Robert Frithiof

Neutrophil primary granule proteins HBP and HNP1–3 boost bacterial phagocytosis by human and murine macrophages

In acute inflammation, infiltrating polymorphonuclear leukocytes (also known as PMNs) release preformed granule proteins having multitudinous effects on the surrounding environment. Here we present what we believe to be a novel role for PMN-derived proteins in bacterial phagocytosis by both human and murine macrophages. Exposure of macrophages to PMN secretion markedly enhanced phagocytosis of IgG-opsonized Staphylococcus aureus both in vitro and in murine models in vivo. PMN secretion activated macrophages, resulting in upregulation of the Fcgamma receptors CD32 and CD64, which then mediated the enhanced phagocytosis of IgG-opsonized bacteria. The phagocytosis-stimulating activity within the PMN secretion was found to be due to proteins released from PMN primary granules; thorough investigation revealed heparin-binding protein (HBP) and human neutrophil peptides 1-3 (HNP1-3) as the mediators of the macrophage response to PMN secretion. The use of blocking antibodies and knockout mice revealed that HBP acts via beta2 integrins, but the receptor for HNP1-3 remained unclear. Mechanistically, HBP and HNP1-3 triggered macrophage release of TNF-alpha and IFN-gamma, which acted in an autocrine loop to enhance expression of CD32 and CD64 and thereby enhance phagocytosis. Thus, we attribute what may be a novel role for PMN granule proteins in regulating the immune response to bacterial infections.

Neutrophil degranulation mediates severe lung damage triggered by streptococcal M1 protein

Streptococcus pyogenes of the M1 serotype can cause streptococcal toxic shock syndrome commonly associated with acute lung injury. The aim of the present study was to investigate the role of neutrophils and their secretion products in M1 protein-induced lung damage. The degranulation of neutrophils by M1 protein was studied in whole blood using marker analysis for individual granule subsets. In mice, M1 protein was injected intravenously and the lung damage was assessed by histology, electron microscopy, cell count in bronchoalveolar lavage fluid and analysis of lung vascular permeability. Comparisons were made in mice with intact white blood count, neutropenic mice and neutropenic mice injected with the secretion of activated neutrophils. In whole blood, M1 protein forms complexes with fibrinogen that bind to β2-integrins on the neutrophil surface, resulting in degranulation of all four subsets of neutrophil granules. Intravenous injection of M1 protein into mice induced neutrophil accumulation in the lung, increase in vascular permeability and acute lung damage. Depletion of neutrophils from the circulation completely abrogated lung injury and vascular leakage. Interestingly, the lung damage was restored by injecting neutrophil secretion. The present data suggest that neutrophil granule proteins are directly responsible for lung damage induced by the streptococcal M1 protein.

Septic shock induces distinct changes in sympathetic nerve activity to the heart and kidney in conscious sheep

Sepsis and septic shock are the chief cause of death in intensive care units, with mortality rates between 30 and 70%. In a large animal model of septic shock, we have demonstrated hypotension, increased cardiac output, and tachycardia, together with renal vasodilatation and renal failure. The changes in cardiac sympathetic nerve activity (CSNA) that may contribute to the tachycardia have not been investigated, and the changes in renal SNA (RSNA) that may mediate the changes in renal blood flow and function are unclear. We therefore recorded CSNA and RSNA during septic shock in conscious sheep. Septic shock was induced by administration of Escherichia coli, which caused a delayed hypotension and an immediate, biphasic increase in heart rate (HR) associated with similar changes in CSNA. After E. coli, RSNA decreased for over 3 h, followed by a sustained increase (180%), whereas renal blood flow progressively increased and remained elevated. There was an initial diuresis, followed by oliguria and decreased creatinine clearance. There were differential changes in the range of the arterial baroreflex curves; it was depressed for HR, increased for CSNA, and unchanged for RSNA. Our findings, recording CSNA for the first time in septic shock, suggest that the increase in SNA to the heart is not driven solely by unloading of baroreceptors and that the increase has an important role to increase HR and cardiac output. There was little correlation between the changes in RSNA and renal blood flow, suggesting that the renal vasodilatation was mediated mainly by other mechanisms.

