Why sickness hurts: A central mechanism for pain induced by peripheral inflammation.

Low-grade systemic inflammation has been implicated in chronic pain, as well as in comorbid diseases like depression and fatigue. We have previously shown that women's pain perception and regulation is more affected by systemic inflammation than that of men. Here we investigated the neural substrates underlying these effects using an fMRI paradigm previously employed in a clinical population. Fifty-one participants (29 women) were injected with 0.6ng/kg lipopolysaccharide (LPS) or saline to induce a peripheral inflammatory response. The subjects were then tested with a pressure pain fMRI paradigm designed to capture descending pain inhibitory activity 2h after injection, and blood was sampled for cytokine analysis. The subjects injected with LPS became more pain sensitive compared to the placebo group, and the heightened pain sensitivity was paralleled by decreased activity in the ventrolateral prefrontal cortex and the rostral anterior cingulate cortex (rACC) compared to placebo; areas involved in descending pain regulation. The LPS group also had higher activity in the anterior insular cortex, an area underpinning affective and interoceptive pain processing. Women displayed overall less pain-evoked rACC activity compared to men, which may have rendered women less resilient to immune provocation, possibly explaining sex differences in LPS-induced pain sensitivity. Our findings elucidate the pain-related brain circuits affected by experimental peripheral inflammation, strengthening the theoretical link between systemic inflammation and weakened pain regulation in chronic pain disorders. The results further suggest a possible mechanism underlying the female predominance in many chronic pain disorders.

Modality and sex differences in pain sensitivity during human endotoxemia.

Systemic inflammation can induce pain hypersensitivity in animal and human experimental models, and has been proposed to be central in clinical pain conditions. Women are overrepresented in many chronic pain conditions, but experimental studies on sex differences in pain regulation during systemic inflammation are still scarce. In two randomized and double blind placebo controlled experiments, we used low doses of lipopolysaccharide (LPS) as an experimental model of systemic inflammation. The first study employed 0.8ng/kg LPS in a within-subject design of 8 individuals (1 woman), and the second study 0.6ng/kg LPS in a between-subject design of 52 participants (29 women). We investigated the effect on (a) pressure, heat, and cold pain thresholds, (b) suprathreshold noxious heat and cold sensitivity, and (c) conditioned pain modulation (CPM), and differences between men and women. LPS induced significantly lower pressure pain thresholds as compared to placebo (mean change with the 0.8ng/kg dose being -64±30kPa P=.04; with the 0.6ng/kg dose -58±55kPa, P<.01, compared to before injection), whereas heat and cold pain thresholds remained unaffected (P's>.70). Suprathreshold noxious pain was not affected by LPS in men (P's⩾.15). However, LPS made women rated suprathreshold noxious heat stimuli as more painful (P=.01), and showed a tendency to rate noxious cold pain as more painful (P=.06) as compared to placebo. Furthermore, LPS impaired conditioned pain modulation, a measure of endogenous pain inhibition, but this effect was also restricted to women (P<.01, for men P=.27). Pain sensitivity correlated positively with plasma IL-6 and IL-8 levels. The results show that inflammation more strongly affects deep pain, rather than cutaneous pain, and suggest that women's pain perception and modulation is more sensitive to immune activation than men's.

Effect of lipopolysaccharide administration on the number, phenotype and content of nuclear molecules in blood microparticles of normal human subjects.

Microparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells and promote inflammation and thrombosis. To characterize the in vivo release of MPs, we used flow cytometry to measure MPs in the blood of 15 healthy volunteers administered bacterial endotoxin (lipopolysaccharide or LPS) in the presence of a low dose of hydrocortisone with or without inhaled nitric oxide. MPs, defined as particles less than 1.0 µm in size, were assessed following labelling for CD42a, CD14 and CD62E or CD144 antibodies to identify MPs from platelets (PMPs), monocytes (MMPs) and endothelial cells (EMPs). In addition, PMPs and MMPs were labelled with anti-HMGB1 and stained with SYTO13 to assess nuclear acid content. Administration of LPS led to an increase in the numbers of PMPs, MMPs and EMPs as defined by CD62E, as well as the number of MMPs and PMPs staining with anti-HMGB1 and SYTO13. Inhalation of NO did not influence these findings. Together, these studies show that LPS can increase levels of blood MPs and influence phenotype, including nuclear content. As such, particles may be a source of HMGB1 and other nuclear molecules in the blood during inflammation.

