Signaling by hemolytically inactive C5b67, an agonist of polymorphonuclear leukocytes

Area of Science:

• Immunology
• Cell Signaling
• Complement System

Background:

• The complement component complex C5b67 (iC5b67) is known to signal human polymorphonuclear leukocytes (PMN).
• Its dual role as an agonist for chemotaxis and antagonist for C5a- and FMLP-stimulated functions requires further investigation into its signaling pathways.

Purpose of the Study:

• To investigate the specific signaling pathways utilized by iC5b67 in human PMN.
• To determine if iC5b67 signals through the C5a receptor or a distinct receptor.
• To elucidate the mechanisms behind iC5b67's agonist and antagonist functions.

Main Methods:

• Transfection of COS cells with G alpha 16 and C5a receptor.
• Measurement of inositol phosphate formation and phospholipase C activity.
• Assessment of GTPase activity in PMN membrane fractions.
• Analysis of intracellular Ca2+ mobilization.
• Evaluation of iC5b67-induced PMN chemotaxis and superoxide production using inhibitors (herbimycin A, wortmannin, chelerythrine).
• Measurement of intracellular cAMP levels.

Main Results:

• iC5b67 failed to directly activate G proteins or C5a receptor-mediated signaling in COS cells.
• iC5b67 stimulated GTPase activity in PMN and mobilized intracellular Ca2+ stores.
• iC5b67-induced PMN chemotaxis involved tyrosine and phosphatidylinositol 3-kinases, but not protein kinase C.
• iC5b67 induced increases in intracellular cAMP, which can inhibit superoxide production.
• iC5b67 antagonized C5a and FMLP receptor-mediated superoxide generation but not PMA-induced production.

Conclusions:

• iC5b67 signals through a unique, likely G protein-linked receptor, distinct from the C5a receptor.
• The signaling pathways for iC5b67's agonist (chemotaxis) and antagonist (superoxide production) functions diverge early in the transduction process.
• iC5b67's antagonistic effect on superoxide production is mediated by an increase in intracellular cAMP.