Discharge properties of cardiac and renal sympathetic nerves and their impaired responses to changes in blood volume in heart failure

Sympathetic nerve activity (SNA) consists of discharges that vary in amplitude and frequency, reflecting the level of recruitment of nerve fibers and the rhythmic generation and entrainment of activity by the central nervous system. It is unknown whether selective changes in these amplitude and frequency components account for organ-specific changes in SNA in response to alterations in blood volume or for the impaired SNA responses to volume changes in heart failure (HF). To address these questions, we measured cardiac SNA (CSNA) and renal SNA (RSNA) simultaneously in conscious, normal sheep and sheep in HF induced by rapid ventricular pacing. Volume expansion decreased CSNA (-62 +/- 10%, P < 0.05) and RSNA (-59 +/- 10%, P < 0.05) equally (n = 6). CSNA decreased as a result of a reduction in burst frequency, whereas RSNA fell because of falls in burst frequency and amplitude. Hemorrhage increased CSNA (+74 +/- 9%, P < 0.05) more than RSNA (+21 +/- 5%, P < 0.09), in both cases because of increased burst frequency, whereas burst amplitude decreased. In HF, burst frequency of CSNA (from 26 +/- 3 to 75 +/- 3 bursts/min) increased more than that of RSNA (from 63 +/- 4 to 79 +/- 4 bursts/min). In HF, volume expansion caused no change in CSNA and an attenuated decrease in RSNA, due entirely to decreased burst amplitude. Hemorrhage did not significantly increase SNA in either nerve in HF. These findings support the concept that the number of sympathetic fibers recruited and their firing frequency are controlled independently. Furthermore, afferent stimuli, such as changes in blood volume, cause organ-specific responses in each of these components, which are also selectively altered in HF.

Toll-Like Receptor 4 Inhibitor TAK-242 Attenuates Acute Kidney Injury in Endotoxemic Sheep

BACKGROUNDnThis study was conducted to investigate the role of toll-like receptor 4 (TLR4) in mediating acute kidney injury in endotoxemic sheep using the selective TLR4 inhibitor TAK-242.nnnMETHODSnA randomized, controlled, experimental study was performed with 20 adult Texel crossbred sheep. Before an Escherichia coli lipopolysaccharide infusion (3 μg · kg(-1) · h(-1) for 24 h), sheep were randomized to receive a bolus dose (2 mg/kg), followed by a continuous infusion (4 mg · kg(-1) · 24 h(-1)) of either TAK-242 (n = 7) or vehicle (n = 7). A third group of lipopolysaccharide-treated sheep (n = 6) received norepinephrine, titrated to maintain baseline arterial blood pressure.nnnRESULTSnEndotoxin infusion established a state of hyperdynamic circulation, with an increased cardiac index, hypotension, and tachycardia. Urine output and creatinine clearance decreased throughout the experiment, together with increasing plasma creatinine, blood urea nitrogen, and arterial lactate concentrations. After 24 h, TLR4 inhibition had significantly (P ≤ 0.001) attenuated the mean ± SEM decrease in arterial pressure (97 ± 3 vs. 71 ± 4 mmHg), urine output (1.16 ± 0.15 vs. 0.13 ± 0.05 ml · kg(-1) · h(-1)), and creatinine clearance (126 ± 13 vs. 20 ± 7 ml/min) compared with vehicle-treated animals. Furthermore, arterial lactate, plasma creatinine, and blood urea nitrogen concentrations were significantly lower in the TAK-242 group versus the vehicle-treated animals. Compared with TLR4 inhibition, norepinephrine caused similar effects on arterial pressure, cardiac index, and heart rate; however, it did not attenuate the decrease in urine output or creatinine clearance.nnnCONCLUSIONSnThese results indicate a critical role for TLR4 in impairing renal function during ovine endotoxemia that is independent of changes in central hemodynamics.

Comparison between the effects on hemodynamic responses of central and peripheral infusions of hypertonic NaCl during hemorrhage in conscious and isoflurane-anesthetized sheep.