Immunomodulation by a combination of nitric oxide and glucocorticoids in a human endotoxin model.

BACKGROUND: inflammatory reactions arise in reaction to a variety of pathogenic insults. The combination of inhaled nitric oxide (iNO) and glucocorticoids (GC) may attenuate endotoxin-induced inflammatory responses. It has been shown that the combination of iNO (30 p.p.m.) and steroids blunted the inflammatory response in a porcine endotoxin model, but not in humans. Therefore, we investigated whether a clinically 'maximal' dose of iNO in combination with GC could modulate the systemic inflammatory response in a human endotoxin model. METHODS: a double-blind, cross-over, placebo-controlled randomized study including 15 healthy Caucasian volunteers (five females, 10 males). Performed at the Intensive Care Unit in a university hospital. iNO 80 p.p.m. or placebo (nitrogen) was started 2h before administration of endotoxin (2 ng/kg). Thirty minutes later, GC (2mg/kg, hydrocortisone) was administered intravenously. Blood samples and clinical signs were collected before and up to 24 h after the endotoxin injection. RESULTS: body temperature and heart rate increased significantly subsequent to endotoxin challenge. The plasma levels of IFN-γ, IL-1ß, IL-2, 4 5, 6, 8, 10, 12, 13 and TNFα were markedly elevated. However, HMGB-1 and sRAGE were unaffected. No difference between placebo/GC and iNO/GC treatment was observed in the clinical or cytokine response, neither was there any difference between the first and the second exposure to endotoxin. CONCLUSIONS: pre-treatment with iNO 80 p.p.m. along with GC (2mg/kg) administrated after the endotoxin challenge could not modulate the systemic inflammatory response in this model of human experimental inflammation.

Adenosine infusion attenuates soluble RAGE in endotoxin-induced inflammation in human volunteers.

AIM: To evaluate possible anti-inflammatory effects of pre-treatment with adenosine in a human experimental inflammatory model. METHODS: The study design was double-blind, crossover, placebo-controlled and randomized. In the Intensive Care Unit of a university hospital, 16 healthy male volunteers were treated for 5.5 h with infusions of adenosine 40 microg kg(-1) min(-1) or placebo. Thirty minutes after the start of adenosine or placebo, 2 ng kg(-1)E-Coli endotoxin was administered. Heart rate, body temperature, blood pressure, plasma cytokines (TNF-alpha, IL-6 and IL-10), soluble RAGE and resistin, exhaled nitric oxide and nitrite/nitrate in urine were determined. RESULTS: Endotoxin elicited the expected clinical signs of an inflammatory reaction (tachycardia, fever) and led to prominent release of the cytokines studied (P < 0.001). Resistin in plasma increased after endotoxin (P < 0.001). After placebo treatment, soluble RAGE (sRAGE) in plasma increased 5 h after the endotoxin challenge (P < 0.001) but not after adenosine. After placebo, orally exhaled NO increased with a peak at 4 h (P < 0.001), although there was no statistically significant difference between the two treatments. Nitrite/nitrate in urine (n = 11) did not differ between adenosine and placebo treatments. CONCLUSION: In conclusion, adenosine infusion starting before endotoxin challenge in humans attenuated sRAGE significantly but otherwise had no clear anti-inflammatory effect. Adenosine as a potential anti-inflammatory treatment in humans needs further study, including use of higher doses. The mechanism underlying the effect of adenosines on sRAGE remains unknown.

