Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47 PHOX. Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47 PHOX may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

Evaluation of antibody responses to pneumococcal vaccines with ELISA and opsonophagocytic assay

Antibodies to a capsular polysaccharide (PS) provide protection against Streptococcus pneumoniae which express the homologous capsular serotype, and pneumococcal vaccines are designed to induce antibodies in the capsular PS. Levels and opsonophagocytic capacity of antibodies to the capsular PS of S. pneumoniae serotype 19F were determined by sera from adults immunized with 23-valent S. pneumoniae capsular PS vaccines. Geometric means of IgG anti-19F antibody level and specific opsonic titer rise significantly after immunization. The level of anticapsular PS antibodies for S. pneumoniae 19F serotype is fairly well correlated (r2=O.63) with the opsonophagocytic activities of sera. However, 3.7% (1/27) of serum samples display strikingly less opsonophagocytic activity than expected on the basis of their antibody level. Thus, antibody level may be of general use in predicting vaccine-induced protection among adults for 19F serotype. However, the opsonic activity data suggest that antibody levels are not always indicative of functional antibody.

In vitro effect of intravenous immunoglobulin supplementation on serum opsonic activity in tracheostomised patients.

An in vitro study of the effect of immunoglobulins (mainly IgG) on opsonophagocytic activity of polymorphs was carried out in 17 tracheostomised patients admitted in medical intensive care unit of our hospital. The opsonic and phagocytic activities were tested against Staphylococcus aureus by modified polymorphonuclear leucocyte overlay method; and serum IgG and serum IgM levels were estimated by single radial immunodiffusion technique. As compared to healthy volunteers, opsonophagocytic activity was significantly lower in tracheostomised patients. However, this activity improved markedly after immunoglobulin supplementation (P less than 0.01). The same degree of enhancement was also observed in normal controls.