ABSTRACT The i.v. infusion of hypertonic NaCl solutions, as in small volume hypertonic NaCl resuscitation, improves cardiovascular function in hypovolemic shock. The mechanism(s) of action of this treatment is(are) not fully elucidated. In this study, we investigate the possible importance of fully functional neurocardiovascular regulation for the effect of intracerebroventricular (i.c.v.) and i.v. administration of hypertonic NaCl on the hemodynamic responses to hemorrhage. Six groups (each n = 6) of adult ewes were subjected to hypotensive hemorrhage during treatment with i.c.v. infusion (20&mgr;L/min) of either artificial cerebrospinal fluid (controls) or 0.5 mol/L NaCl, or i.v. infusion of 1.2 mol/L NaCl (4 mL/kg) when conscious, respectively anesthetized with isoflurane. Thirty minutes into infusion, treatment blood was withdrawn at 0.7 mL/kg per minute from a jugular vein until the mean arterial pressure dropped to a value just below 50 mmHg. In conscious animals, the amount of blood loss needed to lower the mean arterial pressure to less than 50 mmHg was increased by the i.c.v. and i.v. infusions of hypertonic NaCl (24.0 ± 4.6 and 22.4 ± 3.3 mL/kg, respectively), compared with controls receiving i.c.v. infusion of artificial cerebrospinal fluid (14.2 ± 1.4 mL/kg). Isoflurane anesthesia, as such, severely compromised the cardiovascular compensatory mechanisms activated by hemorrhage and reduced the blood loss necessary to cause hypotension (10.2 ± 2.5 mL/kg). Furthermore, anesthesia totally abolished the effect of i.c.v. hypertonic NaCl (10.4 ± 2.2 mL/kg) and blunted the response to i.v. hypertonic NaCl (15.9 ± 2.1 mL/kg) seen in conscious animals. The results show that an intact autonomic cardiovascular control is crucial for the effect of i.c.v. hypertonic saline and indicate that i.v. hypertonic saline exerts some of its action through the central nervous system.

Gut microcirculatory and mitochondrial effects of hyperdynamic endotoxaemic shock and norepinephrine treatment

BACKGROUNDnMicrocirculatory and mitochondrial dysfunction are important factors in the development of septic shock. In this study, we investigated the effects of fluid resuscitated endotoxaemic shock and norepinephrine treatment on intestinal microcirculation and mitochondrial function in sheep.nnnMETHODSnEight anaesthetized sheep received an i.v. infusion of endotoxin. After 24 h, mean arterial pressure (MAP) was restored to baseline levels with a norepinephrine infusion. Five sheep served as sham experiments. Central and regional haemodynamics were monitored, and ileal microcirculation was evaluated with laser Doppler and sidestream dark-field videomicroscopy techniques. Gut mucosal acidosis was assessed by air tonometry, and ileal wall biopsies were analysed for mitochondrial activity.nnnRESULTSnAfter 24 h of endotoxaemia, the animals had developed hyperdynamic shock with systemic and mucosal acidosis. Although superior mesenteric artery (SMA) flow was higher than the baseline values, ileal microcirculatory perfusion and mitochondrial complex I activity decreased. After norepinephrine was started, SMA flow, ileal microcirculation, and mucosal acidosis remained unchanged. Although no statistically significant difference could be demonstrated, norepinephrine increased mitochondrial complex I activity in five of the six animals from which ileal biopsies were taken.nnnCONCLUSIONSnAlthough fluid resuscitated endotoxaemic shock increased regional blood flow, microcirculatory and mitochondrial alterations were still present. Restoring MAP with norepinephrine did not affect ileal microcirculation or mucosal acidosis, indicating that perfusion pressure manipulation is of limited importance to the intestinal microcirculation in established endotoxaemic shock.

Intravenous hypertonic NaCl acts via cerebral sodium‐sensitive and angiotensinergic mechanisms to improve cardiac function in haemorrhaged conscious sheep

Acute NaCl loading as resuscitation in haemorrhagic hypovolaemia is known to induce rapid cardiovascular recovery. Besides an osmotically induced increase in plasma volume the physiological mechanisms of action are unknown. We hypothesized that a CNS mechanism, elicited by increased periventricular [Na+] and mediated by angiotensin II type 1 receptors (AT1), is obligatory for the full effect of hypertonic NaCl. To test this we investigated the cardiovascular responses to haemorrhage and subsequent hypertonic NaCl infusion (7.5% NaCl, 4 ml (kg BW)−1) in six conscious sheep subjected to intracerebroventricular (i.c.v.) infusion of artificial cerebrospinal fluid (aCSF; control), mannitol solution (Man; 75 mmol l−1[Na+], total osmolality 295 mosmol kg−1) or losartan (Los; 1 mg ml−1, AT1 receptor antagonist) at three different occasions. Man normalized (144 ± 6 mmol l−1, mean ±s.d.) the increase in i.c.v. [Na+] seen after aCSF (161 ± 2 mmol l−1). Compared with control, both Man and Los significantly (P < 0.05) attenuated the improvement in mean arterial blood pressure (MAP), cardiac index and mesenteric blood flow (SMBF) in response to intravenous hypertonic NaCl: MAP, rapid response +45 mmHg versus+38 mmHg (Man) and +35 mmHg (Los); after 180 min, +32 mmHg versus+21 mmHg (Man) and +19 mmHg (Los); cardiac index after 180 min, +1.9 l min−1 (m2)−1versus+0.9 l min−1 (m2)−1 (Man) and +0.9 l min−1 (m2)−1 (Los); SMBF rapid response, +981 ml min−1versus+719 ml min−1 (Man) and +744 ml min−1 (Los); after 180 min, +602 ml min−1versus+372 ml min−1 (Man) and +314 ml min−1 (Los). The results suggest that increased periventricular [Na+] and cerebral AT1 receptors contribute, together with plasma volume expansion, to improve systemic haemodynamics after treatment with hypertonic NaCl in haemorrhagic hypovolaemia.