Complement activation, endothelin-1 and neuropeptide Y in relation to the cardiovascular response to endotoxin-induced systemic inflammation in healthy volunteers.

BACKGROUND: Endotoxin is a major stimulus for triggering the host response in septicaemia. The pathophysiology of sepsis involves activation of the vascular endothelium and leukocytes, resulting in the release of various mediators, e.g. cytokines, nitric oxide (NO), endothelin (ET-1) and complement factors. We evaluated the blood levels of complement activation, ET-1 and neuropeptide Y (NPY) in parallel with the haemodynamic and oxygen transport response during human experimental endotoxemia. METHODS: Eleven healthy men had venous, arterial and pulmonary arterial catheters placed for continuous haemodynamic measuring. After 30 min rest endotoxin (E. Coli 4 ng kg(-1), Lot G1) was intravenously administered. Blood samples from pulmonary and arterial catheters were collected hourly over 4 h. RESULTS: Body temperature augmented significantly from baseline values (36.7 +/- 0.7 degrees C, mean +/- SEM) with a maximum after 3.5 h (39.1 +/- 0.3 degrees C, P < 0.001). Cardiac output increased by 100%, systemic vascular resistance decreased by 50%, the oxygen consumption and the tissue oxygen transport increased. Activation of the complement system was indicated by an increase in SC5b-9. Endothelin-1-like immunoreactivity (ET-1-LI) increased over time in arterial blood. NPY-like immunoreactivity (NPY-LI) did not change over time. CONCLUSION: A dose of endotoxin associated with reproducible systemic vasodilation and fever in healthy subjects causes complement activation and increased systemic levels of ET-1-LI, illustrating that the model is a useful tool for inducing moderate systemic inflammation where several mediator systems are activated.

Nicotinamide does not influence cytokines or exhaled NO in human experimental endotoxaemia.

This study examined the hypothesis that nicotinamide could attenuate endotoxin-induced inflammatory responses in humans as indicated by levels of cytokines and nitric oxide. Ten healthy male volunteers participated in a randomised, double-blind, cross-over design with regard to the effects of nicotinamide. The volunteers received orally 4 g nicotinamide or placebo at 14 h and at 2 h preceding the experiment (total dose of 8 g). Endotoxin (E. coli, 2 ng/kg), was administered intravenously. Blood samples and haemodynamic data were collected prior to and up to 6 h after the endotoxin infusion. Orally exhaled NO was measured hourly. Following endotoxin, body temperature increased from baseline 36.3 +/- 0.09 degrees C to a maximum of 38.0 +/- 0.1 degrees C for all (mean +/- SEM, P < 0.001) and heart rate increased from 59 +/- 1.9 to 87.0 +/- 2.6 beats/min after 3 h (mean +/- SEM, P < 0.001). Endotoxin challenge also markedly elevated the TNF-alpha, IL-6, IL-8 and IL-10 concentrations (P < 0.001 versus baseline for all) during the study period. Orally exhaled NO also increased (P < 0.01) compared to baseline. Nicotinamide treatment did not influence the patterns of cytokine and NO response to endotoxin. In conclusion, there was no effect on the inflammatory parameters by oral nicotinamide at a dose of 8 g, limiting the potential use of this agent for anti-inflammatory purpose in man.

Exhaled NO and plasma cGMP increase after endotoxin infusion in healthy volunteers.

