L-selectin tyrosine phosphorylates cbl and induces association of tyrosine-phosphorylated cbl with crkl and grb2.

L-Selectin-mediated rolling of leukocytes on endothelial cells is an important step for lymphocyte homing and an early event in the immune response to pathogens or inflammatory stimuli. We have previously elucidated intracellular signaling cascades upon L-selectin engagement resulting in activation of Ras, Rac and JNK as well as cytoskeletal changes, oxygen release, ceramide synthesis and receptor capping. Activation of the src-tyrosine kinase p56lck is followed by phosphorylation of the L-selectin molecule and MAP-K. Here we show a tyrosine kinase dependent phosphorylation of the Cbl adapter protein after L-selectin engagement in lymphocytes. Phosphorylation of Cbl was absent in Jurkat cells that are pharmacologically treated with tyrosine kinase inhibitors and in lck-deficient JCaM cells. There is an activation induced association of tyrosine phosphorylated Cbl with Grb2 and CrkL, respectively, but not CrkII. Therefore, the adapter protein Cbl plays a role in L-selectin signaling and might modulate immune function by the specific recruitment of signaling molecules to multiprotein complexes.

Surfactant modulates intracellular signaling of the adhesion receptor L-selectin.

Intraalveolar leukocyte accumulation is one of the hallmarks during respiratory distress. In the intraalveolar space, leukocyte activation is mediated by pathogens, cytokines, and different ligands binding to adhesion molecules. Leukocyte stimulation via the adhesion molecule L-selectin is specifically induced by ligands expressed on leukocytes, platelets, endothelial cells, or lipopolysaccharide. Recently, we have demonstrated that leukocyte activation by L-selectin transmits several intracellular signaling cascades resulting in capping and cytoskeletal changes, the activation of kinases and neutral sphingomyelinase, the recruitment of adaptor proteins to the cell membrane, the activation of the small G-proteins Ras and Rac, and the release of oxygen. In the present study, we examined the effects of surfactant on L-selectin-induced signal transduction in leukocytes. Using fluorescence microscopy, we provide evidence that preincubation of leukocytes with surfactant significantly inhibits receptor capping; 28+/-7% of cells show capping after L-selectin stimulation versus 8+/-5% and 3+/-1% of cells after preincubation with Exosurf and Curosurf, respectively (p < 0.05). The activity of the neutral sphingomyelinase in cell lysates is also modulated by surfactant. In addition, we show that the activation of the tyrosine kinase p56lck is diminished by approximately 50% after surfactant treatment. This results in inhibition in tyrosine phosphorylation of certain intracellular proteins. The interaction of the L-selectin molecule with its antibody was not influenced by surfactant as shown by flow cytometry. Surfactant inhibits intracellular signaling events of the L-selectin receptor in leukocytes and might therefore contribute to the modulatory effects of surfactant on immune function.

Neutrophil respiratory burst in term and preterm neonates without signs of infection and in those with increased levels of C-reactive protein.

Developmental immaturities in neonatal host defense predispose the neonates to an increased mortality rate during bacterial infections. Early diagnosis is of great clinical importance, but, especially in neonates, is sometimes very difficult. The ability to generate reactive oxygen species, the so-called respiratory burst, is essential for neutrophils to kill infectious microorganisms. Therefore, changes of respiratory burst may reflect increased susceptibility of neonates to infections and may be useful for the early detection of infections. Superoxide anion production was determined by a flow cytometric method using dihydrorhodamine 123 (DHR) as an oxidative probe after priming of neutrophils with PBS buffer (spontaneous burst), with N-formyl-methionyl-leucyl-phenylalanine (fMLP), or with Escherichia coli. During the study period, the spontaneous percentage of activated cells in whole blood as well as the percentage of activated cells in stimulation with fMLP was lower in adults (n = 100; PBS, 1.0 +/- 0.1%; fMLP, 8.3 +/- 0.9%) compared with neonates without signs of infection (n = 143). Among the latter, the percentage of activated cells (PBS and fMLP assay) varied with respect to gestational age and hours of life: lowest values were measured in preterm newborns with gestational age less than 32 wk and between 25 and 120 h of life. The same correlation to gestational age was true for total neutrophil cell counts. In neonates with increased levels of C-reactive protein during the first 5 d of life (n = 43), the percentages of activated cells after PBS and fMLP incubation were higher than those of neonates without signs of infection. The relationship of neutrophil respiratory burst and neutrophil cell counts to gestational age might reflect at least in part a reason for the increased susceptibility of neonates to infections. Furthermore, determination of respiratory burst may prove to be a new laboratory parameter of neonatal infection.

A new side effect of inhaled nitric oxide in neonates and infants with pulmonary hypertension: functional impairment of the neutrophil respiratory burst.

INTRODUCTION: Inhaled nitric oxide (NO) may be beneficial in the treatment of pulmonary hypertension, both of the newborn and in the adult respiratory distress syndrome. Up to now, serious systemic side effects have not been reported. OBJECTIVE: The effect of inhaled NO on superoxide anion production by neutrophils. DESIGN: Prospective study of a consecutive series of 15 neonates and infants. SETTING: Neonatal and paediatric ICUs with a total of 17 beds (university hospital). MEASUREMENTS AND RESULTS: Superoxide anion production was determined by a flow cytometric method using dihydrorhodamine 123 (DHR) as an oxidative probe after the priming of neutrophils with N-formyl-methionyl- leucylphenylalanine (fMLP) or with Escherichia coli. The generated fluorescence was expressed as relative fluorescence intensity (RFI). Inhalation of NO for more than 24 h reduced the superoxide anion production by neutrophils stimulated with E. coli to below baseline values before NO inhalation (mRFI = 158 +/- 25 vs 222 +/- 24; P = 0.03). This decrease was more pronounced after more than 72 h (mRFI = 133 +/- 17). At this time, superoxide anion production by fMLP-stimulated neutrophils was also decreased (mRFI = 40 +/- 3, vs 57 +/- 5; P = 0.03). The reduced capacity of superoxide production persisted throughout therapy with NO and lasted up to more than 4 days after the end of NO inhalation. CONCLUSION: The results suggest that inhalation of NO in patients with pulmonary hypertension causes reduced superoxide anion production by neutrophils stimulated with E. coli or with fMLP. To determine the clinical importance of this systemic side effect with respect to bacterial infections, a randomized controlled study is necessary.