Hemolytically inactive C5b67 complex: an agonist of polymorphonuclear leukocytes

Abstract

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

In vitroandin vivoresponses of murine granulocytes to human complement-derived, haemolytically inactive C5b67 (iC5b67)

“…Recombinant human C5a was purchased from Sigma. C5b6 and iC5b67 were formed from purified B, D, cobra venom factor, C5, C6 and C7 as described [4].…”

“…In vitro chemotaxis assay Leucocytes were loaded with calcein-AM and the chemotaxis assay was performed using a polyvinylpyrrolidone-free polycarbonate filter (3 mm for PMN, 5mm for eosinophils) and a 96-well chemotaxis chamber (Neuroprobe, Cabin John, MD) [4]. Migrating cells remained adherent to the underside of the filter and were quantified using a fluorescence plate reader (Millipore, Bedford, MA).…”

“…iC5b67 also functions differently from C5a in terms of regulating complement receptors CR1 (CD35) and CR3 (CD11b/CD18) expression of human PMN: namely, iC5b67 does not up-regulate CR1 and CR3 expression, while C5a does [5]. iC5b67 stimulates many second messengers, including calcium, G-protein, GTPase, cAMP, tyrosine kinase and PI-3 kinase [6]. We think that iC5b67 probably signals through a specific receptor, but this has been difficult to prove by standard ligand binding assay because iC5b67 aggregates.…”

“…Third, C5b67 can refold in the fluid phase forming a haemolytically inactive C5b67 complex (iC5b67) [3]. We have found that human iC5b67, which signals from outside the cell, is both an agonist and an antagonist for human polymorphonuclear leucocytes (PMN) in vitro [4]. iC5b67 is a potent chemotactic factor for PMN, while it fails to stimulate superoxide generation and can even antagonize the superoxide production elicited by C5a and N-formyl-Met-Leu-Phe (FMLP).…”

“…Although the chemotactic activities of iC5b67 for human PMN have been well documented in vitro [4,7,8], there have been no data as to whether iC5b67 formed from purified components would function in vivo. To determine if the mouse would be an appropriate model for testing iC5b67 in vivo, we had first to ascertain if murine PMN were able to respond to human iC5b67.…”