Nitric oxide (NO) is believed to be involved in the pathophysiology of sepsis. This study evaluated the activity of the NO pathway in a human endotoxin model. At baseline and after endotoxin, on-line measurements of exhaled NO (eNO) were made using a chemiluminescence technique with a single-breath method. NO-free air was inhaled prior to exhalation against a resistance. NO in orally and nasally exhaled air and in rectal gas was investigated. Plasma nitrite, nitrate, and guanosine 3', 5'-monophosphate (cGMP) and the events after diclophenac administration were also studied. Endotoxin infusion resulted in tachycardia and fever. An early increase in oral eNO concentration was observed and oral eNO decreased after diclophenac administration. NO exhaled nasally, NO in rectum gas and nitrite/nitrate levels remained unchanged over the study period, cGMP increased after 4 h. These findings suggest an early increase in nitric oxide production from the lungs, probably due to increased activity of the constitutive nitric oxide synthase upon endotoxin stimulation. In contrast, nitric oxide production in the upper airways, measured as nasally exhaled nitric oxide and nitric oxide in rectal gas, remained unchanged. Further studies will elucidate if exhaled nitric oxide is a valuable marker of sepsis-induced lung injury and if monitoring of treatment is possible.

Differential release of matrix metalloproteinase-9 and nitric oxide following infusion of endotoxin to human volunteers.

BACKGROUND: Roughly 400.000 cases of sepsis occur every year in the United States only and this is associated with a very high mortality. Bacterial lipopolysaccharide (LPS) triggers systemic inflammatory reactions in sepsis. However, down-stream cellular cascade initiated by LPS is still being elucidated. Nitric oxide (NO) and matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) are known to be induced by LPS. We have investigated the release of NO, MMP-2 and MMP-9 following infusion of LPS to volunteers. METHODS: IPS (2 ng kg-1) was infused to 10 healthy volunteers. Before the experiments were started the subjects had an intravenous catheters placed. An electrocardiogram was also placed and monitored constantly. Body temperature was measured by ear thermometer every 10 min Venous blood was collected and cell-free plasma assayed for the presence of MMP-2 and MMP-9 using zymography and NO using HPLC assay for NO metabolites, nitrite and nitrate. RESULTS: The administration of LPS resulted in increased body temperature and tachycardia. Time-dependent release of MMP-9(30 fold increase from the baseline) peaking at 2 h following infusion of LPS was observed. LPS did not significantly modify the activity of MMP-2 (P > 0.05). Infusion of LPS did not significantly change the levels of nitrite and nitrate (from 60 +/- 11 to 67 +/- 10 micro m, P > 0.05). CONCLUSION: The release of MMP-9, but not MMP-2 or NO, is a sensitive index of endotoxaemia in humans. MMP-9 release may contribute to the pathogenesis of sepsis via its pro-inflammatory effects on the vasculature.

Multi-cellular activation in vivo by endotoxin in humans--limited protection by adenosine infusion.

The influence of adenosine infusion (40 microg/kg/min for 4 h) on inflammatory and hemostatic parameters was investigated in healthy males without (n = 10) or with (n = 11) intravenous endotoxin injection (4 ng/kg). Without endotoxin, adenosine elevated circulating leukocytes and circulating platelet-leukocyte aggregates. Endotoxin activated platelets and leukocytes in vivo. Platelet activation was seen as slightly increased platelet P-selectin expression, decreased platelet counts, and elevated plasma soluble P-selectin (from 39.6 +/- 3.4 to 68.9 +/- 6.6 ng/ml, P<0.01). Leukocyte activation was evidenced by increased CD1 lb expression (from MFI of 0.54 +/- 0.02 to 2.21 +/- 0.17; P<0.01) and plasma elastase levels (from 25.3 +/- 2.5 to 169.3 +/- 22.5 ng/ml: P <0.01). Endotoxin also enhanced platelet and leukocyte responsiveness to in vitro stimulation. Endotoxin induced von Willebrand factor secretion (from 92 +/- 8 units to 265 +/- 19 units at 4 h; P <0.001) and enhanced thrombin generation in vivo. Endotoxin induced leukocytosis and thus increased circulating platelet-leukocyte, mainly platelet-neutrophil, aggregates. Adenosine caused slight attenuation of platelet reactivity to agonist stimulation, enhanced the endotoxin-induced leukocytosis, and detained more platelet-leukocyte aggregates in circulation, but did not attenuate endotoxin-induced neutrophil elastase secretion, von Willebrand factor secretion, or thrombin generation. Thus, endotoxemia induces multi-cellular activation in vivo. Adenosine inhibits leukocyte adhesion and extravasation, and mildly attenuates platelet responsiveness and soluble P-selectin release. Adenosine has the potential of becoming a therapeutic antiinflammatory drug, but an optimal treatment strategy needs to be developed.

