Neutrophils develop rapid proinflammatory response after engulfing Hb-activated platelets under intravascular hemolysis.

Neutrophils maintain immune homeostasis by engulfing apoptotic cells and debris. We describe the rapid activation of neutrophils after engulfing hemoglobin (Hb)-activated platelets, which are abundant in the circulation of hemolytic patients. Neutrophils from healthy individuals after engulfing Hb-activated platelets express elevated CD11b and secrete significant amounts of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, myeloperoxidase (MPO) and elastase within 4-h platelets, but not with free-Hb only in vitro. These neutrophils exhibit early onset of apoptosis and cell death after engulfing Hb-activated platelets, but not with free-Hb only. Further, our data from mice with phenylhydrazine-induced intravascular hemolysis display a gradual decrease in total neutrophil count, but the number of activated neutrophils and neutrophil-platelet aggregates increases, along with the rise of TNF-α, IL-1ß, IL-6 and MPO in circulation. Our data from paroxysmal nocturnal hemoglobinuria (PNH) patients confirmed the observation of decreased total neutrophil counts, but elevated numbers of activated neutrophils, including neutrophil-platelet aggregates, in parallel with elevated expression of TNFA, IL1B and IL6 genes in neutrophils, also increased levels of these cytokines along with MPO in circulation, and this correlated directly with elevated intravascular hemolysis (high free-Hb in plasma). The patients' neutrophils displayed significant localization of intracellular Hb and platelets, unlike the counterparts from healthy individuals. Together, therefore, our observations suggest that Hb-activated platelets, which are abundant in the circulation of patients with hemolytic disorders, including PNH, promotes early onset of neutrophil activation and increases their proinflammatory response and leads to early apoptosis and cell death.

Erythrocyte membrane sulfatide plays a crucial role in the adhesion of sickle erythrocytes to endothelium.

Enhanced adhesion of sickle erythrocytes to the vascular endothelium and subendothelial matrix is fundamental to the development of vascular occlusion in sickle cell disease. Erythrocyte membrane sulfatide is implicated in the pathogenesis of vasoocclusive crises in sickle cell disease (SCD) patients. Because previous evidence linking sulfatide to cell adhesion has largely been circumstantial due to a lack of reagents that specifically target sulfatide, we used two sulfatide-specific strategies to address the role of erythrocyte membrane sulfatide in sickle cell adhesion to the vascular endothelium: a single-chain fragment variable chain (scFv) antibody against sulfatide as well as cerebroside sulfotransferase-deficient mice incapable of synthesising sulfatide. The sickle erythrocytes from mice and humans adhered at a greater extent and at higher shear stresses to activated endothelium than normal erythrocytes, and approximately 60% of the adhesion was prevented by the anti-sulfatide scFv. Similarly, the extent of adhesion of sulfatide-deficient erythrocytes was lower than normal erythrocytes. These findings suggest an important role for membrane sulfatide in sickle cell disease pathophysiology.

Von Willebrand factor present in fibrillar collagen enhances platelet adhesion to collagen and collagen-induced platelet aggregation.

We examined the basis of the differences observed between different collagen preparations in their ability to aggregate platelets and support their adhesion under flow. As in previous studies, we found fibrillar collagen to be 10-fold more potent than acid-soluble collagen in inducing platelet aggregation and found that acid-soluble collagen did not support the adhesion of washed platelets under flow. Further, platelets in whole blood adhered to surfaces coated with either fibrillar or acid-soluble collagen, but thrombi formed faster and grew larger on fibrillar collagen. As a possible basis for this difference, we found that fibrillar collagen, but not acid-soluble collagen, contains a substantial quantity of von Willebrand factor (VWF), as demonstrated by enzyme-linked immunosorbent assay and by the ability of fibrillar collagen to support the adhesion of VWF antibody-coated beads and to agglutinate GPIb-IX-V complex-expressing Chinese hamster ovary cells. Supporting a role for VWF in collagen-induced platelet aggregation, aggregation induced by acid-soluble collagen was greatly enhanced by added VWF. Further, platelet aggregation by fibrillar collagen was partially blocked by a GPIbalpha antibody that inhibits the GPIb-VWF interaction. Taken together, these results suggest that much of the difference in prothrombotic potency of different collagens is directly related to their differences in VWF content. This probably accounts for the different conclusions made regarding the relative importance of different direct and indirect collagen receptors in collagen-dependent platelet functions and further emphasizes the close synergistic roles of the GPIb-IX-V complex and the collagen receptors GPVI and alpha2beta1 in supporting platelet adhesion.

