivity of iNOS, nitrotyrosine, and chlorotyrosine in alveolar wall cells, alveolar macrophages, and neutrophils. Leukocyte depletion by cyclophosphamide and inhibition of leukocyte accumulation in the lungs by NO inhalation did not eliminate the increase in iNOS immunoreactivity in alveolar macrophages after lipopolysaccharide treatment, but nitrotyrosine and chlorotyrosine were not produced in these cells. Tyrosine nitration in response to lipopolysaccharide was associated with increases in neutrophil count in BALF and in myeloperoxidase activity in lung homogenates, whereas NO inhalation suppressed the neutrophil count in BALF and reduced tyrosine nitration and chlorination. These findings suggest that myeloperoxidase pathway has a role in tyrosine nitration in the lungs of lipopolysaccharide-treated rats, and that NO inhalation during early phase of inflammation does not increase but rather decreases tyrosine nitration and chlorination, possibly by reducing neutrophil sequestration.

S-Nitrosothiol Formation in Blood of Lipopolysaccharide-Treated Rats

The administration of the gram-negative bacterial cell wall component lipopolysaccharide (LPS) to experimental animals results in the dramatic up-regulation of the inducible form of nitric oxide synthase (iNOS). The resulting sustained overproduction of nitric oxide (NO) is thought to contribute to the septic shock-like state in these animals. Numerous studies have characterized the kinetics and magnitude of expression of iNOS as well as the production of NO-derived nitrite and nitrate. However, little is known regarding the ability of iNOS-derived NO to interact with physiological substrates such as thiols to yield biologically active S-nitrosothiols during endotoxemia. It has been hypothesized that these relatively stable, vaso-active compounds may serve as a storage system for NO and they may thus play an important role in the pathophysiology associated with endotoxemia. In the present study, we demonstrate that 5 h after i.p. administration of LPS in rats, circulating S-nitrosoalbumin was increased by ∼3.4-fold over control. S-nitrosohemoglobin was increased by ∼25-fold over controls and by threefold over S-nitrosoalbumin. No increase in low molecular weight S-nitrosothiols (i.e., S-nitrosoglutathione and S-nitrosocysteine) could be detected under our experimental conditions. Taken together these data demonstrate that endotoxemia dramatically enhances circulating S-nitrosothiol formation.

Dissociation of temperature changes and anorexia after experimental colitis and LPS administration in rats.

The acute phase of inflammation induces both anorexia and fever. Because several analyses suggest a linkage between the meal size and body temperature, we assessed whether temperature changes were causal to anorexia in situations involving acute inflammation. Specifically, we evaluated whether elevations of body temperature could account for the reduced food intake after induction of experimental colitis [via intrarectal infusions of trinitrobenzene sulfonic acid (TNB)] or injection of 100 micrograms/kg lipopolysaccharide (LPS). Temperature was monitored telemetrically in rats via implanted temperature transmitters. TNB-treated rats demonstrated a 5-day anorexia that resulted specifically from a decrease in meal size. Although TNB-treated rats were hypothermic on the day of treatment, no other body temperature changes were noted. LPS reduced food intake and elevated temperature, but these two effects were uncorrelated temporally. Although these results do not identify the mechanisms of anorexia, the findings indicate clearly that the anorexia associated with the acute inflammatory response is not secondary to fever.

Selective depletion of macrophages prevents pituitary-adrenal activation in response to subpyrogenic, but not to pyrogenic, doses of bacterial endotoxin in rats.

The mechanisms by which bacterial endotoxin [lipopolysaccharide (LPS)] stimulates the hypothalamo-pituitary-adrenal axis (HPAA) have not been elucidated. The present study was designed to investigate the involvement of macrophages in plasma ACTH and corticosterone responses to LPS administration in rats using selective in vivo macrophage depletion. Intraperitoneal administration of subpyrogenic doses of LPS to normal rats resulted in elevated plasma ACTH and corticosterone concentrations, measured 2 h later. The response showed a remarkable steep dose relationship, with minimal effective doses between 0.5-1.5 micrograms (ACTH) and 0.5 micrograms or less (corticosterone)/kg BW. Plasma PRL, LH, and catecholamine (norepinephrine, epinephrine) levels were not significantly changed under the conditions used. Only at 6 h after LPS administration was a small elevation of norepinephrine noted. To deplete macrophages, rats were injected with liposomes encapsulated with dichloromethylene diphosphonate (Cl2MDP). Histochemical (acid phosphatase) and immunocytochemical techniques (monoclonal antibodies to rat macrophages coded ED1 and ED3) were applied to examine the efficiency of macrophage elimination by the Cl2MDP liposomes in cytospins of peritoneal exudates and in sections of the liver and spleen. Since cells of the macrophage lineage are considered to be the main source of IL-1 in the circulation, we also measured circulating levels of immunoreactive interleukin-1 beta (IL-1) concentrations in control and Cl2MDP liposome-treated rats by the use of a newly developed RIA. Reduced numbers of macrophages were seen in peritoneal lavages of Cl2MDP liposome-treated animals, whereas the morphological appearance and numbers of mast cells, granulocytes, and T-cells were unaffected. Similarly, macrophages were effectively eliminated in the spleen, mesenteric lymph nodes, and liver, as inferred from the reduction of macrophage staining in these organs. Plasma IL-1 concentrations could only be detected in response to a pyrogenic (2.5 mg/kg, iv) and not to a subpyrogenic (0.025 mg/kg, ip) dose of LPS. The increase in plasma IL-1 concentrations in response to the pyrogenic dose of LPS, reaching levels of 20-40 ng/ml in control rats, was blunted in animals treated with the Cl2MDP liposomes. Macrophage depletion by Cl2MDP liposomes did not affect either resting plasma corticosterone levels or the corticosterone response to ether exposure. At subpyrogenic doses of LPS, plasma ACTH and corticosterone responses were completely prevented by macrophage depletion. In contrast, at pyrogenic doses of LPS, plasma ACTH and corticosterone responses were not significantly affected by depleting macrophages. These data demonstrate that activation of the HPAA by a subpyrogenic dose of LPS is macrophage dependent. However, macrophage-independent mechanisms mediate activation of the HPAA in response to a pyrogenic dose of LPS.