Endothelin Receptor A Antagonism Attenuates Renal Medullary Blood Flow Impairment in Endotoxemic Pigs

Background Endothelin-1 is a potent endogenous vasoconstrictor that contributes to renal microcirculatory impairment during endotoxemia and sepsis. Here we investigated if the renal circulatory and metabolic effects of endothelin during endotoxemia are mediated through activation of endothelin-A receptors. Methods and Findings A randomized experimental study was performed with anesthetized and mechanically ventilated pigs subjected to Escherichia coli endotoxin infusion for five hours. After two hours the animals were treated with the selective endothelin receptor type A antagonist TBC 3711 (2 mg⋅kg−1, nu200a=u200a8) or served as endotoxin-treated controls (nu200a=u200a8). Renal artery blood flow, diuresis and creatinine clearance decreased in response to endotoxemia. Perfusion in the cortex, as measured by laser doppler flowmetry, was reduced in both groups, but TBC 3711 attenuated the decrease in the medulla (pu200a=u200a0.002). Compared to control, TBC 3711 reduced renal oxygen extraction as well as cortical and medullary lactate/pyruvate ratios (p<0.05) measured by microdialysis. Furthermore, TBC 3711 attenuated the decline in renal cortical interstitial glucose levels (pu200a=u200a0.02) and increased medullary pyruvate levels (pu200a=u200a0.03). Decreased creatinine clearance and oliguria were present in both groups without any significant difference. Conclusions These results suggest that endothelin released during endotoxemia acts via endothelin A receptors to impair renal medullary blood flow causing ischemia. Reduced renal oxygen extraction and cortical levels of lactate by TBC 3711, without effects on cortical blood flow, further suggest additional metabolic effects of endothelin type A receptor activation in this model of endotoxin induced acute kidney injury.

Hypertonic sodium resuscitation after hemorrhage improves hemodynamic function by stimulating cardiac, but not renal, sympathetic nerve activity

Small volume hypertonic saline resuscitation can be beneficial for treating hemorrhagic shock, but the mechanism remains poorly defined. We investigated the effects of hemorrhagic resuscitation with hypertonic saline on cardiac (CSNA) and renal sympathetic nerve activity (RSNA) and the resulting cardiovascular consequences. Studies were performed on conscious sheep instrumented with cardiac (n=7) and renal (n=6) sympathetic nerve recording electrodes and a pulmonary artery flow probe. Hemorrhage (20 ml/kg over 20 min) caused hypotension and tachycardia followed by bradycardia, reduced cardiac output, and abolition of CSNA and RSNA. Resuscitation with intravenous hypertonic saline (1.2 mol/l at 2 ml/kg) caused rapid, dramatic increases in mean arterial pressure, heart rate, and CSNA, but had no effect on RSNA. In contrast, isotonic saline resuscitation (12 ml/kg) had a much delayed and smaller effect on CSNA, less effect on mean arterial pressure, no effect on heart rate, but stimulated RSNA, although the plasma volume expansion was similar. Intracarotid infusion of hypertonic saline (1 ml/min bilaterally, n=5) caused similar changes to intravenous administration, indicating a cerebral component to the effects of hypertonic saline. In further experiments, contractility (maximum change in pressure over time), heart rate, and cardiac output increased significantly more with intravenous hypertonic saline (2 ml/kg) than with Gelofusine (6 ml/kg) after hemorrhage; the effects of hypertonic saline were attenuated by the β-receptor antagonist propranolol (n=6). These results demonstrate a novel neural mechanism for the effects of hypertonic saline resuscitation, comprising cerebral stimulation of CSNA by sodium chloride to improve cardiac output by increasing cardiac contractility and rate and inhibition of RSNA.