A reproductive phase-dependent effect of dietary L-tryptophan on pineal gland and gonad of a nocturnal bird, Indian spotted owlet Athene brama.

Unlike other temperate owls, Indian spotted owlet Athene brama possesses a well-developed pineal gland that secrets moderate amount of hydroxy- (serotonin) and methoxy- (melatonin) indoles in circulation. However, in this study, we have reported the response of this endocrine gland to exogenous L-Tryptophan (precursor of the above indoles), and also its effect on gonads of this nocturnal bird. During breeding phase or pineal inactive phase (March), oral treatment of L-Trp (0.5 mg/100 g Bwt/day) significantly increased the pineal gland wt and plasma melatonin (MEL) level, while decreased the gonadal wt and plasma sex steroids levels (estradiol and progesterone in female and testosterone in male). Interestingly, during reproductively quiescent phase or pineal active phase (August), similar amount of L-Trp significantly decreased the plasma MEL level, while increased the above sex steroid levels in plasma. Finally, the results show a clear reproductive phase-dependent inverse effect of L-Trp on pineal gland and gonads for both sexes of the spotted owlets, and suggest that the therapeutic use of this amino acid would be a great advantage for controlling the reproduction of these economically important birds.

Relationship between the annual rhythms in melatonin and immune system status in the tropical palm squirrel, Funambulus pennanti.

Melatonin (MEL) regulation of seasonal variation in immunity has been studied extensively in temperate mammals. This report is the first on a tropical mammal, the Indian palm squirrel, F. pennanti. In response to the annual environmental cycle, we studied the rhythms of plasma MEL and the immune parameters of total blood leucocytes, absolute blood lymphocytes and blastogenic responses of blood, thymus and spleen lymphocytes. We found that in parallel with MEL all the immune parameters increased during the month of April onward, when natural day length, temperature, humidity and rainfall were increasing. Maximum values occurred during November (reproductively inactive phase) when the values of all the physical factors were comparatively low. Lowest values occurred during January-March (reproductively active phase) when the values of the physical factors were lowest. In order to establish a clear interrelationship between the pineal MEL and the immune system function, we manipulated these squirrels with exogenous MEL (25mg/100 g B wt/day) at 1730 h during their pineal inactive phase (March) while another group was pinealectomized (Px) during November when their pineal was active. The MEL injection significantly increased all the immune parameters, while Px decreased them significantly. Hence, we suggest that MEL is immuno-enhancing for this tropical squirrel, and plays an important role in the maintenance of its immunity in accordance with the seasonal changes in environmental factors and gonadal status.

Pineal gland of a nocturnal bird, Indian spotted owlet, Athene brama: morphological and endocrine observations.

It has been reported that owls (Strigiformes) do not have a pineal gland. However, our light microscopy study revealed an intermediate form of tubulofollicular and solid-type large pineal gland in a tropical owlet, Athene brama. The epithelial cells forming follicles (6-8) in the distal region and the solid cluster of parenchymal cells of different diameters in the proximal region anteriorly tapered with a long cylindrical stalk and continued into commissural organs and choroid plexus. The intrapineal localization of perivascular nerve fibers and blood vessels clearly explained the sympathetic innervation as well as vascularization of this neuroendocrine gland. Further, electron microscopy revealed a developed intracellular structure of the pinealocytes with a large number of mitochondria, Golgi bodies, and granular as well as clear vesicles in the process terminals. The evidence of intrapinealocyte lipid droplets and dense bodies and a moderate amount of melatonin in plasma (ranging from 100-365 pg/mL) during different reproductive phases finally proved a defined secretory activity of the gland in this tropical, nocturnal bird.

Time and reproductive phase-dependent effects of exogenous melatonin on the pineal gland and ovary of a nocturnal bird, the Indian spotted owlet, Athene brama.

In a tropical nocturnal bird, the Indian spotted owlet, Athene brama, the intraperitonial injection of an identical amount (20 mg/100 g b. wt/day) of exogenous melatonin (MEL) for 15 consecutive days increased the pineal weight and plasma MEL level in sexually active birds while it decreased them in inactive birds more potently when injected in the evening (18.30-19.30 h) rather than the morning (0500-0600 h). On the other hand, more efficiently than the morning hour treatment, the evening hour MEL injection decreased the ovary weight and plasma estradiol and progesterone levels both in sexually active and inactive birds, but more potently in active than inactive birds. Thus, the exogenous MEL showed the time and reproductive phase dependent effects on the pineal gland and the ovary of this nocturnal bird.