Local vs. systemic immune and haemostatic response to hip arthroplasty.

Local and systemic immune and haemostatic responses were studied in 10 patients, aged 57-78 years, undergoing elective hip arthroplasty. Cytokines, soluble cytokine receptors, interleukin-1 receptor antagonist, soluble adhesion molecules, antithrombin, fibrin, soluble and fibrin D-dimer were analysed in wound drainage blood and in blood taken from the systemic circulation for up to 24 h post-operatively. Wound drainage blood concentrations of cytokines, interleukin-1 receptor antagonist and soluble cytokine receptors were increased compared with those in the systemic circulation except for the soluble interleukin-6 receptor. In wound drainage blood, soluble tumour necrosis factor receptors (P < 0.05), interleukin-1 receptor antagonist (P < 0.05) and interleukin-6 (P < 0.05-< 0.01) increased during the study period. In blood from the systemic circulation interleukin-6 increased (P < 0.05) while the soluble interleukin-6 receptor decreased (P < 0.05) compared with pre-operative values. Concentrations of soluble adhesion molecules did not change. Wound drainage blood showed marked hypercoagulation. After hip arthroplasty pro-inflammatory cytokines and their inhibitors were mainly confined to the local trauma site. A predominance for inhibitors was noted.

Local wound and systemic coagulation/fibrinolysis responses in hip arthroplasty. Influence of allogeneic and autologous blood transfusion.

22 patients undergoing elective hip arthroplasty were studied. In 12 patients, a closed-loop autotransfusion system, without anticoagulant, was used and 10 had an ordinary wound drainage allowing repeated blood sampling from the wound. Plasma concentrations of antithrombin (AT), fibrin, soluble (SF) and fibrin D-dimer were determined preoperatively, 3, 8, and 24 hours after starting surgery. Wound drainage blood had increased concentrations of SF and fibrin D-dimer and decreased concentrations of AT compared to reference values and systemic concentrations in patients. Plasma concentrations of SF, fibrin D-dimer and AT did not differ between patients receiving retrieved blood and those receiving stored red blood cell concentrates (RBCs). Patients receiving blood transfusions had lower AT concentrations at 8 hours after starting surgery than those not receiving such a transfusion.

Spontaneous lymphocyte proliferation during trauma and infection.

Spontaneous lymphocyte proliferation (SpP), measured in vitro as the rate of [14C]thymidine incorporation in blood lymphocytes, was investigated in non-infected postoperative patients, infected postoperative patients, and healthy volunteers, with 72, 24, and 3 h of lymphocyte culture. With 24-h cultures, infected postoperative patients showed 17-fold higher SpP than non-infected postoperative patients (2527 +/- 1552 versus 151 +/- 77 cpm, mean +/- SD, P less than 0.001) and 37-fold higher SpP than healthy volunteers (P less than 0.001). Postoperative patients without infection had twice as high SpP as healthy volunteers (P less than 0.001). Lymphocytes harvested after 24 h of cell culture showed significantly higher SpP than corresponding values at 72 and 3 h, in patients as well as in healthy volunteers (P less than 0.01). Infected postoperative patients showed a higher SpP than non-infected patients after only 3 h of cell culture (270 +/- 192 versus 48 +/- 10 cpm, P less than 0.001). An inverse correlation was observed between the level of SpP and body temperature in patients with postoperative infection (r = -0.62, P less than 0.05). The results indicate that lymphocytes are activated by uncomplicated surgery and particularly by postoperative infection, and that characteristics of SpP are reproducible in short cell-culture periods, which suggests that in vitro measurements of SpP may be of value in the detection of severe postoperative infection.