Daniel Granfeldt

Capacitative Ca2+ influx and activation of the neutrophil respiratory burst. Different regulation of plasma membrane- and granule-localized NADPH-oxidase

The neutrophil NADPH‐oxidase may be activated in the plasma membrane, resulting in release of oxygen metabolites extracellularly, or in the granule or phagosomal membranes, giving intracellular production of oxidants. An increase in [Ca2+]i mediated through binding of fMLF to its receptor is part of a signaling cascade that activates the plasma membrane‐localized oxidase. In contrast, a rise in [Ca2+]i induced by a Ca2+ ionophore results in activation of the intracellular pool of oxidase. We mimicked fMLF‐induced emptying of intracellular Ca2+ stores with thapsigargin. This induced a pronounced intracellular oxidase activity but no extracellular release of oxidants. The thapsigargin‐induced effect was dependent on capacitative Ca2+ influx, because the effect was inhibited dose‐dependently by EGTA and the Ca2+ channel blocker La3+. At La3+ concentrations between 200 and 400 μM, thapsigargin also induced a massive extracellular production of superoxide anion. No other channel blockers tested induced a similar effect. We conclude that elevation in [Ca2+]i by capacitative Ca2+ influx induces NADPH‐oxidase activation at an intracellular site. Further, activation of the plasma membrane‐localized NADPH‐oxidase is regulated by a more complex Ca2+ signaling, involving capacitative Ca2+ influx and possibly the specific action of La3+‐sensitive Ca2+ channels.

An Intact Cytoskeleton is Required for Prolonged Respiratory Burst Activity During Neutrophil Phagocytosis

The temporal relationship between phagocytosis and respiratory burst activity was investigated. Neutrophil uptake of yeast particles was synchronized and the kinetics of the oxidative burst was determined using an isoluminol/luminol amplified chemiluminescence system. The reactive oxygen species were mainly generated intracellularly (defined as the activity that remained in an luminol-enhanced system in the presence of superoxide dismutase and catalase). Following phago- cytosis, the intracellular response rapidly reached a level close to the maximum and the activity was almost constant for the first 10 to 15 min. The response then slowly declined. The presence of cytochalasin B, an inhibitor of actin polymerization, greatly reduced the respiratory burst activity, and this was true also when the inhibitor was added after completion of uptake of yeast particles. Our results thus show that there is a continuous production of oxygen metabolites long after phagocytosis is completed. There is also a requirement for an intact cytoskeleton for prolonged superoxide production inside the phagosome.

Inhibition of phospholipase A2 abrogates intracellular processing of NADPH-oxidase derived reactive oxygen species in human neutrophils

Upon activation of human neutrophils, superoxide can be produced at two cellular sites; either in the plasma membrane, giving extracellular release of oxidants, or in intracellular organelles, resulting in oxidants being retained in the cell. The involvement of phospholipase A(2) (PLA(2)) in phorbol myristate acetate (PMA)-induced activation of the two pools of NADPH-oxidase was investigated using a variety of PLA(2) inhibitors and the oxidase activity was measured by luminol/isoluminol-amplified chemiluminescence (CL). Two of the seven inhibitors were without effect, two inhibitors inhibited both intra- and extracellular ROS production equally, and three inhibitors inhibited intracellular but not extracellular CL. Using another technique to measure ROS, PHPA oxidation, we found that intracellular ROS production was unaltered with the three last inhibitors, indicating that PLA(2) is not involved in the NADPH-oxidase activity per se, but in the intracellular processing of the radicals necessary for the CL reaction to take place. The PLA(2) inhibitors did not abolish the activity of myeloperoxidase (MPO), an enzyme necessary for intracellular CL to occur. Instead, we suggest that these PLA(2) inhibitors block heterotypic granule fusion and prohibit the colocalization of ROS and MPO needed for intracellular CL activity.

Comorbidities, treatment patterns and cost-of-illness of acromegaly in Sweden: a register-linkage population-based study

OBJECTIVE Acromegaly is a complex endocrine disease with multiple comorbidities. Treatment to obtain biochemical remission includes surgery, medical therapy and radiation. We aimed to describe comorbidities, treatment patterns and cost-of-illness in patients with acromegaly in Sweden. DESIGN A nationwide population-based study. METHODS Patients with acromegaly were identified and followed in national registers in Sweden. Longitudinal treatment patterns were assessed in patients diagnosed between July 2005 and December 2013. The cost-of-illness during 2013 was estimated from a societal perspective among patients diagnosed between 1987 and 2013. RESULTS Among 358 patients with acromegaly (48% men, mean age at diagnosis 50.0 (s.d. 15.3) years) at least one comorbidity was reported in 81% (n = 290). The most common comorbidities were hypertension (40%, n = 142), neoplasms outside the pituitary (30%, n = 109), hypopituitarism (22%, n = 80) and diabetes mellitus (17%, n = 61). Acromegaly treatment was initiated on average 3.7 (s.d. 6.9) months after diagnosis. Among the 301 treated patients, the most common first-line treatments were surgery (60%, n = 180), somatostatin analogues (21%, n = 64) and dopamine agonists (14%, n = 41). After primary surgery, 24% (n = 44) received somatostatin analogues. The annual per-patient cost was €12 000; this was €8700 and €16 000 if diagnosed before or after July 2005, respectively. The cost-of-illness for acromegaly and its comorbidities was 77% from direct costs and 23% from production loss. CONCLUSIONS The prevalence of comorbidity is high in patients with acromegaly. The most common first-line treatment in acromegalic patients was surgery followed by somatostatin analogues. The annual per-patient cost of acromegaly and its comorbidities was €12 000.

Neutrophil secretion induced by an intracellular Ca2+ rise and followed by whole-cell patch-clamp recordings occurs without any selective mobilization of different granule populations

We have investigated calcium-induced secretion in human neutrophils, using a whole-cell patch-clamp technique. Mobilization of subcellular granules to the cell membrane was followed as the change in membrane capacitance (ΔCm). Both the magnitude and the kinetics of the response differed between low and high concentrations of Ca2+. A sustained secretion following a short lag phase was induced by high concentrations of Ca2+ (100 μM and higher). A stable plateau was reached after 5–7 minutes at ΔCm values corresponding to values expected after all specific as well as azurophil granules have been mobilized. Capacitance values of the same magnitude could be obtained also at lower Ca2+ concentrations, but typically no stable plateau was reached within the measuring time. In contrast to previous studies, we were unable to detect any pattern of secretion corresponding to a distinct submaximal response or selective mobilization of granule subsets specified by their Ca2+-sensitivity.